PVRIG antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-human PVRIG antibody or an antigen binding fragment thereof, the anti-human PVRIG antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises antigenic determining regions VH CDR1, VH CDR2 and VH CDR3, and the VL comprises antigenic determining regions VL CDR1, VL CDR2 and VL CDR3. The humanized anti-human PVRIG antibody can be specifically combined with PVRIG, and shows a good clinical anti-tumor application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to novel artificially designed anti-PVRIG antibodies or antigen-binding fragments thereof and applications thereof, more specifically, the present invention relates to antibodies or antigen-binding fragments thereof that can specifically recognize PVRIG, nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions, pharmaceutical uses, kits for detecting PVRIG and applications thereof. Background Art

[0002] Driven by the success of tumor immunotherapy, the research on negative regulatory lymphocyte function receptors is developing rapidly, and immune checkpoint receptors and their immune signaling networks have become an important research field. Among them, the CD112 / CD155 co-inhibitory network has become the focus of tumor immune targeted therapy research in recent years.

[0003] 1. CD112 / CD155 co-inhibitory network

[0004] Different inhibitory and co-stimulatory checkpoint receptors in the co-inhibitory network can regulate each other, and ligands can regulate the activity of lymphocytes by binding to different receptors. In the co-inhibitory pathway, members of the PVR and PVR-like families, including PVRIG (CD112R), TIGIT, CD96, CD155, CD226, and CD112, form a complex signaling network to regulate T cell activation, in which CD112 / CD155 and its receptor form a co-inhibitory network. CD155 can interact with CD226, TIGIT, and CD96, while CD112 interacts with CD226, TIGIT, and CD112R ( Fig.26 ). Competition or synergistic regulation between CD112R (i.e., PVRIG) and other immune receptors and their associated ligands controls immune cell infiltration and activation in the tumor microenvironment. CD112 is also a ligand for CD226, which can activate T cells and NK cells. Compared with CD226, CD112 has a higher affinity for PVRIG, so under normal circumstances, CD112 mainly inhibits the activation of NK and T cells by binding to PVRIG, thereby effectively regulating CD226-mediated cell activation. Anti-CD112R antibodies that block the interaction with CD112 directly limit the inhibitory signaling downstream of CD112R, while promoting greater immune cell activation by increasing the interaction of CD226 with CD112.

[0005] In CD8+T cells activated by specific antigens, co-expression of CD112R (PVRIG) and TIGIT can be observed. PVRIG, as a new immune checkpoint protein expressed on the surface of T cells and NK cells, exhibits binding activity with CD112, but does not bind to CD155 [Zhu Y, Paniccia A, Schulick AC, et al. Identification of CD112R as a novel checkpoint for human T cells. [J]. Journal of Experimental Medicine, 2016, 213 (2): 167-176.]. Blocking the binding of PVRIG (CD112R) and ligand CD112 can lead to the induced expression of TIGIT, which transmits inhibitory signals to NK cells and T cells, while also competing with CD226 for ligands and blocking the stimulatory signals it transmits, thereby achieving negative regulation of T cell function. Compared with CD226, the ligand CD112 on the surface of tumor cells has a high affinity with PVRIG, and can inhibit the cellular function of lymphocytes after binding. Studies have shown that PVRIG is an inhibitory receptor expressed on natural killer cells (NK cells) and T cells, which regulates immune function. We believe that targeted intervention of PVRIG can effectively enhance the anti-tumor response in vitro [Whelan S, Ophir E, Kotturi MF, et al. PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+T-cell Function[J]. Cancer Immunology Research, 2019, 7(2): 257-268.].

[0006] CD112R expression occurs in NK cells, CD8+T cells, and CD4+T cells in various types of solid tumors. High levels of expression are often seen in tumors of the kidney, ovary, lung, prostate, and endometrium, and in T cells and NK cells in acute myeloid leukemia. CD112R expression occurs simultaneously with PD-1 and TIGIT expression on CD8+ and CD4+ TILs and is accompanied by the expression of exhaustion markers on NK cells, including CD96, TIGIT, Tim-3, and PD-1.

[0007] 2. The role of PVRIG (CD112R) / CD112 axis in tumor therapy

[0008] The CD112R / CD112 axis is known to impede the immune function of T cells. The novel CD112R / CD112 pathway is expected to play a role in tumor immunotherapy.

[0009] Existing data indicate that the CD112R-CD112 axis plays an important role in regulating the tumor-killing ability of T cells and NK cells. Recent studies have found that in CD112R-deficient mouse models of melanoma and colon cancer, tumor growth is stopped and tumor immune cell infiltration is increased. In addition, blocking CD112R contributes to the anti-tumor efficacy of NK cells and inhibits tumor growth. In vitro, CD112R or TIGIT blockers can increase the number of IFN-γ+NK cells co-cultured with breast cancer cells. The combination of CD112R and TIGIT monoclonal antibodies can induce additional increases in IFN-γ+NK cells and enhance the cytotoxicity of NK cells. CD112R blockade can effectively inhibit tumor growth and prolong the survival of tumor-bearing mice, which is related to the enhancement of the frequency and cytotoxic potential of tumor-infiltrating NK cells.

[0010] 3. Application requirements

[0011] Specific targeted drugs targeting the (PVRIG) CD112R / CD112 axis have shown promising applications in cancer immunotherapy. Clinical results have shown that PVRIG (CD112R) blockers can not only serve as an adjuvant therapy, but may also become a viable alternative monotherapy. After the first "first-in-class" monoclonal antibody COM701 targeting PVRIG was approved for clinical use, antibodies targeting PVRIG need to be further improved to obtain more new structures of blocking antibodies that can specifically target PVRIG or CD112 with high affinity and inhibit PVRIG (CD112R) / CD112 binding to meet the alternative needs of cancer treatment optimization combination strategies and combination drug strategies. Summary of the invention

[0012] The present invention aims to solve the problems of tumor treatment in the prior art, specifically, to solve the technical problem of improving the efficacy of tumor-targeted antibody molecules. To this end, one object of the present invention is to provide an antibody or antigen-binding fragment that specifically binds to PVRIG, which can effectively bind to PVRIG and can block the binding of PVRIG to its ligand CD112. The anti-PVRIG antibody or antibody fragment provided has been verified to have a high affinity for human PVRIG. And it can effectively block the interaction between human PVRIG and CD112. The anti-PVRIG antibody or antigen-binding fragment thereof provided by the present invention can be used as an independent therapy or in combination with other therapies / or other anti-cancer agents for the treatment of cancer.

[0013] The present invention is accomplished based on the following problems and findings of the inventors:

[0014] Tumor immunotherapy is a huge breakthrough in the field of tumor treatment in recent years. At present, PVRIG is a very important emerging immune checkpoint. For cancer patients, the expression of CD112 is upregulated on the surface of various tumor cells. When CD112 binds to PVRIG on the surface of immune cells, it inhibits the cell activity of T cells and NK cells, and achieves immune escape by inhibiting their anti-tumor function; PVRIG antibody can block the PVRIG / CD112 signaling pathway by binding to PVRIG, restore the immune killing function of T cells and NK cells, and can exert a good therapeutic effect, with good clinical application prospects. In addition, studies have also shown that PVRIG antibody therapy can significantly inhibit tumor growth, and combined with other immune checkpoint inhibitors can achieve better therapeutic effects. At present, a humanized antibody targeting PVRIG is undergoing phase I clinical trials, and has achieved good therapeutic effects.

[0015] Based on this, the inventors have obtained a variety of anti-PVRIG antibodies that can specifically recognize PVRIG after a large number of experiments. The anti-PVRIG antibodies of the present invention can specifically target and bind to the PVRIG receptor, thereby blocking the binding of CD112 and PVRIG, and enhancing the killing effect of lymphocytes (especially T cells and NK cells). Especially for cancer patients, after the PVRIG antibody binds to PVRIG, it can enhance the killing effect of lymphocytes and improve the therapeutic effect of existing antibody drugs for treating cancer.

[0016] The high affinity anti-PVRIG antibodies described herein block TIGIT- and / or CD112R-PVRIG interactions and subsequently restore T cell and NK cell activity. The antibodies of the present invention are highly specific for human PVRIG. These properties make the anti-PVRIG monoclonal antibodies of the present invention valuable candidates for cancer immunotherapy, which can be administered at lower doses and have fewer side effects.

[0017] Furthermore, the antibodies described herein have been found to be highly specific for human or cynomolgus monkey PVRIG protein.

[0018] In a first aspect of the present invention, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically recognize PVRIG.

[0019] According to certain embodiments, the antibody specifically recognizes human PVRIG and binds to the extracellular domain of PVRIG.

[0020] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: a CDR sequence selected from at least one of the following:

[0021] The HCDR sequence of the heavy chain variable region is selected from the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15;

[0022] LCDR sequence of the light chain variable region: selected from the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, or an amino acid sequence that has at least 80% identity with the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0023] According to an embodiment of the present invention, the above-mentioned antibody can specifically target and bind to the PVRIG receptor, blocking the binding between PVRIG and CD112.

[0024] According to an embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0025] In some embodiments, the antibody or antigen-binding fragment thereof that specifically recognizes and binds to PVRIG of the present invention comprises heavy chain complementary determining regions VHCDR1, VHCDR2, and VHCDR3, and light chain complementary determining regions VLCDR1, VLCDR2, and VLCDR3;

[0026] Wherein, the VHCDR1 is selected from the sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 13,

[0027] The VHCDR2 is selected from the sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 14,

[0028] The VHCDR3 is selected from the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 15;

[0029] Wherein, the VLCDR1 is selected from the sequence shown in any one of SEQ ID NO: 4 or SEQ ID NO: 10 and SEQ ID NO: 16,

[0030] The VLCDR2 is selected from the sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 17,

[0031] The VLCDR3 is selected from the sequence shown in any one of SEQ ID NO:6, SEQ ID NO:12, and SEQ ID NO:18.

[0032] According to an embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0033] The antibodies include:

[0034] The VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively; or the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively; or the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively.

[0035] According to an embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0036] The antibodies include:

[0037] The VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively; or the VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively; or the VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively.

[0038] In a second aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to PVRIG, wherein the antibody or antigen-binding fragment comprises a combination of 6 CDR sequences selected from any one of the following:

[0039] i. A set of 6 CDRs, wherein: VHC CDR1 is (SEQ ID NO: 1); VH CDR2 is (SEQ ID NO: 2); VHCDR3 is (SEQ ID NO: 3); VL CDR1 is (SEQ ID NO: 4); VL CDR2 is (SEQ ID NO: 5); and VL CDR3 is (SEQ ID NO: 6);

[0040] ii) a set of 6 CDRs, wherein: the VH CDR1 sequence is (SEQ ID NO: 7); VH CDR2 is (SEQ ID NO: 8); VH CDR3 is (SEQ ID NO: 9); VL CDR1 is (SEQ ID NO: 10); VL CDR2 is (SEQ ID NO: 11); and VL CDR3 is (SEQ ID NO: 12) and

[0041] iii. a group of 6 CDRs, wherein: the VLCDR1 sequence is (SEQ ID NO: 11); VL CDR2 is (SEQ ID NO: 12); VL CDR3 is (SEQ ID NO: 13); VL CDR1 is (SEQ ID NO: 14); VL CDR2 is (SEQ ID NO: 15); and VL CDR3 is (SEQ ID NO: 16).

[0042] Iv. An amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of i or ii or iii.

[0043] According to an embodiment of the present invention, the anti-PVRIG antibody or antigen-binding fragment thereof specifically recognizes the extracellular region of PVRIG. Analogs and derivatives of the antibody and fragment are also within the scope of the present invention.

[0044] In a third aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to PVRIG, comprising a heavy chain variable region (VH) and a light chain variable region (VL).

[0045] According to an embodiment of the present invention, the antibody or its antigen-binding fragment contains: at least one of a heavy chain framework region sequence and a light chain framework region sequence; wherein at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0046] In one embodiment, the heavy chain variable region (VH) of the antibody of the present invention comprises VHCDR1, VHCDR2 and VHCDR3 of any of the aforementioned aspects, and further comprises a heavy chain framework region (VHFRs), wherein the VHFRs comprises VHFR1, VHFR2, VHFR3 and VHFR4 of at least one of the following FR regions:

[0047] In one embodiment, the heavy chain framework region VHFR1 comprises an amino acid sequence having no less than 85% homology to the sequence shown in any one of SEQ ID NOs: 63-70; in one embodiment, the heavy chain framework region VHFR2 comprises an amino acid sequence having no less than 85% homology to the sequence shown in any one of SEQ ID NOs: 71-76; in one embodiment, the heavy chain framework region VHFR3 comprises an amino acid sequence having no less than 85% homology to the sequence shown in any one of SEQ ID NOs: 77-83; in one embodiment, the heavy chain framework region VHFR4 comprises an amino acid sequence having no less than 85% homology to the sequence shown in any one of SEQ ID NOs: 84-91. Each possibility represents an independent embodiment of the present invention.

[0048] In one embodiment, the light chain variable region (VL) of the antibody of the present invention comprises VLCDR1, VLCDR2 and VLCDR3 of any of the aforementioned items, and further comprises a light chain framework region (VLFRs), wherein the VLFRs are selected from VLFR1, VLFR2, VLFR3 and VLFR4 of at least one of the following FR regions.

[0049] In one embodiment, the light chain variable region VLFR1 comprises an amino acid sequence having no less than 85% homology to the sequence shown in any one of SEQ ID NOs: 92-100; in one embodiment, the light chain framework region VLFR2 comprises an amino acid sequence having no less than 85% homology to the sequence shown in any one of SEQ ID NOs: 101-109; in one embodiment, the light chain framework region VLFR3 comprises an amino acid sequence having no less than 85% homology to the sequence shown in any one of SEQ ID NOs: 110-118; in one embodiment, the light chain framework region VLFR4 comprises an amino acid sequence having no less than 85% homology to the sequence shown in any one of SEQ ID NOs: 119-127. Each possibility represents an independent embodiment of the present invention.

[0050] In one embodiment, the antibody has a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35;

[0051] In one embodiment, the antibody has a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 24, 26, 28, 36, 37, 38, 39, 40, 41 or 42;

[0052] In one embodiment, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 23 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 24;

[0053] In one embodiment, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 25 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 26;

[0054] In one embodiment, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 27 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 28;

[0055] In one embodiment, the antibody has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 29 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 36;

[0056] In one embodiment, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 30 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 37;

[0057] In one embodiment, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 31 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 38;

[0058] In one embodiment, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 32 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 39;

[0059] In one embodiment, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 33 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 40;

[0060] In one embodiment, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:34 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:42;

[0061] In one embodiment, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:35 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:39.

[0062] In a fourth aspect, the present invention provides an anti-PVRIG antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups:

[0063] (I) a heavy chain variable region selected from the amino acid sequence of SEQ ID NO: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35, or an analog having at least 85% sequence similarity to the heavy chain variable region sequence of SEQ ID NO: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35, or an amino acid sequence having at least one amino acid substitution compared to the sequence of SEQ ID NO: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35; and / or

[0064] A light chain variable region sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 24, 26, 28, 35, 36, 37, 38, 39, 40, 41 or 42, or an analog comprising at least 85% sequence identity to the light chain variable region sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 24, 26, 28, 35, 36, 37, 38, 39, 40, 41 or 42, or an amino acid sequence having at least one amino acid substitution compared to the sequence of 24, 26, 28, 35, 36, 37, 38, 39, 40, 41 or 42;

[0065] (II) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35; and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41 or 42;

[0066] (III) a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO:23 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO:24; or a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO:25 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO:26; or a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO:27 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO:28;

[0067] (IV) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:29 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:36, or a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:30 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:37, or a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:31 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:38;

[0068] (V) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:32 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:39, or a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:33 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:40 or 41, or a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:34 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:42, or a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:35 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:39.

[0069] According to an embodiment of the present invention, the anti-PVRIG antibody contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody or a mutant thereof.

[0070] In some embodiments, the heavy chain constant regions are all from murine IgG antibodies or mutants thereof.

[0071] In some embodiments, the heavy chain constant regions are all derived from human IgG antibodies or mutants thereof.

[0072] According to certain embodiments, the antibody or antibody fragment comprises a heavy chain constant region selected from the group consisting of mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2, human IgG3 and human IgG4. Each possibility represents a separate embodiment of the invention.

[0073] According to certain embodiments, the human constant region of the chimeric antibody may be selected from human IgG1, human IgG2, human IgG3 and human IgG4.

[0074] In an exemplary embodiment, the heavy chain constant regions are all from human IgG4 antibodies.

[0075] In an exemplary embodiment, the light chain constant regions are all from human kappa chains.

[0076] In an exemplary embodiment, the light chain constant regions are all from human lambda chains.

[0077] According to an embodiment of the present invention, the anti-PVRIG antibody is a monoclonal antibody.

[0078] According to some embodiments of the present invention, the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody.

[0079] According to certain embodiments, the chimeric antibody comprises a portion of a constant region derived from a human.

[0080] According to some embodiments of the present invention, the monoclonal antibody or antigen-binding fragment thereof is a humanized antibody. Humanized antibodies mainly refer to antibodies that are re-expressed after the modification of mouse monoclonal antibodies using gene cloning and DNA recombination technology, and bind to human targets after modification. Most of its amino acid sequences are replaced by human sequences, which basically retains the affinity and specificity of the parent mouse monoclonal antibody, and reduces its heterology, which is beneficial for application in the human body.

[0081] In a fifth aspect, the present invention provides an anti-PVRIG antibody or antigen-binding fragment, which comprises a heavy chain and a light chain, wherein:

[0082] The heavy chain has an amino acid sequence as shown in any one of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, or an amino acid sequence having at least 85% sequence identity with the sequence shown in any one of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, or an amino acid sequence having at least one amino acid substitution; and / or,

[0083] The light chain has an amino acid sequence as shown in any one of SEQ ID NO: 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62, or an amino acid sequence having at least 85% sequence identity compared to the sequence shown in any one of 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62, or an amino acid sequence having at least one amino acid substitution.

[0084] In one embodiment, the anti-PVRIG antibody is a chimeric antibody, wherein the anti-PVRIG chimeric antibody has a heavy chain amino acid sequence as shown in SEQ ID NO: 43, 44 or 45 and a light chain amino acid sequence as shown in SEQ ID NO: 53, 54 and 55.

[0085] In one embodiment, the anti-PVRIG antibody is a humanized anti-PVRIG antibody, which comprises a heavy chain and a light chain, wherein the heavy chain amino acid sequence is any one of SEQ ID NO: 46, 47, 48, 49, 50, 51 or 52, or a sequence having at least one amino acid substitution compared to any one of SEQ ID NO: 46, 47, 48, 49, 50, 51 or 52; the light chain amino acid sequence is any one of SEQ ID NO: 56, 57, 58, 59, 60, 61 or 62, or a sequence having at least one amino acid substitution compared to any one of SEQ ID NO: 56, 57, 58, 59, 60, 61 or 62.

[0086] In some embodiments, the PVRIG antibody is a humanized antibody, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:48 and the light chain comprises the amino acid sequence shown in SEQ ID NO:58.

