Anti-human pvrig antibody and use thereof

Abstract

The present invention relates to the field of biotechnology. Disclosed are an anti-human PVRIG antibody and a use thereof. The provided anti-human PVRIG antibody specifically binds to a human PVRIG antigen, and can be used for treating tumors.

Description

Anti-human PVRIG antibody and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese invention patent application number 202311579153.1, filed on November 23, 2023, entitled “Anti-human PVRIG antibodies and their applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of biotechnology, and in particular, to an anti-human PVRIG antibody and applications thereof. Background Art

[0004] PVRIG (poliovirus receptor-related immunoglobulin domain-containing, also known as CD112R) is a new inhibitory receptor whose ligand is PVRL2 ((poliovirus receptor-related 2, also known as CD112). On the one hand, PVRIG can compete with the activating receptor CD226 for binding to the ligand PVRL2 with higher affinity, thereby weakening T cell activation. On the other hand, after binding to the ligand, it will inhibit the activity of T cells through intracellular inhibitory motifs.

[0005] PVRIG is expressed on activated lymphocytes, particularly on exhausted CD8+ cells and NK cells. Its expression is tumor tissue-specific. Analysis of tumor and normal tissues from different cancer patients revealed that PVRIG expression was significantly higher on tumor-infiltrating lymphocytes than on lymphocytes in peripheral normal tissues from the same patients. Furthermore, PVRIG expression correlated with TIGIT (T-cell immunoreceptor with Ig and ITIM domains) and PD-1 (Programmed cell death protein 1). High PVRIG expression is associated with poor prognosis in cancer patients.

[0006] PVRL2 is primarily expressed on tumor cells, macrophages, and activated dendritic cells (DCs). Its expression is also tumor-specific. Analysis of tumor and normal tissue from different cancer patients revealed that PVRL2 expression was significantly higher on tumor-infiltrating lymphocytes than on peripheral normal tissue from the same patients. High PVRL2 expression is also associated with poor prognosis in cancer patients.

[0007] Application Contents

[0008] The present disclosure provides an anti-human PVRIG antibody.

[0009] In some embodiments, the present disclosure discloses an anti-human PVRIG antibody, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in the variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 100-104, and / or the light chain variable region of the antibody comprising HCDR1, HCDR2 and HCDR3 in the variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID No. No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 99 or 105 having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity in the variable region;

[0010] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0011] A. The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 1, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 2;

[0012] B. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 3, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 4;

[0013] C. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 5, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 6;

[0014] D. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 7, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 8;

[0015] E. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 9, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 10;

[0016] F. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 11, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 12;

[0017] G. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 13, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 14;

[0018] H. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 15, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 16;

[0019] I. The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 17, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 18;

[0020] J. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 19, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 20;

[0021] K. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 21, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 22; or

[0022] L. The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 23, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 24.

[0023] In some embodiments, the anti-human PVRIG antibody of any of the above items, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system;

[0024] In some embodiments, in the anti-human PVRIG antibody of any of the above items, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering system.

[0025] In some embodiments, the anti-human PVRIG antibody of any of the above items, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the IMGT numbering system.

[0026] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0027] a. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.25, HCDR2 comprises the amino acid sequence of SEQ ID NO.26, and HCDR3 comprises the amino acid sequence of SEQ ID NO.27; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.28, LCDR2 comprises the amino acid sequence of SEQ ID NO.29, and LCDR3 comprises the amino acid sequence of SEQ ID NO.30;

[0028] b. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.31, HCDR2 comprises the amino acid sequence of SEQ ID NO.32, and HCDR3 comprises the amino acid sequence of SEQ ID NO.33; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34, LCDR2 comprises the amino acid sequence of SEQ ID NO.35, and LCDR3 comprises the amino acid sequence of SEQ ID NO.36;

[0029] c. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.37, HCDR2 comprises the amino acid sequence of SEQ ID NO.38, and HCDR3 comprises the amino acid sequence of SEQ ID NO.39; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34, LCDR2 comprises the amino acid sequence of SEQ ID NO.35, and LCDR3 comprises the amino acid sequence of SEQ ID NO.36;

[0030] d. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.40, HCDR2 comprises the amino acid sequence of SEQ ID NO.41, and HCDR3 comprises the amino acid sequence of SEQ ID NO.42; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.43, LCDR2 comprises the amino acid sequence of SEQ ID NO.44, and LCDR3 comprises the amino acid sequence of SEQ ID NO.45;

[0031] e. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.46, HCDR2 comprises the amino acid sequence of SEQ ID NO.47, and HCDR3 comprises the amino acid sequence of SEQ ID NO.48; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.49, LCDR2 comprises the amino acid sequence of SEQ ID NO.50, and LCDR3 comprises the amino acid sequence of SEQ ID NO.51;

[0032] f. the heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO.46, HCDR2 comprises the amino acid sequence of SEQ ID NO.52, and HCDR3 comprises the amino acid sequence of SEQ ID NO.48; and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO.49, LCDR2 comprises the amino acid sequence of SEQ ID NO.50, and LCDR3 comprises the amino acid sequence of SEQ ID NO.51;

[0033] g. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.53, HCDR2 comprises the amino acid sequence of SEQ ID NO.54, and HCDR3 comprises the amino acid sequence of SEQ ID NO.55; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.49, LCDR2 comprises the amino acid sequence of SEQ ID NO.56, and LCDR3 comprises the amino acid sequence of SEQ ID NO.57;

[0034] h. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.58, HCDR2 comprises the amino acid sequence of SEQ ID NO.59, and HCDR3 comprises the amino acid sequence of SEQ ID NO.60; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.49, LCDR2 comprises the amino acid sequence of SEQ ID NO.61, and LCDR3 comprises the amino acid sequence of SEQ ID NO.62;

[0035] i. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.63, HCDR2 comprises the amino acid sequence of SEQ ID NO.64, and HCDR3 comprises the amino acid sequence of SEQ ID NO.65; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.66, LCDR2 comprises the amino acid sequence of SEQ ID NO.67, and LCDR3 comprises the amino acid sequence of SEQ ID NO.68;

