PVRIG Binding Protein and Its Pharmaceutical Use

Abstract

The present invention provides PVRIG-binding proteins and pharmaceutical uses thereof, particularly anti-PVRIG single-domain antibodies and anti-PVRIG / TIGIT bispecific antibodies, pharmaceutical compositions containing the antibodies, and methods for treating cancer and pharmaceutical uses thereof.

Description

Technical Field

[0001] This disclosure claims priority to a Chinese patent application (Application No. CN202010174835.4) filed on March 13, 2020.

[0002] This disclosure relates to PVRIG-binding proteins such as anti-PVRIG antibodies and bispecific antibodies formed with anti-TIGIT antibodies, and their use as drugs in the treatment of cancer.

Background Art

[0003] Cancer is the biggest health problem that the current human society has been facing for a long time. Conventional therapies such as surgery, chemotherapy, and radiotherapy have little effect in the treatment of disseminated solid tumors. Tumor immunotherapy is one of the hotspots in the field of tumor treatment, and among them, T cell-based tumor immunotherapy is also at the core. Tumor immunotherapy kills tumors by fully utilizing and mobilizing killer T cells in the body of tumor patients, and can be the most effective and safest method for treating tumors. Currently, tumor immunotherapy has a strong prospect of treating several different types of cancers including disseminated metastatic tumors.

[0004] The activation of T cells in the human body adopts a system including two signal transduction pathways. In addition to providing the first signal by presenting MHC-antigen peptides to T cells by antigen-presenting cells (APCs), a series of co-stimulatory molecules are also required to provide the second signal, which enables the normal immune response of T cells for the first time. This dual signal transduction pathway system plays a very important role in the balance of the body's immune system and strictly regulates the body's different immune responses to self-antigens and non-self-antigens. The lack of the second signal provided by co-stimulatory molecules causes unresponsiveness or persistent specific immune responses of T cells, resulting in immune tolerance. Therefore, the second signal transduction pathway plays a very crucial regulatory role in the whole process of the body's immune response.

[0005] PVRIG, also known as CD112R, is a protein expressed on the cell surface. It belongs to the B7 / CD28 superfamily like TIGIT, CD96, CD226, etc., and plays an important role in the immune system. It contains one extracellular region, one transmembrane region, and one intracellular region. When its ligand PVRL2 (also called CD112) binds to PVRIG, the ITIM domain in the intracellular region of PVRIG is activated so that PVRIG exerts an immunosuppressive effect.

[0006] 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, renal cancer, gastric cancer, endometrial cancer, head and neck cancer, etc. The expression of PVRIG in these cancers is highly correlated with that of TIGIT and PD-1. Similar to PD-1 and TIGIT, PVRIG-positive T cells also show Eomes positivity and Tbet negativity, suggesting that PVRIG is associated with T cell exhaustion. Therefore, PVRIG represents a new immune checkpoint other than PD-1 and TIGIT and may play a role in redundancy. As is clear from in vitro cell experiments and mouse models, knocking out or inhibiting mouse PVRIG can effectively inhibit tumor growth and produce a synergistic effect with PD-1 and TIGIT inhibitors.

[0007] Another target of interest, TIGIT, is highly expressed in lymphocytes including tumor-infiltrating lymphocytes (TILs) and Tregs that infiltrate different types of tumors. It has been demonstrated that the binding of TIGIT signaling to its cognate ligand PVR (also called CD155) directly inhibits the cytotoxicity of NK cells via its cytoplasmic ITIM domain. PVR is also widely expressed in tumors, suggesting that the TIGIT-PVR signaling axis may be a major immune evasion mechanism in cancer.

[0008] However, there has never been a PVRIG / TIGIT bispecific antibody drug used clinically. Compugen's COM701 is a humanized hybridoma antibody against PVRIG that was first approved by the FDA worldwide for clinical use and is currently in Phase I clinical trials and is used to treat cancer. Surface Oncology is also developing the anti-PVRIG antibody SRF-813. Anti-TIGIT antibodies include Genentech's tiragolumab, BMS-986207 co-developed by Ono Pharmaceutical and BMS, Merck Sharp & Dohme's MK-7684, iTeos Therapeutics' EOS-884448, and Arcus Biosciences' AB-154, all of which are in Phase II clinical trials.

[0009] In the prior art, there is still a lack of anti-PVRIG antibodies and anti-PVRIG / TIGIT bispecific antibodies with high affinity, high selectivity, and high biological activity that can inhibit the growth of cancer or tumors in the body. The present disclosure aims to provide antibodies that activate the immune system and treat cancer by blocking the inhibitory pathways of PVRIG and / or TIGIT.

Summary of the Invention

[0010] The present disclosure provides a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), a bispecific antibody thereof and an anti-TIGIT antibody, as well as their coding nucleic acids, vectors, host cells, pharmaceutical compositions, methods for treating cancer, and pharmaceutical uses.

[0011] In a first aspect, the present disclosure provides a PVRIG-binding protein or an anti-PVRIG antibody.

[0012] In some embodiments, 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 called CDR1, CDR2, and CDR3, among which, CDR1 is selected from the amino acid sequences shown in any one of SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64 or amino acid sequences having 3, 2, 1 or more amino acid differences therefrom, and / or CDR2 is selected from the amino acid sequences shown in any one of SEQ ID NO: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65 or amino acid sequences having 3, 2, 1 or more amino acid differences therefrom, and / or CDR3 is selected from the amino acid sequences shown in any one of 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 or amino acid sequences having 3, 2, 1 or more amino acid differences therefrom. Among them, SEQ ID NO: 7-21, 150, 151 follow the Kabat numbering rule, SEQ ID NO: 22-36 follow the Chothia numbering rule, SEQ ID NO: 37-51 follow the IMGT numbering rule, and SEQ ID NO: 52-66 follow the AbM numbering rule.

[0013] In some embodiments, the PVRIG-binding protein comprises at least one immunoglobulin single variable domain, and the at least one immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO:2 and positions 75 to 79, or CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO:3 and positions 80 to 84, or CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO:4 and positions 86 to 90, or CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO:5 and positions 91 to 95, or CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO:6 and positions 96 to 100, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, and in some specific embodiments, the CDR is determined according to the Kabat numbering rule.

[0014] In some embodiments, according to the Kabat numbering rule, the immunoglobulin single variable domain of the PVRIG-binding protein comprises three complementarity-determining regions, namely CDR1, CDR2, and CDR3, among which, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:7, 8, and 9 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:7, 8, and 150 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:10, 11, and 12 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:10, 11, and 151 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:13, 14, and 15 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:16, 17, and 18 respectively, or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, 20, and 21, respectively.

[0015] In some embodiments, according to the Chothia numbering convention, the immunoglobulin single variable domain of the PVRIG binding protein contains three complementarity-determining regions, CDR1, CDR2, and CDR3, among which, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 22, 23, and 24, respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 25, 26, and 27, respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 28, 29, and 30, respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 31, 32, and 33, respectively, or the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 34, 35, and 36, respectively.

[0016] In some embodiments, according to the IMGT numbering convention, the immunoglobulin single variable domain of the PVRIG binding protein contains three complementarity-determining regions, CDR1, CDR2, and CDR3, among which, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 37, 38, and 39, respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 40, 41, and 42, respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 43, 44, and 45, respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 46, 47, and 48, respectively, or the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 49, 50, and 51, respectively.

[0017] In some embodiments, according to the AbM numbering rule, the immunoglobulin single variable domain of the PVRIG binding protein comprises three complementarity determining regions, namely CDR1, CDR2 and CDR3, among which, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:52, 53, and 54 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:55, 56, and 57 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:58, 59, and 60 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:61, 62, and 63 respectively, or the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:64, 65, and 66 respectively.

[0018] In some embodiments, a PVRIG binding protein comprising an immunoglobulin single variable domain is provided, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, and according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively shown in SEQ ID NO:7, 8 and 9, or shown in SEQ ID NO:7, 8 and 150, or shown in SEQ ID NO:10, 11 and 12, or shown in SEQ ID NO:10, 11 and 151, or shown in SEQ ID NO:13, 14 and 15, or shown in SEQ ID NO:16, 17 and 18, or shown in SEQ ID NO:19, 20 and 21.

[0019] In some embodiments, 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.

[0020] In some embodiments, 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 NOs: 2-6, or has at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In some embodiments, the PVRIG-binding protein is a mutant protein having 3, 2, 1 or more amino acid differences in CDR1 of the PVRIG-binding protein, and / or 3, 2, 1 or more amino acid differences in CDR2, and / or 3, 2, 1 or more amino acid differences in CDR3.

[0021] In some embodiments, an anti-PVRIG antibody comprising CDR1, CDR2, and CDR3 in the above PVRIG-binding protein is provided. It may be humanized and / or affinity matured. In some specific embodiments, the amino acid sequence of the anti-PVRIG antibody is shown in any one of SEQ ID NOs: 2-6, 75-84, 86-100, or has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto. In some specific embodiments, the anti-PVRIG single domain antibody is connected to the Fc region of human IgG1, IgG2, IgG3, IgG4, for example, connected to the Fc region of IgG4 having S228P, F234A, L235A and / or K447A mutations (for example, shown in SEQ ID NO: 101 or 153).

[0022] In some embodiments, 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 a humanized and / or affinity matured VHH.

[0023] In some embodiments, the PVRIG-binding protein of the present disclosure comprises an antibody.

[0024] In some embodiments, the PVRIG-binding protein of the present disclosure is an antibody (e.g., VHH).

[0025] In some embodiments, the PVRIG-binding protein of the present disclosure is a camel antibody, a humanized antibody, or a fully human antibody.

[0026] In some embodiments, 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 the VHH is shown in any one of SEQ ID NOs: 2 to 6, or has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0027] In some embodiments, the PVRIG-binding protein of the present disclosure or the immunoglobulin single variable domain therein is a humanized antibody, and the framework region of the antibody is a heavy chain framework region of a human germline template, such as IGHV3-7, specifically, for example, IGHV3-7*01 or IGHV3-30*02.

[0028] In some specific embodiments, the amino acid sequence of the anti-PVRIG humanized antibody of the present disclosure is shown in any one of SEQ ID NOs: 75 to 84, 86 to 100, or has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0029] In some embodiments, the PVRIG-binding protein of the present disclosure comprises or is a humanized antibody comprising a heavy chain framework region of a human germline template.

[0030] In some embodiments, the heavy chain framework region of the human germline template is IGHV3-7*01 or IGHV3-30*02.

[0031] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain of the humanized antibody is shown in any one of SEQ ID NOs: 75-84 and 86-100, or has at least 90%, at least 95%, at least 98% or at least 99% sequence identity therewith.

[0032] In some embodiments, 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, IgG4. In some specific embodiments, the human immunoglobulin Fc region is the Fc region of human IgG4. In some specific embodiments, the human immunoglobulin Fc region is the Fc region of human IgG1. The Fc region may have mutations such as amino acid mutations of S228P, F234A, L235A and / or K447A (e.g., as shown in SEQ ID NO: 101 or 153).

[0033] In some embodiments, in the PVRIG-binding protein of the present disclosure, the immunoglobulin single variable domain capable of specifically binding to PVRIG is connected to the immunoglobulin Fc region directly or by a linker. The linker may have a length of 1-20, 1-30, 1-40, 1-50 or more amino acids and may be a non-functional amino acid sequence without secondary or higher structures. The linker may be a flexible linker such as GS, GAP, ASGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, YGNGT, (YGNGT)2, (YGNGT)3, (YGNGT)4, (YGNGT)5, (YGNGT)6, etc.

[0034] In some specific embodiments, the Fc region in the PVRIG-binding protein of the present disclosure enables the PVRIG-binding protein to form a dimer molecule containing 2 or 4 PVRIG-binding domains. Such a PVRIG-binding protein is also called a bivalent or tetravalent PVRIG-binding protein. The dimer is, for example, a homodimer.

[0035] The PVRIG-binding protein or anti-PVRIG antibody of the present disclosure has at least one of the following characteristics: (a) The KD value of the binding to human PVRIG is less than 1×10 -7 M. (b) It blocks the interaction between PVRIG and its ligand (e.g., PVRL2). (c) It relieves the inhibitory effect of dendritic cells on T cells and activates T cells. (d) It relieves the inhibitory effect of tumor cells on NK cells. (e) It inhibits tumor growth.

[0036] The KD value of the binding of the PVRIG-binding protein or anti-PVRIG antibody of the present disclosure to PVRIG is less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, less than 1×10 -10 M and may also be less.

[0037] 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%.

[0038] The PVRIG-binding protein or anti-PVRIG antibody of the present disclosure may be a monomer, and / or PEGylated, and / or glycosylated, and / or albumin-conjugated or fused, and / or Fc-fused, and / or hydroxyethylated, and / or de-O-glycosylated.

[0039] In a second aspect, the present disclosure provides a bispecific antibody against PVRIG.

[0040] In some embodiments, a bispecific antibody is provided that includes a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to PVRIG.

[0041] In some embodiments, the first antigen-binding domain of the bispecific antibody of the present disclosure specifically binds to PVRIG, and the first antigen-binding domain includes at least one immunoglobulin single variable domain (e.g., VHH), and the at least one immunoglobulin single variable domain (e.g., VHH) includes three complementarity-determining regions called CDR1, CDR2, and CDR3, wherein CDR1 is selected from the amino acid sequences shown in any one of SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64 or an amino acid sequence having 3, 2, 1, or more amino acid differences therefrom, and / or CDR2 is selected from the amino acid sequences shown in any one of SEQ ID NO: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65 or an amino acid sequence having 3, 2, 1, or more amino acid differences therefrom, and / or CDR3 is selected from the amino acid sequences shown in any one of 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 or an amino acid sequence having 3, 2, 1, or more amino acid differences therefrom. Among them, SEQ ID NOs: 7-21 follow the Kabat numbering rule, SEQ ID NOs: 22-36 follow the Chothia numbering rule, SEQ ID NOs: 37-51 follow the IMGT numbering rule, and SEQ ID NOs: 52-66 follow the AbM numbering rule.

[0042] In some embodiments, 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 CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO: 2, 75 to 79, or comprises CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO: 3, 80 to 84, or comprises CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO: 4, 86 to 90, or comprises CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO: 5, 91 to 95, or comprises CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NO: 6, 96 to 100, and the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the CDRs are determined according to the Kabat numbering rule.

[0043] In some specific embodiments, according to the Kabat numbering rule, the first antigen-binding domain (such as VHH) that specifically binds to PVRIG comprises three complementarity-determining regions, namely CDR1, CDR2, and CDR3, among which, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, 8, and 9 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, 8, and 150 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 10, 11, and 12 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 10, 11, and 151 respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 13, 14, and 15 respectively, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 16, 17, and 18, respectively, or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, 20, and 21, respectively.