[0087] In some embodiments, the PVRIG antibody is a humanized antibody, wherein the heavy chain comprises the heavy chain with an amino acid sequence as shown in SEQ ID NO:49 and the light chain comprises the light chain with an amino acid sequence as shown in SEQ ID NO:59.

[0088] In some embodiments, the PVRIG antibody is a humanized antibody, wherein the heavy chain comprises the heavy chain with an amino acid sequence as shown in SEQ ID NO: 50 and the light chain comprises the light chain with an amino acid sequence as shown in SEQ ID NO: 60 or 61.

[0089] In some embodiments, the PVRIG antibody is a humanized antibody, wherein the heavy chain comprises the heavy chain with an amino acid sequence as shown in SEQ ID NO:51 and the light chain comprises the light chain with an amino acid sequence as shown in SEQ ID NO:62.

[0090] In some embodiments, the PVRIG antibody is a humanized antibody, wherein the heavy chain comprises the heavy chain with an amino acid sequence as shown in SEQ ID NO:52 and the light chain comprises the light chain with an amino acid sequence as shown in SEQ ID NO:59,

[0091] According to one embodiment of the present invention, the antibody is an anti-human PVRIG antibody, and the anti-human PVRIG antibody or an antigen-binding fragment thereof has a cross-reaction with monkey PVRIG, but has no binding activity with mouse PVRIG.

[0092] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof is a monoclonal antibody. A monoclonal antibody is a highly uniform antibody produced by a single B cell clone and directed only against a specific antigen epitope. For example, the antibody of the embodiment of the present invention specifically binds only to PVRIG.

[0093] In one embodiment, the antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds to human PVRIG and is selected from the group consisting of Fv, Fab, Fab', scFv and F(ab') 2 , at least one of scFv-Fc fusion protein, scFv-Fv fusion protein, Fv fragment, and minimum recognition unit, or the anti-human PVRIG antibody is selected from a single-chain antibody, a multimeric antibody, a CDR-grafted antibody or a small molecule antibody.

[0094] In a sixth aspect, the present invention provides an isolated anti-human PVRIG antibody or antigen-binding fragment thereof having at least one of the following characteristics:

[0095] (I) the epitope of the human PVRIG protein bound is identical to, completely overlaps with, or partially overlaps with the anti-PVRIG antibody or antigen-binding fragment thereof as described in any one of the aforementioned aspects;

[0096] (II) competing with the anti-PVRIG antibody or antigen-binding fragment thereof described in any one of the preceding aspects for binding to an epitope of the human CD112R protein.

[0097] In a seventh aspect, the present invention provides an isolated anti-PVRIG antibody or antigen-binding fragment thereof having one or more of the following characteristics:

[0098] (1) Binds to human / cynomolgus monkey CD112R protein with high affinity;

[0099] (2) Inhibit or block the binding of human CD112R protein to human CD112 or cells expressing human CD112.

[0100] In an eighth aspect, the present invention provides an immunoconjugate comprising the anti-PVRIT antibody or antigen-binding fragment thereof or the isolated antibody or antigen-binding fragment according to any of the preceding aspects.

[0101] In a ninth aspect, the present invention provides an isolated polynucleotide. According to an embodiment of the present invention, the isolated polynucleotide encodes the anti-PVRIG antibody or antigen-binding fragment thereof according to any one of aspects 1 to 5. Thus, the polypeptide encoded by the polynucleotide can specifically bind to PVRIG.

[0102] According to an embodiment of the present invention, the isolated polynucleotide encodes the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the isolated polynucleotide encodes the light chain variable region or heavy chain variable region of the anti-human PVRIG antibody or antigen-binding fragment thereof described in the preceding item. Or according to an embodiment of the present invention, the isolated polynucleotide encodes the aforementioned light chain or heavy chain.

[0103] In some embodiments, the polynucleotide sequence encoding the heavy chain variable region VH of the anti-human PVRIG antibody or antigen-binding fragment thereof described in the preceding paragraph comprises: a sequence selected from any one of SEQ ID NOs: 128-137, or a variant thereof having at least 85% identity with any one of SEQ ID NOs: 128-137. Or in some embodiments, the polynucleotide sequence encoding the light chain variable region of the anti-human PVRIG antibody or antigen-binding fragment thereof described in the preceding paragraph comprises: a sequence selected from any one of SEQ ID NOs: 138-147, or a variant thereof having at least 85% identity with any one of SEQ ID NOs: 138-147.

[0104] In a tenth aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the vector comprises one or more polynucleotides of the ninth aspect.

[0105] According to an embodiment of the present invention, the vector comprises the aforementioned two polynucleotides, and the vector encodes a VL region and a VH region, and the VL region and the VH region together bind to PVRIG.

[0106] In the eleventh aspect, the present invention provides a vector pair. According to an embodiment of the present invention, each of the vector pairs comprises one of the polynucleotides described in the eighth aspect, wherein the vector pair jointly encodes a VL region and a VH region, and the VL region and the VH region together bind to PVRIG.

[0107] In a twelfth aspect, the present invention provides a host cell. According to an embodiment of the present invention, the host cell comprises the vector described in the ninth aspect or the vector pair described in the tenth aspect.

[0108] In the thirteenth aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof, the aforementioned polynucleotide, the aforementioned vector, the aforementioned vector pair, or the aforementioned host cell; and optional pharmaceutically acceptable excipients. Thus, the composition can specifically bind to PVRIG, and has a good tumor inhibition effect and good drug efficacy.

[0109] In a fourteenth aspect, the present invention provides a use. According to an embodiment of the present invention, the use includes: use of the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof, the aforementioned polynucleotide, the aforementioned vector, the aforementioned vector pair, the aforementioned host cell or the aforementioned composition, conjugate in the preparation of cancer diagnostic reagents and cancer therapeutic drugs.

[0110] According to an embodiment of the present invention, the cancer includes breast cancer, colorectal cancer, female reproductive system cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, liver cancer (HCC), endometrial cancer, pancreatic cancer, gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cell carcinoma (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 cell carcinoma, high MSI cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, myeloma.

[0111] In a fifteenth aspect, the present invention provides a method for preventing or treating a subject suffering from the disease. According to an embodiment of the present invention, the method comprises administering an effective amount of the aforementioned anti-PVRIG antibody or antigen-binding fragment thereof or the aforementioned pharmaceutical composition to the subject.

[0112] In a sixteenth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the aforementioned anti-PVRIG antibody or antigen-binding fragment thereof or an isolated antibody or antigen-binding fragment. Two or more (e.g., two, three or four) of any antibodies or antigen-binding fragments described herein may be present in any combination in a pharmaceutical kit, and the pharmaceutical kit may be prepared in any manner known in the art.

[0113] In a seventeenth aspect, the present invention provides a method for detecting PVRIG in a sample. According to an embodiment of the present invention, the method comprises:

[0114] (a) contacting the sample with the aforementioned antibody or antigen-binding fragment thereof; and

[0115] (b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and PVRIG;

[0116] Optionally, the antibody is detectably labeled.

[0117] The antibody or antibody fragment provided by the present invention can specifically bind to the PVRIG antigen, and can significantly enhance the killing effect of human PBMC, and exhibits a good anti-tumor effect in a mouse model. Other features and advantages of the present invention will be described in detail in the following specific embodiments.

[0118] According to the above embodiments of the present invention, the inventors found that the antibodies of the embodiments of the present invention can bind to and activate PVRIG with a KD less than 2.123 x 10 -10 M affinity, high stability, strong affinity, higher biological activity, showing good clinical anti-tumor application prospects, enriching the drug selection of targeted drugs for cancer treatment, and has good commercial prospects.

[0119] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Various other advantages and benefits will become apparent to those skilled in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Also, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings,

[0121] Figure 1 The results of the ESLIA binding activity analysis of the PVRIG mouse monoclonal antibody binding to the PVRIG protein in Example 3.1 are shown.

[0122] Figure 2 Example 3.2 Results of ELISA blocking activity analysis of PVRIG mouse monoclonal antibody blocking PVRIG protein binding to CD112.

[0123] Figure 3 Example 3.3 Results of FACS binding activity analysis of PVRIG mouse monoclonal antibody binding to PVRIG on the surface of CHO-K1-PVRIG cells.

[0124] Figure 4 Example 3.4 Results of FACS activity analysis of PVRIG mouse monoclonal antibody blocking the binding of CHO-K1-CD112 to PVRIG at the cellular level

[0125] Figure 5 Example 5.1 ELISA binding activity analysis results of PVRIG chimeric antibody ELISA binding to PVRIG protein

[0126] Figure 6Example 5.2 ELISA blocking activity analysis results of PVRIG chimeric antibody blocking the binding of PVRIG to CD112

[0127] Figure 7 Example 5.3 Results of FACS binding activity analysis of PVRIG chimeric antibody cell level binding to PVRIG on CHO-K1-PVRIG cell surface

[0128] Figure 8 Example 5.4 Results of FACS blocking activity analysis of PVRIG chimeric antibody blocking the binding of CD112 and PVRIG on the surface of CHO-K1-CD112 cells at the cellular level

[0129] Fig. 9 Example 5.5 Affinity analysis results of antibodies BT-5A8-CHI, BT-29D5-CHI, BT-26C4-CHI and antigen PVRIG-HIS detected based on surface plasmon resonance (SPR) technology

[0130] Figure 10. Example 5.5 Affinity analysis results of antibody BT-29D5-CHI and antigen PVRIG-HIS detected based on surface plasmon resonance (SPR) technology

[0131] Fig.11 Example 5.5 Affinity analysis results of antibody BT-26C4-CHI and antigen PVRIG-HIS detected based on surface plasmon resonance (SPR) technology

[0132] Fig.12 Example 7.1 ELISA binding activity analysis results of PVRIG humanized antibody binding to PVRIG protein

[0133] Fig.13 Example 7.2 Results of ELISA blocking activity analysis of PVRIG humanized antibody blocking CD112 binding to PVRIG protein

[0134] Fig.14 Example 7.3 FACS analysis of PVRIG humanized antibody binding activity to PVRIG on the surface of CHO-K1-PVRIG cells

[0135] Binding activity analysis results

[0136] Fig.15 Example 7.4 FACS blocking activity results of VRIG humanized antibody blocking the binding activity of CHO-K1-CD112 and PVRIG at the cell level

[0137] Fig.16Example 7.5 Detection of antibodies based on biolayer interferometry (BLI) technology Detection of the binding kinetics of PVRIG humanized antibody BT-5A8-12 and recombinant human PVRIG-His based on BLI technology.

[0138] Fig.17 Example 7.5 Antibody detection based on biolayer interferometry (BLI) technology The binding kinetics of PVRIG humanized antibody BT-5A8-11 and recombinant human PVRIG-His were detected based on BLI technology.

[0139] Fig.18 Example 7.5 Antibody detection based on biolayer interferometry (BLI) technology The binding kinetics of PVRIG humanized anti-BT-5A8-32 and recombinant human PVRIG-His were detected based on BLI technology.

[0140] Fig.19 Example 7.5 Antibody detection based on biolayer interferometry (BLI) technology The binding kinetics of PVRIG humanized anti-BT-5A8-42 and recombinant human PVRIG-His were detected based on BLI technology.

[0141] Fig. 20 Example 7.5 Antibody detection based on biolayer interferometry (BLI) technology The binding kinetics of PVRIG humanized antibody BT-29D5-41 and recombinant human PVRIG-His were detected based on BLI technology.

[0142] Fig.21 Example 7.5 Antibody detection based on biolayer interferometry (BLI) technology The binding kinetics of PVRIG humanized antibody BT-29D5-44 and recombinant human PVRIG-His were detected based on BLI technology.

[0143] Fig. 22 Example 7.5 Antibody detection based on biolayer interferometry (BLI) technology The binding kinetics of PVRIG humanized antibody BT-29D5-43 and recombinant human PVRIG-His were detected based on BLI technology.

[0144] Fig.23 .ELISA analysis of the binding activity of humanized PVRIG antibody to cynomolgus monkey PVRIG protein

[0145] Fig.24 .ELISA analysis of the binding activity of humanized PVRIG antibody to mouse PVRIG protein

[0146] Fig.25 . The eukaryotic expression vector PHR-X- of Example 4.

[0147] Fig.26 Background Technology Section CD112 / CD155 Co-inhibitory Network DETAILED DESCRIPTION

[0148] The present invention provides a monoclonal antibody (mAb) specific for human PVRIG, i.e., an anti-human PVRIG mAb. The present invention also provides the production of the antibody and the use of the antibody as a therapeutic agent. Specifically, the anti-human PVRIG mAb of the present invention can be used to enhance anti-tumor killing activity and can be used as a diagnostic reagent. In certain embodiments, the present invention provides an antibody specific for PVRIG for efficiently restoring the immune activity of immune cells against cancer cells overexpressing PVRIG.

[0149] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0150] definition

[0151] For a better understanding of the present invention, some terms are first defined. Other definitions are listed throughout the detailed description. Unless otherwise indicated, the implementation of the present invention will adopt conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, which are all within the technical scope of this area.

[0152] Abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids. As used herein (including the claims), singular forms include their corresponding plural forms unless the context clearly dictates otherwise.

[0153] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1% and ±2%.

[0154] ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0155] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0156] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when terms are clearly indicated to the contrary, such as "only one" or "exactly one" or when "consisting of..." is used in the claims, it will refer to only one number listed or one element of the list.

[0157] As used herein, the words "a" and "an" should be understood to mean "at least one" unless expressly stated to the contrary.

[0158] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0159] As used herein, the term "antigen" refers to a molecule or part of a molecule that can induce antibody formation and is specifically bound by an antibody. An antigen may have one or more epitopes. The specific binding mentioned above means that the antigen reacts with its corresponding antibody in a highly selective manner and does not react with a large number of other antibodies that may be evoked by other antigens. According to certain embodiments of the present invention, the antigen is a PVRIG protein. "PVRIG antibodies" include variants, subtypes, species homologs of human PVRIG or PVRIG of other species and analogs having at least one common epitope of PVRIG, unless otherwise specified. The term includes unprocessed full-length PVRIG and any form of PVRIG produced as a result of processing in cells. The term covers "full-length" unprocessed PVRO\IG and any form of PVRIG or any fragment thereof produced by intracellular processing, such as splice variants or allelic variants.

[0160] In one embodiment, PVRIG refers to full-length PVRIG from human or cynomolgus monkey or a fragment thereof (such as a mature fragment thereof lacking a signal peptide).

[0161] As used herein, the PVRIG protein is a single-pass type I membrane glycoprotein with two Ig-like C2-type domains and one Ig-like V-type domain. This protein is one of the plasma membrane components of adherens junctions. It also acts as an entry receptor for certain mutant strains of herpes simplex virus and pseudorabies virus and is involved in the cell-to-cell spread of these viruses.

[0162] antigen

[0163] The antigen of the present invention specifically refers to PVRIG or CD112R.

[0164] Structure and expression of PVRIG (CD112R)

[0165] PVRIG is a co-inhibitory receptor belonging to the poliovirus receptor (PVR) family, originally known as PVR-related Ig domain (PVRIG). Human PVRIG (poliovirus receptor-related immunoglobin domain-containing protein), also known as CD112 receptor (CD112R), is a transmembrane protein of approximately 34kda in the poliovirus receptor-like protein (PVR) family. This gene encodes a transmembrane protein consisting of an extracellular IgV domain, a transmembrane domain, and a long intracellular domain. CD112R (PVRIG) is a novel B7 / CD28-like immune checkpoint protein.

[0166]

[0167] Exemplary PVRIGs according to the invention are shown in the following SwissPort, UniPort and GenBank codes or accession numbers: Gene ID: 5819, Q92692, I68093, NP_001036189.1, NP_002847.1 and #Q92692.

[0168] The antibody or fragment thereof according to the present invention binds to an epitope in PVRIG, specifically, the antibody binds to an epitope within the extracellular domain (extracellular part) of the PVRIG protein.

[0169] The term "ligand"

[0170] Herein, the ligand refers to CD112, the ligand of the specific antigen PVRIG of the PVRIG antibody of the present invention.

[0171] CD112, also known as connexin-2 or PVR-related protein 2 (PVRL2), "immunoglobulin domain associated with PVR", "CD112 receptor", "immunoglobulin domain associated with poliovirus receptor", "Nectin-2 receptor", "C7orfl5", "transmembrane protein PVRIG" are all used interchangeably and are members of the connexin family and are associated with tumor angiogenesis, growth and metastasis. CD112 includes an extracellular region with three Ig-like domains (two IgC and a distal IgV domain), a cytoplasmic tail and a transmembrane region. The cytoplasmic tail has a conserved afadin-binding motif (Glu / Ala-X-Tyr-Val) that can bind to the PDZ domain, thereby helping the connexin to connect to F-actin. High levels of CD112 expression are associated with tumor progression and poor prognosis in most cancer patients.

[0172] The term "percent (%) amino acid sequence identity" or simply "identity", "homology" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after the amino acid sequences are aligned (and gaps are introduced, if necessary) to obtain the maximum percentage sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to obtain maximum alignment over the entire length of the compared sequences.

[0173] The term "immune response" refers to the actions of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines and complement), which result in the selective damage, destruction or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0174] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-effect relationship, usually initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another part of the cell. Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a series of reactions that lead to signal transduction. The penultimate process usually involves nuclear events, resulting in changes in gene expression.

[0175] The terms "activity" or "biological activity", or the terms "biological property" or "biological characteristics" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize CD112R activity in vivo or in vitro, IC 50 , the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., usually with non-human homologs of the targeted peptide, or with cross-reactivity of other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics are observed, measured or evaluated using techniques known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from different sources (including humans, primates or any other source).

[0176] The term "specificity" refers to the ability to determine the presence or absence of a protein in a heterogeneous population of proteins and / or other organisms. Thus, under specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0177] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.

[0178] The identification or determination of the complementary determining region (CDR) from a given heavy chain or light chain variable sequence is generally performed using one of several methods known in the art. For example, such determination is performed according to Kabat (Wu TT and Kabat EA, J Exp Med, 1970; 132: 211–50) and IMGT (Lefranc MP et al., Dev Comp Immunol, 2003, 27: 55-77).

[0179] When the term "CDR having a certain sequence" or similar terms is used, it includes the option wherein the CDR comprises the specified sequence as well as the option wherein the CDR consists of the specified sequence.

[0180] The antigenic specificity of an antibody is based on the hypervariable region (HVR), i.e., the unique CDR sequences of both the light chain and the heavy chain that together form the antigen binding site. The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0181] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.

[0182] The term "monoclonal antibody" or "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecule preparation of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0183] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring the production of the antibody by any particular method. mAbs can be obtained by methods known to those skilled in the art. For example, the monoclonal antibodies used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., Nature 1975,256,495, or can be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567)

[0184] In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the production of the antibody by any particular method. The term "full-length antibody" refers to an immunoglobulin molecule that contains at least four peptide chains when naturally present: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementary determining regions (CDRs) and regions separated by more conservative framework regions (FRs). Each VH or VL region is composed of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0185] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of antibodies, generally including at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody.