[0036] j. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.69, the HCDR2 comprises the amino acid sequence of SEQ ID NO.70, and the HCDR3 comprises the amino acid sequence of SEQ ID NO.71; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.72, the LCDR2 comprises the amino acid sequence of SEQ ID NO.29, and the LCDR3 comprises the amino acid sequence of SEQ ID NO.73;

[0037] k. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.74, HCDR2 comprises the amino acid sequence of SEQ ID NO.75, and HCDR3 comprises the amino acid sequence of SEQ ID NO.76; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.28, LCDR2 comprises the amino acid sequence of SEQ ID NO.77, and LCDR3 comprises the amino acid sequence of SEQ ID NO.78; or

[0038] 1. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.79, HCDR2 comprises the amino acid sequence of SEQ ID NO.80, and HCDR3 comprises the amino acid sequence of SEQ ID NO.81; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.82, LCDR2 comprises the amino acid sequence of SEQ ID NO.83, and LCDR3 comprises the amino acid sequence of SEQ ID NO.68.

[0039] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0040] A. the anti-human PVRIG antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, respectively; or

[0041] B. The anti-human PVRIG antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO.33, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, respectively.

[0042] In some embodiments, the anti-human PVRIG antibody as described in any of the above items is a murine antibody, a chimeric antibody or a humanized antibody.

[0043] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0044] The heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 100-104), and / or the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 99 or 105).

[0045] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0046] The heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 100-104), and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 99 or 105).

[0047] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0048] (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 2;

[0049] (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 4;

[0050] (c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 6;

[0051] (d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 8;

[0052] (e) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 10;

[0053] (f) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.12;

[0054] (g) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 13, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 14;

[0055] (h) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 15, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 16;

[0056] (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 17, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 18;

[0057] (j) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 19, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 20;

[0058] (k) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 21, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 22;

[0059] (1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 23, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 24;

[0060] (m) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.104, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.99;

[0061] (n) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 100, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 99;

[0062] (o) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 100, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 105;

[0063] (p) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.101, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.99;

[0064] (q) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.101, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.105;

[0065] (r) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.102, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.99;

[0066] (s) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 102, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 105;

[0067] (t) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.103, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.99; or

[0068] (u) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 103, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 105.

[0069] In some embodiments, the anti-human PVRIG antibody as described in any one of the above items, wherein the anti-human PVRIG antibody comprises a heavy chain variable region and a light chain variable region, wherein,

[0070] (a) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 1, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 2;

[0071] (b) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 3, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 4;

[0072] (c) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 5, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 6;

[0073] (d) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 7, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 8;

[0074] (e) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 9, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 10;

[0075] (f) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 11, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 12;

[0076] (g) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 13, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 14;

[0077] (h) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 15, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 16;

[0078] (i) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 17, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 18;

[0079] (j) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 19, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 20;

[0080] (k) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 21, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 22;

[0081] (1) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 23, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 24;

[0082] (m) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 104, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 99;

[0083] (n) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 100, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 99;

[0084] (o) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 100, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 105;

[0085] (p) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 101, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 99;

[0086] (q) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 101, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 105;

[0087] (r) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 102, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 99;

[0088] (s) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 102, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 105;

[0089] (t) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 103, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 99; or

[0090] (u) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 103, and the light chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO. 105.

[0091] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, (a) the antibody includes the heavy chain variable region shown in SEQ ID NO.1, and the light chain variable region shown in SEQ ID NO.2; (b) the antibody includes the heavy chain variable region shown in SEQ ID NO.3, and the light chain variable region shown in SEQ ID NO.4; or (c) the antibody includes the heavy chain variable region shown in SEQ ID NO.101, and the light chain variable region shown in SEQ ID NO.99.

[0092] In some embodiments, the anti-human PVRIG antibody of any of the above items comprises a constant region.

[0093] In some embodiments, the anti-human PVRIG antibody according to any one of the above items, the heavy chain constant region of the antibody is selected from the heavy chain constant region of IgG1, IgG2, IgG3 and IgG4, and / or the light chain constant region is selected from the κ or λ chain constant region;

[0094] In some embodiments, in the anti-human PVRIG antibody as described in any of the above items, the species origin of the constant region is mouse or human;

[0095] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, the heavy chain constant region is a mouse IgG2a constant region, a mouse IgG1 constant region, a human IgG1 constant region or a human IgG4 constant region, and / or the light chain constant region is a mouse κ constant region or a human κ constant region.

[0096] In some embodiments, the anti-human PVRIG antibody as described in any of the above items is a full-length antibody or an antigen-binding fragment selected from any one of F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv and scFv.

[0097] In some embodiments, disclosed are antibodies that compete for binding to human PVRIG with any of the aforementioned anti-human PVRIG antibodies, or antibodies that bind to the same epitope as any of the aforementioned anti-human PVRIG antibodies.

[0098] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein the anti-human PVRIG antibody has at least one of the following characteristics:

[0099] A. The anti-human PVRIG antibody can specifically bind to human PVRIG; in some embodiments, the anti-human PVRIG antibody can bind to human PVRIG protein with an EC50 value of ≤10 nM (e.g., ≤10 nM, ≤5 nM, ≤4.50 nM, ≤3.00 nM, ≤2.00 nM, ≤1.00 nM, ≤0.9 nM, ≤0.8 nM, ≤0.7 nM, ≤0.6 nM, ≤0.5 nM, ≤0.4 nM, ≤0.3 nM, ≤0.2 nM, ≤0.1 nM, ≤0.05 nM, ≤0.01 nM, ≤0.005 nM, ≤0.001 nM or less), wherein the EC50 value is determined by flow cytometry; in some embodiments, the EC50 value is determined by the method of Example 2 of the present application;