[0044] In some specific embodiments, according to the Chothia numbering rule, the first antigen-binding domain (e.g., VHH) that specifically binds to PVRIG includes three complementarity-determining regions, namely CDR1, CDR2, and CDR3, among which, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 22, 23, and 24, respectively, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 25, 26, and 27, respectively, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 28, 29, and 30, respectively, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 31, 32, and 33, respectively, or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 34, 35, and 36, respectively.

[0045] In some specific embodiments, according to the IMGT numbering rule, the first antigen-binding domain (e.g., VHH) that specifically binds to PVRIG includes three complementarity-determining regions, namely CDR1, CDR2, and CDR3, among which, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 37, 38, and 39, respectively, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 40, 41, and 42, respectively, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 43, 44, and 45, respectively, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 46, 47, and 48, respectively, or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 49, 50, and 51, respectively.

[0046] In some specific embodiments, according to the AbM numbering rule, the first antigen-binding domain (e.g., VHH) that specifically binds to PVRIG includes three complementarity-determining regions, namely CDR1, CDR2, and CDR3. Among them, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 52, 53, and 54, respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 55, 56, and 57, respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 58, 59, and 60, respectively, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 61, 62, and 63, respectively, or the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 64, 65, and 66, respectively.

[0047] In some embodiments, the first antigen-binding domain (e.g., VHH) of the bispecific antibody of the present disclosure includes the amino acid sequence shown in any one of SEQ ID NOs: 2-6, 75-84, 86-100, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto.

[0048] In some embodiments, in the bispecific antibody of the present disclosure, the first antigen-binding domain is the first antibody which is VHH, the second antigen-binding domain is the second antibody which includes a heavy chain (HC) and a light chain (LC), the VHH is located at the N-terminus and / or C-terminus of the heavy chain or light chain of the second antibody as the first antibody.

[0049] In some specific embodiments, the bispecific antibody of the present disclosure comprises a first antibody of one second antibody and two VHHs, wherein the second antibody comprises two HCs and two LCs, and the VH of one HC and the VL of one LC of the second antibody form an antigen-binding site, and the VH of the other HC and the VL of the other LC form an antigen-binding site.

[0050] In some specific embodiments, the first antibody of one VHH in the bispecific antibody of the present disclosure is located at the N-terminus of the heavy chain or light chain of the second antibody, and the first antibody of the other VHH is located at the C-terminus of the heavy chain or light chain of the second antibody.

[0051] In some specific embodiments, 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 two light chains of the second antibody respectively, or the first antibody of each VHH is located at the C-terminus of the two heavy chains or two light chains of the second antibody respectively.

[0052] In some specific embodiments, 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 of the first antibody respectively, or the first antibody of each VHH is located at the C-terminus of the two heavy chains of the first antibody respectively.

[0053] In some specific embodiments, the first antibody may be connected to 1, 2, 3, 4, 5, 6, 7, or 8 second antibodies of VHH. The second antibodies of VHH may be the same or different, and any of them may be connected to the N-terminus of the heavy chain of the first antibody, or any of them may be connected to the C-terminus of the heavy chain of the first antibody, or any of them may be connected to the N-terminus of the light chain of the first antibody, or any of them may be connected to the C-terminus of the light chain of the first antibody, or to any combination of the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain of the first antibody.

[0054] In some specific embodiments, 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 (G m S n ) x or (GGNGT) x or (YGNGT) x selected from the amino acid sequences shown, where m and n are each independently selected from integers of 1 to 8 (for example, 1, 2, 3, 4, 5, 6, 7 or 8), and x is independently selected from integers of 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20). For example, the linker is an amino acid sequence shown by G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6.

[0055] In some embodiments, 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 constant region (CL). The second antibody may be a full-length antibody.

[0056] In some embodiments, the heavy chain of the second antibody of the bispecific antibody of the present disclosure is an IgG isotype such as IgG1, IgG2, IgG3 or IgG4, for example, the IgG1 isotype, and / or the light chain of the second antibody is the Kappa isotype.

[0057] In some embodiments, the two HCs of the second antibody of the bispecific antibody of the present disclosure include the same CDR, and / or the two LCs include the same CDR. In some specific embodiments, the two HCs of the second antibody include the same VH, and / or the two LCs include 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.

[0058] In some embodiments, the first antibodies of the two VHHs of the bispecific antibody of the present disclosure have identical or different amino acid sequences. For example, the first antibodies of the two VHHs have the same amino acid sequence.

[0059] In some embodiments, the bispecific antibody of the present disclosure comprises two first polypeptide chains and two second polypeptide chains. For each polypeptide chain, a) the first polypeptide chain independently comprises the heavy chain (HC) of the first antibody and the second antibody of the VHH, respectively; b) the second polypeptide chain independently comprises the light chain (LC) of the second antibody, respectively, wherein the VHH is connected to the N-terminus and / or C-terminus of the HC of the first antibody by a linker. Or, i) the first polypeptide chain independently comprises the heavy chain (HC) of the second antibody, respectively; ii) the second polypeptide chain independently comprises the light chain (LC) of the first antibody and the second antibody of the VHH, respectively, wherein the VHH is directly or connected to the N-terminus and / or C-terminus of the LC of the second antibody by a linker.

[0060] In some specific embodiments, the bispecific antibody of the present disclosure comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0061] In some embodiments, 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 hereby incorporated by reference in their entirety. For example, the TIGIT antibody may be any one 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).

[0062] In some embodiments, the second antigen-binding domain of the bispecific antibody of the present disclosure is a second antibody. The anti-TIGIT antibody in WO2019062832 is incorporated herein as the second antibody. In said second antibody, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 115, 116, and 117, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 118, 119, and 120, respectively, or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 121, 122, and 123, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 124, 125, and 126, respectively, or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 127, 128, and 129, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 130, 131, and 132, respectively, or The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 133, 134, and 135 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 136, 137, and 138 respectively, or The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 139, 140, and 141 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 142, 143, and 144 respectively.

[0063] In some specific embodiments, the first antigen-binding domain or the first antibody (e.g., VHH) in the bispecific antibody of the present disclosure contains CDR1, CDR2, and CDR3 shown in SEQ ID NO: 7, 8, 9, or contains CDR1, CDR2, and CDR3 shown in SEQ ID NO: 7, 8, 150. The heavy chain variable region of the second antigen-binding domain or the second antibody contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 121, 122, and 123 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 124, 125, and 126 respectively.

[0064] In some specific embodiments, the first antigen-binding domain or the first antibody (e.g., VHH) in the bispecific antibody of the present disclosure contains CDR1, CDR2, and CDR3 shown in SEQ ID NO: 10, 11, 12, or contains CDR1, CDR2, and CDR3 shown in SEQ ID NO: 10, 11, 151. The heavy chain variable region of the second antigen-binding domain or the second antibody contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 121, 122, and 123 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 124, 125, and 126 respectively.

[0065] In some specific embodiments, the first antibody of the VHH in the bispecific antibody of the present disclosure comprises the amino acid sequence shown in any one of SEQ ID NO: 6, 79, 81, 92, 98, 99, or a sequence having 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 thereto, and the second antibody comprises VH shown in any one of SEQ ID NO: 145 to 147, VL shown in any one of SEQ ID NO: 148 to 149, or HC shown in SEQ ID NO: 102 and LC shown in SEQ ID NO: 103, or a sequence having 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 to the VH, HC, VL, or LC.

[0066] In some specific embodiments, the bispecific antibody of the present disclosure comprises a first polypeptide chain shown in SEQ ID NO: 104 and a second polypeptide chain shown in SEQ ID NO: 103, comprises a first polypeptide chain shown in SEQ ID NO: 105 and a second polypeptide chain shown in SEQ ID NO: 103, comprises a first polypeptide chain shown in SEQ ID NO: 102 and a second polypeptide chain shown in SEQ ID NO: 106, comprises a first polypeptide chain shown in SEQ ID NO: 102 and a second polypeptide chain shown in SEQ ID NO: 107, comprises a first polypeptide chain shown in any one of SEQ ID NO: 108 to 112, 114 and a second polypeptide chain shown in SEQ ID NO: 103, or a variant having 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 to the first polypeptide chain and / or the second polypeptide chain.

[0067] In some embodiments, the Fc regions of the PVRIG-binding protein, anti-PVRIG antibody, and bispecific antibody of the present disclosure with anti-TIGIT have mutations and include one or more amino acid mutations selected from the following: i) A mutation that changes the number of cysteine residues in the hinge region of CH1 to facilitate the association of the light chain and heavy chain, or to improve or decrease the stability of the antibody; ii) A mutation that enhances the binding to FcγRIIIa to enhance ADCC, and weakens the binding to FcγRIIb, such as 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 299T, 297N, or any combination thereof; iii) A mutation that extends the biological half-life, such as T252L, T254S, T256F, 428L, 434A, 434S, 428L / 434S, or any combination thereof; iv) One or more amino acid mutations at positions 234, 235, 236, 237, 297, 318, 320, and 322, or any combination thereof, that retain the antigen-binding ability of the parental antibody while changing the affinity of the antibody for effector ligands; v) One or more amino acid mutations at positions 329, 331, and 322, or any combination thereof, such that the antibody has modified C1q binding and / or reduces or abolishes complement-dependent cytotoxicity (CDC); vi) One or more amino acid mutations in positions 231-239, or any combination thereof, that change the complement fixation ability of the antibody; vii) One or more amino acid mutations of 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 or 439 that improve the ability of ADCC and / or improve the affinity of the antibody for the Fcγ receptor, or any combination thereof, viii) Amino acid mutations of S228P, F234A, L235A and / or K447A, ix) Amino acid mutations of S354C, E356D, M358L and / or T366W.

[0068] In some embodiments, provided are antibodies that competitively bind to the same epitope as the PVRIG-binding protein, PVRIG / TIGIT-binding protein, anti-PVRIG single-domain antibody, and anti-PVRIG / anti-TIGIT bispecific antibody of the present disclosure.

[0069] In some embodiments, the present disclosure provides a PVRIG / TIGIT-binding protein comprising a first antigen-binding domain that specifically binds to PVRIG and a second antigen-binding domain that specifically binds to TIGIT.

[0070] The first antigen-binding domain that specifically binds to PVRIG comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain CDR1, CDR2, CDR3 in the sequence shown in any one of SEQ ID NO:3, 80-84, or CDR1, CDR2, CDR3 in the sequence shown in any one of SEQ ID NO:2, 75-79, or CDR1, CDR2, CDR3, or in the sequence shown in any one of SEQ ID NOs: 4, 86 - 90 CDR1, CDR2, CDR3, or in the sequence shown in any one of SEQ ID NOs: 5, 91 - 95 comprises CDR1, CDR2, CDR3 in the sequence shown in any one of SEQ ID NOs: 6, 96 - 100, wherein said CDR1, CDR2, CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.

[0071] According to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domain are respectively shown in SEQ ID NOs: 7, 8 and 9, or shown in SEQ ID NOs: 7, 8 and 150, or shown in SEQ ID NOs: 10, 11 and 12, or shown in SEQ ID NOs: 10, 11 and 151, or shown in SEQ ID NOs: 13, 14 and 15, or shown in SEQ ID NOs: 16, 17 and 18, or shown in SEQ ID NOs: 19, 20 and 21.

[0072] In a specific embodiment, the first antigen - binding domain of said PVRIG / TIGIT - binding protein is shown in any one of SEQ ID NOs: 2 - 6, 75 - 84 and 86 - 100, or comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity therewith.

[0073] In a specific embodiment, the second antigen - binding domain of said PVRIG / TIGIT - binding protein comprises a heavy - chain variable region (VH) and a light - chain variable region (VL), wherein, The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 115, 116, and 117 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 118, 119, and 120 respectively. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 121, 122, and 123 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 124, 125, and 126 respectively. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 127, 128, and 129 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 130, 131, and 132 respectively. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 133, 134, and 135 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 136, 137, and 138 respectively, or The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 139, 140, and 141 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 142, 143, and 144 respectively.

[0074] In a specific embodiment, the second antigen-binding domain of the PVRIG / TIGIT binding protein contains a full-length heavy chain (HC) and a full-length light chain (LC). Specifically, the full-length heavy chain is of the IgG1 or IgG4 isotype, and the full-length light chain is of the Kappa isotype. More specifically, the heavy chain sequence is shown in SEQ ID NO: 102 or has at least 90% sequence identity thereto, and the light chain sequence is shown in SEQ ID NO: 103 or has at least 90% sequence identity thereto.

[0075] In a specific embodiment of the PVRIG / TIGIT binding protein of the present disclosure, the VHH of the first antigen-binding domain that specifically binds to PVRIG is located at the N-terminus of the heavy-chain variable region or the full-length heavy chain of the second antigen-binding domain that specifically binds to TIGIT, the VHH of the first antigen-binding domain that specifically binds to PVRIG is located at the C-terminus of the heavy-chain variable region or the full-length heavy chain of the second antigen-binding domain that specifically binds to TIGIT, the VHH of the first antigen-binding domain that specifically binds to PVRIG is located at the N-terminus of the light-chain variable region or the full-length light chain of the second antigen-binding domain that specifically binds to TIGIT, and / or the VHH of the first antigen-binding domain that specifically binds to PVRIG is located at the C-terminus of the light-chain variable region or the full-length light chain of the second antigen-binding domain that specifically binds to TIGIT.

[0076] In a specific embodiment of the PVRIG / TIGIT binding protein of the present disclosure, the VHH of the first antigen-binding domain that specifically binds to PVRIG and the second antigen-binding domain that specifically binds to TIGIT are connected directly or by a linker, preferably, the linker has an amino acid sequence shown by (G4S) x wherein x is independently selected from integers of 1 to 20, more preferably, the linker is an amino acid sequence shown by (G4S)2 or (G4S)3.

[0077] In some embodiments, the PVRIG / TIGIT binding protein provided by the present disclosure includes a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain includes an amino acid sequence shown by any one of SEQ ID NOs: 108 to 112 and 114, the second polypeptide chain includes an amino acid sequence shown by SEQ ID NO: 103, or The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 104 or 105, the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 103, or the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 102, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 106 or 107.

[0078] In a third aspect, the present disclosure provides a polynucleotide encoding the PVRIG-binding protein, PVRIG / TIGIT-binding protein, anti-PVRIG antibody (e.g., VHH) or anti-PVRIG / TIGIT bispecific antibody. The polynucleotide may be DNA or RNA.

[0079] In some embodiments, provided is a polynucleotide composition comprising a first nucleic acid encoding a VH or HC comprising the anti-PVRIG / TIGIT bispecific antibody of the present disclosure and a second nucleic acid encoding a VL or LC comprising the anti-PVRIG / TIGIT bispecific antibody of the present disclosure.