[0186] The term "antigen-binding fragment" (or simply antibody portion) of an antibody herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) a Fd fragment consisting of VH and CH1; (iv) a Fv fragment consisting of a single-arm VL and VH of an antibody; (v) a dAb fragment consisting of VH (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity determining region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by recombinant methods via a synthetic linker that makes the two into a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule (called single-chain Fc (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These single-chain antibodies are also intended to be included in the meaning of the term. Also included are any other types of molecules into which these antibody reactive parts have been physically inserted. These molecules can be provided by any known technology including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant technology. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0187] The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. The Fab' fragment is produced by cleaving the disulfide bond at the hinge cysteine ​​of the F(ab')2 pepsin digest. Additional chemical coupling of antibody fragments is known to those of ordinary skill in the art. Fab and F(ab')2 fragments lack the fragment crystallizable (Fc) region of the intact antibody, are cleared more rapidly from the circulation of the animal, and may have less nonspecific tissue binding than intact antibodies (see, e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

[0188] As is generally understood in the art, an "Fc" region is a fragment crystallizable constant region of an antibody that does not contain an antigen-specific binding region. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments, derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody. IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

[0189] The isotype of the heavy chain (gamma, alpha, delta, epsilon, or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE, or IgM, respectively). The light chain has two isotypes (kappa or lambda). Both isotypes are present in all antibody classes.

[0190] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0191] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0192] The term "bivalent antibody" contains two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific.

[0193] The term "mouse antibody" or "hybridoma antibody" in the present disclosure refers to a monoclonal antibody against human CD112R prepared according to the knowledge and skills in the art. During preparation, a test subject is injected with CD112R antigen, and then a hybridoma expressing an antibody with a desired sequence or functional properties is isolated.

[0194] The term "chimeric antibody" is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from antibodies such as rodents ("parent antibodies"), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibodies are less likely to induce adverse immune responses in human subjects than the parent rodent antibodies.

[0195] The term "humanized antibody" refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

[0196] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat sugar chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat sugar chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0197] An "isotype" antibody refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modifications have been made to alter Fc function, such as to enhance or reduce effector function or binding to Fc receptors.

[0198] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also referred to as the EU index, such as Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0199] The term "epitope" refers to a protein determinant that is capable of specific binding to an antibody. Epitopes are usually composed of various chemically active surface molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that in the presence of denaturing solvents, the binding to the former but not to the latter is lost.

[0200] The term "cross-reaction" described herein refers to the combination of antigen fragments of the same target molecule of human, monkey, and / or mouse (mouse or rat). Therefore, "cross-reaction" should be understood as the inter-species reaction with the same molecule X expressed in different species. The cross-reaction specificity of the monoclonal antibody recognizing human CD112R, monkey, and / or mouse CD112R (mouse or rat) can be determined by FACS analysis.

[0201] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ) represents the equilibrium dissociation constant, which is the dissociation rate constant and the association rate constant (k dis and k on ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0202] The term "cross-reactivity" as described herein refers to the binding of antigenic fragments of the same target molecule of human, monkey, and / or murine origin (mouse or rat). Therefore, "cross-reactivity" should be understood as an interspecies reaction with the same molecule X expressed in different species. The cross-reactivity specificity of monoclonal antibodies recognizing human PVRIG (CD112R), monkey, and / or murine PVRIG (CD112R) (mouse or rat) can be determined by FACS analysis.

[0203] The term "binding affinity" is used herein as a measure of the strength of non-covalent interactions between two molecules (e.g., an antibody or its fragment, and an antigen). The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules (e.g., an antibody or its fragment, and an antigen) via monovalent interactions can be quantitatively determined by measuring the dissociation constant (KD). The affinity of molecule X to its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. A specific method for measuring affinity is the ForteBio kinetic binding assay herein. By standard assays known in the art, such as SPR, the binding kinetics and binding affinity of antibodies can also be evaluated. Competitive binding assays can be performed, wherein the binding of an antibody to a target and the binding of another ligand (e.g., another antibody) to the target to the target are compared. It should be noted that affinity can be quantified using known methods such as surface plasmon resonance (SPR) (described in Scarano S, Mascini M, Turner AP, Minunni M., Surface plasmon resonance imaging for affinity-based biosensors, Biosens Bioelectron. 2010, 25:957-66) and can be calculated using, for example, the dissociation constant Kd, such that a lower Kd reflects a higher

[0204] When the binding activity of the antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functional conservative variants" of an antibody) that do not significantly change its biological activity. The term "binding compound" refers to both the affinity of an antibody and its binding fragment.

[0205] The term "not binding" to a protein or cell means that it does not bind to the protein or cell, or does not bind to it with high affinity, i.e., the K of the binding protein or cell is D 1.0×10 -6 M or more, more preferably 1.0×10 -5 M or more, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or more, more preferably 1.0×10-2 M or higher.

[0206] The term "high affinity" for IgG antibodies refers to the K D 1.0×10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or less, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype refers to a K D For 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0207] The terms "nucleic acid", "polynucleotide", "nucleic acid molecule" and "polynucleotide molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless expressly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al.

[0208] "Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). After introduction into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors".

[0209] "Activation," "stimulation," and "treatment" as applied to a cell or receptor may have the same meaning, eg, a cell or receptor is activated, stimulated, or treated with a ligand, unless the context dictates otherwise or clearly dictates.

[0210] "Ligands" include natural and synthetic ligands, such as cytokines, cytokine variants, analogs, mutant proteins, and binding compounds derived from antibodies. "Ligands" also include small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" can refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response / reaction", such as the response of cells, tissues, organs or organisms, includes changes in biochemical or physiological behavior (such as concentration, density, adhesion or migration within a biological compartment, gene expression rate or differentiation state), where the change is related to activation, stimulation or treatment, or to internal mechanisms such as genetic programming. In the present invention, the ligand refers to the ligand CD112 of PVRIG.

[0211] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation or composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0212] The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancer may be benign (also known as a benign tumor), pre-malignant or malignant. Cancer cells may be solid cancer cells or leukemia cancer cells. The term "tumor" as used herein refers to one or more cells comprising a cancer. The term "tumor growth" is used herein to refer to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.

[0213] The present invention uses recombinant human PVRIG-human IgG4 Fc fusion protein containing human PVRIG extracellular region gene to BALA / C female mice, and screens four strains of mouse antibodies that can specifically bind to human PVRIG protein through hybridoma fusion technology. The antibody fragment can specifically target and bind to PVRIG receptor, block the binding of PVRIG and PCRIG, and enhance the killing effect of lymphocytes (especially T cells and NK cells). Especially for cancer patients, after the PVRIG antibody binds to PVRIG, the killing effect of lymphocytes can be enhanced, and the efficacy of existing antibody drugs can be improved.

[0214] Anti-PVRIG antibody

[0215] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to PVRIG. The term "anti-PVRIG antibody", "anti-CD112R antibody", "anti-CD112R", "CD112R antibody" or "antibody that binds to CD112R" refers to an antibody that can bind to PVRIG protein (also known as CD112R protein) with sufficient affinity. white) or a fragment thereof, which can be used as a diagnostic agent and / or a therapeutic agent targeting PVRIG.

[0216] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that can specifically recognize and bind to PVRIG protein. In some embodiments, the present invention provides an antibody that blocks the binding of PVRIG to CD112 protein. In some embodiments, the present invention provides an antibody that enhances the activation of CD226, T cells and / or NK cells.

[0217] In some embodiments, the antibodies of the invention specifically bind to human or cynomolgus monkey PVRIG (CD112R) protein. In some embodiments, the antibodies of the invention bind to human PVRIG (CD112R) and block the interaction between human PVRIG (CD112R) and human CD112 protein.

[0218] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically recognizes the extracellular region of PVRIG.

[0219] Of course, these conservative amino acid substitutions will not change the biological function of the antibody or antigen-binding fragment. In some specific embodiments, these conservative amino acid substitutions can occur in amino acids other than the CDR region in the heavy chain variable region and the light chain variable region.

[0220] The variable region sequences of exemplary PVRIG antibodies of the present invention are shown in Table 1.

[0221] Table 1 Variable regions of exemplary PVRIG antibodies of the invention

[0222]

[0223]

[0224]

[0225] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PVRIG, which comprises three CDRs of the heavy chain variable region shown in any one of SEQ ID NOs: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35 and three CDRs of the light chain variable region sequence shown in any one of SEQ ID NOs: 24, 26, 28, 36, 37, 38, 39, 40, 41 or 42.

[0226] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, wherein the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 23 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 24. In at least one embodiment, the antibody that specifically binds to PVRIG is a chimeric antibody.

[0227] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, wherein the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 25 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 26. In at least one embodiment, the antibody that specifically binds to PVRIG is a chimeric antibody.

[0228] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, wherein the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 271 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 28.

[0229] In at least one embodiment, the antibody that specifically binds PVRIG is a chimeric antibody.

[0230] The anti-PVRIG antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of these "chimeric" antibodies, as long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). In addition, complementarity determining regions (CDRs) may be grafted to alter certain properties of the antibody molecule, including affinity or specificity. Non-limiting examples of CDR grafting are disclosed in U.S. Pat. No. 5,225,539. Chimeric antibodies are molecules whose different portions are derived from different animal species, such as those having variable regions derived from murine mAbs and human immunoglobulin constant regions. The antibody with the variable region framework residues substantially coming from human antibody (being called acceptor antibody) and the CDR substantially coming from mouse antibody (being called donor antibody) is also referred to as humanized antibody.Chimeric antibody is mainly used for reducing immunogenicity in application and improving yield in production, for example, in the case where murine mAb has higher yield from hybridoma production but higher immunogenicity in humans, human / murine chimeric mAb is used.Chimeric antibodies and their production methods are known in the art (for example PCT patent application WO 86 / 01533, WO 97 / 02671, WO 90 / 07861, WO 92 / 22653 and U.S. Patent Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539).

[0231] In some embodiments ，The present invention provides a monoclonal antibody or antigen-binding fragment comprising a PVRIG antigen-binding domain (ABD), wherein the antigen-binding domain comprises three CDRs of a light chain (i.e., VLCDRs) and three CDRs of a heavy chain (i.e., VHCDRs), wherein the ABD has at least 90% sequence identity or similarity with the ABD of a monoclonal mouse antibody comprising the following chains: (i) a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 23 and a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 24 (identified herein as clone BT-5A8-CHI); or (ii) a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 25 and a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 26 (identified herein as clone BT-29D5 CHI); or (ii) a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 27 and a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 28 (identified herein as clone BT-29D5CHI).

[0232] In at least some embodiments, the present invention provides an antibody or antibody fragment comprising heavy chain complementarity determining regions (VHCDRs) and light chain complementarity determining regions (VLCDRs).

[0233] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR sequence selected from at least one of the following:

[0234] The heavy chain variable region comprises a VHCDR sequence: an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR sequence selected from at least one of the following: an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0235] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which comprises heavy chain complementary determining regions VHCDR1, VHCDR2 and VHCDR3, and light chain complementary determining regions VLCDR1, VLCDR2 and

[0236] VLCDR3; wherein the VHCDR1 is selected from the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 7 or SEQ ID NO: 13, the VHCDR2 is selected from the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 14, and the VHCDR3 is selected from the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 15;

[0237] In some embodiments, the VLCDR1 is selected from the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:16, the VLCDR2 is selected from the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:11 or SEQ ID NO:17; the VLCDR3 is selected from the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:18.

[0238] According to an embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment may further include at least one of the following additional technical features: in at least some embodiments, the antibody includes VHCDR1, VHCDR2, and VHCDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively;

[0239] In at least some embodiments, the antibody comprises the VHCDR1, VHCDR2, and VHCDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively;

[0240] In at least some embodiments, the antibody comprises the VHCDR1, VHCDR2, and VHCDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively;

[0241] In at least some embodiments, the antibody comprises the VLCDR1, VLCDR2, and VLCDR3 sequences shown as the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively;

[0242] In at least some embodiments, the antibody comprises VLCDR1, VLCDR2, VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively; or

[0243] In at least some embodiments, the antibody comprises VLCDR1, VLCDR2, and VLCDR3 sequences having amino acid sequences as shown in SEQ ID NOs: 16, 17, and 18, respectively.

[0244] In at least some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which comprises or consists of a combination of CDRs of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 having amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, such as the group of 6 CDRs contained in the exemplary chimeric antibody BT-5A8 CHI, humanized antibodies BT-5A8-12, BT-5A8-11, and BT-5A8-32, wherein: VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0245] In at least some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which comprises or consists of a combination of CDRs of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 having amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively, such as a group of 6 CDRs contained in the exemplary chimeric antibody BT-29D5 CHI, 29D5-2 (H4B L1A), 29D5-3 (H4B L4A), 29D5-4 (H4C L3A), and 29D5-5 (H3A L2A), wherein: VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 respectively comprise CDRs having amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. The amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0246] In at least some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, comprising a CDR combination of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, or a group thereof, such as the exemplary chimeric antibody BT-26C4 CHI, which comprises a CDR combination selected from the following VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 or a group thereof, comprising a group of 6 CDRs, wherein: the VH CDR1 sequence is SEQ ID NO: 13; VH CDR2 is SEQ ID NO: 14; VH CDR3 is SEQ ID NO: 15; VL CDR1 is SEQ ID NO: 16; VL CDR2 is SEQ ID NO: 17; and V CDR3 is SEQ ID NO:18.

[0247] In some embodiments, the sequences of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are determined based on various CDR definitions known in the art, such as the Kabat definition, the Chothia definition, or the IMGT definition. The sequence combinations of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of exemplary anti-PVRIG antibodies of the present invention are shown in Table 2.

[0248] Table 2 Sequences of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3.

[0249]

[0250]

[0251]

[0252] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of antibodies and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. The inventors can obtain the CDR regions of the light chain variable region sequences of the above antibodies (such as SEQ ID NO: 4-6, SEQ ID NO: 10-12, SEQ ID NO: 16-18) and the CDR regions of the heavy chain variable region sequences (such as SEQ ID NO: 1-3, SEQ ID NO: 7-9, SEQ ID NO: 13-15) through the antibody sequence comparison database (NCBI, IMGT) or related software.

[0253] Unless otherwise specified, the CDRs of the antibodies of the present invention can be determined by those skilled in the art according to any

[0254] The boundaries are determined by any scheme (e.g. different assignment systems or combinations).

[0255] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies using specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0256] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved in antigen binding. Using at least two of Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, by the structure of the antibody and protein folding, the residues of the rest of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0257] In some implementations, according to an embodiment of the present invention, the antibody or its antigen-binding fragment contains: at least one of a heavy chain framework region (VHFRs) sequence consisting of VHFR1, VHFR2, VHFR3 and VHFR4 and a light chain framework region (VLFRs) sequence consisting of VLFR1, VLFR2, VLFR3 and VLFR4; wherein at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0258] In some embodiments, the anti-PVRIG antibody of the present invention comprises a VHFR1 selected from any one of SEQ ID NOs: 63-70; in some embodiments, the anti-PVRIG antibody of the present invention comprises a VHFR2 selected from any one of SEQ ID NOs: 71-76; in some embodiments, the anti-PVRIG antibody of the present invention comprises a VHFR3 selected from any one of SEQ ID NOs: 77-83; in some embodiments, the anti-PVRIG antibody of the present invention comprises a VHFR4 selected from any one of SEQ ID NOs: 84-91;

[0259] The heavy chain framework regions (VHFRs) of the anti-PVRIG antibodies of the present invention are shown in Table 3.

[0260] In some embodiments, the PVRIG antibody of the present invention comprises a VLFR1 selected from any one of SEQ ID NOs: 92-100; In some embodiments, the PVRIG antibody of the present invention comprises a VLFR2 selected from any one of SEQ ID NOs: 101-109;

[0261] In some embodiments, the PVRIG antibody of the present invention comprises a VLFR3 selected from any one of SEQ ID NOs: 110-118; in some embodiments, the PVRIG antibody of the present invention comprises a VLFR4 selected from any one of SEQ ID NOs: 119-127.

[0262] Table 3. Exemplary heavy chain framework regions (VHFRs) of PVRIG antibodies of the invention

[0263]

[0264]

[0265] In some embodiments, the PVRIG antibody of the present invention comprises a VLFR1 selected from any one of SEQ ID NOs: 92-100; in some embodiments, the PVRIG antibody of the present invention comprises a VLFR2 selected from any one of SEQ ID NOs: 101-109; in some embodiments, the PVRIG antibody of the present invention comprises a VLFR3 selected from any one of SEQ ID NOs: 110-118; in some embodiments, the PVRIG antibody of the present invention comprises a VLFR4 selected from any one of SEQ ID NOs: 119-127. The light chain framework regions (VLFRs) of the PVRIG antibody of the present invention are shown in Table 4.

[0266] Table 4 Exemplary light chain framework regions (VLFRs) of PVRIG antibodies of the invention

[0267]

[0268]

[0269]

[0270] In one aspect, the present invention provides an anti-human PVRIG antibody or an antigen-binding fragment thereof, comprising a heavy chain and a light chain. In one embodiment, the heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 43-52. In one embodiment, the light chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 53-62. The amino acid sequences of the heavy chain and light chain of exemplary anti-PVRIG antibodies of the present invention are shown in Table 5, respectively.

[0271] Table 5 Amino acid sequences of exemplary heavy and light chains of exemplary anti-PVRIG antibodies of the invention

[0272]

[0273]

[0274]

[0275]

[0276] In some embodiments, the anti-human PVRIG antibody is a chimeric antibody. The chimeric antibody or antigen-binding fragment thereof described in the present invention comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:43, and a light chain with an amino acid sequence as shown in SEQ ID NO:53.

[0277] In some embodiments, the anti-human PVRIG antibody is a chimeric antibody. The chimeric antibody or antigen-binding fragment thereof described in the present invention comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:44, and a light chain with an amino acid sequence as shown in SEQ ID NO:54.

[0278] In some embodiments, the anti-human PVRIG antibody is a chimeric antibody. The chimeric antibody or antigen-binding fragment thereof described in the present invention comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:45, and a light chain with an amino acid sequence as shown in SEQ ID NO:55.

[0279] In some embodiments, the anti-PVRIG antibody is a humanized antibody, the humanized anti-human PRVIG antibody of the present invention, which comprises a heavy chain and a light chain. In some embodiments, the heavy chain of the humanized anti-human PRVIG antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 46, 47 or 48. In some embodiments, the light chain of the humanized anti-human PRVIG antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 56, 57 or 58.

[0280] In some embodiments, the anti-human PVRIG antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:46, and a light chain having an amino acid sequence as shown in SEQ ID NO:56;

[0281] In some embodiments, the anti-human PVRIG antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:47, and a light chain having an amino acid sequence as shown in SEQ ID NO:57;

[0282] In some embodiments, the anti-human PVRIG antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO:48, and a light chain having an amino acid sequence as shown in SEQ ID NO:58;

[0283] In some embodiments, the anti-human PVRIG antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:49, and a light chain having an amino acid sequence as shown in SEQ ID NO:59;

[0284] In some embodiments, the anti-human PVRIG antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 50, and a light chain having an amino acid sequence as shown in SEQ ID NO: 60 or 61;

[0285] In some embodiments, the anti-human PVRIG antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:51, and a light chain having an amino acid sequence as shown in SEQ ID NO:62;

[0286] In some embodiments, the anti-human PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises: a heavy chain with an amino acid sequence as shown in SEQ ID NO:52, and a light chain with an amino acid sequence as shown in SEQ ID NO:63.