[0100] B. The anti-human PVRIG antibody can specifically bind to monkey PVRIG; in some embodiments, the anti-human PVRIG antibody can bind to monkey PVRIG protein with an EC50 value of ≤10 nM (e.g., ≤10 nM, ≤5 nM, ≤4.50 nM, ≤3.00 nM, ≤2.00 nM, ≤1.00 nM, ≤0.9 nM, ≤0.8 nM, ≤0.7 nM, ≤0.6 nM, ≤0.5 nM, ≤0.4 nM, ≤0.3 nM, ≤0.2 nM, ≤0.1 nM, ≤0.05 nM, ≤0.01 nM, ≤0.005 nM, ≤0.001 nM or less), wherein the EC50 value is determined by flow cytometry; in some embodiments, the EC50 value is determined by the method of Example 5 of the present application;

[0101] C. The anti-human PVRIG antibody can specifically bind to mouse PVRIG; in some embodiments, the anti-human PVRIG antibody can bind to mouse PVRIG protein with an EC50 value of ≤10 nM (e.g., ≤10 nM, ≤5 nM, ≤4.50 nM, ≤3.00 nM, ≤2.00 nM, ≤1.00 nM, ≤0.9 nM, ≤0.8 nM, ≤0.7 nM, ≤0.6 nM, ≤0.5 nM, ≤0.4 nM, ≤0.3 nM, ≤0.2 nM, ≤0.1 nM, ≤0.05 nM, ≤0.01 nM, ≤0.005 nM, ≤0.001 nM or less), wherein the EC50 value is determined by flow cytometry; in some embodiments, the EC50 value is determined by the method of Example 5 of the present application; and / or

[0102] D. The anti-human PVRIG antibody can inhibit the binding activity of PVRIG and PVRL2.

[0103] The present disclosure also provides a multispecific antibody comprising any of the anti-human PVRIG antibodies described above. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a trispecific antibody.

[0104] The present disclosure also provides an antibody conjugate, comprising the anti-human PVRIG antibody described in any of the preceding items. In some embodiments, the antibody conjugate further comprises a therapeutic agent or imaging agent conjugated to the antibody; in some embodiments, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; in some embodiments, the antibody conjugate further comprises a solid phase support conjugated to the antibody; in some embodiments, the antibody conjugate further comprises a label conjugated to the antibody; in some embodiments, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent agent, and a nanoparticle label; in some embodiments, the label is colloidal gold.

[0105] The present disclosure also provides an isolated nucleic acid encoding the anti-human PVRIG antibody described in any one of the above items.

[0106] The present disclosure also provides a cell containing any of the above nucleic acids.

[0107] The present disclosure also provides a pharmaceutical composition comprising: the anti-human PVRIG antibody, multispecific antibody, antibody conjugate, cell or nucleic acid described in any of the foregoing items. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

[0108] The present disclosure also provides the use of any of the aforementioned anti-human PVRIG antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids, or pharmaceutical compositions for the preparation of a product having at least one of the following uses: detecting human PVRIG expression or treating a disease associated with elevated human PVRIG expression. In some embodiments, the disease associated with elevated human PVRIG expression is a tumor. In some embodiments, the tumor is selected from the group consisting of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and hematologic cancer.

[0109] In some embodiments, the present disclosure provides a method for treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of any of the aforementioned anti-human PVRIG antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids, or pharmaceutical compositions. In some embodiments, the disease is associated with high human PVRIG expression. In some embodiments, the disease is a tumor. In some embodiments, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and hematologic cancer.

[0110] In another aspect, the present disclosure further provides any of the aforementioned anti-human PVRIG antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids, or pharmaceutical compositions for use as a medicament. In some embodiments, the medicament is used to treat a disease. In some embodiments, the medicament is used to detect human PVRIG expression or treat a disease associated with high human PVRIG expression. In some embodiments, the disease associated with high human PVRIG expression is a tumor. In some embodiments, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and hematologic cancer.

[0111] In some embodiments, the treatment described in any of the above further comprises administering to the subject an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent.

[0112] The present disclosure also provides a method for detecting or measuring human PVRIG, comprising the step of detecting or measuring human PVRIG using the anti-human PVRIG antibody or antibody conjugate as described in any one of the preceding items.

[0113] The present disclosure also provides a kit comprising the anti-human PVRIG antibody or antibody conjugate as described in any of the preceding items; in some embodiments, the kit is used for detecting or measuring human PVRIG.

[0114] The present disclosure also provides a method for preparing the anti-human PVRIG antibody as described in any of the preceding items, comprising the steps of culturing the cell as described in any of the preceding items, and then isolating and purifying the anti-human PVRIG antibody.

[0115] The anti-human PVRIG antibodies disclosed in the present disclosure can specifically bind to human PVRIG; in some embodiments, the anti-human PVRIG antibodies can specifically bind to mouse PVRIG and / or monkey PVRIG; the anti-human PVRIG antibodies can inhibit the binding activity of PVRIG to PVRL2; and / or the anti-human PVRIG antibodies can inhibit tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0117] Figure 1 shows the results of ELISA testing of the binding of the disclosed anti-human PVRIG antibodies (CG004-01, CG004-02, CG004-04, CG004-05, CG004-06, and CG004-07) to hPVRIG;

[0118] FIG2 is a graph showing the ELISA test results of the anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, and CG004-14) disclosed herein binding to hPVRIG;

[0119] FIG3 is a graph showing the FACS detection results of the binding of the disclosed anti-human PVRIG antibodies (CG004-01, CG004-02, CG004-04, CG004-05, CG004-07, R2247) to hPVRIG;

[0120] FIG4 is a graph showing the FACS detection results of the binding of the disclosed anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, R2247) to hPVRIG;

[0121] FIG5 shows the experimental results of the anti-human PVRIG antibodies (CG004-01, CG004-02, CG004-04, CG004-05, CG004-06, CG004-07, and R2247) disclosed herein blocking the binding of human PVRIG to its ligand;