[0080] In a fourth aspect, the present disclosure provides an expression vector or expression vector composition comprising the polynucleotide or polynucleotide composition described above, and the expression vector may be a eukaryotic cell expression vector, a prokaryotic cell expression vector, or a viral vector.

[0081] In some embodiments, provided is an expression vector composition comprising a first expression vector comprising the first nucleic acid in the above polynucleotide composition and a second expression vector comprising the second nucleic acid in the above polynucleotide composition.

[0082] In a fifth aspect, the present disclosure provides a host cell transformed with the expression vector, expression vector composition described above, or comprising the expression vector, expression vector composition, which may be a eukaryotic cell or a prokaryotic cell.

[0083] In some embodiments, 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.

[0084] In a sixth aspect, the present disclosure provides a preparation method including expressing a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody in the above-described host cell, and isolating and recovering the PVRIG-binding protein, the anti-PVRIG antibody (e.g., VHH), or the anti-PVRIG / TIGIT bispecific antibody from the host cell.

[0085] In a specific embodiment, the present disclosure the polynucleotide step of the present disclosure in the host cell of the present disclosure, and isolating the PVRIG-binding protein, PVRIG / TIGIT-binding protein, anti-PVRIG antibody, or antigen-binding fragment thereof expressed from the host cell, and provides a method for preparing a PVRIG-binding protein, PVRIG / TIGIT-binding protein, anti-PVRIG antibody, or antigen-binding fragment thereof.

[0086] In a seventh aspect, the present disclosure provides a composition (e.g., a pharmaceutical composition) including a therapeutically effective amount of the above-described PVRIG-binding protein, anti-PVRIG antibody (e.g., VHH), anti-PVRIG / TIGIT bispecific antibody, or PVRIG / TIGIT-binding protein, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0087] In some embodiments, the composition (e.g., pharmaceutical composition) comprises a PVRIG-binding protein or an anti-PVRIG antibody (e.g., VHH) of the present disclosure, and an anti-TIGIT antibody. The TIGIT antibody may be any of the above-mentioned anti-TIGIT antibodies such as the anti-TIGIT antibodies in Table 23 and Table 24 of the present disclosure. The composition may also comprise a pharmaceutically acceptable excipient, diluent or carrier.

[0088] In some specific embodiments, the anti-TIGIT antibody comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 121, 122, and 123, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 124, 125, and 126, respectively.

[0089] In some specific embodiments, the pharmaceutical composition per unit dose may comprise 0.01 to 99% by weight of a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody, or the content of the PVRIG-binding protein, anti-PVRIG antibody (e.g., VHH), or anti-PVRIG / TIGIT bispecific antibody in the pharmaceutical composition per unit dose is 0.1 to 2000 mg, or 1 to 1000 mg.

[0090] In an eighth aspect, provided is the use of any one or any combination of a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), an anti-PVRIG / TIGIT bispecific antibody, a PVRIG / TIGIT-binding protein of the present disclosure, and a polynucleotide encoding a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), an anti-PVRIG / TIGIT bispecific antibody, a PVRIG / TIGIT-binding protein of the present disclosure in the preparation of a method, medicament, or pharmaceutical composition for diagnosing, treating, or preventing a disease (e.g., used for treating or preventing a proliferative disorder (e.g., cancer or tumor) or delaying the progression of a related disorder).

[0091] In some embodiments, the Targetadministering a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure, Target to provide a method for treating or alleviating a disorder, wherein the disorder is cancer.

[0092] In some embodiments, the Target administering a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure, Target to provide a method for activating cytotoxic T lymphocytes (CTLs), wherein a subgroup of the Target CTLs is activated.

[0093] In some embodiments, the Target administering a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure, Target to provide a method for activating NK cells, wherein a subgroup of the Target NK cells is activated.

[0094] In some embodiments, the Target administering a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure, Target to provide a method for activating γδ T cells, wherein a subgroup of the Target γδ T cells is activated.

[0095] In some embodiments, the Target administering a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure, Target to provide a method for activating Th1 cells, wherein a subgroup of the Target Th1 cells is activated.

[0096] In some embodiments, the Targetadministering a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure, Target to provide a method for activating, reducing, or eliminating the number and / or activity of at least one type of regulatory T cell (Treg) in the body.

[0097] In some embodiments, the Target administering a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure, Target to provide a method for increasing the production of interferon-γ and / or the secretion of pro-inflammatory cytokines in the body.

[0098] In some embodiments, the Target administering a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure, Target to provide a method for inhibiting the interaction between PVRIG and PVLR2 in the body.

[0099] In some embodiments, the Target or Target administering a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure, Target to provide a method for treating.

[0100] In some specific embodiments, the above Target disorder is a proliferative disorder (e.g., cancer or tumor), or the above Targetsuffers from a proliferative disorder (e.g., cancer or tumor). The cancer or tumor is selected from prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, 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 cell carcinoma and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T cell acute lymphoblastic leukemia (T-ALL), diffuse large B cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel Cells cancer, high MSI cancer, KRAS mutant tumor, adult T cell leukemia / lymphoma, and myelodysplastic syndrome (MDS) or a combination thereof. The above disorder may be related to abnormal expression of PVRIG and / or TIGIT. In some specific embodiments, the cancer or tumor is selected from triple-negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merkel cell cancer, high MSI cancer, KRAS mutant tumor, adult T cell leukemia / lymphoma, and myelodysplastic syndrome (MDS) or a combination thereof. In some specific embodiments, the cancer or tumor is selected from triple-negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merkel cell, and high MSI cancer or a combination thereof. In some embodiments, the above Target has a condition related to PVRIG and / or TIGIT. In some specific forms, Target the condition of includes cancers that express or do not express PVRIG, and further includes non-metastatic or non-invasive, and invasive or metastatic cancers, among which the PVRIG expression of immune cells, stromal cells, or cells in which the lesion has occurred inhibits the anti-tumor response and the anti-invasive immune response. The methods of the present disclosure are particularly suitable for the treatment of angiogenic tumors.

[0101] In some embodiments, the Target or Target comprises administering to the a PVRIG-binding protein, an anti-PVRIG antibody (e.g., VHH), or an anti-PVRIG / TIGIT bispecific antibody of the present disclosure. TargetProvided is a method for treating or preventing infection or sepsis. In some specific forms, the infection is a pathogen infection, characterized by varying degrees of dysfunction of virus-specific T cell responses, such as HIV, HCV, HBV. In some specific forms, the sepsis includes severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, septic syndrome.

[0102] In some embodiments, provided is the use of the above PVRIG-binding protein, PVRIG / TIGIT-binding protein, anti-PVRIG antibody or its antigen-binding fragment, polynucleotide, or composition of the present disclosure in the treatment or delay of a disease, preferably, the disease is a proliferative disease, more preferably, the proliferative disease is cancer, even more preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, blood cancer or any other disease or condition characterized by uncontrolled cell proliferation.

[0103] In some embodiments, a) Target contacting tissue from [source] with the PVRIG-binding protein or anti-PVRIG antibody of the present disclosure; and b) determining the presence of overexpression of PVRIG in the tissue as an indicator of the presence of a disease or disorder, to provide a method for diagnosing a disease.

[0104] The tissue may be a blood sample or a solid tumor biopsy sample. The PVRIG-binding protein or anti-PVRIG antibody may be labeled, and further, a second labeled antibody that binds to the PVRIG-binding protein or anti-PVRIG antibody may be contacted with the sample. In some specific embodiments, the PVRIG-binding protein or anti-PVRIG antibody is labeled with a radioisotope, a dye (e.g., having a biotin-streptavidin complex), a contrast agent, a fluorescent compound or molecule, and a reinforcing agent (e.g., a paramagnetic ion) used in magnetic resonance imaging (MRI). In some specific embodiments, the disease or disorder is the cancer or tumor, infection or sepsis described above.

[0105] In a ninth aspect, the present disclosure provides a use for detecting a PVRIG-binding protein.

[0106] The present disclosure provides a composition for detecting PVRIG, comprising a PVRIG-binding protein or an anti-PVRIG antibody. The present disclosure further provides a method, system or device for detecting PVRIG in vivo or in vitro, including using a PVRIG-binding protein or an anti-PVRIG antibody.

[0107] In some embodiments, an in vitro detection method, system or device may include, for example, (1) contacting a sample with a PVRIG-binding protein or an anti-PVRIG antibody, (2) detecting a complex formed between the PVRIG-binding protein or anti-PVRIG antibody and the sample, and / or (3) contacting a reference sample (e.g., 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. For example, compared with that in a control sample or Target in, a change in complex formation (e.g., a statistically significant change) in the sample or Target in indicates the presence of PVRIG in the sample.

[0108] In some other embodiments, an in vivo detection method, system or device is (1) Targetadministering a PVRIG-binding protein or an anti-PVRIG antibody thereto, and (2) detecting complex formation between the PVRIG-binding protein or anti-PVRIG antibody and Target may be included. Detection may include determining the location or time of complex formation. Antibodies that bind to PVRIG may be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include a variety of enzymes, cofactor families, fluorescent substances, luminescent substances, and radioactive substances. Complex formation between a PVRIG-binding protein or anti-PVRIG antibody and PVRIG can be detected by measuring or visualizing an antibody that binds or does not bind to PVRIG. For example, conventional detection assays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry can be used. In some embodiments, the presence of PVRIG in a sample is analyzed by a competitive immunoassay that uses a standard substance labeled with a detectable substance and an unlabeled PVRIG-binding protein or anti-PVRIG antibody. The biological sample to be detected or measured may be a tissue cell, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

[0109] In some embodiments, for detection purposes, the PVRIG-binding protein or anti-PVRIG antibody of the present disclosure can be labeled with a fluorophore and a chromophore.

[0110] In some embodiments, a kit is further provided, the kit includes a protein that binds to PVRIG or an anti-PVRIG antibody, and may further include a diagnostic manual. The kit may further include at least one additional reagent such as a labeling agent or a further diagnostic agent. For in vivo use, the antibody can be prepared as a pharmaceutical composition.

[0111] The PVRIG antibody and anti-PVRIG / TIGIT bispecific antibody provided by the embodiments of the present disclosure have high specificity and high affinity for PVRIG and / or TIGIT, significantly reduce the immunogenicity of the humanized antibody, completely retain excellent in vitro and in vivo activities, have good pharmacokinetic properties in rats and humans, have a long half-life, high bioavailability, good long-term stability, no obvious abnormal chemical modifications, no obvious aggregation at high concentrations, have high purity and thermal stability, enhance the activities of T cells and NK cells, and have a good effect on inhibiting the occurrence and progression of tumors. Further aspects of the present invention are described below: [Item 1] A PVRIG-binding protein comprising at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 3, 80-84, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 2, 75-79, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 4, 86-90, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 5, 91-95, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 6, 96-100, and the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, preferably, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively shown in SEQ ID NO: 10, 11 and 12 or 151, or shown in SEQ ID NO: 7, 8 and 9 or 150, or shown in SEQ ID NO: 13, 14 and 15, or shown in SEQ ID NO: 16, 17 and 18, or shown in SEQ ID NO: 19, 20 and 21, a PVRIG-binding protein. [Item 2] The immunoglobulin single variable domain of the PVRIG-binding protein is VHH, preferably, a humanized and / or affinity matured VHH, more preferably, the humanized and / or affinity matured VHH comprises the heavy chain framework region IGHV3-7*01 or IGHV3-30*02 of the human germline template, the PVRIG-binding protein according to Item 1. [Item 3] The amino acid sequences of the immunoglobulin single variable domain are respectively shown in any one of SEQ ID NO: 3, 80-84, or shown in any one of SEQ ID NO: 2, 75-79, or shown in any one of SEQ ID NO: 4, 86-90, or shown in any one of SEQ ID NOs: 5, 91 to 95, or shown in any one of SEQ ID NOs: 6, 96 to 100, or the PVRIG-binding protein according to claim 1 or 2, having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with any one of the said sequences. [Claim 4] further comprising a human immunoglobulin Fc region, preferably, the Fc region is the Fc region of human IgG1 or IgG4, more preferably, the Fc region of human IgG4 has S228P, F234A, L235A and / or K447A mutations, and the PVRIG-binding protein according to any one of claims 1 to 3. [Claim 5] A PVRIG / TIGIT-binding protein comprising a first antigen-binding domain that specifically binds to PVRIG and a second antigen-binding domain that specifically binds to TIGIT, wherein the first antigen-binding domain that specifically binds to PVRIG comprises at least one immunoglobulin single variable domain, and the immunoglobulin single variable domain is CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NOs: 3, 80 to 84, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NOs: 2, 75 to 79, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NOs: 4, 86 to 90, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NOs: 5, 91 to 95, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NOs: 6, 96 to 100, wherein the CDR1, CDR2, CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, preferably, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively shown in SEQ ID NOs: 10, 11 and 12 or 151, or shown in SEQ ID NOs: 7, 8 and 9 or 150, or shown in SEQ ID NOs: 13, 14 and 15, or shown in SEQ ID NOs: 16, 17 and 18, or The PVRIG / TIGIT binding proteins shown in SEQ ID NOs: 19, 20, and 21. [Item 6] The amino acid sequences of the immunoglobulin single variable domains in the first antigen-binding domain are each shown in any one of SEQ ID NO: 3, 80-84, or shown in any one of SEQ ID NO: 2, 75-79, or shown in any one of SEQ ID NO: 4, 86-90, or shown in any one of SEQ ID NO: 5, 91-95, or shown in any one of SEQ ID NO: 6, 96-100, or The PVRIG / TIGIT binding protein according to item 5, having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with any one of the above sequences. [Item 7] The second antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), among which the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 121, 122, and 123 respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 124, 125, and 126 respectively, or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 115, 116, and 117 respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 118, 119, and 120 respectively, or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 127, 128, and 129 respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 130, 131, and 132 respectively, or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 133, 134, and 135 respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 136, 137, and 138 respectively, or The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 139, 140, and 141 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 142, 143, and 144 respectively. The PVRIG / TIGIT binding protein according to any one of items 5 or 6. [Item 8] The heavy chain variable region of the second antigen-binding domain that specifically binds to TIGIT is shown by any one of SEQ ID NO: 145 to 147, or includes an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto. The light chain variable region is shown by any one of SEQ ID NO: 148 to 149, or includes an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto. The PVRIG / TIGIT binding protein according to item 7. [Item 9] The second antigen-binding domain that specifically binds to TIGIT includes a full-length heavy chain (HC) and a full-length light chain (LC). Preferably, the full-length heavy chain is of the IgG1 or IgG4 isotype, and the full-length light chain is of the Kappa isotype. More preferably, the heavy chain sequence is shown in SEQ ID NO: 102, or has at least 90% sequence identity thereto, and the light chain sequence is shown in SEQ ID NO: 103, or has at least 90% sequence identity thereto. The PVRIG / TIGIT binding protein according to item 8. [Item 10] The second antigen-binding domain that specifically binds to TIGIT includes a heavy chain variable region (VH) and a light chain variable region (VL), among which The immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG is located at the N-terminus of the heavy chain variable region of the second antigen-binding domain that specifically binds to TIGIT. The immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG is located at the C-terminus of the heavy chain variable region of the second antigen-binding domain that specifically binds to TIGIT. The immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG is located at the N-terminus of the light chain variable region of the second antigen-binding domain that specifically binds to TIGIT, and / or The immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG is located at the C-terminus of the light chain variable region of the second antigen-binding domain that specifically binds to TIGIT, and is the PVRIG / TIGIT binding protein according to any one of items 5 to 9. [Item 11] The immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG and the second antigen-binding domain that specifically binds to TIGIT are connected directly or by a linker, Preferably, the linker has an amino acid sequence represented by (G4S)x, wherein x is independently selected from integers of 1 to 20, More preferably, the linker is the amino acid sequence represented by (G4S)2 or (G4S)3, and is the PVRIG / TIGIT binding protein according to item 10. [Item 12] It includes 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 NOs: 108 to 112 and 114, 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, and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 106 or 107, and is the PVRIG / TIGIT binding protein according to any one of items 5 to 11. [Item 13] It includes the immunoglobulin single variable domain according to any one of items 1 to 3 or 5 to 6, Preferably, it further includes the second antigen-binding domain that specifically binds to TIGIT according to item 7, and is an anti-PVRIG antibody or its antigen-binding fragment. [Item 14] A polynucleotide encoding the PVRIG binding protein according to any one of items 1 to 4, the PVRIG / TIGIT binding protein according to any one of items 5 to 12, or the anti-PVRIG antibody or its antigen-binding fragment according to item 13. [Item 15] A host cell containing the polynucleotide according to item 14. [Item 16] The step of expressing the polynucleotide according to item 14 in the host cell according to item 15, and The step of isolating the PVRIG-binding protein, PVRIG / TIGIT-binding protein, or anti-PVRIG antibody or antigen-binding fragment thereof expressed from the host cell; A method for preparing a PVRIG-binding protein, PVRIG / TIGIT-binding protein, or anti-PVRIG antibody or antigen-binding fragment thereof, comprising: [Item 17] A pharmaceutical composition comprising the PVRIG-binding protein according to any one of Items 1 to 4, the PVRIG / TIGIT-binding protein according to any one of Items 5 to 12, or the anti-PVRIG antibody or antigen-binding fragment thereof according to Item 13, and a pharmaceutically acceptable excipient, diluent, or carrier. [Item 18] The step of administering an effective amount of the PVRIG-binding protein according to any one of Items 1 to 4, the PVRIG / TIGIT-binding protein according to any one of Items 5 to 12, or the anti-PVRIG antibody or antigen-binding fragment thereof according to Item 13, the polynucleotide according to Item 14, or the pharmaceutical composition according to Item 17, or any combination thereof, to a subject for treating or delaying a disease; Preferably, the disease is a proliferative disease; More preferably, the proliferative disease is cancer; Even more preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, blood cancer, or any other disease or condition characterized by uncontrolled cell growth, a method for treating or delaying the progression of the disease. [Item 19] A method for activating NK cells, γδ T cells, and / or Th1 cells, comprising the step of administering an effective amount of the PVRIG-binding protein according to any one of Items 1 to 4, the PVRIG / TIGIT-binding protein according to any one of Items 5 to 12, or the anti-PVRIG antibody or antigen-binding fragment thereof according to Item 13, the polynucleotide according to Item 14, or the pharmaceutical composition according to Item 17, or any combination thereof, to a subject as needed. [Item 20] A method for increasing the production of interferon-γ and / or the secretion of pro-inflammatory cytokines in a subject, comprising the step of administering, as needed, an effective amount of the PVRIG-binding protein according to any one of items 1 to 4, the PVRIG / TIGIT-binding protein according to any one of items 5 to 12, or the anti-PVRIG antibody or antigen-binding fragment thereof according to item 13, the polynucleotide according to item 14, or the pharmaceutical composition according to item 17, or any combination thereof. [Item 21] Use of the PVRIG-binding protein according to any one of items 1 to 4, the PVRIG / TIGIT-binding protein according to any one of items 5 to 12, or the anti-PVRIG antibody or antigen-binding fragment thereof according to item 13, the polynucleotide according to item 14, or the pharmaceutical composition according to item 17, in the treatment or delay of a disease, preferably, the disease is a proliferative disease, more preferably, the proliferative disease is cancer, even more preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, blood cancer or any other disease or condition characterized by uncontrolled cell growth.