[0287] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is a murine antibody, a chimeric antibody,

[0288] humanized antibodies, or fully human antibodies, or antigen-binding fragments thereof.

[0289] Any suitable method for producing antibodies can be used to produce the antibodies of the present invention. Any suitable form of PVRIG can be used as an immunogen (antigen) for producing antibodies. By way of example and not limitation, any PVRIG variant or fragment thereof can be used as an immunogen.

[0290] In some embodiments, hybridoma cells producing murine monoclonal anti-human PVRIG antibodies can be produced by methods known in the art.

[0291] Antibodies derived from rodents (such as mice) may cause unwanted antibody immunogenicity when used as therapeutic drugs in vivo. Repeated use causes the human body to produce an immune response against the therapeutic antibody, which at least leads to loss of therapeutic efficacy, and in severe cases, leads to potentially lethal allergic reactions. One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies, in which the mouse variable region is fused to the human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, the retention of the complete rodent variable region in the chimeric antibody may still cause harmful immunogenicity in patients. Transplanting the complementary determining region (CDR) loop of the rodent variable domain to the human framework (i.e., humanization) has been used to further minimize the rodent sequence (Jones et al. (1986) Nature 321: 522; Verhoeyen et al. (1988) Science 239: 1534).

[0292] In some embodiments, chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the prepared mouse monoclonal hybridoma antibody. DNA encoding heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0293] In some embodiments, the chimeric PVRIG antibody of the present invention can be prepared by effectively linking the variable regions of the heavy and light chains of immunoglobulins derived from hybridomas to the constant regions of human IgG using methods known in the art (see, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy chains and chimeric light chains. In some embodiments, the constant region contained in the chimeric antibody of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably human IgG4.

[0294] In some embodiments, the chimeric PVRIG antibodies of the present invention can be obtained by "mixing and matching" chimeric light chain and chimeric heavy chain expression plasmids to transfect expression cells such as CHO cells, and the PVRIG binding of such "mixed and matched" antibodies can be tested using the above-mentioned binding assays and other conventional binding assays (e.g., ELISA).

[0295] The present invention mixes and pairs various chimeric heavy chain and light chain expression plasmids to transfect expression cells to obtain three kinds of anti-human

[0296] PVRIG chimeric antibody.

[0297] In some embodiments, the amino acid sequences of the light chain and heavy chain variable regions and CDRs of exemplary anti-human PVRIG chimeric antibodies of the present invention are shown in Table 6.

[0298] Table 6: CDR and variable region amino acid sequences of anti-PVRIG chimeric antibodies (KABAT scheme)

[0299]

[0300] In some embodiments, the CDR regions and framework regions of exemplary anti-PVRIG chimeric antibodies of the present invention are as shown in Table 7.

[0301] Table 7 CDR and FR regions included in the variable region of chimeric antibodies

[0302]

[0303]

[0304] The numbering and pairing of the light and heavy chain variable regions are shown in Tables 8 and 9.

[0305] Table 8. Anti-PVRIG chimeric antibodies of the present invention, their numbers and light and heavy chain variable region pairings

[0306]

[0307]

[0308] Table 9 Anti-PVRIG chimeric antibodies of the present invention, their numbers and light and heavy chain combinations

[0309]

[0310] In one embodiment, the anti-PVRIG antibody of the present invention is a chimeric antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 43 and a variable light chain having an amino acid sequence as shown in SEQ ID NO: 53. In at least one embodiment, it comprises a variable heavy chain having an amino acid sequence as shown in SEQ ID NO: 23 and a variable light chain as shown in SEQ ID NO: 24.

[0311] In at least one embodiment, the antibody is BT-26C4-CHI, wherein the heavy chain variable region is shown in SEQ ID NO:23, and the light chain variable region is shown in SEQ ID NO:24.

[0312] In at least one embodiment, the anti-PVRIG antibody of the present invention is a chimeric antibody, whose heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and further comprises VHFR1, VHFR2, VHFR3 and VHFR4 as shown in SEQ ID NOs: 63, 71, 77 and 84, respectively; its light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; and further comprises VLFR1, VLFR2, VLFR3 and VLFR4 as shown in SEQ ID NOs: 92, 101, 110 and 119, respectively (identified herein as clone BT-5A8-CHI).

[0313] The VH and VL of an exemplary chimeric anti-PVRIG antibody BT-5A8-CHI are:

[0314] BT-5A8-CHI VH:

[0315] QIQLQQSGAELMKPGASVKISCLATGHTFSTYWIEWVKQRPGHGLEWIGEILPGSGSTNYDEKFKDKATFTADTSSNTA YMQLSSSLTSEDSAVYYCASFFFAYWGQGTLVTVSA(SEQ ID NO:23)

[0316] VHCDR1: GHTFSTYW (SEQ ID NO: 1), VHCDR2: ILPGSGST (SEQ ID NO: 2), VHCDR3: ASFFFAY (SEQ ID NO: 3);

[0317] BT-5A8-CHI VL:

[0318] DIVMTQSPSSLSVSAGEKVTLSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTL TISSVQAEDLAVYFCQNAHSYPLTFGAGTKLELK(SEQ ID NO:24);

[0319] VLCDR1: QSLLNSGNQKNY (SEQ ID NO: 4), VLCDR2: GAS (SEQ ID NO:5), VLCDR3: QNAHSYPLT (SEQ ID NO: 6).

[0320] In one embodiment, the anti-PVRIG antibody of the present invention is a chimeric antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 44 and a light chain having an amino acid sequence as shown in SEQ ID NO: 54, a variable heavy chain having an amino acid sequence as shown in SEQ ID NO: 25, and a variable light chain having an amino acid sequence as shown in SEQ ID NO: 26. In at least one embodiment, the antibody is BT-5A8-CHI, wherein the heavy chain variable region is shown in SEQ ID NO: 25, and the light chain variable region is shown in SEQ ID NO: 26.

[0321] In at least one embodiment, the anti-PVRIG antibody of the present invention is a chimeric antibody, whose heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NOs: 7, 8 and 9, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 as shown in SEQ ID NOs: 64, 72, 78 and 85, respectively; its light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively; and further comprises VLFR1, VLFR2, VLFR3 and VLFR4 as shown in SEQ ID NOs: 93, 102, 111 and 120, respectively (identified herein as clone BT-29D5-CHI), for example, the VH and VL of the exemplary chimeric anti-PVRIG antibody BT-29D5-CHI are:

[0322] BT-29D5-CHI VH

[0323] QVLLKESGPGLVAPSQSLSITCTVSGFSLTSYGVQWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSQ SQVFLKMNSLRTDDTAMYYCARGGNYNYAGAMDYWGQGTSVTVSS(SEQ ID NO:25),

[0324] VHCDR1:GFSLTSYG (SEQ ID NO:7), VHCDR2:IWAGGST (SEQ ID NO:8), VHCDR3:ARGGNYNYAGAMDY (SEQ ID NO:9);

[0325] BT-29D5-CHI VL:

[0326] DILLTQSPAILSVSPGERVSFSCRASQYIGTSIHWYQQRANGSPRLLIKYASESISGIPSRFSGSGSGTDFTLTI NSVESEDIADYYCQQSNSWPMYTFGGGTKLGIK(SEQ ID NO:26)VLCDR1:QYIGTS (SEQ ID NO:10), VLCDR2:YAS (SEQ ID NO:11), VLCDR3:QQSNSWPMYT (SEQ ID NO: 12).

[0327] In one embodiment, the anti-PVRIG antibody of the present invention is a chimeric antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 45 and a light chain having an amino acid sequence as shown in SEQ ID NO: 55, wherein the heavy chain and the light chain and the heavy chain respectively comprise a variable heavy chain having an amino acid sequence as shown in SEQ ID NO: 27 and a variable light chain having an amino acid sequence as shown in SEQ ID NO: 28;

[0328] In at least one embodiment, the antibody is BT-29D5-CHI, wherein the sequence of the heavy chain variable region shown in SEQ ID NO:27 is as follows: the sequence of the light chain variable region shown in SEQ ID NO:28.

[0329] In at least one embodiment, the anti-PVRIG antibody of the present invention is a chimeric antibody, whose heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 13, 14 and 15, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 with amino acid sequences as shown in SEQ ID NOs: 65, 73, 79 and 86, respectively; its light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 16, 17 and 18, respectively; and further comprises VLFR1, VLFR2, VLFR3 and VLFR4 with amino acid sequences as shown in SEQ ID NOs: 94, 103, 112 and 121, respectively (identified herein as clone BT-26C4-CHI).

[0330] BT-26C4-CHI VH:

[0331] QVQLQQPGAELVMPGASVKMSCKASGYTFTDYWMHWVKQRPGQGLEWIGAIDISDSNTSYNQKFKGKATLTVDES SSTAYMQLSSLTSEDSAVYYCARYYYGSSYGMDYWGQGTSVTVSS (SEQ ID NO: 27);

[0332] VHCDR1: GYTFTDYW (SEQ ID NO: 13), VHCDR2: IDISDSNT (SEQ ID NO: 14), VHCDR3: ARYYYGSSYGMDY(SEQ ID NO:15);

[0333] BT-26C4-CHI VL:

[0334] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGAPSRFSGSGSGTEYSLTI SNLEQEDIATYFCQQGKTLPPTFGGGTKLEIK(SEQ ID NO:28);

[0335] VLCDR1:QDISNY (SEQ ID NO:16), VLCDR2:YTS (SEQ ID NO:17), VLCDR3:QQGKTLPPT (SEQ ID NO: 18).

[0336] In some preferred embodiments, the present invention provides a PVRIG antibody, which is a humanized antibody having a heavy chain with an amino acid sequence as shown in any one of SEQ ID NOs: 46-52 and a light chain with an amino acid sequence as shown in any one of SEQ ID NOs: 56-62.

[0337] In at least one exemplary embodiment, VHCDR1, VHCDR2 and VHCDR3 of the humanized antibody of the present invention have the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and VLCDR1, VLCDR2 and VLCDR3 have the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. (An exemplary embodiment is identified herein as clones BT-5A8-12, BT-5A8-11 and BT-5A8-32).

[0338] In one embodiment, the anti-PVRIG antibody of the present invention is a humanized antibody, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 47 and a light chain with an amino acid sequence as shown in SEQ ID NO: 57; the heavy chain comprises a heavy chain variable region with an amino acid sequence of SEQ ID NO: 29 and the light chain comprises a light chain variable region with an amino acid sequence of SEQ ID NO: 36; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 with amino acid sequences as shown in SEQ ID NOs: 66, 74, 80 and 87, respectively; the light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively; and further comprises amino acid sequences as shown in SEQ ID NOs: VLFR1, VLFR2, VLFR3 and VLFR4 as shown in NOs: 95, 104, 113 and 122 (an exemplary embodiment is identified herein as clone BT-5A8-12 (H1B, L1A)).

[0339] In at least some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO:46, and the light chain amino acid sequence is shown in SEQ ID NO:56; in at least some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is the sequence shown in SEQ ID NO:29, and the light chain variable region is the sequence shown in SEQ ID NO:36.

[0340] In at least some embodiments, the antibody is antibody BT-5A8-12, the heavy chain amino acid sequence of antibody BT-5A8-12 is the sequence shown in SEQ ID NO: 46, the light chain amino acid sequence is the sequence shown in SEQ ID NO: 56; the heavy chain variable region is the sequence shown in SEQ ID NO: 29, and the light chain variable region is the sequence shown in SEQ ID NO: 36. In at least some preferred embodiments, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of antibody BT-5A8-12 is shown in SEQ ID NO: 129; the nucleotide sequence encoding the amino acid sequence of the light chain variable region of antibody BT-5A8-12 is shown in SEQ ID NO: 139.

[0341] In one embodiment, the anti-PVRIG antibody of the present invention is a humanized antibody, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 47 and a light chain with an amino acid sequence as shown in SEQ ID NO: 57; the heavy chain comprises a heavy chain variable region with an amino acid sequence of SEQ ID NO: 30 and the light chain comprises a light chain variable region with an amino acid sequence of SEQ ID NO: 37; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 with amino acid sequences as shown in SEQ ID NOs: 66, 74, 80 and 88, respectively; the light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively; and further comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively. VLFR1, VLFR2, VLFR3 and VLFR4 as shown in NOs: 96, 105, 114 and 123 (identified herein as clone BT-5A8-11 (H1A, L2A) in an exemplary embodiment).

[0342] In at least some embodiments, the antibody or antibody fragment includes a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO:47, and the light chain amino acid sequence is shown in SEQ ID NO:57; a heavy chain variable region and a light chain variable region, the heavy chain variable region is the sequence shown in SEQ ID NO:30, and the light chain variable region is the sequence shown in SEQ ID NO:37.

[0343] In at least some preferred embodiments, the antibody is antibody BT-5A8-11, the heavy chain variable region of the antibody BT-5A8-11 is the sequence shown in SEQ ID NO: 30, and the light chain variable region is the sequence shown in SEQ ID NO: 37. In at least some preferred embodiments, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of antibody BT-5A8-11 is shown in SEQ ID NO: 130; the nucleotide sequence encoding the light chain amino acid sequence of antibody BT-29D5-CHI is shown in SEQ ID NO: 140.

[0344] In one embodiment, the anti-PVRIG antibody of the present invention is a humanized antibody, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 48 and a light chain with an amino acid sequence as shown in SEQ ID NO: 58; the heavy chain comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 31 and the light chain comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 38; its heavy chain variable region (VH) comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 with amino acid sequences as shown in SEQ ID NOs: 67, 75, 81 and 87, respectively; its light chain variable region (VL) comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively; and further comprises amino acid sequences as shown in SEQ ID NOs: VLFR1, VLFR2, VLFR3 and VLFR4 as shown in NOs: 95, 104, 113 and 124 (an exemplary embodiment is identified herein as clone BT-5A8-32 (H3A, L2B)).

[0345] In at least some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO:48, and the light chain amino acid sequence is shown in SEQ ID NO:58; in at least some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is the sequence shown in SEQ ID NO:31, and the light chain variable region is the sequence shown in SEQ ID NO:38.

[0346] In at least some preferred embodiments, the antibody is antibody BT-5A8-32, the heavy chain variable region of the antibody BT-5A8-32 is the sequence shown in SEQ ID NO: 31, and the light chain variable region is the sequence shown in SEQ ID NO: 38. In at least some preferred embodiments, the nucleotide sequence encoding the heavy chain amino acid sequence of the heavy chain variable region of antibody BT-5A8-32 is shown in SEQ ID NO: 131; the nucleotide sequence encoding the light chain amino acid sequence is shown in SEQ ID NO: 141

[0347] Humanized antibodies of the present invention The variable region sequence of BT-5A8-12 is:

[0348] BT-5A8-12 VH(H1A:):

[0349] <h2 style=";text-align:left;direction:ltr">EVQLVQSGAEVKKPGATVKISCKATGHTFSTYWIEWVQQAPGKGLEWIGEILPGSGSTNYAEKFQGRVTITADTS TDTAYMELSSLRSEDTAVYYCASFFFAYWGQGTLVTVSS(SEQ ID NO:29),<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0350] <h2 style=";text-align:left;direction:ltr"> BT-5A8-12 VL(L2A):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0351] <h2 style=";text-align:left;direction:ltr"> DIVMTQTPLSLSVTPGQPASISCKSSQSLLNSGNQKNYLAWYLQKPGQPPQLLIYGASTRESGVPDRFSGSGSGT DFTLKISRVEAEDVGVYYCQNAHSYPLTFGQGTKLEIK(SEQ ID NO:36),<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0352] <h2 style=";text-align:left;direction:ltr"> The variable region sequence of the humanized antibody BT-5A8-11 of the present invention is: <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0353] <h2 style=";text-align:left;direction:ltr"> BT-5A8-11 VH(H1B):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0354] <h2 style=";text-align:left;direction:ltr"> EVQLVQSGAEVKKPGATVKISCKATGHTFSTYWIEWVQQAPGKGLEWIGEILPGSGSTNYAEKFQGRVTITADTS TDTAYMELSSLRSEDTAVYYCASFFFAYWGRGTLVTVSS(SEQ ID NO:30),<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0355] <h2 style=";text-align:left;direction:ltr"> BT-5A8-11 VL(L1A):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0356] <h2 style=";text-align:left;direction:ltr"> DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQKNYLAWYQQKPGKVPKLLIYGASTRESGVPSRFSGSGSGT DFTLTISSLQPEDVATYYCQNAHSYPLTFGQGTKVEIK(SEQ ID NO:37),<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0357] <h2 style=";text-align:left;direction:ltr"> The variable region sequence of the humanized antibody BT-5A8-32 of the present invention is: <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0358] <h2 style=";text-align:left;direction:ltr"> BT-5A8-32 VH(H3A):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0359] QVQLVESGGGVVQPGRSLRLSCAATGHTFSTYWIEWVRQAPGKGLEWIGEILPGSGSTNYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCASFFFAYWGQGTLVTVSS(SEQ ID NO:31),

[0360] BT-5A8-32 VH(L2B):

[0361] DIVMTQTPLSLSVTPGQPASISCKSSQSLLNSGNQKNYLAWYLQKPGQPPQLLIYGASTRESGVPDRFSGSGSGT DFTLKISRVEAEDVGVYYCQNAHSYPLTFGQGTRLEIK (SEQ ID NO: 38).

[0362] In one embodiment, the anti-PVRIG antibody of the present invention is a humanized antibody, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 49 and a light chain with an amino acid sequence as shown in SEQ ID NO: 59; the heavy chain comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 32 and the light chain comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 39; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 with amino acid sequences as shown in SEQ ID NOs: 68, 72, 82 and 89, respectively; the light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 10, 11 and 12, respectively; and further comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 11, 12 and 13, respectively. VLFR1, VLFR2, VLFR3 and VLFR4 shown in NOs: 97, 106, 115 and 125 (an exemplary embodiment is identified herein as clone 29D5-42 (H4A, L2A), abbreviated as 29D5-1 (H4A, L2A).

[0363] In at least some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO:49, and the light chain amino acid sequence is shown in SEQ ID NO:59; in at least some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is the sequence shown in SEQ ID NO:32, and the light chain variable region is the sequence shown in SEQ ID NO:39.

[0364] In at least some preferred embodiments, the antibody is antibody BT-29D5-42, the heavy chain variable region of the antibody BT-29D5-42 is the sequence shown in SEQ ID NO: 32, and the light chain variable region is the sequence shown in SEQ ID NO: 39. In at least some preferred embodiments, the heavy chain variable region amino acid sequence of antibody BT-29D542 corresponds to a nucleotide sequence encoding the heavy chain amino acid sequence as shown in SEQ ID NO: 133; the light chain amino acid sequence of antibody BT-29D-42 is shown in SEQ ID NO: 59, and the nucleotide sequence encoding the light chain amino acid sequence is shown in SEQ ID NO: 143.