[0122] FIG6 shows the experimental results of the anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, and R2247) disclosed herein blocking the binding of human PVRIG to its ligand;

[0123] FIG7 shows the binding experimental results of the anti-human PVRIG antibodies (CG004-01, CG004-04, CG004-05, CG004-06, CG004-07, R2247) disclosed herein and cynoPVRIG;

[0124] FIG8 shows the binding experimental results of the anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, R2247) disclosed herein and cynoPVRIG;

[0125] FIG9 shows the binding experimental results of the disclosed anti-human PVRIG antibodies (CG004-01, CG004-04, CG004-05, CG004-06, CG004-07, R2247) with mouse PVRIG;

[0126] FIG10 shows the binding experimental results of the anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, R2247) disclosed herein and mouse PVRIG;

[0127] FIG11 is a graph showing the results of a reporter gene experiment in which the anti-human PVRIG antibody disclosed herein inhibits the interaction between PVRIG and PVRL2;

[0128] FIG12 is a graph showing the results of the T cell activation activity of the anti-human PVRIG antibody disclosed herein;

[0129] FIG13 shows the binding assay results of the disclosed anti-human PVRIG humanized antibodies (R3942-R3950) to 293T-hPVRIG cells;

[0130] FIG14 is a graph showing the binding experimental results of the disclosed anti-human PVRIG humanized antibodies (R3942-R3950) to 293T-cyno PVRIG cells. DETAILED DESCRIPTION

[0131] In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure are clearly and completely described below, but the description and embodiments should not be construed as limiting the scope of the present disclosure. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially. Unless clearly defined in the present disclosure, the technical terms and scientific terms used in the present disclosure have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0132] In the present disclosure, "a / an", "a", "said", "above", and "aforementioned" include plural references unless the context clearly indicates otherwise. For example, "an antibody" means one antibody or more than one antibody. In addition, 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 technical features indicated. The term "plurality" means at least two, such as 2, 3, etc., unless otherwise clearly and specifically indicated otherwise.

[0133] Unless the context clearly indicates otherwise, in the specification and claims of the present disclosure, the words "comprise", "have", etc. should be understood as meaning "include", rather than in an exclusive or exhaustive sense; that is, in the sense of "including but not limited to". For example, "comprise A" means "including A, but not limited to A".

[0134] "About" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless expressly stated otherwise in the examples or elsewhere in the specification, "about" means within one standard deviation, or up to 5%, as is customary in the art.

[0135] "Optional," "optionally," "optional," or "optionally" means that the subsequently described event or circumstance may but need not occur, that is, includes instances where the event or circumstance occurs or does not occur, unless the context clearly indicates otherwise.

[0136] The term "and / or" refers to each of the two specified features or a combination thereof, including or excluding the other specified feature. For example, "A and / or B" is intended to include any of the following situations: A and B, A or B, A (alone) and B (alone); "A, B and / or C" is intended to include any of the following situations: A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0137] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0138] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and modified amino acids thereof, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Common natural amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, carboxyl, amino, and R groups), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics are chemical compounds that have a structure that differs from the general chemical structure of an amino acid but function in a manner similar to that of a naturally occurring amino acid.

[0139] The term "antibody" in this disclosure is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies), murine antibodies, chimeric antibodies, humanized antibodies, human antibodies, full-length antibodies, antigen-binding fragments (also referred to as antigen-binding portions, or antibody fragments), etc., as long as they exhibit the desired antigen-binding activity.

[0140] "Native antibodies" are naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two light chains and two heavy chains bound by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain of an IgG antibody has a heavy chain variable region (VH, also called a variable heavy domain or heavy chain variable domain), followed by a heavy chain constant region (CH, also called a constant heavy domain), which includes three constant domains (CH1, CH2, and CH3); similarly, from the N-terminus to the C-terminus, each light chain has a light chain variable region (VL, also called a variable light domain or light chain variable domain), followed by a light chain constant region (CL, also called a constant light domain). Depending on whether the antibody contains α, δ, ε, γ and μ heavy chains, antibodies can be divided into five isotypes: IgA, IgD, IgE, IgG and IgM. Isotype antibodies can be further divided into different subclasses. For example, IgG isotypes include four subtypes: IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain) and IgG4 (γ4 heavy chain). IgA isotypes are divided into two subtypes: IgA1 (α1 heavy chain) and IgA2 (α2 heavy chain).

[0141] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure, or an antibody having heavy chains including an Fc region.

[0142] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody can be purified to greater than 90% or 99% purity, which can be performed and assayed by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0143] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding to an antigen. The heavy chain variable region (VH) and light chain variable region (VL) of a natural antibody each contain four conserved framework regions (FRs) and three hypervariable regions (also called complementarity determining regions, HVRs or CDRs).

[0144] The terms "complementarity determining region," "hypervariable region," or "CDR" refer to the major regions within the variable domain that contribute to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH comprises three CDR regions: HCDR1, HCDR2, and HCDR3; VL comprises three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is typically composed of three CDRs and four FRs arranged in the following order (from amino-terminus to carboxyl-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid sequence boundaries of CDRs can be determined by methods well known to those skilled in the art, for example: "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art.

[0145] The term "light chain" includes the light chain variable region (VL) and the light chain constant region (CL). The VL is located at the amino terminus of the light chain, and the CL is located at the carboxyl terminus of the light chain. The light chain can be a kappa chain or a lambda chain, etc.

[0146] The term "heavy chain" includes the heavy chain variable region (VH) and the heavy chain constant region (CH). The heavy chain variable region is located at the amino terminus of the heavy chain, and the heavy chain constant region is located at the carboxyl amino terminus of the heavy chain. The IgG antibody heavy chain constant region includes three constant region domains: CH1, CH2, and CH3. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgD, IgM, and IgE.

[0147] The term "constant region" or "constant domain" refers to the carboxyl terminal portion of the light chain and the heavy chain, which is not directly involved in the binding of the antibody to the antigen. Compared with VH and VL, the constant region has a relatively conserved amino acid sequence.