Brief Description of the Drawings

[0112]

Figure 1

Figure 2

Figure 3

Figure 4A - 4B

Figure 5A - 5B

Figure 6A - 6E

Figure 7A - 7E

Figure 8

Figure 9A - 9B

Figure 10A - 10B

Modes for Carrying Out the Invention

[0113] I. Terms To make the present disclosure more easily understood, several technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this specification, all other technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art. The three-letter codes and one-letter codes of amino acids used in the present disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0114] "PVRIG" or "PVRIG protein" or "PVRIG polypeptide" includes any such protein or its variants, conjugates or fragments, optionally including the known or wild-type PVRIG described in this specification, and any naturally occurring splice variants, amino acid variants or isoforms, particularly the soluble extracellular domain (ECD) fragment of PVRIG (not limited thereto). The ECD in this specification is as defined in Patent WO2016134333. The complete human PVRIG sequence can be found under GenBank accession number AAH73861.1.

[0115] The "PVRIG-binding protein" refers to any protein that can specifically bind to PVRIG or any molecule containing said protein. The PVRIG-binding protein may include an antibody defined in the present disclosure against PVRIG, an antigen-binding fragment thereof, or a conjugate thereof. The PVRIG-binding protein also encompasses immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. The "PVRIG-binding protein" of the present disclosure may include at least one immunoglobulin single variable domain (e.g., VHH) that binds to PVRIG. In some embodiments, the "PVRIG-binding protein" may include 2, 3, 4 or more immunoglobulin single variable domains (e.g., VHH) that bind to PVRIG. The PVRIG-binding protein of the present disclosure, in addition to including the immunoglobulin single variable domain of PVRIG, may further include a linker and / or a half-life extension moiety (e.g., an immunoglobulin single variable domain that binds to serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, the "PVRIG-binding protein" of the present disclosure includes immunoglobulins that bind to different antigens (e.g., a first antibody that binds to a first antigen (e.g., PVRIG) and a second antibody that binds to a second antigen (e.g., TIGIT), optionally including a third antibody that binds to a third antigen, and further optionally including a fourth antibody that binds to a fourth antigen, also encompassing bispecific / multispecific antibodies).

[0116] "TIGIT" or "TIGIT protein" or "TIGIT polypeptide" may optionally include any such protein or its variants, conjugates or fragments, including but not limited to the known or wild-type TIGIT described herein, and any naturally occurring splice variants, amino acid variants or isoforms. The complete TIGIT sequence can be found under GenBank accession number AAI01289.1.

[0117] "Binding to PVRIG" means being able to interact with PVRIG or its epitope, and the PVRIG or its epitope may be of human origin. "Binding to TIGIT" means being able to interact with TIGIT or its epitope, and the TIGIT or its epitope may be of human origin. "Antigen-binding site" refers to a discontinuous three-dimensional spatial site on an antigen recognized by the antibody or antigen-binding fragment of the present disclosure.

[0118] "Antibody" or "immunoglobulin" in a broad sense includes conventional antibodies (tetrapeptide chain structure antibodies in which two identical heavy chains and two identical light chains are connected by interchain disulfide bonds), and Fab, Fv, sFv, F(ab’)2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies peptibody, domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies) and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv). Therefore, the term "antibody" as used in this disclosure includes full-length antibodies, their single chains and any part, domain or fragment having antigen-binding activity, and multispecific antibodies (including but not limited to antigen-binding domains or fragments that are VHH domains or VH / VL domains respectively) containing their single chains and any part, domain or fragment having antigen-binding activity. Conventional antibodies or immunoglobulins usually have a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are connected by interchain disulfide bonds. Since the amino acid composition and sequence order of the heavy chain constant region are different, their antigenicity is also different. Thus, immunoglobulins can be divided into five types, or are called immunoglobulin isotypes IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain respectively. Igs of the same class can be further divided into different subclasses according to the differences in the amino acid composition of their hinge regions and the number and positions of the heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, IgG4. Light chains are divided into κ chains or λ chains according to the differences in the constant regions. Each class of the five classes of Igs can have either κ (kappa) chains or λ (lambda) chains. In some embodiments, the antibodies of the present disclosure specifically or substantially specifically bind to PVRIG and / or TIGIT.

[0119] The "antibody" of the present disclosure includes, but is not limited to: (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of VH and CH1 domains; (iii) an F(ab')2 fragment which is a bivalent fragment containing two connected Fab fragments; (vii) a single-chain Fv molecule (scFv) in which the VH domain and the VL domain are connected by a peptide linker that enables two domains to bind to form an antigen-binding site (Bird et al., 1988, Science 242:423-426, and Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, which are hereby incorporated by reference in their entirety); (iv) a "bispecific antibody" or "trispecific antibody" which is a multivalent or multispecific fragment constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479, WO94 / 13804, and Holliger et al., 1993, Proc. Natl. Acad. Sci. 90:6444-6448, all of which are hereby incorporated by reference in their entirety); (v) a "domain antibody" or "dAb" including an immunoglobulin single variable domain derived from rodents (e.g., as disclosed in WO00 / 29004), other species such as nurse sharks, and camelid V-HH dAbs (which may be referred to as "immunoglobulin single variable domains"); (vi) SMIP (small molecule immunopharmaceutical), camel antibodies, nanobodies, and IgNAR; (vii) humanized antibodies of (i) to (vi) above.

[0120] Unless otherwise specified, the antibodies of the present disclosure generally use the Kabat numbering system. The EU numbers in Kabat are generally also used for constant domains and / or Fc domains.

[0121] The antibodies of the present disclosure may be polyclonal, monoclonal, heterologous, allogeneic, syngeneic, or modified forms thereof, and monoclonal antibodies are particularly applicable to a plurality of examples. Generally, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" broadly refers to products such as cells or nucleic acids, proteins, or vectors, where the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or modifying natural nucleic acids or proteins, or where the cells are derived from cells thus modified. For example, recombinant cells express genes that do not exist in the natural (non-recombinant) cell form, or express natural genes that were originally abnormally expressed, under-expressed, or not expressed at all.

[0122] "Monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody molecules that contain only one species of antigen-binding site capable of immunoreacting with a specific antigen epitope, and "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody molecules that contain 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 immunoreact.

[0123] "Antigen" refers to a molecule for immunizing an immunocompetent vertebrate to produce antibodies that recognize the antigen, or for screening an expression library (e.g., particularly a phage, yeast, or ribosome display library). As used herein, an antigen is more broadly defined and is generally expected to include a target molecule specifically recognized by an antibody, and thus includes a part or mimetic of a molecule used in an immunization process for producing an antibody or in screening a library for selecting an antibody.

[0124] "Array" (e.g., in terms such as "immunoglobulin array", "antibody array", "single variable domain array", "VHH array" or "protein array") should generally be understood to include not only the relevant amino acid sequence but also the nucleic acid sequence or nucleotide sequence encoding said sequence, unless further limited interpretation is required in the present disclosure.

[0125] "Polynucleotide" or "nucleic acid" refers to nucleotide chains 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. Polynucleotides may include modified nucleotides such as, for example, methylated nucleotides and their analogs. Modifications to the nucleotide structure can be imparted before or after association of the strands, if present. Polynucleotides may further include analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomer sugars, epimeric sugars (e.g., arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose), acyclic analogs and nucleobase-free nucleotide analogs such as methyl riboside.

[0126] "Identity" or "identicalness" refers to the similarity of sequences between two polynucleotide sequences or between two polypeptides. When the positions in the two sequences being compared are occupied by the same base or amino acid monomer subunit, for example, when each position of two DNA molecules is occupied by adenine, the molecules are identical at that position. The percentage of identity between two sequences is a function obtained by dividing the number of matching or identical positions shared by the two sequences by the number of positions being compared and multiplying by 100. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are identical, the two sequences are 60% identical. Generally, comparison is made when the maximum percentage of identity is obtained by aligning the two sequences.

[0127] The "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of other parts of the protein. Generally, a domain bears a single functional property of the protein and, in many cases, may be added, removed, or transferred to other proteins without losing the function of other parts and / or domains of the protein.

[0128] The "immunoglobulin domain" refers to the globular region of an antibody chain (for example, the chain of a normal tetrapeptide chain structure antibody or the chain of a heavy chain antibody), or a polypeptide substantially composed of such a globular region. The immunoglobulin domain is characterized by maintaining the folding properties of the immunoglobulin of the antibody molecule and being composed of a two-layer sandwich of about seven anti-parallel β-sheet strands arranged in two β-sheets and stabilized by optionally conserved disulfide bonds.

[0129] The term "immunoglobulin variable domain" refers to an immunoglobulin domain substantially composed of four "framework regions" respectively called "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4" in the art and hereinafter, and the said framework regions are separated by three "complementary determining regions" or "CDRs" respectively called "complementary determining region 1" or "CDR1", "complementary determining region 2" or "CDR2", and "complementary determining region 3" or "CDR3" in the art and hereinafter. Therefore, the general structure or sequence of an immunoglobulin variable domain may be shown as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Since the immunoglobulin variable domain has an antigen-binding site, it confers specificity for an antigen.

[0130] The term "antibody framework (FR)" refers to a part of the variable domain that is used as a stent for the antigen-binding loops (CDRs) of the variable domain.

[0131] Regarding the determination or definition of "CDR", the clear description of CDR and the identification of residues including the binding site of the antibody can be achieved by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex. This can be realized by any of various techniques known to those skilled in the art, such as X-ray crystallography. Various analytical methods can be used for the identification of CDR, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, and conformational definition. The Kabat numbering system is a standard for numbering residues in antibodies and is generally 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 positions of some structural loop regions (see, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; and Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses a set of computer programs produced by the Oxford Molecular Group that modeled the antibody structure (see, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies", Oxford, UK; Oxford Molecular Ltd).The AbM numbering system models the tertiary structure of an antibody from a primary sequence using a combination of a knowledge database and ab initio methods (see "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" described in Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). 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). In the conformation definition, the positions of the CDRs can be identified as residues that make an enthalpic contribution to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Note that other CDR boundary definitions do not necessarily strictly follow one of the above methods, but they can be shortened or extended according to predictions or experimental results that overlap with at least a part of the Kabat CDRs and that certain residues or groups of residues do not significantly affect antigen binding. As used herein, a CDR can refer to a CDR defined by any method known in the art (including combinations of methods). The methods used herein can use CDRs defined by any of these methods. In any given embodiment that includes more than one CDR, the CDRs can be defined by any of the Kabat, Chothia, extended, AbM, IMGT, contact, and / or conformation definitions.