[0365] In one embodiment, the anti-PVRIG antibody of the present invention is a humanized antibody, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 50 and a light chain with an amino acid sequence as shown in SEQ ID NO: 60; the heavy chain comprises a heavy chain variable region with an amino acid sequence of SEQ ID NO: 33 and the light chain comprises a light chain variable region with an amino acid sequence of SEQ ID NO: 40; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 with amino acid sequences as shown in SEQ ID NOs: 69, 72, 82 and 90, respectively; the light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 10, 11 and 12, respectively; and further comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 11, 12 and 13, respectively. VLFR1, VLFR2, VLFR3 and VLFR4 shown in NOs: 98, 107, 116 and 126 (an exemplary embodiment is identified herein as clone 29D5-41 (H4B, L1A), abbreviated as 29D5-2 (H4B, L1A).

[0366] In at least some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO:50, and the light chain amino acid sequence is shown in SEQ ID NO:60; in at least some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is the sequence shown in SEQ ID NO:33 or, the light chain variable region is the sequence shown in SEQ ID NO:40.

[0367] In at least some preferred embodiments, the antibody is antibody BT-29D5-41, the heavy chain variable region of the antibody BT-29D5-41 is the sequence shown in SEQ ID NO: 33, and the light chain variable region is the sequence shown in SEQ ID NO: 40. In at least some preferred embodiments, the nucleotide sequence encoding the heavy chain amino acid sequence of the heavy chain variable region of antibody BT-29D5-41 is shown in SEQ ID NO: 134; the nucleotide sequence encoding the light chain amino acid sequence of the light chain variable region of antibody BT-29D5- is shown in SEQ ID NO: 144.

[0368] In one embodiment, the anti-PVRIG antibody of the present invention is a humanized antibody, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 50 and a light chain with an amino acid sequence as shown in SEQ ID NO: 61; the heavy chain comprises a heavy chain variable region with an amino acid sequence of SEQ ID NO: 33 and the light chain comprises a light chain variable region with an amino acid sequence of SEQ ID NO: 41; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 with amino acid sequences as shown in SEQ ID NOs: 69, 72, 82 and 90, respectively; the light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 10, 11 and 12, respectively; and further comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 11, 12 and 13, respectively. VLFR1, VLFR2, VLFR3 and VLFR4 shown in NOs: 99, 108, 117 and 126 (an exemplary embodiment is identified herein as clone 29D5-44 (H4B, L4A), abbreviated as 29D5-3 (H4B, L4A).

[0369] In at least some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO:50, and the light chain amino acid sequence is shown in SEQ ID NO:61; in at least some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is the sequence shown in SEQ ID NO:33, and the light chain variable region is the sequence shown in SEQ ID NO:41.

[0370] In at least some preferred embodiments, the antibody is antibody BT-29D5-44, the heavy chain variable region of the antibody BT-29D5-44 is the sequence shown in SEQ ID NO: 33, and the light chain variable region is the sequence shown in SEQ ID NO: 41. In at least some preferred embodiments, the amino acid sequence of the heavy chain variable region of antibody BT-29D5-44 corresponds to the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 134; the amino acid sequence of the light chain variable region of antibody BT-29D5-32 corresponds to the nucleotide sequence encoding the light chain amino acid sequence as shown in SEQ ID NO: 145.

[0371] In one embodiment, the anti-PVRIG antibody of the present invention is a humanized antibody, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 51 and a light chain with an amino acid sequence as shown in SEQ ID NO: 62; the heavy chain comprises a heavy chain variable region with an amino acid sequence of SEQ ID NO: 34 and the light chain comprises a light chain variable region with an amino acid sequence of SEQ ID NO: 42; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 with amino acid sequences as shown in SEQ ID NOs: 69, 72, 82 and 91, respectively; the light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 10, 11 and 12, respectively; and further comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 10, 11 and 12, respectively. VLFR1, VLFR2, VLFR3 and VLFR4 shown in NOs: 100, 109, 118 and 127 (an exemplary embodiment is identified herein as clone 29D5-43 (H4C, L3A), abbreviated as 29D5-4 (H4C, L3A).

[0372] In at least some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO:51, and the light chain amino acid sequence is shown in SEQ ID NO:62; in at least some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is the sequence shown in SEQ ID NO:34, and the light chain variable region is the sequence shown in SEQ ID NO:42.

[0373] In at least some preferred embodiments, the antibody is BT-29D5-43, the heavy chain variable region of the antibody BT-29D5-43 is the sequence shown in SEQ ID NO: 34, and the light chain variable region is the sequence shown in SEQ ID NO: 42. In at least some preferred embodiments, the amino acid sequence of the heavy chain variable region of the antibody BT-29D5-43 corresponding to the nucleotide sequence encoding the heavy chain amino acid sequence is shown in SEQ ID NO: 135; the amino acid sequence of the light chain variable region of the antibody BT-29D5-43 corresponding to the nucleotide sequence encoding the light chain amino acid sequence is shown in SEQ ID NO: 146.

[0374] In one embodiment, the anti-PVRIG antibody of the present invention is a humanized antibody, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 52 and a light chain with an amino acid sequence as shown in SEQ ID NO: 59; the heavy chain comprises a heavy chain variable region with an amino acid sequence of SEQ ID NO: 35 and the light chain comprises a light chain variable region with an amino acid sequence of SEQ ID NO: 39; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively, and VHFR1, VHFR2, VHFR3 and VHFR4 with amino acid sequences as shown in SEQ ID NOs: 70, 76, 83 and 90, respectively; the light chain variable region comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 10, 11 and 12, respectively; and further comprises VLCDR1, VLVDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 11, 12 and 13, respectively. VLFR1, VLFR2, VLFR3 and VLFR4 as shown in NOs: 97, 106, 115 and 125 (an exemplary embodiment is identified herein as clone 29D5-32 (H3A L2A), abbreviated as 29D5-5 (H3A, L2A).

[0375] In at least some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO:52, and the light chain amino acid sequence is shown in SEQ ID NO:59; in at least some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is the sequence shown in SEQ ID NO:35, and the light chain variable region is the sequence shown in SEQ ID NO:39.

[0376] In at least some preferred embodiments, the antibody is BT-29D5-32, the heavy chain variable region of the antibody BT-29D5-32 is the sequence shown in SEQ ID NO: 35, and the light chain variable region is the sequence shown in SEQ ID NO: 39. In at least some preferred embodiments, the heavy chain variable region amino acid sequence of the antibody BT-29D5-42 corresponds to a nucleotide sequence encoding the heavy chain amino acid sequence as shown in SEQ ID NO: 136; the light chain variable region amino acid sequence of the antibody BT-29D5-42 corresponds to a nucleotide sequence encoding the light chain amino acid sequence as shown in SEQ ID NO: 144.

[0377] The exemplary humanized anti-PVRIG antibodies 29D5-1 (also known as 29D5-42 (29D5-1, H4A, L2A)), 29D5-2 (29D5-41, 29D5-42, H4B, L1A)), 29D5-3 (29D5-44, (H4B L4A)), 29D5-4 (29D5-43, (H4C L3A)) and 29D5-5 (29D5-32, (H3A L2A)) of the present invention have VHCDR1, VHCDR2 and VHCDR3 comprising the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and VLCDR1, VLCDR2 and VLCDR3 comprise the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0378] The variable region sequences of humanized antibodies 29D5-1 (H4A, L2A), 29D5-2 (H4B L1A), 29D5-3 (H4B L4A), 29D5-4 (H4CL3A) and 29D5-5 (H3A L2A) are:

[0379] 29D5-1(H4A, L2A)

[0380] VH of H4A:

[0381] QVQLQESGPGLVKPSGTLSLTCAVSGFSLTSYGVQWVRQPPGKGLEWLGVIWAGGSTNYNPSLKSRVTISVDKSK NQFSLKLSSVTAADTAVYYCARGGNYNYAGAMDYWGRGTLVTVSS (SEQ ID NO: 32);

[0382] VL of L2A:

[0383] DIVMTQTPLSLSVTPGQPASISCRASQYIGTSIHWYLQKPGQSPQLLIKYASESISGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCQQSNSWPMYTFGPGTKVDIK(SEQ ID NO: 39).

[0384] 29D5-2 (H4B, L1A)

[0385] VH of H4B:

[0386] QVQLQESGPGLVKPSGTLSLTCAVSGFSLTSYGVQWVRQPPGKGLEWLGVIWAGGSTNYNPSLKSRVTISVDKSK NQFSLKLSSVTAADTAVYYCARGGNYNYAGAMDYWGQGTMVTVSS(SEQ ID NO: 33); VL of L1A:

[0387] DIQMTQSPSSLSASVGDRVTITCRASQYIGTSIHWYQQKPGKVPKLLIKYASESISGVPSRFSGSGSGTDFTLTI SSLQPEDVATYYCQQSNSWPMYTFGPGTKVDIK(SEQ ID NO: 40).

[0388] 29D5-3: (H4B, L4A)

[0389] VH of H4B:

[0390] QVQLQESGPGLVKPSGTLSLTCAVSGFSLTSYGVQWVRQPPGKGLEWLGVIWAGGSTNYNPSLKSRVTISVDKSK NQFSLKLSSVTAADTAVYYCARGGNYNYAGAMDYWGQGTMVTVSS(SEQ ID NO: 33);

[0391] VL of L4A:

[0392] DIVMTQSPDSLAVSLGERATINCRASQYIGTSIHWYQQKPGQPPKLLIKYASESISGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQQSNSWPMYTFGPGTKVDIK(SEQ ID NO: 41).

[0393] 29D5-4: (H4C, L3A)

[0394] VH of H4C:

[0395] QVQLQESGPGLVKPSGTLSLTCAVSGFSLTSYGVQWVRQPPGKGLEWLGVIWAGGSTNYNPSLKSRVTISVDKSK NQFSLKLSSVTAADTAVYYCARGGNYNYAGAMDYWGQGTTVTVSS(SEQ ID NO:34);

[0396] VL of L3A:

[0397] EIVLTQSPATLSLSPGERATLSCRASQYIGTSIHWYQQKPGQAPRLLIKYASESITGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQSNSWPMYTFGQGTKLEIK (SEQ ID NO: 42);

[0398] 29D5-5(H3A,L2A)

[0399] VH of H3A:

[0400] QVQLVESGGGVVQPGRSLRLSCAVSGFSLTSYGVQWVRQAPGKGLEWLGVIWAGGSTNYADSVKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCARGGNYNYAGAMDYWGQGTMVTVSS(SEQ ID NO:35);

[0401] VL of L2A:

[0402] DIVMTQTPLSLSVTPGQPASISCRASQYIGTSIHWYLQKPGQSPQLLIKYASESISGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCQQSNSWPMYTFGPGTKVDIK (SEQ ID NO: 39).

[0403] The variable regions of the exemplary anti-PVRIG humanized antibodies of the present invention include CDR regions and FR regions, as shown in Tables 10 and 11.

[0404] Table 10 CDR regions and FR regions included in exemplary humanized antibodies BT-5A8-12, BT-5A8-11 and BT-5A8-32

[0405]

[0406]

[0407] Table 11 CDR and FR regions included in exemplary humanized antibodies 29D5-1 (H4A, L2A), 29D5-2 (H4B L1A), 29D5-3 (H4B L4A), 29D5-4 (H4C L3A) and 29D5-5 (H3A L2A)

[0408]

[0409]

[0410] The humanized antibodies of the present invention can be prepared by inserting mouse CDR regions into human germline framework regions using methods known in the art. See U.S. Pat. No. 5,225,539 of Winter et al. and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 of Queen et al. The variable region combinations of the exemplary humanized anti-PVRIG antibodies of the present invention are shown in Table 12.

[0411] Table 12 Variable region combinations of humanized anti-PVRIG antibodies of the present invention

[0412]

[0413]

[0414] In some embodiments, the anti-PVRIG antibody of the invention is an IgG antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody or a modified form thereof, as described in the following sections.

[0415] In some embodiments, the anti-PVRIG antibodies of the present invention comprise any one of a κ light chain and a λ light chain. In some embodiments, the anti-PVRIG antibodies of the present invention comprise a κ light chain. In some embodiments, the anti-PVRIG antibodies of the present invention comprise a human κ-type light chain.

[0416] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0417] In some embodiments, the anti-PVRIG chimeric antibodies and anti-PVRIG humanized antibodies of the present invention are IgG antibodies.

[0418] In some embodiments, the anti-PVRIG chimeric antibody and anti-PVRIG humanized antibody of the present invention are antibodies having a human IgG4 heavy chain constant region.

[0419] The light chain and heavy chain amino acid sequences of the PVRIG chimeric antibodies BT-5A8-CHI, BT-29D5-CHI and BT-26C4-CHI of the present invention are shown below, and the sequences outside the variable regions of the remaining chimeric antibodies LC and HC are the same as the corresponding sequences of BT-5A8-CHI, BT-29D5-CHI and BT-26C4-CHI, among which the humanized antibodies subjected to biological tests are all IgG4 heavy chains.

[0420] The amino acid sequence number combinations of the light chains and heavy chains of the humanized anti-human PVRIG antibodies BT-5A8-12, BT-5A8-11, BT-5A8-32, BT-29D5-42, BT-29D5-41, BT-29D5-44, and BT-29D5-43 of the present invention are shown in Table 13.

[0421] Table 13 Combinations of amino acid sequence numbers of the light and heavy chains of the chimeric antibodies BT-5A8-CHI, BT-29D5-CHI, BT-26C4-CHI and the humanized anti-human PVRIG antibodies BT-5A8-12, BT-5A8-11, BT-5A8-32, BT-29D5-42, BT-29D5-41, BT-29D5-44, BT-29D5-43 of the present invention

[0422]

[0423]

[0424] The sequence of the antibody BT-5A8-CHI heavy chain shown in SEQ ID NO:43 is:

[0425] QIQLQQSGAELMKPGASVKISCLATGHTFSTYWIEWVKQRPGHGLEWIGEILPGSGSTNYDEKFKDKATFTADTSSNTAYMQLSSLTSEDSAVYYCASFFFAYWGQGTLVT VSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:43)(IgG4),

[0426] The sequence of the antibody BT-5A8-CHI light chain shown in SEQ ID NO: 24 is:

[0427] DIVMTQSPSSLSVSAGEKVTLSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYFCQNAHSYPLTFGAGTKL ELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0428] The nucleic acid sequence encoding the amino acid sequence of the heavy chain variable region of antibody BT-5A8-CHI is shown in SEQ ID NO:128, see Example 4.

[0429] The amino acid sequence of the heavy chain of antibody BT-29D5-CHI is shown in SEQ ID NO:44: EVQLVQSGAEVKKPGATVKISCKATGHTFSTYWIEWVQQAPGKGLEWIGEILPGSGSTNYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCASFFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4)(SEQ ID NO:44),

[0430] The nucleic acid sequence encoding the amino acid sequence of the heavy chain variable region of antibody BT-29D5-CHI is shown in SEQ ID NO:132 (SEQ ID NO:132), see Example 4.

[0431] The amino acid sequence of the heavy chain of antibody BT-26C4-CHI is shown in SEQ ID NO:45: QVQLQQPGAELVMPGASVKMSCKASGYTFTDYWMHWVKQRPGQGLEWIGAIDISDSNTSYNQKFKGKATLTVDESSSTAYMQLSSLTSEDSAVYYCARYYYGSSYGMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:45)

[0432] The nucleic acid sequence encoding the amino acid sequence of the heavy chain variable region of antibody BT-26C4-CHI is shown in SEQ ID NO: 137: (SEQ ID NO: 137), see Example 4.

[0433] The light chain and heavy chain amino acid sequences of the PVRIG humanized antibodies BT-5A8-12, BT-5A8-11, BT-5A8-32, BT-29D5-42, BT-29D5-41, BT-29D5-44, and BT-29D5-43 of the present invention are shown below, and the sequences outside the variable regions of the remaining humanized antibodies LC and HC are the same as the corresponding sequences of BT-5A8-12, BT-5A8-11, BT-5A8-32, BT-29D5-42, BT-29D5-41, BT-29D5-44, and BT-29D5-43, wherein the humanized antibodies subjected to biological tests are all IgG4 heavy chains.

[0434] The amino acid sequence of the heavy chain variable region of antibody BT-5A8-12 is shown in SEQ ID NO:29.

[0435] Antibody BT-5A8-12 heavy chain amino acid sequence as SEQ ID NO:46 shows: EVQLVQSGAEVKKPGATVKISCKATGHTFSTYWIEWVQQAPGKGLEWIGEILPGSGSTNYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCASFFFAYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(IgG4)(SEQ ID NO:46).

[0436] The nucleic acid sequence encoding the heavy chain amino acid sequence of antibody BT-5A8-12 is shown in SEQ ID NO: 131: (SEQ ID NO: 129), see Example 6.

[0437] The amino acid sequence of the heavy chain variable region of antibody BT-5A8-11 is shown in SEQ ID NO:30.

[0438] Antibody BT-5A8-11 heavy chain amino acid sequence as SEQ ID IgG4 shown in NO:47): EVQLVQSGAEVKKPGATVKISCKATGHTFSTYWIEWVQQAPGKGLEWIGEILPGSGSTNYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCASFFF AYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(IgG4 S228P mutation) (SEQ ID NO:47).

[0439] The nucleic acid sequence encoding the heavy chain amino acid sequence of antibody BT-5A8-11 is shown in SEQ ID NO: 130: (SEQ ID NO: 130).

[0440] The amino acid sequence of the heavy chain variable region of antibody BT-5A8-32 is shown in SEQ ID NO:31;

[0441] Antibody BT-5A8-32 heavy chain amino acid sequence as SEQ ID Shown in NO:48 (IgG4): QVQLVESGGGVVQPGRSLRLSCAATGHTFSTYWIEWVRQAPGKGLEWIGEILPGSGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASFFFA YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(IgG4)(SEQ ID NO:48).

[0442] The nucleic acid sequence encoding the heavy chain amino acid sequence of antibody BT-5A8-32 is shown in SEQ ID NO: 131 (SEQ ID NO: 131), see Example 6.

[0443] The amino acid sequence of the heavy chain variable region of antibody BT-29D5-42 is shown in SEQ ID NO:32.

[0444] Antibody BT-29D5-42 heavy chain amino acid sequence as SEQ ID NO:49 shows: QVQLQESGPGLVKPSGTLSLTCAVSGFSLTSYGVQWVRQPPGKGLEWLGVIWAGGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARGGNYNYAGAMD YWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(IgG4)(SEQ ID NO:49).

[0445] The nucleic acid sequence encoding the heavy chain amino acid sequence of antibody BT-29D5-42 is shown in SEQ ID NO: 133 (SEQ ID NO: 133), see Example 6.

[0446] The amino acid sequence of the heavy chain variable region of antibody BT-29D5-41 is shown in SEQ ID NO:33.

[0447] Antibody BT-29D5-41 heavy chain amino acid sequence as SEQ ID NO:50 shown): QVQLQESGPGLVKPSGTLSLTCAVSGFSLTSYGVQWVRQPPGKGLEWLGVIWAGGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARGGNYNYAGAMDYWGQGT MVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(IgG4)(SEQ ID NO:50).