[0148] The term "antigen-binding fragment" refers to a portion that contains an intact antibody, but unlike an intact antibody, this portion can specifically bind to the antigen bound by the intact antibody. Some examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dsFv, (dsFv)2, single-chain Fab (scFab), single-chain antibodies (such as scFv), diabodies, linear antibodies, and multispecific antibodies formed by antigen-binding fragments. Among them, "Fab" is a monovalent fragment composed of VL, VH, CL and CH1 domains; "Fv" consists of VH and VL; "Fab'" is a Fab fragment containing part of the hinge region; "F(ab')2" contains two F domains connected by a disulfide bond at the hinge region. ab' fragment; "scFab" is a polypeptide consisting of VH, CH1, VL, CL and a linker, wherein the antibody domains and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; "scFv" is a fusion protein comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are linked by a peptide linker. The scFv is head-connected and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv herein can have the VL and VH variable regions in any order, for example, from the N-terminus to the C-terminus of the scFv polypeptide, it can contain: a) VL-linker-VH or b) VH-linker-VL; "dsFv" is a disulfide-stabilized Fv fragment; (dsFv)2 is a dimerized dsFv; "Fab'-SH" is a cysteine ​​residue in the hinge region of the Fab' fragment carrying a free thiol group.

[0149] The term "Fc region" refers to the C-terminal region of an antibody heavy chain, including native antibody Fc regions and Fc region variants. In some embodiments, the Fc region of a human IgG heavy chain extends from the amino acid residue at position Cys226 to its carboxyl terminus. In some embodiments, the Fc region of a human IgG heavy chain extends from Pro230 to its carboxyl terminus. The C-terminus of the Fc region may also be, for example, a C-terminal lysine (residue 447 according to the EU numbering system) or a C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system) missing from the Fc region. In some embodiments, the composition of a complete antibody may include an antibody population that has lost the K447 residue and / or the G446+K447 residues. In some embodiments, the composition of a complete antibody may include an antibody population that does not lose the K447 residue and / or the G446+K447 residues. In some embodiments, the composition of a complete antibody has an antibody population that is a mixture of antibodies with and without the K447 residue and / or the G446+K447 residues. The Fc regions used in the antibodies described herein include those of human IgG1, IgG2 (IgG2a, IgG2b), IgG3, and IgG4. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0150] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular species, and the remainder of the heavy and / or light chain is derived from another species.

[0151] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be achieved by retaining the CDR regions of a non-human antibody and replacing the rest of the antibody with its human counterpart (i.e., the framework regions of the heavy chain variable region, the framework regions of the light chain variable region, and the human antibody constant region).

[0152] The terms "human antibody," "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably to refer to antibodies in which the variable and constant region sequences are human. Human antibodies include antibodies derived from human genes but with alterations such as changes in cysteine ​​residues or glycosylation sites. Human antibodies can also be recombinantly produced in non-human cells. Human antibodies include antibodies obtained from transgenic mice expressing human immunoglobulin heavy and light chain loci.

[0153] The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as disclosed herein, "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is typically expressed as a dissociation constant (KD). Affinity can be measured by conventional methods known in the art. The terms "kassoc" or "ka" refer to the on-rate of a specific antibody-antigen interaction, and the terms "kdis" or "kd" refer to the off-rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka), typically expressed as molar concentration (M). The KD value of an antibody can be measured using methods well known in the art. Methods for measuring antibody KD include using a biosensor system such as a system measuring surface plasmon resonance, or measuring affinity in a solution by solution equilibrium titration (SET).

[0154] The term "affinity matured" refers to an antibody that has one or more amino acid residue changes in one or more CDRs of the antibody that result in an improved affinity of the antibody for the antigen compared to the parent antibody. In some embodiments, affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies can be generated by methods known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in: Barbas et al., PNAS, 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995) and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0155] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence mutated Fc region). Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0156] The term "monoclonal antibody" refers to a substantially homogeneous antibody population, i.e., the amino acid sequences of the antibody molecules contained in the population are identical (except for possible naturally occurring mutations that may be present in small amounts). In contrast, polyclonal antibodies typically comprise different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes and / or different antigens. "Monoclonal" refers to an antibody obtained from a substantially homogeneous antibody population. Monoclonal antibodies can be produced by methods well known in the art, for example, by the hybridoma method (Kohler et al., (1975) Nature 256:495), by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), by isolation from phage antibody libraries (Clackson et al., (1991) Nature 352:624-628 and Marks et al., (1991) J. Mol. Biol. 222:581-597), and by using transgenic animals containing all or part of the human immunoglobulin loci to produce monoclonal antibodies (see Presta (2005) J. Allergy Clin. Immunol. 116:731).

[0157] The term "antigen" refers to a protein that can selectively bind to an antigen binding protein (eg, an antibody). An antigen may have one or more epitopes that interact with different antigen binding proteins (eg, antibodies).

[0158] The term "epitope" refers to an area (area or region) on an antigen that is capable of specific binding to an antibody. An epitope is generally composed of an antigenic determinant of a special chemical group having a certain structure. An epitope can be formed by continuous amino acid residues (linear epitope) or by non-continuous amino acid residues (conformational epitope), for example, non-continuous amino acid residues that are spatially close due to the folding of the antigen. In some embodiments, the epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acid residues in a unique spatial conformation. The epitope can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structure determination methods (e.g., nuclear magnetic resonance spectroscopy). Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be achieved using methods well known in the art, including, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0159] An "antibody that binds to the same epitope as a reference antibody" or an "antibody that competes for binding with a reference antibody" refers to an antibody that blocks the binding of the reference antibody to the antigen by 50% or more in a competition assay, or an antibody whose binding to the antigen is blocked by the reference antibody by 50% or more. To determine whether a test antibody binds to the same epitope as a reference antibody, it can be tested under saturating conditions to determine whether the reference antibody is allowed to bind to the antigen. For example, after removing excess reference antibody, the ability of the test antibody to bind to the antigen is assessed. If the test antibody is able to bind to the antigen after saturating binding of the reference antibody, then the test antibody binds to a different epitope than the reference antibody. However, if the test antibody is unable to bind to the antigen after saturating binding of the reference antibody, then the test antibody binds to the same epitope as the reference antibody. To confirm whether the test antibody binds to the same epitope or is merely sterically hindered from binding, routine experiments (e.g., peptide mutations and detection using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative method known in the art) can be used. If, in both settings (i.e., both antibodies are used as saturated antibodies), only the saturated antibody is able to bind to the antigen, then it can be concluded that the test antibody and the reference antibody compete for binding to the antigen. In some embodiments, in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502), if an excess amount of one antibody (e.g., a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold amount) inhibits the binding of the other antibody to the antigen by at least 50%, at least 75%, at least 90%, or even 99% or more, then the two antibodies are considered to bind to the same or overlapping epitope.