[0132] "Immunoglobulin single variable domain" is used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including VH, VHH or VL domains) that can form a functional antigen-binding site when it does not normally interact with other variable domains (for example, when there is no VH / VL interaction required between the VH and VL domains of a normal four-chain monoclonal antibody). Examples of "immunoglobulin single variable domain" include nanobodies (including camelized VH such as VHH, humanized VHH and / or camelized human VH), IgNAR, domains, antibodies as VH domains or derived from VH domains (single domain antibodies) (e.g., dAbs (商標) ) and antibodies as VL domains or derived from VL domains (single domain antibodies) (e.g., dAbs (商標) ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are usually preferred. As one specific example of an immunoglobulin single variable domain, a "VHH domain" (or abbreviated as "VHH") defined as follows can be mentioned.

[0133] The "VHH domain" is also called a heavy-chain single-domain antibody, VHH, VHH antibody fragment, VHH antibody, or nanobody, and is the variable domain of an antigen-binding immunoglobulin called a "heavy-chain antibody" (i.e., "antibody lacking a light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy-chain variable domain (referred to as the "VH domain" in this disclosure) and the light-chain variable domain (referred to as the "VL domain" in this disclosure) present in a normal tetrapeptide-chain structure antibody. The VHH domain specifically binds to an epitope without requiring other antigen-binding domains (this is the opposite of the VH or VL domain in a normal tetrapeptide-chain structure antibody, where the epitope is recognized by both the VL domain and the VH domain). The VHH domain is a small, stable, and efficient antigen recognition unit formed by a single immunoglobulin domain. The terms "heavy-chain single-domain antibody", "VHH domain", "VHH", "VHH domain", "VHH antibody fragment", "VHH antibody", and "domain" (Nanobody is a trademark of Ablynx N.V., Ghent, Belgium) may be used interchangeably. The "VHH domain" includes, but is not limited to, natural antibodies produced by camelids, and may be humanized after antibody production by camelids, or may be screened by phage display technology.

[0134] As is known in the art for VH domains and VHH domains, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions based on Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering). This generally means that Kabat-based numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or rules include Chothia, IMGT, AbM.

[0135] The total number of amino acid residues in the VHH domain is usually in the range of 110 - 120, and often between 112 - 115. However, it should be noted that small and long sequences can also be suitable for the purposes described in this disclosure.

[0136] The VHH domain (either alone or as part of a larger polypeptide) offers many significant advantages over using normal VH and VL domains, scFv or normal antibody fragments (e.g., Fab- or F(ab’)2-fragments): - It only needs to bind to the antigen with high affinity and high selectivity by a single domain, without the need for two separate domains to be present, nor the need to ensure that the two domains are present in a proper spatial conformation and arrangement (e.g., scFv generally requires the use of a specially designed linker). - The VHH domain can be expressed by a single gene and does not require folding or modification after translation. - The VHH domain can be easily engineered into multivalent and multispecific formats. - The VHH domain is highly soluble and has no tendency to aggregate. - The VHH domain is highly stable against heat, pH, proteases and other denaturing agents or conditions, so it is not necessary to use refrigeration equipment during preparation, storage or transportation, achieving cost and time savings and environmental friendliness. - The VHH domain is easy to prepare, relatively inexpensive, and this is also the case even at the scale required for production. - The VHH domain is relatively small (about 15 kDa or 1 / 10 the size of a normal IgG) compared to a normal tetrapeptide chain structure antibody and its antigen-binding fragment, and thus shows high tissue penetration and can be administered at high doses compared to a normal tetrapeptide chain structure antibody and its antigen-binding fragment. - The VHH domain can exhibit so-called cavity-binding properties (especially by the CDR3 loop extended more than a normal VH domain), and can reach targets and epitopes that cannot be reached by a normal tetrapeptide chain structure antibody and its antigen-binding fragment.

[0137] Methods for obtaining VHHs that bind to a specific antigen or epitope have been previously disclosed in documents such as 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, 17 June, 2009 and WO94 / 04678.

[0138] "Fc variant" or "variant Fc" means a protein containing an amino acid modification in the Fc domain. The Fc variants of the present disclosure are defined based on the amino acid modifications that constitute them. Thus, for example, S228P or 228P is an Fc variant having a proline substitution at position 228 relative to the parental Fc polypeptide, and the number is based on the EU index. The identity of the WT amino acid may not be specified, and in this case the variant is called 228P.

[0139] Examples of "humanization" include cases where the VHH domain derived from a camelid can be "humanized" by substituting one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a human normal tetrapeptide chain structure antibody (in the present disclosure, also referred to as "sequence optimization", and "sequence optimization" may include, in addition to humanization, other modifications added to the sequence by one or more mutations that give improved properties to the VHH, such as removal of potential post-translational modification sites). The humanized VHH domain may include one or more complete human framework region sequences, and in some specific embodiments, may include the human framework region sequence of IGHV3. Still other examples of "humanization" include antibodies produced by transplanting mouse CDR sequences into the human antibody variable region framework, i.e., the framework sequences of different types of human germline antibodies. The strong antibody response of the antibody variable region induced by the chimeric antibody having a large amount of mouse protein components can be overcome. Examples of humanization methods include, for example, resurfacing of protein surface amino acids and the antibody humanization universal framework grafting method (CDR grafting to a universal framework) of "transplanting" the CDR to other "stents" (including but not limited to human stents or non-immunoglobulin stents). The stents and techniques suitable for the transplantation of the CDR are known in the art. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (obtained at the Internet www.mrccpe.com.ac.uk / vbase), and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies of the present disclosure further include humanized antibodies after affinity maturation of the CDR by phage display. Also, in order to avoid a decrease in activity associated with a decrease in immunogenicity, the activity can be maintained by performing the least number of reverse mutations or revertant mutations on the human antibody variable region framework sequence.

[0140] An "affinity matured" antibody has one or more changes in one or more CDRs, and these changes result in an increase in the affinity for the antigen compared to the respective parent antibody. Affinity matured antibodies can be prepared, for example, by methods known in the art 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. Mol. Biol. 226(3):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0141] Typically, the antibodies of the present disclosure have a dissociation constant (KD), preferably measured in a Biacore or KinExA or Fortibio assay, of 10 -7 ~10 -10 mol / liter (M), more preferably 10 -8 ~10 -10 mol / liter, even more preferably 10 -9 ~10 -10 or less, and / or bind to an antigen (i.e., PVRIG) with an association constant (KA) of at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, even more preferably at least 10 -10 M. 10 -4Any KD value greater than M is generally considered to exhibit non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be measured by any suitable known method, including, for example, the surface plasmon resonance (SPR) assay, Scatchard assay, and / or competitive binding assay (such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assay) described in the present disclosure.

[0142] "Epitope" or "antigenic determinant", which may be used interchangeably, refers to any antigenic determinant in an antigen to which the paratope of an antibody binds. Antigenic determinants usually contain chemically active surface groups of molecules such as, for example, amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope usually contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation and may be a "linear" epitope or a "conformational" epitope. In a linear epitope, all interaction sites between a protein and an interacting molecule (such as an antibody) are linearly present along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites are present across protein amino acid residues that are separated from each other. The epitopes of an antigen can be identified by many well-known epitope mapping techniques in the art (for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996), US4708871). Antibodies can be screened from the binding competition for the same epitope by conventional techniques known to those skilled in the art. For example, by performing competition and cross-competition studies, antibodies that compete or cross-compete for binding to an antigen can be obtained (for high-throughput screening methods, see, for example, WO03 / 48731). Therefore, by conventional techniques known to those skilled in the art, antibodies and antigen-binding fragments thereof that competitively bind to the same epitope on PVRIG as the antibody molecules of the present disclosure can be obtained.

[0143] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a given antigen. Usually, when recombinant human PVRIG, TIGIT or its epitope is used as an analyte and an antibody is used as a ligand and measured by surface plasmon resonance (SPR) technology in an instrument, the antibody binds to a given antigen or its epitope with an equilibrium dissociation constant (KD) of less than about 10 -7 M or less or a smaller value, and the binding affinity for the given antigen or its epitope is at least twice the binding affinity for a non-specific antigen other than the given antigen (or its epitope) or an antigen closely related thereto (for example, BSA, etc.). "Antibody that recognizes an antigen" may be used interchangeably with "antibody that specifically binds" herein.

[0144] "Binding affinity" is used herein as a measure of the strength of non-covalent interactions between two molecules (e.g., an antibody or a portion thereof and an antigen) and is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). For example, the KD can be determined by measuring the kinetics of complex formation and dissociation using surface plasmon resonance (SPR) methods (Biacore). The rate constants corresponding to the binding and dissociation of the monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. KD is related to ka and kd by the equation KD = kd / ka. The value of the dissociation constant can be determined directly by known methods (see Caceci et al., 1984, Byte 9:340-362; and Wong & Lohman, 1993, PNAS 90:5428-5432). Other standard assays for evaluating the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, and other assays listed elsewhere in this specification. Similarly, the specificity of an interaction can be evaluated by determining and comparing the KD value of the target interaction (e.g., the specific interaction between an antibody and an antigen) and the KD value of a non-target interaction (e.g., a known control antibody that does not bind to PVRIG). In some embodiments, the affinity of the anti-PVRIG antibodies of the present disclosure to bind to their target is at least 2-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or 10,000-fold greater than the affinity to bind to other molecules that are not PVRIG, although the definitions herein are not limiting.

[0145] "Conservative modifications" apply to amino acid and nucleotide sequences. For a particular nucleotide sequence, conservative modifications refer to the substitution of nucleic acids that encode the same or substantially the same amino acid sequence, or, when the nucleotide does not encode an amino acid sequence, the substitution of substantially the same nucleotide sequence. For an amino acid sequence, conservative modifications refer to the ability to frequently vary the amino acid sequence without changing the biological activity of the protein by substituting amino acids in the protein with other amino acids having similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.). As is known to those skilled in the art, generally, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change its biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224, (4th edition)).

[0146] "Amino acid mutations" include substitutions, deletions, insertions, modifications, and any combination thereof of amino acids so as to achieve the final construct and for the final construct to have desired properties such as enhanced stability, improved activity, etc. Deletions and insertions of amino acid sequences include deletions at the amino acid and / or carboxy termini and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. For example, in order to change the binding properties of an anti-PVRIG antibody, non-conservative amino acids can be substituted, i.e., one amino acid can be substituted with another amino acid having different structures and / or chemical properties. Preferred amino acid substitutions include substituting hydrophobic amino acids with hydrophilic amino acids. Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, etc. Amino acid mutations can occur in the CDR region, FR region, or Fc region of an antibody.

[0147] Regarding amino acid mutations in the Fc region, they can be used to introduce mutations into the wild-type Fc sequence of the antibodies of the present disclosure to alter Fc-mediated related activities, and the mutations include, but are not limited to, a) mutations that alter Fc-mediated CDC activity, b) mutations that alter Fc-mediated ADCC activity, or c) mutations that alter the in vivo half-life mediated by FcRn (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). Specifically, it includes changing the number of cysteine residues in the hinge region by modifying the hinge region of CH1, for example, increasing or decreasing it (see US5,677,425, which is incorporated herein by reference in its entirety). Mutations that enhance binding to FcγRIIIa (causing enhanced ADCC), introducing mutations that weaken binding to FcγRIIb, 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 herein by reference in their entirety).By performing Fc modification, its biological half-life can be extended, and for example, one or more of the following mutations can be introduced: T252L, T254S, T256F (see US 6,277,375); to extend the biological half-life, the antibody can be altered in the CH1 or CL region to include a salvage receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG (see US 5,869,046, US 6,121,022); additional mutations for extending 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 herein by reference in their entirety). The Fc region can be altered by substituting at least one amino acid residue, thereby altering the effector function of the antibody. For example, by substituting one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322, the affinity of the antibody for an effector ligand can be altered while retaining the antigen-binding ability of the parental antibody. The effector ligand with altered affinity can be, for example, an Fc receptor of complement or the C1 component (see US 5,624,821, US 5,648,260, which are incorporated herein by reference in their entirety). Altering one or more amino acid residues at positions 231 and 239 of the amino acid changes the complement fixation ability of the antibody (see WO 94 / 29351, which is incorporated herein by reference in its entirety).To enhance the ability of an antibody to mediate antibody-dependent cell cytotoxicity (ADCC) and / or improve the affinity of the antibody for Fcγ receptors, the Fc region is modified by modifying one or more amino acids at the following positions: 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 or 439 (see WO00 / 42072, which is incorporated herein by reference in its entirety). Also, the binding sites for FcγRI, FcγRII, FcγRIII and FcRn on human IgG1 have been mapped and variants with improved binding are described (see Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 have been shown to improve binding to FcyRIII. Combinatorial variants such as T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A have been shown to improve binding to FcγRIII. Mutations such as M252Y / S254T / T256E or M428L / N434S also improve binding to FcRn and extend the circulating half-life of the antibody (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316).

[0148] Modifications of the antibodies of the present disclosure include, for example, pegylation (PEGylation) or the addition of other water-soluble moieties to extend the half-life. "PEGylation" refers to connecting at least one PEG molecule to another molecule (e.g., a therapeutic protein). For example, PEG is a linear or branched polyether with one end connected to a hydroxyl group and having a general structure such as HO-(CH2CH2O) n -CH2CH2-OH. To conjugate PEG to a molecule (polypeptide, polysaccharide, polynucleotide, and small organic molecule), PEG can be activated by preparing several or two derivatives of PEG having functional groups at the ends. A common method for PEG conjugation of proteins is to activate PEG with a functional group suitable for reaction with lysine and the N-terminal amino acid group. In particular, the common reactive groups involved in conjugation are the α or ε amino groups of lysine. By the reaction of the pegylation linker with the protein, the PEG moiety is mainly connected to sites such as the α amino group at the N-terminus of the protein, the ε amino group on the side chain of the lysine residue, or the imidazolyl group on the side chain of the histidine residue. Since most recombinant proteins have a single α and multiple ε amino groups and imidazolyl groups, many positional isomers can be produced based on the chemical properties of the linker.

[0149] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. Mammalian expression systems will cause glycosylation of the antibody, 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 produce antibodies by expanding the culture in a serum-free medium in a bioreactor. The culture medium in which the antibody is secreted can be purified and collected by conventional techniques. The antibody can be concentrated by filtration by conventional methods. Soluble mixtures and multimers may be removed by conventional methods such as molecular sieving, ion exchange, etc. The resulting product must be immediately frozen, for example, at -70 °C, or lyophilized.