[0448] The nucleic acid sequence encoding the amino acid sequence of the heavy chain variable region of antibody BT-29D5-41 is shown in SEQ ID NO: 135: (SEQ ID NO: 134), see Example 6.

[0449] The amino acid sequence of the heavy chain variable region of antibody BT-29D5-44 is shown in SEQ ID NO: 33.

[0450] Antibody BT-29D5-44 heavy chain amino acid sequence as SEQ ID NO:50 shows: QVQLQESGPGLVKPSGTLSLTCAVSGFSLTSYGVQWVRQPPGKGLEWLGVIWAGGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARGGNYNYAGAMD YWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(IgG4)(SEQ ID NO:50).

[0451] The nucleic acid sequence encoding the amino acid sequence of the heavy chain variable region of antibody BT-29D5-44 is shown in SEQ ID NO: 134, see Example 6.

[0452] The amino acid sequence of the heavy chain variable region of antibody BT-29D5-43 is shown in SEQ ID NO:34:

[0453] The amino acid sequence of the heavy chain of antibody BT-29D5-43 is shown in SEQ ID NO:51:

[0454] QVQLQESGPGLVKPSGTLSLTCAVSGFSLTSYGVQWVRQPPGKGLEWLGVIWAGGSTNYNPSLKSRVTISVDKSKNQFS

[0455] LKLSSVTAADTAVYYCARGGNYNYAGAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT

[0456] VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG

[0457] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG

[0458] KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(IgG4)(SEQ IDNO:51).

[0459] The nucleic acid sequence encoding the amino acid sequence of the heavy chain variable region of antibody BT-29D5-43 is shown in SEQ ID NO: 135 (SEQ ID NO: 135), see Example 6.

[0460] The amino acid sequence of the heavy chain variable region of antibody BT-29D5-32 is shown in SEQ ID NO: 35.

[0461] Antibody BT-29D5-32 heavy chain amino acid sequence as SEQ ID NO:52 shows: QVQLVESGGGVVQPGRSLRLSCAVSGFSLTSYGVQWVRQAPGKGLEWLGVIWAGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNYNYAGAMD YWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(IgG4)(SEQ ID NO:52).

[0462] The nucleic acid sequence encoding the amino acid sequence of the heavy chain variable region of antibody BT-29D5-32 is shown in SEQ ID NO: 136: (SEQ ID NO: 136), see Example 6. In one embodiment, the antigen-binding fragment of the antibody of the present invention is an antibody fragment that specifically binds to human PVRIG.

[0463] Examples of antigen-binding fragments include, but are not limited to, Fv fragments, disulfide-stabilized Fv fragments (dsFv), Fab fragments, (Fab)2, scFv-Fc fusion proteins, scFv-Fv fusion proteins, and Fv-Fc fusion proteins.

[0464] In one embodiment, the antibody or antigen-binding fragment thereof described in the present invention is at least one of the minimum recognition units.

[0465] In one embodiment, the anti-human PVRIG antibody is a single chain antibody.

[0466] In one embodiment, the anti-human PVRIG antibody is a multimeric antibody.

[0467] In one embodiment, the anti-human PVRIG antibody is a CDR-grafted antibody.

[0468] In one embodiment, the anti-human PVRIG antibody is a small molecule antibody.

[0469] In one embodiment, the antigen-binding fragment of the anti-human PVRIG antibody forms a multispecific antibody, a single domain antibody, a VHH nanobody, a domain antibody, or a bivalent domain antibody.

[0470] These molecules may be provided by any known technique including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant technology.

[0471] Nucleic acid molecules

[0472] The present invention also provides a kind of separable nucleic acid, the nucleic acid encoding the above-mentioned antibody or antigen binding fragment. Separable nucleic acid refers to that nucleic acid can be separated from other substances basically, and does not contain material or chemical substance basically. Nucleic acid can be present in intact cells, cell lysate, or in partially purified or basically pure form. Nucleic acid is "separated" or "basically pure" when it can be purified from other cellular components or other impurities, such as other cellular nucleic acids or proteins by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and other techniques well known in the art.

[0473] In the process of preparing or obtaining these antibodies or antigen-binding fragments thereof, nucleic acid molecules expressing these antibodies or antigen-binding fragments thereof can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies or antigen-binding fragments thereof.

[0474] The embodiments of the present invention also provide nucleic acids comprising polynucleotides encoding polypeptides of the present invention, wherein the polypeptides comprise anti-human PVRIG immunoglobulin heavy chains or immunoglobulin light chains, heavy chain variable regions or light chain variable regions. The nucleic acid sequences encoding the heavy chain variable regions and light chain variable regions of exemplary anti-PVRIG antibodies of the present invention are shown in Tables 14 and 15. The immunoglobulin heavy chain or immunoglobulin light chain comprises the CDRs shown in Table 2, or has a variable region sequence as shown in Table 1. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to PVRIG (e.g., human PVRIG).

[0475] Table 14 Nucleic acid sequences encoding the variable regions of exemplary anti-PVRIG antibodies (chimeric antibodies) of the present invention

[0476]

[0477]

[0478] Table 15 Nucleic acid sequences encoding the variable regions of exemplary anti-PVRIG antibodies (humanized antibodies) of the present invention

[0479]

[0480]

[0481]

[0482]

[0483] In one embodiment, the present invention provides a polynucleotide encoding: (1) an antibody or an antigen-binding fragment thereof that specifically recognizes PVRIG as described in the aforementioned aspects, (2) the light chain variable region or the heavy chain variable region of (1), and (3) at least one sequence of the light chain or heavy chain of (1).

[0484] In one embodiment, the polynucleotide sequence encodes an antibody heavy chain variable region, wherein the polynucleotide sequence comprises: a sequence selected from any one of SEQ ID NOs: 62-70 or a variant thereof having at least 85% identity with any one of SEQ ID NOs: 62-70. In one embodiment, the polynucleotide sequence encodes an antibody light chain variable region, wherein the polynucleotide sequence comprises: a sequence selected from any one of SEQ ID NOs: 71-79 or a variant thereof having at least 85% identity with any one of SEQ ID NOs: 62-70.

[0485] In some embodiments, the polynucleotide molecules encoding the antibodies of the present invention include polynucleotide molecules that have been mutated by nucleotide deletion, insertion or substitution, but still have at least 85% sequence homology with the coding regions corresponding to the CDRs depicted in the sequences described above, at least 80% sequence homology, including 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity, or any percentage of sequence identity between the above adjacent percentages, such as 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 96%, 97%, 98%, 99% or 100%.

[0486] In some preferred embodiments, the nucleic acid molecule is species optimized and is more easily expressed in mammalian cells.

[0487] Variants, analogs, derivatives

[0488] Based on the polynucleotide molecules encoding the antibodies of the present invention generated by nucleotide deletion, insertion or substitution mutation, in some embodiments, the present invention also provides conservative amino acid variants of the antibody molecules according to the present invention. Analogs and derivatives of the above-mentioned isolated antibodies and antigen-binding fragments are also within the scope of the present invention, including but not limited to conservative and non-conservative substitutions, insertions and deletions of amino acids within the sequence. These modifications and resulting antibody analogs or variants are within the scope of the present invention as long as they provide or even improve the binding of the antibody to human PVRIG.

[0489] It should be emphasized that the variant chain sequence is determined by sequencing using specific primers. Using different sequencing methods for the same sequence may produce slightly different sequences, especially in the sequence ends, due to technical problems and different primers.

[0490] As used herein, the term "antibody variant" refers to any molecule comprising an antibody of the invention. For example, a fusion protein in which the antibody or antigen-binding fragment thereof is linked to another chemical moiety is considered an antibody variant. 。

[0491] In some embodiments, the amino acid changes include amino acid deletions, insertions, or substitutions.

[0492] In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4 or 5 amino acid mutations in the CDR regions by amino acid deletion, insertion or substitution when compared to the CDR regions depicted in the specific sequence. In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4 or 5 amino acid mutations in the framework regions by amino acid deletion, insertion or substitution when compared to the framework regions in the specific sequence. Each possibility represents an independent embodiment of the present invention.

[0493] According to certain embodiments, the antibody or antigen-binding fragment comprises CDRs having light and heavy chain regions as defined above, wherein at least one amino acid is replaced. According to a specific embodiment, the antibody or antibody fragment comprises CDRs as defined above, wherein at least one amino acid is replaced.

[0494] Herein, the at least one amino acid substitution mentioned may be one amino acid substitution, two amino acid substitutions, three amino acid substitutions, four amino acid substitutions or five amino acid substitutions, or even more. According to a preferred embodiment of the present invention, the amino acid substitution is preferably a conservative amino acid substitution. Herein, a conservative amino acid substitution is an amino acid substitution that does not impair the binding of the antibody to the PVRIG antigen.

[0495] In some embodiments, the anti-PVRIG antibodies or antigen-binding fragments thereof of the invention include those having amino acid sequences that have been mutated by amino acid deletions, insertions or substitutions, but still have at least 80% sequence identity with the above antibodies (particularly in the CDR regions depicted in the above sequences). The at least 80% sequence identity of the present invention includes any of about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, and any value between two values. The variants may contain conservative changes. Each possibility represents an independent embodiment of the present invention.

[0496] Sequence identity is the amount of amino acids or nucleotides that are exactly matched between two different sequences. Sequence similarity allows conservative substitutions of amino acids to be determined as identical amino acids. The polynucleotide sequences described herein can be codon optimized for expression in specific cells such as human cells. Codon optimization does not change the encoded amino acid sequence of the antibody chain, but can, for example, improve expression in cells.

[0497] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific amino acid sequence as shown in this specification, after aligning the candidate sequence with the specific amino acid sequence as shown in this specification and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a considerable degree of identity, such as identity, relative to the antibody molecules and sequences thereof specifically disclosed herein.

[0498] Conservative amino acid substitutions include replacing one amino acid with another amino acid having the same type of functional group or side chain, such as aliphatic, aromatic, positively charged, negatively charged functional groups or side chains. These substitutions may enhance oral bioavailability, permeability and targeting to a particular cell population, immunogenicity, etc. Professional and technical personnel will recognize that changes, additions or deletions made to a peptide, polypeptide or protein sequence, each replacement, deletion or addition of a single amino acid or a small proportion of amino acids in the encoded sequence, where the changes result in amino acids being replaced by chemically similar amino acids, are "conservatively modified variants". Conservative substitution tables that provide functionally similar amino acids are well known in the art. For example, according to a table known in the art, the following 6 groups each contain amino acids that are conservatively substituted with each other:

[0499] 1) Alanine (A), serine (S), threonine (T);

[0500] 2) Aspartic acid (D), Glutamic acid (E);

[0501] 3) Asparagine (N), glutamine (Q);

[0502] 4) Arginine (R), Lysine (K);

[0503] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0504] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0505] It should be emphasized that the sequence of the variant chain is determined by sequencing using specific primers. Using different sequencing methods for the same sequence, different primers due to technical problems may produce slightly different sequences, especially in the end of the sequence. "Conservative amino acid substitution" refers to the replacement of an amino acid by another amino acid residue that is biologically, chemically or structurally similar. Biologically similar means that the substitution does not destroy the biological activity of the PVRIG antibody or the PVRIG antigen. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine or serine, or have side chains of similar size. Chemical similarity means that the amino acids have the same charge or are hydrophilic or hydrophobic. For example, hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other. Or polar amino acids are used, such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, etc.

[0506] Variants according to the present invention can also be made to retain the overall molecular structure of the encoded protein. Given the properties of the individual amino acids that constitute the disclosed protein product, the skilled person will recognize some reasonable substitutions. Amino acid substitutions, i.e., "conservative substitutions," can be made, for example, on the basis of similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. When used in this article, the term "antibody analog" refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.

[0507] When used herein, the term "antibody variant" refers to any molecule comprising an antibody of the invention. For example, a fusion protein in which the antibody or antigen-binding fragment thereof is linked to another chemical moiety is considered an antibody variant.

[0508] Variants according to the present invention can also be made to retain the overall molecular structure of the encoded protein. Given the properties of the individual amino acids that constitute the disclosed protein product, the skilled person will recognize some reasonable substitutions. Amino acid substitutions, i.e., "conservative substitutions," can be made, for example, on the basis of similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. When used in this article, the term "antibody analog" refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.

[0509] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR having one, two or three conservative amino acid substitutions, insertions or deletions with three CDRs of the heavy chain variable region shown in any one of SEQ ID NOs: 23, 25, 27, 29, 30 or 31, 32, 33, 34 or 35, or the light chain variable region comprises a CDR having one, two or three conservative amino acid substitutions, insertions or deletions with three CDRs of the light chain variable region sequence shown in any one of SEQ ID NOs: 24, 26, 28, 36, 37, 38, 39, 40, 41 or 42.

[0510] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, whose heavy chain variable region comprises an analog or derivative having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35.

[0511] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, whose light chain variable region comprises an analog or derivative having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 24, 26, 28, 36, 37, 38, 39, 40, 41 or 42.

[0512] These conservative amino acid substitutions will not change the biological function of the antibody or antigen-binding fragment. In some specific embodiments, these conservative amino acid substitutions may occur in amino acids other than the CDR region in the heavy chain variable region and the light chain variable region. In some embodiments, the heavy chain variable region sequence of the antibody or antigen-binding fragment is selected from the amino acid sequence shown in any one of SEQ ID NO: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35, and has conservative amino acid substitutions. In some embodiments, the light chain variable region sequence of the antibody or antigen-binding fragment has conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 24, 26, 28, 36, 37, 38, 39, 40, 41 or 42.

[0513] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention heterosexually binds to PVRIG, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 29, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 36.

[0514] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention heterosexually binds to PVRIG, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO:30, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO:37.

[0515] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention heterosexually binds to PVRIG, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO:31, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO:38.

[0516] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention heterosexually binds to PVRIG, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO:32, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO:39.

[0517] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention heterosexually binds to PVRIG, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 9, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:33, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 9, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:40.

[0518] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention heterosexually binds to PVRIG, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 33, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 41.

[0519] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention heterosexually binds to PVRIG, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO:34, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO:42.

[0520] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention heterosexually binds to PVRIG, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 35, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 39.

[0521] In some embodiments, the present invention provides an antibody or an antigenic fragment thereof that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three complementarity determining regions (CDRs) of a heavy chain variable region (VH) of SEQ ID NO: 29 and three CDRs of a light chain variable region (VL) of SEQ ID NO: 36, or provides an analog or derivative having at least 80% sequence identity with the antibody or fragment sequence or comprising no more than one, two or three amino acid substitutions, deletions or additions; or

[0522] In some embodiments, the present invention provides an antibody or an antigenic fragment thereof that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 30 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 37, or provides an analog or derivative having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity with the antibody or fragment sequence or comprising no more than one, two or three amino acid substitutions, deletions or additions.

[0523] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 32 and a light chain variable region sequence shown in SEQ ID NO: 39, or provides an analog having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence similarity to the light chain variable region sequence or comprising no more than one, two or three amino acid substitutions, deletions or additions.

[0524] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 33 and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 40 or 41, or provides an analog or derivative having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity with the antibody or fragment sequence or containing no more than one, two or three amino acid substitutions, deletions or additions.

[0525] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:23.

[0526] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:23.

[0527] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:23.

[0528] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:23.

[0529] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 25.

[0530] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:25.

[0531] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 25.

[0532] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 25.

[0533] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:27.

[0534] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:27.

[0535] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:27.

[0536] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 27.

[0537] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:24.

[0538] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:24.

[0539] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:24.

[0540] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:24.

[0541] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:26.

[0542] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:26.

[0543] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:26.

[0544] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:26.

[0545] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:28.

[0546] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:28.

[0547] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:28.

[0548] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a chimeric antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 28.

[0549] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:29.

[0550] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:29.

[0551] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0552] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0553] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 36.

[0554] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:36.

[0555] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:36.

[0556] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:36.

[0557] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:36.

[0558] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:30.

[0559] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:30.

[0560] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 30.

[0561] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:30.

[0562] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody, whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 29, and whose light chain variable region comprises an analog or derivative having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 37.

[0563] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:37.

[0564] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:37.

[0565] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:37.

[0566] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody, whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30, and whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 37.

[0567] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:31.

[0568] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:31.

[0569] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 31.

[0570] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:31.

[0571] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:38.

[0572] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:38.

[0573] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:38.

[0574] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:38.

[0575] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody, whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 31, and whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 38.

[0576] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:32.

[0577] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:32.

[0578] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:32.

[0579] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:32.

[0580] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0581] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0582] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0583] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0584] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody, whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 32, and whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39.

[0585] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:33.

[0586] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:33.

[0587] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 33.

[0588] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33.

[0589] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:40.

[0590] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:40.

[0591] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:40.

[0592] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:40.

[0593] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody, whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 33, and whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 40.

[0594] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:33.

[0595] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:33.

[0596] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:33.

[0597] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33.

[0598] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:41.

[0599] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:41.

[0600] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:41.

[0601] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:41.

[0602] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody, whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 33, and whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 41.

[0603] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:34.

[0604] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:34.

[0605] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 34.

[0606] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:34.

[0607] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:42.

[0608] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:42.

[0609] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:42.

[0610] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:42.

[0611] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody, whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, and whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 42.

[0612] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:35.

[0613] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:35.

[0614] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 35.

[0615] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:35.

[0616] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0617] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0618] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0619] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0620] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, which is a humanized antibody, whose heavy chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 35, and whose light chain variable region comprises an analog or derivative having at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39.

[0621] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three complementarity determining regions (CDRs) of the heavy chain variable region (VH) of SEQ ID NO: 29 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 36, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention; or

[0622] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 30 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 37, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention; or

[0623] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 31 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 38, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention; or

[0624] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 32 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 39, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention; or

[0625] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 33 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 40 or 41, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention; or

[0626] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 34 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 42, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention; or

[0627] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 35 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 39, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention; or

[0628] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 23 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 24, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention; or

[0629] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 25 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 26, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention; or

[0630] In some embodiments, the present invention provides an antibody or antibody fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain variable region (VH) of SEQ ID NO: 27 and three CDRs of the light chain variable region (VL) of SEQ ID NO: 28, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence.

[0631] In some embodiments, an antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having one amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, an antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having two amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO: 29.

[0632] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:29.

[0633] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:29.

[0634] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:36.

[0635] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:36.

[0636] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:36.

[0637] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having five amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:36.

[0638] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:30.

[0639] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:30.

[0640] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:30.

[0641] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:30.

[0642] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:37.

[0643] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:37.

[0644] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:37.

[0645] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:37.

[0646] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:31.

[0647] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:31.

[0648] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:31.

[0649] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:31.

[0650] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:38.

[0651] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:38.

[0652] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:38.

[0653] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:38.

[0654] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:32.

[0655] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:32.

[0656] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:32.

[0657] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:32.

[0658] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:39.

[0659] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:39.

[0660] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:39.

[0661] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:39.

[0662] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:33.

[0663] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:33.