[0160] The term "specific binding" or "specifically binds" refers to the non-random binding between two molecules, for example, an antibody binds to its antigen or epitope with a higher affinity than to other antigens or epitopes. -7 M or less (e.g., about 1×10 -8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or about 1×10 -12 The antibody binds to the antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of 1% or less. In some embodiments, the KD of the antibody binding to the antigen is 10%, 1% or less of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD values ​​can be measured using methods known in the art, such as by Surface plasmon resonance assay measurement. Antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species, such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzee (Pan troglodytes (chimpanzee, chimp), or marmoset (Callithrix jacchus (common marmoset, marmoset).

[0161] The terms "anti-human PVRIG antibody" and "antibody that binds to PVRIG" refer to an antibody that is capable of binding to PVRIG with sufficient affinity. In one embodiment, the extent of binding of the anti-human PVRIG antibody to non-PVRIG proteins is less than about 10%, 1%, or less of the binding to PVRIG, and the binding can be In some embodiments, the antibody binds to PVRIG with a dissociation constant (KD) of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM or less. In some embodiments, the anti-human PVRIG antibody binds to a conserved PVRIG epitope in PVRIG from different species.

[0162] In some embodiments, the anti-human PVRIG antibody according to any of the above items comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, and / or the light chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in the variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID No. No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 has LCDR1, LCDR2 and LCDR3 in the variable region with at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the anti-human PVRIG antibody of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the variable region having at least 95% sequence identity to SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, and / or the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in the variable region having at least 95% sequence identity to SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24. In some embodiments, the anti-human PVRIG antibody of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the variable region having at least 98% sequence identity to SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, and / or the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in the variable region having at least 98% sequence identity to SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24. In some embodiments, the anti-human PVRIG antibody of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the variable region having at least 100% sequence identity to SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, and / or the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in the variable region having at least 100% sequence identity to SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24.

[0163] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system; in some embodiments, the anti-human PVRIG antibody as described in any of the above items, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

[0164] In some embodiments, the anti-human PVRIG antibody of any one of the above items, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system, wherein

[0165] a. The HCDR1 of the heavy chain variable region is an amino acid sequence of SEQ ID NO.25, the HCDR2 is an amino acid sequence of SEQ ID NO.26, and the HCDR3 is an amino acid sequence of SEQ ID NO.27; and the LCDR1 of the light chain variable region is an amino acid sequence of SEQ ID NO.28, the LCDR2 is an amino acid sequence of SEQ ID NO.29, and the LCDR3 is an amino acid sequence of SEQ ID NO.30;

[0166] b. The HCDR1 of the heavy chain variable region is an amino acid sequence of SEQ ID NO.31, the HCDR2 is an amino acid sequence of SEQ ID NO.32, and the HCDR3 is an amino acid sequence of SEQ ID NO.33; and the LCDR1 of the light chain variable region is an amino acid sequence of SEQ ID NO.34, the LCDR2 is an amino acid sequence of SEQ ID NO.35, and the LCDR3 is an amino acid sequence of SEQ ID NO.36;

[0167] c. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.37, the amino acid sequence of HCDR2 is SEQ ID NO.38, and the amino acid sequence of HCDR3 is SEQ ID NO.39; and the amino acid sequence of LCDR1 of the light chain variable region is SEQ ID NO.34, the amino acid sequence of LCDR2 is SEQ ID NO.35, and the amino acid sequence of LCDR3 is SEQ ID NO.36;

[0168] d. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.40, the amino acid sequence of HCDR2 is SEQ ID NO.41, and the amino acid sequence of HCDR3 is SEQ ID NO.42; and the amino acid sequence of LCDR1 of the light chain variable region is SEQ ID NO.43, the amino acid sequence of LCDR2 is SEQ ID NO.44, and the amino acid sequence of LCDR3 is SEQ ID NO.45;

[0169] e. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.46, the amino acid sequence of HCDR2 is SEQ ID NO.47, and the amino acid sequence of HCDR3 is SEQ ID NO.48; and the amino acid sequence of LCDR1 of the light chain variable region is SEQ ID NO.49, the amino acid sequence of LCDR2 is SEQ ID NO.50, and the amino acid sequence of LCDR3 is SEQ ID NO.51;

[0170] f. The HCDR1 of the heavy chain variable region is an amino acid sequence of SEQ ID NO.46, the HCDR2 is an amino acid sequence of SEQ ID NO.52, and the HCDR3 is an amino acid sequence of SEQ ID NO.48; and the LCDR1 of the light chain variable region is an amino acid sequence of SEQ ID NO.49, the LCDR2 is an amino acid sequence of SEQ ID NO.50, and the LCDR3 is an amino acid sequence of SEQ ID NO.51;

[0171] g. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.53, the amino acid sequence of HCDR2 is SEQ ID NO.54, and the amino acid sequence of HCDR3 is SEQ ID NO.55; and the amino acid sequence of LCDR1 of the light chain variable region is SEQ ID NO.49, the amino acid sequence of LCDR2 is SEQ ID NO.56, and the amino acid sequence of LCDR3 is SEQ ID NO.57;