[0150] "Administration", "administration" and "treatment", when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, refer to contact between an exogenous drug, therapeutic agent, diagnostic agent or composition and an animal, human, subject , cells, tissues, organs or biological fluids, for example, treatment, pharmacokinetics, diagnosis, research and experimental methods. Treatment of cells includes contact between a reagent and the cells, and contact between the reagent and a fluid, wherein the fluid contacts the cells. "Administration", "administration" and "treatment" also mean treating cells, for example, in vitro and ex vivo, by a reagent, a diagnostic, a binding composition, or through another type of cell. When applied to humans, veterinary medicine or research subjects, it means therapeutic treatment, prevention or prophylactic measures, research and diagnostic applications.

[0151] "Treatment" means administering an oral or topical therapeutic agent containing any one of the antibodies of the present disclosure or a pharmaceutical composition thereof as a therapeutic agent, subject to a subject who already suffers from, is at risk of suffering from, has a tendency to suffer from one or more proliferative diseases or symptoms thereof, and the known therapeutic agent has a therapeutic effect on these symptoms. Usually, the subject Alternatively, a therapeutically effective amount of a therapeutic agent is administered to the individual or group to effectively alleviate one or more disease symptoms, induce regression of such symptoms, and inhibit such symptoms from progressing to any clinically measurable degree. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as the "therapeutically effective amount") can vary depending on multiple factors such as the subject disease state, age and weight, and the subject ability of the agent to produce the required therapeutic effect. Whether the disease symptoms have been reduced can be evaluated by any clinical detection method commonly used by physicians and other professional healthcare providers to assess the severity or progression of the said symptoms. Embodiments of the present disclosure (e.g., treatment methods or products) may be ineffective against the alleviation of certain subject target disease symptoms, but are statistically significant in a number of subject cases, as confirmed by any statistical testing method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test, to be able to reduce the target disease symptoms.

[0152] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or syndromes of a medical disorder. An effective amount further refers to an amount sufficient to permit or facilitate a diagnosis. subject The effective amount used for subject can vary depending on factors such as the disorder being treated, the subject overall health status, the method, route, and dosage of administration, and the severity of side effects. The effective amount may also be the maximum volume or dosing schedule that avoids significant side effects or toxic effects. The subject of the present disclosure may be for animals or humans.

[0153] "Host cell" includes each cell or cell culture and may be or already be a receptor for a vector for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny are not necessarily exactly the same as the original parent cell (in terms of morphology or genomic DNA complement). Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell", "cell line" and "cell culture" may be used interchangeably, and all such names include their progeny. It should also be understood that due to intentional or unintentional mutations, not all progeny may be exactly the same in DNA content. Mutant progeny having the same function or biological activity as those selected from the originally transformed cells are included.

[0154] "Vector" means a construct capable of delivering one or more genes or sequences of interest in a host cell and, in some embodiments, expressing it. 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 complexed with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and eukaryotic cells such as producer cells.

[0155] "Optional" or "optionally" means that the event or situation described thereafter may occur, but not necessarily, and the expression includes both the case where the event or situation occurs and the case where it does not occur. For example, "optionally includes one to three antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may be present, but not necessarily.

[0156] "Pharmaceutical composition" means a mixture of one or more antibodies or antigen-binding fragments or their physiologically / pharmaceutically acceptable salts or prodrugs described in this specification and other chemical components, and other components such as physiologically / pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is for promoting administration to a living body, contributing to the absorption of the active ingredient, and further exerting biological activity.

[0157] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and subject does not react with the immune system. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline aqueous solution, water, emulsions such as oil / water emulsions, and various wetting agents. In some embodiments, the diluent used for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are prepared by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0158] The "PVRIG-binding protein" or "PVRIG antibody" of the present disclosure can contain one or more effector molecules, for example, by conjugation. The said "effector molecule" includes, for example, anti-tumor agents, drugs, toxins, bioactive proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and their fragments (such as DNA, RNA and their fragments), radionuclides, especially radioactive iodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy. When the effector molecule is a polymer, it is usually a synthetic or naturally occurring polymer such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer, or a branched or unbranched polysaccharide such as a homopolysaccharide or heteropolysaccharide. Specific optional substituents that can be present in the above synthetic polymers include one or more hydroxy groups, methyl groups or methoxy groups. Specific examples of synthetic polymers include especially optionally substituted poly(ethylene glycol) such as optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or its derivatives, methoxypoly(ethylene glycol) or its derivatives. Specific naturally occurring polymers include lactose, amylose, glucan, glycogen or their derivatives. In one embodiment, the polymer is albumin or its fragment, such as human serum albumin or its fragment. The conjugation method of the polymer with the PVRIG-binding protein or PVRIG antibody can be realized by conventional methods.

[0159] Hereinafter, it will be further described in conjunction with examples, but these examples do not limit the scope of the present disclosure.

[0160] In the examples or test examples, for experimental methods where specific conditions are not specified, generally follow normal conditions or the conditions recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Current Protocols in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents for which specific sources are not specified are commercially available normal reagents.

Example

[0161] Example 1. PVRIG Protein Sequence and Preparation The purified commercial protein reagents of human PVRIG with his tag (h-PVRIG-his), human PVRIG with Fc tag of mouse IgG2a (h-PVRIG-mIgG2a Fc), and mouse PVRIG with Fc tag of human IgG1 (m-PVRIG-hIgG1 Fc) were purchased from Acrobiosystems, and their sequences are shown in Table 1.

[0162]

Table 1

[0163] The recombinant protein sequence of cynomolgus monkey PVRIG with his tag (cyno-PVRIG-his) is as follows: TPEVWVQVQMEATELSSFTVHCGFLGPGSISLVTVSWGGPDGAGGTKLAVLHPELGTRQWAPARQARWETQSSISLALEDSGASSPFANTTFCCKFASFPEGSWESCGSLPPSSDPGLSAPPTPVPILRADHHHHHH (SEQ ID NO:1)

[0164] Recombinant protein was expressed by transient transfection by a conventional method in HEK293 cells, and the supernatant was collected and purified by Ni-NTA. As detected, cyno-PVRIG-his was obtained.

[0165] Example 2. Production of anti-human PVRIG single-domain antibody The anti-human PVRIG monoclonal single-domain antibody was produced by immunizing camels. The immunizing antigen was human PVRIG recombinant protein with a his tag (h-PVRIG-his). It was emulsified with Freund's adjuvant (Sigma, Lot No.: F5881 / F5506), and initially, complete Freund's adjuvant (CFA) was used, and incomplete Freund's adjuvant (IFA) was used for the remaining booster immunizations. The immunization injection times were on days 0, 14, 28, and 42. Blood was collected on day 56 for blood detection, and camel serum was detected by the ELISA method to determine the antibody titer in the camel serum.

[0166] 200 mL of camel peripheral blood was collected, PBMCs in it were isolated, RNA in the cells was extracted with Trizol and reverse transcribed into cDNA. The gene of the single-domain antibody variable region was amplified by the PCR method and cloned into a phage vector to construct a phage library of anti-human PVRIG single-domain antibody.

[0167] The phage library was diluted with BSA for blocking, incubated with magnetic beads Dynabeads (M-280, invitrogen), and phages after negative selection and incubation were collected. Dynabeads were coated and blocked with biotin-labeled human PVRIG with a his tag, the phage suspension collected after negative selection was incubated with the said Dynabeads, and the phages were eluted with trypsin. Three rounds of screening were performed, 400 clones screened in the third round were selected for sequencing, and the heavy chain sequences of 5 single-domain antibodies among them are shown in Table 2, and the CDRs with different numbering rules are shown in Table 3.

[0168]

Table 2

[0169]

Table 3

[0170] Example 3. Preparation of full-length anti-PVRIG antibody The heavy-chain variable regions of the five antibodies of Example 2 were connected to the human IgG4 heavy-chain Fc region to construct a full-length anti-PVRIG antibody. Among them, the heavy-chain Fc region contains a hinge region and has S228P, F234A, L235A, K447A mutations (Eu naming system). The anti-PVRIG antibody CPA.7.021 shown in WO2016134333 was screened from an antibody phage library, its subtype is IgG1, and it can preferably bind 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 to the human IgG4 heavy-chain constant region (having S228P, F234A, L235A, K447A mutations) and the human kappa light-chain constant region to construct a positive antibody Tab5.

[0171] The full-length sequences of the five antibodies and the positive antibody are shown in Table 4.

Table 4-1

Table 4-2

[0172] The above array was synthesized, digested with BamHI and XhoI, and then inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20) via the BamHI / XhoI restriction enzyme cleavage site. HEK293 cells (Life Technologies Cat. No. 11625019) were transfected at a ratio of 1:2 using the expression vector and the transfection reagent PEI (Polysciences Inc., Cat. No. 23966), and placed in a CO2 incubator and incubated for 4 to 5 days. After the expressed antibody was recovered by centrifugation, the antibody was purified and detected by a conventional method, and the target antibody was obtained.

[0173] Example 4. Binding experiment between anti-PVRIG antibody and PVRIG recombinant protein The ELISA experiment was used to detect the binding characteristics of the anti-PVRIG antibody. The plate was directly coated with the his-tagged PVRIG recombinant protein. After adding the antibody, the binding activity of the antibody to the antigen was detected by adding a secondary antibody (HRP-conjugated anti-primary antibody Fc antibody) and the HRP substrate TMB.

[0174] Coat the PVRIG protein of human, cynomolgus monkey or mouse at a concentration of 1 μg / mL, 100 μL per well, in a 96-well microtiter plate, and incubate overnight at 4°C. Wash three times with the washing solution, 250 μL per well. To ensure thorough washing, shake for 10 seconds each time when washing. Add 300 μL / well of blocking solution (PBS + 0.05% Tween20 + 1% BSA), and incubate at room temperature for 1 hour. Wash three times with the washing solution, 250 μL per well. To ensure thorough washing, shake for 10 seconds each time when washing. Add 100 μL of the anti-PVRIG test antibody diluted with the diluent per well. Incubate at 37°C for 1 hour. Wash three times with the washing solution, 250 μL per well. Add 100 μL of HRP-labeled anti-human IgG secondary antibody (Sigma, A8667) per well. Incubate at 37°C for 1 hour. Wash three times with the washing solution, 250 μL per well. Add 100 μL of TMB per well, and react in the dark for 15 minutes. Add 50 μL of 0.16 M sulfuric acid per well. Read the OD value at 450 nm using a Thermo MμLtiSkan Fc microplate reader, and calculate the binding EC 50 value of the anti-PVRIG antibody to PVRIG. All antibodies have a strong binding ability to the PVRIG recombinant protein of human or cynomolgus monkey, but did not bind to the PVRIG recombinant protein of mouse.

[0175]

Table 5

[0176] Example 5. Binding experiment of anti-PVRIG antibody and cells expressing PVRIG Detect the binding characteristics of the anti-PVRIG antibody by flow cytometry (FACS). Construct cell lines overexpressing PVRIG of human or cynomolgus monkey, and after adding the antibody, add the secondary antibody to detect the binding activity of the antibody to the antigen.

[0177] An expression plasmid having the PVRIG gene sequence of human or cynomolgus monkey was transfected into HEK293 cells, and monoclonal cell lines that were stably transfected with overexpression were obtained by antibiotic screening and the limiting dilution method. 2×105 overexpressing cells were inoculated per well in a 96-well plate. Centrifuged at 300 g for 5 minutes, the supernatant was removed, 100 μL of the test antibody was added, and incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, washed 3 times with 200 μL of washing solution (PBS + 2% FBS), 100 μL of Alexa Fluor 488-labeled anti-human IgG secondary antibody (Invitrogen, A-11013) diluted 1:500 was added, and incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and washed 3 times with 200 μL of washing solution (PBS + 2% FBS). The cells were resuspended in 100 μL of PBS and detected by a flow cytometer (BD FACS Calibur or BD FACS Canto_ II). All the antibodies had a strong binding ability to human or cynomolgus monkey PVRIG expressed on the cell surface, were significantly superior to the positive antibody Tab5, and Tab5 did not bind completely to cynomolgus monkey PVRIG.

[0178]

Table 6

[0179] Example 6. Blocking experiment on the binding of anti-PVRIG antibody to PVRIG and PVRL2 In this experiment, an in vitro blocking experiment was used to detect the blocking ability of the screened anti-PVRIG antibody against the binding of human PVRIG and its ligand, human PVRL2. Specifically, a human PVRIG recombinant protein with a mouse IgG2a Fc tag (h-PVRIG-mIgG2a Fc) was coated on a 96-well microtiter plate. After adding the anti-PVRIG antibody and allowing sufficient binding to occupy the epitope, his-tagged PVRL2 (PV2-H52E2, AcroBiosystem) was added. By detecting the his-tag, the binding amount between PVRIG and PVRL2 was calculated, and the IC 50 value of the anti-PVRIG antibody against the PVRIG active site was calculated.

[0180] The 96-well microtiter plate was coated with h-PVRIG-mIgG2a Fc protein at a concentration of 1 μg / mL, 100 μL per well, and incubated overnight at 4°C. It was washed three times with the washing solution, and the volume per well was 250 μL. To ensure thorough washing, it was shaken for 10 seconds each time during washing. Blocking solution was added at 300 μL / well and incubated at room temperature for 1 hour. It was washed three times with the washing solution, and the volume per well was 250 μL. To ensure thorough washing, it was shaken for 10 seconds each time during washing. 50 μL of the diluted anti-PVRIG test antibody and 50 μL of his-tagged ligand PVRL2 were added per well and incubated at 37°C for 1 hour. It was washed three times with the washing solution, and the volume per well was 250 μL. 100 μL of HRP-labeled anti-his-tag secondary antibody (Genscrpit) diluted 1:2000 was added per well. It was incubated at 37°C for 1 hour. It was washed three times with the washing solution, and the volume per well was 250 μL. 100 μL of TMB was added per well and reacted in the dark for 15 minutes. 50 μL of 0.16 M sulfuric acid was added per well. The OD value at 450 nm was read using a Thermo MμLtiSkan Fc microplate reader, and the IC50 of the anti-PVRIG antibody against the binding of PVRIG and PVRL2 was calculated.

[0181] As is clear from the results, all the detected antibodies can strongly inhibit the binding of human PVRIG and human PVRL2.

[0182]

Table 7

[0183] Example 7. Measurement of the affinity between anti-PVRIG antibody and PVRIG The Protein A biosensor (Fortebio, #18-5010) was immersed in 200 μL of KB buffer (PBS, pH 7.4, 0.02% tween-20, 0.1% BSA) for 60 seconds for wetting treatment. Then, the anti-PVRIG antibody was diluted to 10 μg / mL with the KB buffer, and the sensor was placed in 200 μL of the solution until the reading reached 1.2 nm. The sensor was immersed in the KB buffer for 100 seconds to elute the excess antibody. His-tagged human PVRIG was diluted between 64 nM and 4 nM with a two-fold gradient using the KB buffer. The sensor was placed in the solution and allowed to bind for 300 seconds. The sensor was placed in the KB buffer and dissociated for 600 seconds. Fitting was performed by the dynamic 1:1 binding method, and the affinity between the anti-PVRIG antibody and human PVRIG is shown in Table 8.