[0664] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:33.

[0665] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:33.

[0666] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:39.

[0667] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having two amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO: 40 or 41.

[0668] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 40 or 41.

[0669] In some embodiments, an antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having five amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 40 or 41.

[0670] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:34.

[0671] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:34.

[0672] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:34.

[0673] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a heavy chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:34.

[0674] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having an amino acid substitution, deletion or addition compared to the amino acid sequence shown in SEQ ID NO:42.

[0675] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having two amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:42.

[0676] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having three amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:42.

[0677] In some embodiments, the antibody or antigen-binding fragment that specifically binds to PVRIG comprises a light chain variable region having five amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO:42.

[0678] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to PVRIG, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain CDR and a heavy chain framework region (VHFRs), and the VL comprises a light chain CDR and a light chain framework region (VLFRs); wherein at least a portion of at least one of the heavy chain framework region (VHFRs) sequence and the light chain framework region (VLFRs) sequence is derived from a human antibody.

[0679] The inventors can obtain the CDR region of the heavy chain variable region sequence of the above-mentioned antibody (as shown in SEQ ID NO: 1-3, SEQ ID NO: 7-9, SEQ ID NO: 13-15) and the CDR region of the light chain variable region sequence (as shown in SEQ ID NO: 4-6, SEQ ID NO: 10-12, SEQ ID NO: 16-18) through the antibody sequence comparison database (NCBI, IMGT) or related software. In some embodiments, an antibody or antigen-binding fragment is provided, wherein the heavy chain variable region sequence of the antibody or antigen-binding fragment has a conservative amino acid substitution compared with the amino acid sequence shown in any one of SEQ ID NO: 29-35. In some embodiments, the light chain variable region sequence of the antibody or antigen-binding fragment has a conservative amino acid substitution compared with the amino acid sequence shown in any one of SEQ ID NO: 36-42. Of course, these conservative amino acid substitutions will not change the biological function of the antibody or antigen-binding fragment. In some specific embodiments, these conservative amino acid substitutions can occur in amino acids other than the CDR region in the heavy chain variable region and the light chain variable region.

[0680] In some embodiments,The present invention provides a monoclonal antibody or antibody fragment comprising a PVRIG antigen binding domain (ABD), which may have at least 90% sequence identity or similarity with the corresponding ABD of antibody clones BT-5A8-CHI, BT-29D5-CHI and BT-26C4-CHI: (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 24 (identified herein as clone BT-5A8-CHI); or (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 26 (identified herein as clone BT-29D5-CHI); or (iii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 27 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 28. The variable light chain of NO:28 (identified herein as clone BT-26C4-CHI) has an ABD domain with at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, at least 99% sequence identity or similarity, or 100% sequence identity.

[0681] The present invention also provides the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-PVRIG humanized antibody. Since there are different ways to combine mouse antibodies and humanized antibodies (for example, different amino acid substitutions can be used to modify the sequence), the heavy chain and light chain of the antibody can have more than one form of humanized sequence.

[0682] In other embodiments, the antibodies or antigen-binding fragments provided by the present invention comprise a heavy chain of a VHCDR having a conservative amino acid substitution, i.e., a heavy chain of a VHCDR having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; or a light chain of a VLCDR having a conservative amino acid substitution, i.e., a light chain of a VLCDR having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 The light chain of a VLCDR having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence shown in any one of NO:18.

[0683] In some embodiments, the antibodies or antigen-binding fragments provided by the present invention comprise a VHCDR having conservative amino acid substitutions, i.e., a heavy chain of a VHCDR comprising an amino acid sequence that is at least 90%, 95%, 97% or 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; in some embodiments, the antibodies or antigen-binding fragments provided by the present invention comprise a VLCDR having conservative amino acid substitutions, i.e., a light chain of a VLCDR comprising an amino acid sequence that is at least 90%, 95%, 97% or 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0684] In at least some embodiments, the present invention provides an anti-PVRIG antibody or antibody fragment comprising heavy chain complementary determining regions VHDR1, VHCDR2 and VHCDR3, and light chain complementary determining regions VLCDR1, VLCDR2 and VLCDR3.

[0685] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively;

[0686] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively;

[0687] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively;

[0688] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively;

[0689] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively;

[0690] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively;

[0691] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHCDR1, VHCDR2, and VHCDR3 sequences as shown in the amino acids of SEQ ID NOs: 13, 14, and 15, respectively;

[0692] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively;

[0693] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHCDR1, VHCDR2, and VHCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively;

[0694] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0695] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0696] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0697] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively;

[0698] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively;

[0699] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively;

[0700] In at least some embodiments, the antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VLCDR1, VLCDR2, and VLCDR3 sequences of the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively;

[0701] In at least some embodiments, the antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VLCDR1, VLCDR2, and VLCDR3 sequences shown in the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively;

[0702] In at least some embodiments, the antibodies or antibody fragments comprise amino acid sequences that are at least 98% identical to the VLCDR1, VLCDR2, and VLCDR3 sequences shown in the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively.

[0703] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 63, 71, 77 and 84, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 92, 101, 110 and 119.

[0704] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 63, 71, 77 and 84, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 92, 101, 110 and 119.

[0705] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 63, 71, 77 and 84, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 92, 101, 110 and 119.

[0706] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% or 99% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 63, 71, 88, and 84, respectively, and an amino acid sequence that is at least 98% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 92, 101, 110, and 119.

[0707] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 64, 72, 78 and 85, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 93, 102, 111 and 120.

[0708] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 64, 72, 78, and 85, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 93, 102, 111, and 120.

[0709] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 64, 72, 78 and 85, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 93, 102, 111 and 120.

[0710] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% or 99% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 64, 72, 78, and 85, respectively, and an amino acid sequence that is at least 98% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 93, 102, 111, and 120.

[0711] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 65, 73, 79 and 86, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 94, 103, 112 and 121.

[0712] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 65, 73, 79 and 86, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 94, 103, 112 and 121.

[0713] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 65, 73, 79 and 86, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 94, 103, 112 and 121.

[0714] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 65, 73, 79 and 86, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 94, 103, 112 and 121.

[0715] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 66, 74, 80 and 87, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 95, 104, 113 and 122.

[0716] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 66, 74, 80, and 87, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 95, 104, 113, and 122.

[0717] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 66, 74, 80 and 87, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 95, 104, 113 and 122.

[0718] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 66, 74, 80 and 87, respectively, and an amino acid sequence that is at least 98% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 95, 104, 113 and 122.

[0719] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 66, 74, 80 and 88, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 96, 105, 114 and 123.

[0720] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 66, 74, 80 and 88, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 96, 105, 114 and 123.

[0721] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 66, 74, 80 and 88, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 96, 105, 114 and 123.

[0722] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 66, 74, 80 and 88, respectively, and an amino acid sequence that is at least 98% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 96, 105, 114 and 123.

[0723] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 67, 75, 81 and 87, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 95, 104, 113 and 124.

[0724] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 67, 75, 81, and 87, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 95, 104, 113, and 124.

[0725] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 67, 75, 81, and 87, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 95, 104, 113, and 124.

[0726] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 67, 75, 81, and 87, respectively, and an amino acid sequence that is at least 98% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 95, 104, 113, and 124.

[0727] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 68, 72, 82 and 89, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 97, 106, 115 and 125.

[0728] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 68, 72, 82 and 89, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 97, 106, 115 and 125.

[0729] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3 and V5 sequences shown in the amino acids of SEQ ID NOs: 68, 72, 82 and 89, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 97, 106, 115 and 125.

[0730] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHFR1, VHFR2, VHFR3 and V5 sequences shown in the amino acids of SEQ ID NOs: 68, 72, 82 and 89, respectively, and an amino acid sequence that is at least 985% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 97, 106, 115 and 125.

[0731] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82 and 90, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 98, 107, 116 and 126.

[0732] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82, and 90, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 98, 107, 116, and 126.

[0733] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82 and 90, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 98, 107, 116 and 126.

[0734] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82, and 90, respectively, and an amino acid sequence that is at least 98% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 98, 107, 116, and 126.

[0735] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82 and 90, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 99, 108, 117 and 126.

[0736] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82, and 90, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 99, 108, 117, and 126.

[0737] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82, and 90, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 99, 108, 117, and 126.

[0738] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82, and 90, respectively, and an amino acid sequence that is at least 98% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 99, 108, 117, and 126.

[0739] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82 and 91, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 100, 109, 118 and 127.

[0740] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82 and 91, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 100, 109, 118 and 127.

[0741] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82 and 91, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 100, 109, 118 and 127.

[0742] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 69, 72, 82 and 91, respectively, and an amino acid sequence that is at least 98% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 100, 109, 118 and 127.

[0743] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 85% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 70, 76, 83 and 90, respectively, and an amino acid sequence that is at least 85% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 97, 106, 115 and 125.

[0744] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 90% identical to the VHFR1, VHFR2, VHFR3, and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 70, 76, 83, and 90, respectively, and an amino acid sequence that is at least 90% identical to the VLFR1, VLFR2, VLFR3, and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 97, 106, 115, and 125.

[0745] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 95% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 70, 76, 83 and 90, respectively, and an amino acid sequence that is at least 95% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 97, 106, 115 and 125.

[0746] In at least some embodiments, the anti-PVRIG antibody or antibody fragment comprises an amino acid sequence that is at least 98% identical to the VHFR1, VHFR2, VHFR3 and VHFR4 sequences shown in the amino acids of SEQ ID NOs: 70, 76, 83 and 90, respectively, and an amino acid sequence that is at least 98% identical to the VLFR1, VLFR2, VLFR3 and VLFR4 sequences shown in the amino acid sequences of SEQ ID NOs: 97, 106, 115 and 125.

[0747] In some aspects, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:43, 44 or 45; in one embodiment, wherein the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:53, 54 or 55.

[0748] In some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:46, 47, 48, 49, 50, 51 or 52; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 99.5% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:56, 57, 58, 59, 60, 61 or 62.

[0749] In some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:43; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:43; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:43; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:43.

[0750] In some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:44; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:44; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:44; in some embodiments, the heavy chain of the antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:44.

[0751] In some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:45; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:45; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:45; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:45.

[0752] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:53; in some embodiments, the light chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:53; in some embodiments, the light chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:53; in some embodiments, the light chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:53.

[0753] In some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:54; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:54; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:54; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:54.

[0754] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:55; in some embodiments, the light chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:55; in some embodiments, the light chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:55; in some embodiments, the light chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:55.

[0755] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:46; in some embodiments, the heavy chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:46; in some embodiments, the heavy chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:46; in some embodiments, the heavy chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:46.

[0756] In some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:56; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:56; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:56; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:56.

[0757] In some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:47; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:47; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:47; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:47.

[0758] In some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:57; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:57; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:57; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:57.

[0759] In some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:48; in some embodiments, the heavy chain of the antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:48; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:48; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:48.

[0760] In some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:58; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:58; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:58; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:58.

[0761] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:49; in some embodiments, the heavy chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:49; in some embodiments, the heavy chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:49; in some embodiments, the heavy chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:49.

[0762] In some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:59; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:59; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:59; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:59.

[0763] In some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:50; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:50; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:50; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:50.

[0764] In some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:60 or 61; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:60 or 61; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:60 or 61; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:60 or 61.

[0765] In some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:59; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:59; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:59; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:59.

[0766] In some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:51; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:51; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:51; in some embodiments, the heavy chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:51.

[0767] In some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:62; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:62; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:62; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:62.

[0768] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:52; in some embodiments, the heavy chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:52; in some embodiments, the heavy chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:52; in some embodiments, the heavy chain of the anti-PVRIG antibody or antigen-binding fragment thereof described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:52.

[0769] In some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:59; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:59; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:59; in some embodiments, the light chain of the anti-PVRIG antibody or its antigen-binding fragment described in the present invention comprises an amino acid sequence having at least 98%-99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:59.

[0770] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the anti-PVRIG antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 43 and three CDRs of the light chain (L) of SEQ ID NO: 53, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention.

[0771] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 44 and three CDRs of the light chain (L) of SEQ ID NO: 54, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention.

[0772] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 45 and three CDRs of the light chain (L) of SEQ ID NO: 55, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention.

[0773] In some aspects, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 46 and three CDRs of the light chain (L) of SEQ ID NO: 56, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention.

[0774] In some aspects, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 47 and three CDRs of the light chain (L) of SEQ ID NO: 57, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence.

[0775] In some aspects, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 48 and three CDRs of the light chain (L) of SEQ ID NO: 58, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention.

[0776] In some aspects, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 49 and three CDRs of the light chain (L) of SEQ ID NO: 59, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention.

[0777] In some aspects, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 50 and three CDRs of the light chain (L) of SEQ ID NO: 60 or 61, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence; each possibility represents an independent embodiment of the present invention.

[0778] In some aspects, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 51 and three CDRs of the light chain (L) of SEQ ID NO: 62, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence. Each possibility represents an independent embodiment of the present invention.

[0779] In some aspects, the present invention provides an antibody or antigen-binding fragment that specifically recognizes PVRIG, wherein the antibody or antigen-binding fragment comprises three CDRs of the heavy chain (H) of SEQ ID NO: 52 and three CDRs of the light chain (L) of SEQ ID NO: 59, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence. Each possibility represents an independent embodiment of the present invention.

[0780] Because there are different ways to humanize mouse antibodies (eg, the sequence can be modified with different amino acid substitutions), the antibody heavy and light chains may have more than one version of the humanized sequence.

[0781] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers.

[0782] The above-mentioned antibodies or antibody fragments are separable. The term "separable" means that, in addition to the provided antibody, it does not substantially contain other antibodies or antibody fragments with different antigenic specificities. Moreover, the separable antibody or antibody fragment may be substantially free of other cellular materials and / or chemicals.

[0783] Therefore, in some aspects, the antibody provided is an isolated antibody that has been separated from antibodies with different specificities. The isolated antibody can be a monoclonal antibody. The isolated antibody can be a recombinant monoclonal antibody. However, the isolated antibody that specifically binds to a target epitope, isotype or variant can have cross-reactivity with other related antigens, such as from other species (e.g., species homologs).

[0784] Functional activity

[0785] The antibodies or antibody fragments provided by the present invention can bind to the PVRIG protein with high affinity.

[0786] According to certain embodiments, the PVRIG antibody is a chimeric antibody, wherein the antibody or antigen-binding fragment thereof binds to human PVRIG with an ELISIA binding EC50 of no more than 300 ng / ml. According to other embodiments, the antibody or antibody fragment binds to human PVRIG with an ELISIA binding EC50 of no more than 300 ng / ml, an ELISIA binding EC50 of no more than 150 ng / ml, an ELISIA binding EC50 of no more than 100 ng / ml, an ELISIA binding EC50 of no more than 50 ng / ml, or an ELISIA binding EC50 of no more than 30 ng / ml, or even higher affinity.

[0787] According to certain embodiments, the PVRIG antibody is a chimeric antibody, which antibody or fragment thereof blocks the binding of PVRIG to CD112 with an ELISA blocking IC50 of no more than 3000 ng / ml, or an ELISA blocking IC50 of no more than 2000 ng / ml, an ELISA blocking IC50 of no more than 1500 ng / ml, an ELISA blocking IC50 of no more than 1000 ng / ml, or an ELISA blocking IC50 of no more than 800 ng / ml, or an ELISA blocking IC50 of no more than 500 ng / ml.

[0788] According to other embodiments, the PVRIG antibody is a chimeric antibody, and the antibody or antibody fragment binds to CHO-K1-PVRIG cell surface PVRIG with a FACS binding EC50 of no more than 600 ng / ml, a FACS binding EC50 of no more than 500 ng / ml, a FACS binding EC50 of no more than 400 ng / ml, a FACS binding EC50 of no more than 300 ng / ml, a FACS binding EC50 of no more than 200 ng / ml, or even higher affinity.

[0789] According to other embodiments, the PVRIG antibody is a chimeric antibody, and the antibody or antibody fragment blocks the binding of CHO-K1-CD112 to PVRIG with a FACS blocking IC50 of no more than 600 ng / ml, a FACS blocking IC50 of no more than 500 ng / ml, a FACS blocking IC50 of no more than 400 ng / ml, a FACS blocking IC50 of no more than 350 ng / ml, a FACS blocking IC50 of no more than 300 ng / ml, a FACS blocking IC50 of no more than 250 ng / ml, a FACS blocking IC50 of no more than 200 ng / ml, or even higher blocking activity.

[0790] According to certain embodiments, the PVRIG antibody is a chimeric antibody, wherein the antibody or antibody fragment is 10 -9 M to 10-11 According to certain embodiments, the antibody or antibody fragment binds to human PVRIG with an affinity in the range of 10 -9 M to 10 -10 According to certain embodiments, the antibody or antibody fragment binds to human PVRIG with an affinity in the range of 10 -10 M to 10 -11 Each possibility represents a separate embodiment of the present invention.

[0791] According to certain embodiments, the PVRIG antibody is a humanized antibody, which antibody or fragment thereof binds to human PVRIG with an ELISIA binding EC50 of no more than 400 ng / ml, or binds to human PVRIG with an ELISIA binding EC50 of no more than 300 ng / ml, an ELISIA binding EC50 of no more than 200 ng / ml, an ELISIA binding EC50 of no more than 150 ng / ml, an ELISIA binding EC50 of no more than 100 ng / ml, an ELISIA binding EC50 of no more than 50 ng / ml, an ELISIA binding EC50 of no more than 30 ng / ml, or even higher affinity.

[0792] According to certain embodiments, the PVRIG antibody is a humanized antibody, which antibody or fragment thereof blocks the binding of PVRIG to CD112 with an ELISA blocking IC50 of no more than 2000 ng / ml, an ELISA blocking IC50 of no more than 15000 ng / ml, an ELISA blocking IC50 of no more than 1000 ng / ml, or an ELISA blocking IC50 of no more than 800 ng / ml, or an ELISA blocking IC50 of no more than 500 ng / ml.

[0793] According to other embodiments, the PVRIG antibody is a humanized antibody, and the antibody or antibody fragment binds to CHO-K1-PVRIG cell surface PVRIG with a FACS binding EC50 of no more than 10000 ng / ml, a FACS binding EC50 of no more than 8000 ng / ml, a FACS binding EC50 of no more than 5000 ng / ml, a FACS binding EC50 of no more than 300 0 ng / ml, a FACS binding EC50 of no more than 2500 ng / ml, a FACS binding EC50 of no more than 2000 ng / ml, a FACS binding EC50 of no more than 1500 ng / ml, a FACS binding EC50 of no more than 1000 ng / ml, a FACS binding EC50 of no more than 500 ng / ml, or even higher affinity.

[0794] According to other embodiments, the PVRIG antibody is a humanized antibody, and the antibody or antibody fragment blocks CHO-K1-CD112 from binding to human PVRIG with a FACS blocking IC50 of no more than 1000 ng / ml, a FACS blocking IC50 of no more than 800 ng / ml, a FACS blocking IC50 of no more than 500 ng / ml, a FACS blocking IC50 of no more than 400 ng / ml, a FACS blocking IC50 of no more than 350 ng / ml, a FACS blocking IC50 of no more than 300 ng / ml, a FACS blocking IC50 of no more than 250 ng / ml, a FACS blocking IC50 of no more than 200 ng / ml, or even higher blocking activity.