[0172] h. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.58, the amino acid sequence of HCDR2 is SEQ ID NO.59, and the amino acid sequence of HCDR3 is SEQ ID NO.60; and the amino acid sequence of LCDR1 of the light chain variable region is SEQ ID NO.49, the amino acid sequence of LCDR2 is SEQ ID NO.61, and the amino acid sequence of LCDR3 is SEQ ID NO.62;

[0173] i. The HCDR1 of the heavy chain variable region is an amino acid sequence of SEQ ID NO.63, the HCDR2 is an amino acid sequence of SEQ ID NO.64, and the HCDR3 is an amino acid sequence of SEQ ID NO.65; and the LCDR1 of the light chain variable region is an amino acid sequence of SEQ ID NO.66, the LCDR2 is an amino acid sequence of SEQ ID NO.67, and the LCDR3 is an amino acid sequence of SEQ ID NO.68;

[0174] j. The HCDR1 of the heavy chain variable region is an amino acid sequence of SEQ ID NO.69, the HCDR2 is an amino acid sequence of SEQ ID NO.70, and the HCDR3 is an amino acid sequence of SEQ ID NO.71; and the LCDR1 of the light chain variable region is an amino acid sequence of SEQ ID NO.72, the LCDR2 is an amino acid sequence of SEQ ID NO.29, and the LCDR3 is an amino acid sequence of SEQ ID NO.73;

[0175] k. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.74, the amino acid sequence of HCDR2 is SEQ ID NO.75, and the amino acid sequence of HCDR3 is SEQ ID NO.76; and the amino acid sequence of LCDR1 of the light chain variable region is SEQ ID NO.28, the amino acid sequence of LCDR2 is SEQ ID NO.77, and the amino acid sequence of LCDR3 is SEQ ID NO.78; or

[0176] 1. The HCDR1 of the heavy chain variable region is an amino acid sequence of SEQ ID NO.79, the HCDR2 is an amino acid sequence of SEQ ID NO.80, and the HCDR3 is an amino acid sequence of SEQ ID NO.81; and the LCDR1 of the light chain variable region is an amino acid sequence of SEQ ID NO.82, the LCDR2 is an amino acid sequence of SEQ ID NO.83, and the LCDR3 is an amino acid sequence of SEQ ID NO.68.

[0177] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the IMGT numbering system; in some embodiments, the HCDR1 of the heavy chain variable region comprises the amino acid sequence of GNTFSDSY (SEQ ID NO.84), HCDR2 comprises the amino acid sequence of INPYNGDT (SEQ ID NO.85), and HCDR3 comprises the amino acid sequence of ARSNWDVGNCFDY (SEQ ID NO.86); and the LCDR1 of the light chain variable region comprises the amino acid sequence of QDISNY (SEQ ID NO.87), LCDR2 comprises the amino acid sequence of YTS (SEQ ID NO.88), and LCDR3 comprises the amino acid sequence of SEQ ID NO.30.

[0178] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Chothia numbering system; in some embodiments, the HCDR1 of the heavy chain variable region comprises the amino acid sequence of GNTFSDS (SEQ ID NO.89), HCDR2 comprises the amino acid sequence of NPYN (SEQ ID NO.90), and HCDR3 comprises the amino acid sequence of SEQ ID NO.27; and the LCDR1 of the light chain variable region is such as the amino acid sequence of SEQ ID NO.28, LCDR2 is such as the amino acid sequence of SEQ ID NO.29, and LCDR3 is such as the amino acid sequence of SEQ ID NO.30.

[0179] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Contact numbering system; in some embodiments, the HCDR1 of the heavy chain variable region includes the amino acid sequence of HQGYSLPRT (SEQ ID NO.91), HCDR2 includes the amino acid sequence of WIGIINPYNGDTY (SEQ ID NO.92), and HCDR3 includes the amino acid sequence of ARSNWDVGNCFD (SEQ ID NO.93); and the LCDR1 of the light chain variable region includes the amino acid sequence of SNYLNWY (SEQ ID NO.94), LCDR2 includes the amino acid sequence of LLIYYTSRLH (SEQ ID NO.95), and LCDR3 includes the amino acid sequence of HQGYSLPR (SEQ ID NO.96).

[0180] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the AbM numbering system; in some embodiments, the HCDR1 of the heavy chain variable region comprises the amino acid sequence of GNTFSDSYMN (SEQ ID NO.97), HCDR2 comprises the amino acid sequence of IINPYNGDTY (SEQ ID NO.98), and HCDR3 comprises the amino acid sequence of SEQ ID NO.27; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.28, LCDR2 comprises the amino acid sequence of SEQ ID NO.29, and LCDR3 comprises the amino acid sequence of SEQ ID NO.30.

[0181] The binding of the anti-human PVRIG antibodies provided herein to human PVRIG can also be expressed as a "half-maximal effective concentration" (EC50) value. Generally, a lower EC50 indicates better affinity, indicating that the antibody can bind to the antigen at a lower concentration. EC50 values ​​can be determined using binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometric fluorescence sorting, and other binding assays).

[0182] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this disclosure to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages. Nucleic acids can be synthetic, naturally occurring, and non-naturally occurring, such as non-natural nucleic acids that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to a reference nucleic acid. Including, but not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA) modified nucleic acids. "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acid includes nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. "Nucleic acid encoding an anti-human PVRIG antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of the antibody (or fragments thereof), including one or more nucleic acid molecules in a single vector or separate vectors, and one or more nucleic acid molecules present at one or more locations in a host cell. In some embodiments, the nucleic acid sequence also includes conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. For example, degenerate codon substitutions can be obtained by generating a sequence 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., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0183] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0184] The terms "identity" and "sequence identity" refer to the extent (percentage) to which the amino acids (amino acid sequence identity) or nucleic acids (nucleic acid sequence identity) of the two sequences are identical at equivalent positions when the two sequences are optimally aligned. To achieve optimal alignment of the two sequences, gaps may be introduced, if necessary, to obtain the maximum percentage sequence identity. The percentage amino acid sequence identity can be determined using various methods well known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA software well known in the art. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.