[0184] As is clear from the results, all the detected antibodies have a high affinity for human PVRIG.

[0185]

Table 8

[0186] Example 8. Activity experiment of anti-PVRIG antibody reporter gene cells First, construct the plvx-OS8 (G418-resistant) plasmid, infect 293F cells, screen with G418, detect the expression of clone cell OS8 by flow cytometry and detect the activation of Jurkat cells by OS8, select moderately activated clones to obtain the 293F-OS8 cell line, construct the plvx-PVRL2 plasmid, use it to infect 293F-OS8 cells, screen the clone with the highest PVRL2 expression level by flow cytometry to obtain the 293F-OS8-PVRL2 cell line.

[0187] Next, construct plvx-NFAT-Luc (Hygromycin-resistant), package it into lentivirus, infect Jurkat E6.1 cells, add Hygromycin to screen for resistant clones, stimulate the clones with OKT3, screen the clones with moderately high Luciferase signals to obtain the Jurkat-NFAT-Luc cell line, construct the plvx-PVRIG (Puromycin-resistant) vector, package it into lentivirus, infect Jurkat-NFAT-Luc cells, screen the clone with the highest PVRIG expression level by flow cytometry to obtain the Jurkat-NFAT-Luc-PVRIG cell line.

[0188] 1E4 Jurkat-NFAT-Luc-PVRIG cells were incubated with the test antibody at 37°C for 20 minutes. 1E5 293F-OS8-PVRL2 cells were added and incubated at 37°C for 5 hours. The supernatant was removed by centrifugation, Luciferase buffer (Promega, E6130) was added to lyse the cells, and the fluorescence value was detected. The EC50 value was calculated to evaluate the in vitro cell activity of the anti-PVRIG antibody. The experimental results are shown in Figure 1 and Table 9.

[0189] As is clear from the results, all of the detected antibodies were strong in their ability to activate Luciferase in Jurkat cells, with activities 3.7 - 18.5 times that of the positive antibodies, and it was proven that these antibodies could bind to PVRIG and block the binding of PVRL2 and PVRIG.

[0190]

Table 9

[0191] Example 9. NK cell killing experiment of anti - PVRIG antibody PVRIG is expressed in NK cells, while PVRL2 is expressed in many tumor cells (including K562 cells). By blocking the binding of PVRL2 and PVRIG, anti - PVRIG antibodies can relieve the inhibitory effect of tumor cells on the activity of NK cells.

[0192] The cultured NK92 cell line (NK cells from a human malignant non - Hodgkin lymphoma patient) was washed twice with a washing solution (RPMI 1640, 5% FBS, containing 10 ng / mL of IL - 2), and resuspended to a density of 2×10 6 cells / mL. 50 μL (a total of 1×10 5 cells) of NK92 cells were added to each well of a 96 - well plate. 50 μL of the test antibody at 20 nM or 100 nM was added and incubated at 37°C for 30 minutes. After washing twice with the washing solution, it was resuspended to a density of 2×10 5 cells / mL. 50 μL (a total of 1×10 4Human chronic myelogenous leukemia K562 cells (number not specified) were added, and the ratio of the number of NK92 cells to K562 cells was set to 10:1. Incubation was carried out at 37°C for 4 hours. The killing activity was measured using the CytoTox-Glo cytotoxicity system (Promega, G9292). First, 50 μL of AAF-Glo reagent was added, and incubation was carried out at room temperature for 15 minutes to measure the fluorescence of K562 cells killed by NK92 cells. Then, 50 μL of lysis solution was added, and incubation was carried out at room temperature for 15 minutes to lyse all the cells in the well and measure the fluorescence of all the cells. Three control groups were prepared, namely a sample containing only the culture medium (control group 1), a sample containing only NK92 cells (control group 2), and a 150 μL sample containing only K562 cells (control group 3), and the same operations were performed.

[0193] Based on the following formula, the killing activity was calculated. Killing activity (%) = {[(R - BG) - (T - BG) - (E - BG)] / [(TL - BGL) - (T - BG)]} × 100 Among them, R is the fluorescence value after adding AAF-Glo, BG is the fluorescence value after adding AAF-Glo to control group 1, E is the fluorescence value after adding AAF-Glo to control group 2, T is the fluorescence value after adding AAF-Glo to control group 3, TL is the fluorescence value after adding lysis solution to control group 3, and BGL is the fluorescence value after adding lysis solution to control group 1.

[0194] The experimental results are shown in Figure 2 and Table 10, indicating that all the detected anti-PVRIG antibodies can clearly activate NK92 cells and kill K562 cells.

[0195]

Table 10

[0196] Example 10. Mixed lymphocyte reaction (MLR) experiment of anti-PVRIG antibody PVRIG is expressed in T cells, while PVRL2 is expressed in dendritic cells (DC cells). By blocking the binding of PVRL2 and PVRIG, anti-PVRIG antibodies can relieve the inhibitory effect of dendritic cells on T cells and activate T cells.

[0197] The mixed lymphocyte reaction refers to the situation where lymphocytes with normal functions from two unrelated individuals are mixed and cultured in vitro. Since the major histocompatibility antigens are different, they can stimulate the proliferation of each other's T cells. PBMCs are isolated from peripheral blood of the first individual, and the cells are cultured in RPMI 1640 medium containing 10% FBS. Cytokines are added at a final concentration of 50 ng / mL of GM-CSF (Peprotech, 300-03-100UG) and 50 ng / mL of IL-4 (Peprotech, 200-04-100UG). Fresh medium containing cytokines is added every 2 - 3 days, and after culturing for 6 days, 1 μg / mL of LPS (Sigma, L2880-25MG) is added and incubated for 24 hours, and then differentiated and mature DC cells are collected. PBMCs are isolated from peripheral blood of the second individual, and CD3+ T cells are isolated from them using the EasySep Human CD3+ T Cell Isolation Kit (Stemcell, 17952). The densities of CD3+ T cells and DC cells are adjusted, and 1×10 5 CD3+ T cells per well and 2×10 4 DC cells are added. The test antibody is added, incubated at 37 °C for 120 h, the supernatant is taken, and the IFNγ content in the supernatant is detected using an ELISA kit (R&D, DY202).

[0198] The experimental results are shown in Figure 3 and Table 11, indicating that all detected anti-PVRIG antibodies can significantly activate T cells to secrete IFNγ compared with the control antibody IgG4. Moreover, at low doses (e.g., 4 nM, 20 nM), the antibodies of the present disclosure are more effective than the positive control Tab5.

[0199]

Table 11

[0200] Example 11. Humanization of Anti-PVRIG Antibody Based on the typical structures of the VH of the obtained camel single-domain antibodies 20, 30, 38, 39 and 151, the heavy-chain variable region sequences were compared with the antibody GermLine database to obtain highly homologous human germline templates. The framework regions of the camel single-domain antibodies were replaced with the heavy-chain framework regions of the human germline templates, while retaining the CDRs (by the Kabat numbering system), and further recombined with the Fc region of human IgG (IgG4 Fc with S228P, F234A, L235A, K447A mutations). Based on the three-dimensional structure of the camel single-domain antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important influence on the conformation of the variable region were reverted to mutations, and the chemically unstable amino acid residues in the CDR region were optimized to produce a series of humanized single-domain antibodies. The human germline templates and humanized antibody heavy-chain variable region sequences of each single-domain antibody are shown in Tables 12-16.

[0201] [Table 12]

[0202] According to Table 12, antibodies 20H1-20H5 contain CDR1 shown in TDCMG (SEQ ID NO:7), CDR2 shown in HIDSDGIPRYVDSVKG (SEQ ID NO:8), and CDR3 shown in GFKFDEDYCAPND (SEQ ID NO:150).

[0203] [Table 13]

[0204] According to Table 13, antibodies 30H1 - 30H5 contain CDR1 shown in GDCMG (SEQ ID NO:10), CDR2 shown in TIDNAGRIKYADSVKG (SEQ ID NO:11), and CDR3 shown in GWTFGGQCSPAD (SEQ ID NO:151).

[0205]

Table 14

[0206]

Table 15

[0207]

Table 16

[0208] The above - mentioned humanized antibody heavy - chain variable region was connected to the human IgG4 heavy - chain Fc region to construct a full - length anti - PVRIG antibody. Among them, the heavy - chain Fc region contains a hinge region and has S228P, F234A, L235A, K447A mutations. >Human IgG4 heavy - chain Fc region (S228P / F234A / L235A / K447A) ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGA (SEQ ID NO:101) >Human IgG4 heavy - chain Fc region (S228P / K447A) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:153)

[0209] Antibody expression and purification were carried out according to the conventional method, and upon detection, the target antibody was obtained.

[0210] Example 12. Binding experiment between humanized anti-PVRIG antibody and cells expressing PVRIG According to the method of Example 5, the binding between the humanized anti-PVRIG antibody and human or cynomolgus monkey PVRIG was detected by flow cytometry. The experimental results are shown in Table 17.

[0211] [Table 17]

[0212] Example 13. Affinity measurement between humanized anti-PVRIG antibody and PVRIG According to the method of Example 7, the affinity between the humanized anti-PVRIG antibody and human PVRIG was detected. The results are shown in Table 18. All the antibodies listed in the table have high affinity for human PVRIG.

[0213] [Table 18]

[0214] Example 14. Activity experiment of humanized anti-PVRIG antibody reporter gene cells According to the method of Example 8, the activity of the humanized anti-PVRIG antibody in reporter gene cells was detected. The experimental results are shown in FIGS. 4A to 4B and Table 19. All the antibodies listed in the table have the ability to activate Jurkat cells.

[0215]

Table 19

[0216] Example 15. Experiment on the killing ability of NK cells activated by humanized anti-PVRIG antibody According to the method of Example 9, the activation ability of the humanized anti-PVRIG antibody on NK cells was detected. The experimental results are shown in FIGS. 5A to 5B and Tables 20 to 21. As is clear from the results, all the humanized anti-PVRIG antibodies of the present disclosure have a remarkable ability to activate NK cells and promote the killing of target cells K562 by NK cells.

[0217]

Table 20

[0218]

Table 21

[0219] Example 16. Preparation of anti-PVRIG / TIGIT bispecific antibody To investigate the effect on the antibody function of anti-PVRIG / TIGIT bispecific antibodies with different structures, the anti-PVRIG single-domain antibody 151 is connected to the N-terminus or C-terminus of the heavy chain or light chain of the anti-TIGIT antibody 1708 by a GGGGSGGGGS (SEQ ID NO: 152) linker. Four anti-PVRIG / TIGIT bispecific antibodies are formed and named 1708-151-1, 1708-151-2, 1708-151-3, and 1708-151-4, which are connected to the N-terminus of the heavy chain, C-terminus of the heavy chain, N-terminus of the light chain, and C-terminus of the light chain of 1708 corresponding to 151, respectively. The anti-TIGIT antibody 1708 adopts the human IgG4 subtype and has a mutation of S228P (Eu nomenclature system). The sequences of the anti-TIGIT antibody 1708 and the bispecific antibodies formed by it and 151 are shown in Table 22. The sequence information of the anti-TIGIT antibody is shown in Tables 23-24. The TIGIT antibody in WO2019062832A is incorporated herein in its entirety.

[0220]

Table 22-1

[0221]

Table 22-2

[0222]

Table 23

[0223]

Table 24

[0224] Transient transfection, expression, and purification of the antibody were performed according to the conventional method, and upon identification, the full-length anti-PVRIG / TIGIT bispecific antibody of the present disclosure was obtained. The expression level and purity of the bispecific antibody are shown in Table 25. The nanobody was conjugated to a general monoclonal antibody, and good expression levels and purity were shown at both the N-terminus and C-terminus of the heavy and light chains.

[0225]

Table 25

[0226] Example 17. Experiment on the binding of anti-PVRIG / TIGIT bispecific antibody to PVRIG and TIGIT and the blockade against the corresponding ligands A) Binding of bispecific antibodies with different arrangements to human PVRIG and blockade against ligand PVRL2 Experiments were carried out according to the methods of Example 4, Example 5, and Example 6, and the results are shown in Table 26. As is clear from the results, the bispecific antibodies with different arrangements were substantially identical in terms of binding to human PVRIG recombinant protein and overexpressed human PVRIG cells, and blockade against the binding of PVRL2 and PVRIG, with no difference.

[0227]

Table 26

[0228] B) Binding of bispecific antibodies with different arrangements to human TIGIT and blockade against ligand PVR Experiments were carried out according to the methods of Example 4, Example 5, and Example 6 (substituting the corresponding receptor and ligand with human TIGIT and human PVR), and the results are shown in Table 27. As is clear from the results, the bispecific antibodies with different arrangements and the anti-TIGIT antibody were substantially identical in terms of binding to human TIGIT recombinant protein and overexpressed human TIGIT cells, and blockade against the binding of TIGIT and its ligand PVR, with no difference. The connection method of anti-PVRIG antibody 151 did not substantially affect the binding of the anti-TIGIT antibody and TIGIT.

[0229]

Table 27

[0230] When the data in Tables 24 to 25 were summarized, the anti-PVRIG antibody retained the binding to PVRIG and TIGIT and the blockade against the ligand regardless of being connected to the N-terminus or C-terminus of the heavy chain or light chain of the anti-TIGIT antibody, and showed good expression levels and purity.

[0231] Example 18. Preparation of humanized anti-PVRIG / TIGIT bispecific antibody Different humanized anti-PVRIG antibodies (20H5, 30H2, 39H2, 151H7, 151H8) were connected to the N-terminus of the heavy chain of the anti-TIGIT antibody 1708, that is, bispecific antibodies were constructed using a bispecific antibody structure similar to 1708-151-1, and the sequences are shown in Table 28.

[0232]

Table 28-1

Table 28-2

[0233] According to the conventional method, transient transfection, expression and purification of the antibody were carried out and identified, and the target bispecific antibody was obtained.

[0234] Example 19. Binding of humanized anti-PVRIG / TIGIT bispecific antibody to PVRIG and TIGIT and blockade against the corresponding ligand According to the methods of Examples 4, 5, and 6, the binding of humanized anti-PVRIG / TIGIT bispecific antibodies to human and cynomolgus monkey PVRIG and the blockade of human PVRIG from its ligand were detected. The results are shown in Table 29 and Figures 6A-6E. As is clear from the results, each humanized bispecific antibody can bind to human PVRIG and block the binding of PVRIG to PVRL2. 1708-151H8 showed weak binding to cynomolgus monkey PVRIG.