[0795] According to certain embodiments, the PVRIG antibody is a humanized PVRIG antibody, wherein the antibody or fragment thereof has a molecular weight of at least 5×10 -9 According to other embodiments, the antibody or antibody fragment recognizes human PVRIG with an affinity of no greater than 3×10 -9 M, not higher than 10 -9 M, no higher than 3x10 -10 M, not higher than 10 -10 M or even higher affinity binds to human PVRIG.

[0796] According to certain embodiments, the antibody or antibody fragment is 10 -9 M to 10 -11 According to certain embodiments, the antibody or antibody fragment binds to human PVRIG with an affinity of KD value in the range of 10 -9 M to 10 -10 According to certain embodiments, the antibody or antibody fragment binds to human PVRIG with an affinity within the MKD value range of 10 -10 M to 10 -11 Affinity binding to human PVRIG with KD values ​​in the range of M. Each possibility represents a separate embodiment of the present invention.

[0797] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that can specifically recognize and bind to a PVRIG protein, comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region sequence of the antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 23, 25, 27, 29, 30, 31, 32, 33, 34, or 35. In some embodiments, the light chain variable region sequence of the antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 24, 26, 28, 36, 37, 38, 39, 40, 41, or 42.

[0798] In a preferred embodiment, in the detection of the binding kinetics of the anti-PVRIG chimeric antibody of the present invention and recombinant human PVRIG-His based on the biolayer interferometry (BLI) technology, BiacoreX100 is used as an SPR-dependent biosensor for detection. The KD of the PVRIG humanized antibody of the present invention binding to the hPVRIG-His protein is measured to be no higher than 10 -8 M, more preferably not higher than 10 -9 M, more preferably not higher than 10 -10 M.

[0799] In a preferred embodiment, in the detection of the binding kinetics of the anti-PVRIG anti-PVRIG humanized antibody of the present invention and recombinant human PVRIG-His based on the biolayer interferometry (BLI) technology, BiacoreX100 is used as an SPR-dependent biosensor for detection. It is measured that the KD of the PVRIG humanized antibody of the present invention binding to the hPVRIG-His protein is not higher than 10 -9 M, preferably not higher than 10 -10 M.

[0800] Expression vector, recombinant cells

[0801] The present invention also provides a vector comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell introduced with a recombinant vector (i.e., such that the host cell contains a polynucleotide and / or a vector containing the polynucleotide), and a recombinant antibody polypeptide or fragment thereof produced by recombinant technology. The present invention provides a vector comprising one or more polynucleotides described in the foregoing aspects.

[0802] In an embodiment, the vector comprises the two polynucleotides described above, and the vector encodes a VL region and a VH region, and the VL region and the VH region together bind to PVRIG.

[0803] As used herein, a "vector" is any construct capable of delivering one or more target polynucleotides to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more target polynucleotides as encoded polypeptides in a host cell into which the expression vector has been introduced. Therefore, in an expression vector, the target polynucleotide is positioned in the vector for expression by being operably connected to a regulatory element such as a promoter, an enhancer, and / or a polyadenylic acid tail, and the regulatory element is located at or near or on both sides of the integration site of the target polynucleotide in the vector or in the genome of the host cell, so that the target polynucleotide will be translated in the host cell into which the expression vector has been introduced.

[0804] Herein, "operably linked" means connecting the foreign gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the heavy and light chains of the antibody can be inserted into different vectors separately and independently.

[0805] The present invention provides host cells transformed with the vectors described above. The host cell can be a prokaryotic or eukaryotic cell. Preferably, the eukaryotic cell is selected from: protist cells, animal cells, plant cells and fungal cells. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS cells. The preferred host cell is CHO.

[0806] In some preferred embodiments, mammalian host cells are used to express and produce the antibodies of the present disclosure.

[0807] PVRIG antibody or antibody fragment. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes. Preferably, mammalian cell lines with exogenous expression vectors are used, including any normal lethal or normal or abnormal immortal animal or human cells, for example, SP2 / 0 myeloma cells, CHO cells, HeLa cells, PER.C6 cells, COS cells, HKB11 cells, NS0 cells. For example, a variety of suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas. Suitable promoters contained in the host cells can be constitutive, cell type specific, stage specific and / or regulatable or adjustable. Available promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP pollll promoters, constitutive MPSV promoters, constitutive CMV promoters and promoter-enhancer combinations known in the art.

[0808] The vector can be introduced into a host cell by methods known in the art, such as electroporation, chemical transfection (such as DEAE-dextran), transformation, transfection, infection and / or transduction (such as with a recombinant virus), to construct a recombinant cell, and then utilize these recombinant cells to express the antibody or antigen-binding fragment provided by the present invention. The corresponding antibody can be obtained by culturing the recombinant cell. Therefore, the non-limiting embodiments of the vector include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0809] Methods for preparing anti-PVRIG antibodies and antibody expression

[0810] In some embodiments, isolated fragments of human PVRIG can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be generated in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of antigenic peptides or proteins. In some embodiments, the antigenic peptides or proteins are injected with at least one adjuvant. In some embodiments, the antigenic peptides or proteins can be conjugated to an agent that is immunogenic in the species to be immunized. The antigenic peptides or proteins can be injected into the animal multiple times.

[0811] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, a recombinantly expressed polypeptide or a chemically synthesized polypeptide (e.g., a fragment of human PVRIG). The preparation may further comprise an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0812] In some embodiments, the preparation steps of the anti-PVRIG chimeric antibody of the present invention include: obtaining the H and K chain variable regions of the cell line obtained from the mouse monoclonal antibody by reverse transcription PCR through restriction endonucleases and ligation, such as sequencing; connecting the H and K chain variable region coding sequences to a eukaryotic expression vector by restriction endonucleases and ligation, respectively, and purifying the protein after protein expression using a mammalian cell expression system.

[0813] In some embodiments, the preparation steps of the anti-PVRIG humanized antibody of the present invention include: (1) humanizing the variable region sequence of the PVRIG mouse monoclonal antibody, and using bioinformatics analysis to determine the humanized framework region with the highest similarity to the heavy chain sequence of the mouse monoclonal antibody, and the humanized framework region with the highest similarity to the light chain sequence of the mouse monoclonal antibody; (3) transplanting the CDR sequences of the light and heavy chain variable regions of the mouse monoclonal antibody into the humanized framework region with the highest similarity; (4) using BLAST analysis to determine the humanized framework region with the highest similarity to the mouse monoclonal antibodies BT-5A8, BT-29D5, and BT-26C 4, the homologous structure with the highest structural similarity of the variable region sequence of antibody light and heavy chains was selected, and a three-dimensional structural model was established after comparing the antibody light and heavy chain variable region sequences (5). Subsequently, the key amino acids were found through the three-dimensional structure for back mutation, and finally the humanized heavy chain variable region sequence and the humanized light chain variable region sequence were obtained. (6) The H and K chain variable regions of the cell line obtained from the humanized anti-PVRIG antibody were obtained by reverse transcription PCR through restriction endonucleases and ligation, and sequencing; the H and K chain variable region coding sequences were connected to the eukaryotic expression vector by restriction endonucleases and ligation, and the protein was expressed and purified using a mammalian cell expression system.

[0814] In some embodiments, the method of the present invention for preparing an anti-human PVRIG antibody or an antigen-binding fragment thereof comprises:

[0815] 1) culturing the host cell described in the above aspects under appropriate conditions;

[0816] 2) Isolating and recovering the antibody or antigen-binding fragment thereof that specifically binds to PVRIG.

[0817] In one embodiment, the present invention provides a method for preparing an anti-CD112R antibody, wherein the method comprises, when an expression vector is introduced into a mammalian host cell, by culturing the host cell for a sufficient period of time to allow the antibody to be expressed in the host cell, or more preferably the antibody is secreted into a culture medium in which the host cell grows, to produce the antibody. Standard protein purification methods can be used to recover the antibody from the culture medium.

[0818] It is likely that antibodies expressed by different cell lines or expressed in transgenic animals have different glycosylation from one another. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodi...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human PVRIG, comprising a heavy chain variable region and a light chain variable region, It is characterized in that Comprising at least one of the following CDR sequences: HCDR sequence of the heavy chain variable region: the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 1; LCDR sequence of the light chain variable region: the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:

18.

2. The antibody or antigen-binding fragment according to claim 1, It is characterized in that The antibody or antigen-binding fragment comprises heavy chain complementary determining regions VHCDR1, VHCDR2 and VHCDR3, and light chain complementary determining regions VLCDR1, VLCDR2 and VLCDR3; wherein the heavy chain complementary determining regions VHCDR1, VHCDR2 and VHCDR3, and the light chain complementary determining regions VLCDR1, VLCDR2 and VLCDR3 are respectively selected from any one of the following groups: (1) the VHCDR1 is selected from the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 13, (2) the VHCDR2 is selected from the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 14, (3) the VHCDR3 is selected from the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 15; (4) the VLCDR1 is selected from the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10, SEQ ID NO: 16, (5) the VLCDR2 is selected from the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 17, (6) the VLCDR3 is selected from the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 18; (7) the VHCDR comprises VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the VLCDR comprises VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; (8) the VHCDR comprises VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; the VLCDR comprises VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively; (9) the VHCDRs include SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; the VLCDRs include SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; (10) An amino acid sequence combination having at least 80% identity with the amino acid sequence shown in any one of (1) to (9).

3. A PVRIG antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region and the light chain variable region are selected from any one of the following groups: (a) the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35, or comprises an analog having at least 85% sequence similarity to the heavy chain variable region sequence of the amino acid sequence of SEQ ID NOs: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35, or comprises an amino acid sequence having at least one amino acid substitution compared to the sequence of any one of SEQ ID NOs: 23, 25, 27, 29, 30, 31, 32, 33, 34 or 35; The light chain variable region comprises a light chain variable region sequence selected from the amino acid sequence of any one of SEQ ID NOs: 24, 26, 28, 35, 36, 37, 38, 39, 40, 41 or 42, or comprises an analog having at least 85% sequence identity with the light chain variable region sequence selected from the amino acid sequence of any one of SEQ ID NOs: 24, 26, 28, 35, 36, 37, 38, 39, 40, 41 or 42, or has an amino acid sequence having at least one amino acid substitution compared to the sequence of 24, 26, 28, 35, 36, 37, 38, 39, 40, 41 or 42; (b) the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35, and the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41 or 42; (c) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24, (d) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26; (e) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; (f) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 36, (g) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37, (h) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38; (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39; (j) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 40 or 41, (k) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42, (l) the heavy chain variable region or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:35 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

39. (m) a heavy chain variable region and a light chain variable region that have at least 85% homology to the heavy chain variable region amino acid sequence and the light chain variable amino acid sequence of any one of (a) to (l).

4. A PVRIG antibody comprising a heavy chain (HC) and a light chain (LC), Its characteristics are: The heavy chain comprises any one of the following: (a1) a heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, or an amino acid sequence having at least 85% sequence identity with an amino acid sequence as shown in any one of SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, or an amino acid sequence having at least one amino acid substitution compared to the sequence shown in SEQ ID NO:43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, (b1) the heavy chain has an amino acid sequence as shown in SEQ ID NO: 43, 44 or 45, or an amino acid sequence having at least one amino acid substitution compared to the sequence as shown in SEQ ID NO: 43, 44 or 45; (c1) a heavy chain comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in any one of SEQ ID NO: 46, 47, 48, 49, 50, 51 or 52; (d1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:46, (e1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 47, (f1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:48, (g) a heavy chain having an amino acid sequence shown in SEQ ID NO:49, (h1) a heavy chain with an amino acid sequence as shown in SEQ ID NO:51, (i1) a heavy chain with an amino acid sequence as shown in SEQ ID NO:52; Or, further optionally, the light chain comprises any one of the following groups: (a2) a light chain comprising an amino acid sequence as shown in any one of SEQ ID NO:53, 54, 55, 56, 57, 58, 59, 60, 61 or 62, or an amino acid sequence having at least 85% sequence identity with an amino acid sequence as shown in any one of SEQ ID NO:53, 54, 55, 56, 57, 58, 59, 60, 61 or 62, or an amino acid sequence having at least one amino acid substitution compared to the sequence shown in SEQ ID NO:53, 54, 55, 56, 57, 58, 59, 60, 61 or 62; or (b2) the light chain has an amino acid sequence as shown in SEQ ID NO: 53, 54 or 55, or an amino acid sequence having at least one amino acid substitution compared to the sequence as shown in SEQ ID NO: 53, 54 or 55; (c2) a light chain comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in any one of SEQ ID NO:, 56, 57, 58, 59, 60, 61 or 62; or (d2) a light chain comprising the amino acid sequence shown in SEQ ID NO:58, (e) or a light chain with an amino acid sequence shown in SEQ ID NO:59, (f) a light chain with an amino acid sequence shown in SEQ ID NO:60; (g2)) a light chain with an amino acid sequence as shown in SEQ ID NO:62, (h) a light chain with an amino acid sequence as shown in SEQ ID NO:59, (i2) A light chain having an amino acid sequence as shown in SEQ ID NO:

61.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, It is characterized in that The antibody contains at least one of a heavy chain constant region and a light chain constant region, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof; Optionally, the heavy chain constant regions are all from mouse IgG antibodies or mutants thereof, and the light chain constant regions are from mouse κ chains or λ chains, or mouse κ chains, Optionally, the heavy chain constant regions are all derived from human IgG antibodies or mutants thereof, and the light chain constant regions are derived from human κ chains or human λ chains; or Optionally, the heavy chain constant regions are all derived from human IgG4 antibodies or mutants thereof, and the light chain constant regions are derived from human κ chains.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, It is characterized in that, The antibody is an anti-human PVRIG antibody or an antigen-binding fragment thereof, which has a cross-reaction with monkey PVRIG but has no binding activity with mouse PVRIG.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, It is characterized in that The anti-human PVRIG antibody or antigen-binding fragment thereof is a monoclonal antibody.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv or sdAb, and at least one of the minimum recognition units, or the anti-human PVRIG antibody is selected from a single-chain antibody, a multimeric antibody, a CDR-grafted antibody or a small molecule antibody.

9. An isolated anti-human PVRIG antibody or antigen-binding fragment thereof having at least one of the following properties: (I) the epitope of the human PVRIG protein bound is identical to, completely overlaps with, or partially overlaps with the epitope of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; (II) competing with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 for binding to the epitope of human CD112R protein.

10. An isolated polynucleotide encoding: (1) The antibody or antigen-binding fragment thereof that specifically binds to PVRIG according to any one of claims 1 to 8, (2) the light chain variable region or the heavy chain variable region of (1), and (3) at least one sequence encoding the light chain or heavy chain of (1); in, Optional, The polynucleotide sequence encoding the antibody heavy chain variable region comprises: a sequence selected from any one of SEQ ID NOs: 128-137 or a variant thereof having at least 85% identity with any one of SEQ ID NOs: 128-137, or a polynucleotide sequence encoding the antibody light chain variable region, wherein the polynucleotide sequence comprises: a sequence selected from any one of SEQ ID NOs: 135-148 or a variant thereof having at least 85% identity with any one of SEQ ID NOs: 138-147.

11. A vector comprising the isolated polynucleotide of claim 10, optionally The vector comprises the two polynucleotides of claim 9, and the vector encodes a VL region and a VH region, and the VL region and the VH region together bind to PVRIG.

12. A vector pair, It is characterized in that Each vector pair comprises one of the polynucleotides of claim 8, wherein the vector pair together encodes a VL region and a VH region that together bind to PVRIG.

13. A host cell comprising the polynucleotide of claim 10, the vector of claim 11, or the vector pair of claim 12.

14. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the isolated antibody or antigen-binding fragment thereof according to claim 9 conjugated to a therapeutic or diagnostic agent.

15. A composition comprising: 1) the antibody or antigen-binding fragment thereof that specifically binds to PVRIG according to any one of claims 1 to 8, the isolated anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 9, the polynucleotide according to claim 10, the vector according to claim 11, the vector pair according to claim 12, or the host cell according to claim 13; or the immunoconjugate according to claim 14, and 2) Optional pharmaceutically acceptable excipients.

16. A method for preparing an anti-human PVRIG antibody or an antigen-binding fragment thereof, include: (1) Screening of PVRIG mouse monoclonal antibodies using hybridoma technology; (2) cDNA reverse transcription amplification of the H and K chain variable regions of the PVRIG mouse monoclonal antibody obtained in (1); (3) connecting the H and K chain variable regions obtained in (2) to a eukaryotic expression vector to express the chimeric antibody protein; (4) purifying to obtain anti-human PVRIG chimeric antibody; (5) humanizing the anti-human PVRIG chimeric antibody obtained in (4) to obtain a humanized anti-PVRIG antibody; wherein: The humanized design includes transplanting the CDR sequence of the anti-human PVRIG chimeric antibody into the human germline framework in the light chain sequence and heavy chain sequence with the highest degree of similarity, establishing a three-dimensional structural model after aligning the antibody light and heavy chain variable region sequences, finding key amino acids for back mutation, and obtaining a humanized heavy chain variable region sequence and a humanized light chain variable region sequence.

17. A drug combination, comprising an antibody specifically binding to PVRIG or an antigen-binding fragment thereof as described in any one of claims 1 to 8, an isolated anti-human PVRIG antibody or an antigen-binding fragment thereof as described in claim 9, a polynucleotide as described in claim 10, a vector as described in claim 11, a vector pair as described in claim 12, or a host cell as described in claim 13; or an immunoconjugate as described in claim 14, a composition as described in claim 15, and one or more additional therapeutic agents; preferably, the therapeutic agent is selected from one or more of an anti-PD-1 antibody, a PD-L1 antibody, and an anti-TIGIT antibody; more preferably, the therapeutic agent is an anti-TIGIT antibody, or a combination of an anti-TIGIT antibody and a PD-1 antibody.

18. The antibody or antigen-binding fragment thereof specifically binding to PVRIG according to any one of claims 1 to 8, the polynucleotide according to claim 11, the vector according to claim 12, the vector pair according to claim 13, or the host cell according to claim 14; or the immunoconjugate according to claim 15, and the use of the composition according to claim 17 in the preparation of diagnostic reagents for CD112R-mediated diseases or disorders or cancers and in the preparation of drugs for treating cancer.

19. The use according to claim 18, It is characterized in that The cancers include breast cancer, colorectal cancer, female reproductive system cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, liver cancer (HCC), endometrial cancer, pancreatic cancer, gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cell carcinoma (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 cell carcinoma, high MSI cancer, KRAS mutant tumors, adult T-cell leukemia / lymphoma, and myeloma.

20. A kit, It is characterized in that The invention comprises an antibody or antigen-binding fragment thereof that specifically binds to PVRIG as described in any one of claims 1 to 8, an isolated anti-human PVRIG antibody or antigen-binding fragment thereof as described in claim 9, or an isolated antibody or antigen-binding fragment thereof as described in claim 12, or a pharmaceutical composition as described in claim 17.

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