[0235] [Table 29]

[0236] Similar to Examples 4, 5, and 6, the binding of humanized anti-PVRIG / TIGIT bispecific antibodies to human and cynomolgus monkey TIGIT and the blockade of the binding between human TIGIT and its ligand were detected. Among them, the PVRIG protein was replaced with TIGIT, and PVRL2 was replaced with PVR. The results are shown in Table 30 and Figures 7A-7E. As is clear from the results, each bispecific antibody can bind to human and cynomolgus monkey TIGIT and block the binding of TIGIT to PVR.

[0237] [Table 30]

[0238] The affinity of the humanized bispecific antibody with human PVRIG, cynomolgus monkey PVRIG, and human TIGIT was detected by Biacore. After capturing the humanized bispecific antibody on a Protein A biosensor chip (GE lifesciences, 29127557) of a Biacore instrument (Biacore X100, GE), a series of concentration gradients of human PVRIG antigen (AcroBiosystem, PVG-H52H4), cynomolgus monkey PVRIG antigen (SEQ ID NO:1), or human TIGIT antigen (AcroBiosystem, TIT-H52H3) were flowed over the chip surface. The reaction signals were detected in real time by a Biacore instrument (Biacore X100, GE) to obtain binding and dissociation curves. The data obtained in the experiment were fitted using the software BiacoreX100 evaluation software 2.0 GE in a (1:1) Binding model to obtain affinity values, which are shown in Table 31.

[0239] [Table 31]

[0240] Example 20. Mixed lymphocyte reaction (MLR) experiment of humanized anti-PVRIG / TIGIT bispecific antibody According to the method of Example 10, the activation ability of the humanized anti-PVRIG / TIGIT bispecific antibody on T cells was detected. The experimental results are shown in Figure 8 and Table 32. As is clear from the results, the humanized anti-PVRIG / TIGIT bispecific antibody 1708-151H8 has a significant ability to activate T cells and promotes T cells to secrete IFNγ. Importantly, the activity of the bispecific antibody is stronger than that when using the anti-PVRIG antibody 151H8 alone or the anti-TIGIT antibody 1708 alone.

[0241] [Table 32]

[0242] Example 21. Evaluation of the antitumor effect of an anti-PVRIG / TIGIT bispecific antibody in a mouse subcutaneous xenograft tumor model of human melanoma A375 mixed with human PBMCs To further investigate the effect of the bispecific antibody subtype on animal pharmacodynamics, in addition to the IgG4 subtype bispecific antibody described above, the corresponding IgG1 subtype antibody was synthesized and used in the animal pharmacodynamic test. Other antibody sequences not described above used in this experiment are shown in Table 33.

[0243]

Table 33

[0244] Female NCG mice, 4 - 8 weeks old, weighing approximately 18 - 22 g, were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. All NCG mice were cultured under the conditions of an IVC constant temperature and pressure system, which is an SPF-level animal breeding room. A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). A375 cells in the exponential growth phase were collected, resuspended in HBSS until the appropriate concentration was reached, and used for subcutaneous tumor inoculation of NCG mice. The A375 cells used for co-culture needed to be treated with Mitomycin C for 2 h and then washed 3 times with PBS. Normal human peripheral blood was collected, and human PBMCs were isolated by density gradient centrifugation and counted. Then, PBMCs were resuspended in RPMI1640 medium (containing IL2 and 10% FBS) at a concentration of 3×10 6 cells / mL and co-cultured with A375 cells treated with Mitomycin C. After 6 days of co-culture, PBMCs were collected, and at the same time, freshly digested A375 cells were collected. Each mouse was injected with 5×10 5 cells of PBMCs and 4×10 6Individual A375 cells were inoculated with an inoculation volume of 0.2 mL / mouse (containing 50% Matrigel) and subcutaneously inoculated on the right side of female NCG mice. The mice were randomly grouped and administered based on their body weight. The detailed administration method, dosage, and administration route are shown in Table 34, and the day of grouped administration is designated as Day 0. Since the molecular weights of the anti-PVRIG antibody and the anti-TIGIT antibody are different, the dosage was ensured to guarantee that the anti-PVRIG antibody and the anti-TIGIT antibody had the same starting molar concentration.

[0245]

Table 34

[0246] After the start of administration, the body weight and tumor volume of the mice were measured twice a week. The experimental results are shown in Tables 35 - 36 and Figures 9A - 9B, respectively.

[0247]

Table 35

[0248]

Table 36

[0249] At the end of the experiment (Day 26 after administration), compared with the control group, there was no significant difference in the single-agent group of anti-PVRIG antibody 151. The tumor volume decreased in the single-agent group of anti-TIGIT antibody 1708-IgG1, the combination group of anti-PVRIG antibody 151 and anti-TIGIT antibody 1708-IgG1, and the bispecific antibody group of 1708-151-IgG1. The bispecific antibody group of 1708-151-IgG4 could completely inhibit tumor growth and was significantly different from other groups (shown in Figure 9B).

[0250] The mice were randomly grouped and administered based on their body weight. The detailed administration method, dosage, and administration route are shown in Table 37, and the day of grouped administration is designated as Day 0.

[0251]

Table 37

[0252] After the start of administration, the body weight and tumor volume of the mice were measured twice a week. The experimental results are shown in Tables 38 - 39 and Figures 10A - 10B, respectively.

[0253]

Table 38

[0254]

Table 39

[0255] At the end of the experiment (the 28th day after administration), compared with the control group, both the dual - antibody groups of 1708 - 30H2 IgG4 and 1708 - 151H7 IgG4 could effectively inhibit tumor growth at low doses, showing a significant difference from the control group (shown in Figures 10A and 10B).

[0256] As described above, the specific embodiments of the present disclosure have been explained. However, those skilled in the art should understand that these are merely exemplary explanations, and various changes and modifications can be made to these embodiments without departing from the principles and gist of the present invention. Therefore, the scope of the present disclosure is limited by the appended claims.

Claims

1. A PVRIG-binding protein comprising at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 3, 80-84, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 2, 75-79, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 4, 86-90, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 5, 91-95, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 6, 96-100, and The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, a PVRIG-binding protein.

2. According to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively shown in SEQ ID NO: 10, 11 and 12 or 151, or shown in SEQ ID NO: 7, 8 and 9 or 150, or shown in SEQ ID NO: 13, 14 and 15, or shown in SEQ ID NO: 16, 17 and 18, or shown in SEQ ID NO: 19, 20 and 21, The PVRIG-binding protein according to claim 1.

3. The immunoglobulin single variable domain of the PVRIG-binding protein is VHH, The humanized and / or affinity matured VHH comprises the heavy chain framework region IGHV3-7*01 or IGHV3-30*02 of the human germline template, the PVRIG-binding protein according to claim 1 or 2.

4. The amino acid sequences of the immunoglobulin single variable domain are respectively shown in any one of SEQ ID NO: 3, 80-84, or shown in any one of SEQ ID NO: 2, 75-79, or shown in any one of SEQ ID NO: 4, 86-90, or shown in any one of SEQ ID NO: 5, 91-95, or shown in any one of SEQ ID NO: 6, 96-100, or The PVRIG-binding protein according to any one of claims 1 to 3, having at least 80% sequence identity with any one of the said sequences.

5. Further comprising a human immunoglobulin Fc region, The Fc region is the Fc region of human IgG1 or IgG4, the PVRIG-binding protein according to any one of claims 1 to 4.

6. A PVRIG / TIGIT-binding protein comprising a first antigen-binding domain that specifically binds to PVRIG and a second antigen-binding domain that specifically binds to TIGIT, 1) The first antigen-binding domain that specifically binds to PVRIG comprises at least one immunoglobulin single variable domain, and the immunoglobulin single variable domain, CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 3, 80 to 84, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 2, 75 to 79, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 4, 86 to 90, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 5, 91 to 95, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NO: 6, 96 to 100, The CDR1, CDR2, CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; and, 2) The second antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), among which, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 121, 122 and 123 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 124, 125 and 126 respectively, A PVRIG / TIGIT-binding protein.

7. The first antigen-binding domain that specifically binds to PVRIG comprises at least one immunoglobulin single variable domain, and the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively shown in SEQ ID NO: 10, 11 and 12 or 151, or shown in SEQ ID NO: 7, 8, and 9 or 150, or shown in SEQ ID NO: 13, 14, and 15, or shown in SEQ ID NO: 16, 17, and 18, or shown in SEQ ID NO: 19, 20, and 21, the PVRIG / TIGIT binding protein according to claim 6.

8. The amino acid sequences of the immunoglobulin single variable domains in the first antigen-binding domain are respectively shown in any one of SEQ ID NO: 3, 80 - 84, or shown in any one of SEQ ID NO: 2, 75 - 79, or shown in any one of SEQ ID NO: 4, 86 - 90, or shown in any one of SEQ ID NO: 5, 91 - 95, or shown in any one of SEQ ID NO: 6, 96 - 100, or the PVRIG / TIGIT binding protein according to claim 6 or 7, having at least 80% sequence identity with any one of the above sequences.

9. The heavy chain variable region of the second antigen-binding domain that specifically binds to TIGIT is shown in any one of SEQ ID NO: 145 - 147, or includes an amino acid sequence having at least 90% sequence identity therewith, and the light chain variable region is shown in any one of SEQ ID NO: 148 - 149, or includes an amino acid sequence having at least 90% sequence identity therewith, the PVRIG / TIGIT binding protein according to any one of claims 6 to 8.

10. The second antigen-binding domain that specifically binds to TIGIT includes a full-length heavy chain (HC) and a full-length light chain (LC), the heavy chain sequence is shown in SEQ ID NO: 102, or has at least 90% sequence identity therewith, and the light chain sequence is shown in SEQ ID NO: 103, or has at least 90% sequence identity therewith, the PVRIG / TIGIT binding protein according to any one of claims 6 to 9.

11. The second antigen-binding domain that specifically binds to TIGIT includes a heavy chain variable region (VH) and a light chain variable region (VL), among which the immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG is located at the N-terminus of the heavy chain variable region of the second antigen-binding domain that specifically binds to TIGIT, The immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG is located at the C-terminus of the heavy chain variable region of the second antigen-binding domain that specifically binds to TIGIT. The immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG is located at the N-terminus of the light chain variable region of the second antigen-binding domain that specifically binds to TIGIT, and / or The immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG is located at the C-terminus of the light chain variable region of the second antigen-binding domain that specifically binds to TIGIT. The PVRIG / TIGIT binding protein according to any one of claims 6 to 10.

12. The immunoglobulin single variable domain of the first antigen-binding domain that specifically binds to PVRIG and the second antigen-binding domain that specifically binds to TIGIT are connected directly or by a linker. The PVRIG / TIGIT binding protein according to claim 11.

13. Comprising a first polypeptide chain and a second polypeptide chain, wherein The first polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 108 to 112 and 114, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 103, or The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 104 or 105, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 103, or The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 102, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 106 or 107. The PVRIG / TIGIT binding protein according to any one of claims 6 to 12.

14. An anti-PVRIG antibody or an antigen-binding fragment thereof comprising at least one immunoglobulin single variable domain, 1) The immunoglobulin single variable domain is CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NOs: 3, 80 to 84, or CDR1, CDR2 and CDR3 in the sequence shown in any one of SEQ ID NOs: 2, 75 to 79, or CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NOs: 4, 86 - 90, or CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NOs: 5, 91 - 95, or CDR1, CDR2, and CDR3 in the sequence shown in any one of SEQ ID NOs: 6, 96 - 100, and the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; or 2) The amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are respectively shown in SEQ ID NOs: 10, 11, and 12 or 151, or shown in SEQ ID NOs: 7, 8, and 9 or 150, or shown in SEQ ID NOs: 13, 14, and 15, or shown in SEQ ID NOs: 16, 17, and 18, or shown in SEQ ID NOs: 19, 20, and 21, an anti - PVRIG antibody or an antigen - binding fragment thereof.

15. The anti - PVRIG antibody or an antigen - binding fragment thereof further comprises a second antigen - binding domain that specifically binds to TIGIT, and the second antigen - binding domain that specifically binds to TIGIT comprises a heavy - chain variable region (VH) and a light - chain variable region (VL), wherein the heavy - chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 121, 122, and 123 respectively, and the light - chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 124, 125, and 126 respectively. The anti - PVRIG antibody or an antigen - binding fragment thereof according to claim 14.

16. A polynucleotide encoding a PVRIG - binding protein according to any one of claims 1 - 5, a PVRIG / TIGIT - binding protein according to any one of claims 6 - 13, or an anti - PVRIG antibody or an antigen - binding fragment thereof according to claim 14 or 15.

17. A host cell comprising the polynucleotide according to claim 16.

18. The step of expressing the polynucleotide according to claim 16 in the host cell according to claim 17, and the step of isolating the PVRIG - binding protein, PVRIG / TIGIT - binding protein, or anti - PVRIG antibody or an antigen - binding fragment thereof expressed from the host cell. A method for preparing a PVRIG-binding protein, a PVRIG / TIGIT-binding protein, or an anti-PVRIG antibody or an antigen-binding fragment thereof, which comprises

19. A pharmaceutical composition comprising the PVRIG-binding protein according to any one of claims 1 to 5, the PVRIG / TIGIT-binding protein according to any one of claims 6 to 13, or the anti-PVRIG antibody or an antigen-binding fragment thereof according to claim 14 or 15, and a pharmaceutically acceptable excipient, diluent or carrier.

20. A pharmaceutical composition for treating or delaying cancer in a subject, comprising the PVRIG-binding protein according to any one of claims 1 to 5, the PVRIG / TIGIT-binding protein according to any one of claims 6 to 13, or the anti-PVRIG antibody or an antigen-binding fragment thereof according to claim 14 or 15, the polynucleotide according to claim 16, or the pharmaceutical composition according to claim 19, or any combination thereof.

21. The pharmaceutical composition according to claim 20, wherein the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, blood cancer or any other disease or condition characterized by uncontrolled cell proliferation.

22. A pharmaceutical composition for activating NK cells, γδT cells and / or Th1 cells in a subject in need thereof, comprising the PVRIG-binding protein according to any one of claims 1 to 5, the PVRIG / TIGIT-binding protein according to any one of claims 6 to 13, or the anti-PVRIG antibody or an antigen-binding fragment thereof according to claim 14 or 15, the polynucleotide according to claim 16, or the pharmaceutical composition according to claim 19, or any combination thereof.

23. A pharmaceutical composition for increasing the production of interferon-γ and / or the secretion of pro-inflammatory cytokines in a subject in need thereof, comprising the PVRIG-binding protein according to any one of claims 1 to 5, the PVRIG / TIGIT-binding protein according to any one of claims 6 to 13, or the anti-PVRIG antibody or an antigen-binding fragment thereof according to claim 14 or 15, the polynucleotide according to claim 16, or the pharmaceutical composition according to claim 19, or any combination thereof.

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