[0185] The terms "antibody conjugate" and "antibody conjugate" refer to an antibody conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents). In some embodiments of the present disclosure, the "antibody conjugate" includes a conjugate formed by conjugating an anti-human PVRIG antibody to a therapeutic agent or an imaging agent. In an optional embodiment, the antibody conjugate is formed by conjugating an anti-human PVRIG antibody to a therapeutic agent via a linker; in an optional embodiment, the therapeutic agent is a cytotoxic agent; in an optional embodiment, the therapeutic agent can be selected from toxins, chemotherapeutic agents, antibiotics, radioactive isotopes and nucleolytic enzymes. In the present disclosure, "cytotoxic agent" refers to a substance that inhibits or prevents the function of a cell and / or causes cell death or destruction; "toxin" refers to a substance that can have a deleterious effect on the growth or proliferation of cells (such as maytansines, calicheamicin, taxanes, vincristine, colchicine, auristatin, etc.); "chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer. In an optional embodiment, the antibody conjugate also includes biotin or a biotin derivative conjugated to the antibody. In an optional embodiment, the antibody conjugate further comprises a marker coupled to the antibody. In an optional embodiment, the above-mentioned marker refers to a class of substances having characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, through which qualitative or quantitative detection of the corresponding target can be achieved. In an optional embodiment, the marker is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle marker. In actual use, those skilled in the art can select other suitable markers according to the detection conditions or actual needs. No matter what marker is used, it belongs to the protection scope of the present disclosure. In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).In an alternative embodiment, the enzyme includes but is not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphate glucose dehydrogenase. In an alternative embodiment, the radioactive isotope includes but is not limited to. 212 Bi, 131 I. 111 In, 90 Y. 186 Re、 211 At 125 I. 188 Re、 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18F. In an alternative embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives. In an alternative embodiment, the nanoparticle marker includes, but is not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles. In an alternative embodiment, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex. In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold or colloidal silver. In an alternative embodiment, the colloidal metal is colloidal gold. In an alternative embodiment, the antibody conjugate may further include a solid phase carrier coupled to the antibody. In the antibody conjugate, the antibody is coupled to a solid phase carrier. In an alternative embodiment, the solid phase carrier is selected from microspheres, plates, and membranes. In an alternative embodiment, the solid phase includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon and nitrocellulose membranes. In an alternative embodiment, the solid phase carrier is a nitrocellulose membrane.

[0186] The term "linker" or "linker" refers to a connecting unit that connects two polypeptide fragments, which usually has a certain degree of flexibility, and the use of the linker does not cause the loss of the original function of the protein domain. The linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. In some embodiments, the linker is selected from (GxS)y linkers, wherein x is selected from an integer of 1-5 and y is selected from an integer of 0-6. In some embodiments, the linker is selected from (GxS)y linkers, wherein x is selected from an integer of 1-5 and y is selected from an integer of 1-6.

[0187] The term "conservatively modified variants" or "conservative substitutions" of an amino acid refers to replacing an amino acid in a sequence with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophilicity / hydrophobicity, backbone configuration, and rigidity, etc.), but does not alter the biological activity of the protein. It is known to those skilled in the art that, under normal circumstances, single amino acid substitutions in non-essential regions of a polypeptide generally do not alter biological activity (see, e.g., Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). The term "conservatively modified variants" or "conservative substitutions" of a nucleic acid refers to nucleic acids that encode the same or substantially the same amino acid sequence, including nucleic acids that have substantially the same sequence when the nucleic acid does not encode the amino acid sequence. Due to the degeneracy of the genetic code, any given protein can be encoded by multiple functionally identical nucleic acids. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where a codon specifies alanine, the codon can be changed to any of the corresponding codons without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Every nucleic acid sequence encoding a polypeptide in the present disclosure encompasses every possible silent variation of the nucleic acid. Each codon in a nucleic acid (except AUG, which is generally the only codon for methionine; and TGG, which is generally the only codon for tryptophan) can be modified to produce a functionally identical molecule.

[0188] The term "vector" is a vehicle capable of transporting genetic elements (e.g., nucleic acids) connected thereto. A vector can be used to transform, transduce, or transfect a host cell so that the genetic elements it carries are expressed in the host cell. Exemplarily, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. In some embodiments, the vector is a "plasmid," which is a circular double-stranded DNA loop to which additional DNA segments can be attached. In some embodiments, the vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), and the DNA segment can be connected to the viral genome. The vector can contain a variety of elements that control expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. The vector can also contain a replication initiation site. The vector can also include components that assist its entry into the cell, including, but not limited to, viral particles, liposomes, or protein coats. The vector can be an expression vector. In some embodiments, a vector (eg, an expression vector) contains a nucleic acid sequence encoding an antibody of the present disclosure, a promoter (eg, SV40, CMV, EF 1α), and may further contain at least one selection marker.

[0189] The term "expression vector" or "expression construct" is a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and / or controls (along with the host cell) the expression of one or more coding regions operably linked thereto. Expression vectors may include, but are not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of the coding region operably linked thereto.

[0190] The term "operably linked" refers to a connection relationship in which the components to which the term refers allow them to perform their inherent functions under appropriate conditions. For example, a control sequence "operably linked" to a protein coding sequence refers to a sequence that is linked thereto and achieves expression of the protein coding sequence under conditions compatible with the transcriptional activity of the control sequence.

[0191] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and offspring derived therefrom, without regard to the number of passages. Offspring may not be identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initial transformed cells are included herein. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataeaminuta, Pichia lindneri), Pichia truncatula, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Hansenula polymorpha, polymorpha), Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Pichia, any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa.

[0192] The term "pharmaceutical composition" refers to a mixture containing one or more drugs (eg, the antibodies of the present disclosure) with other components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0193] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0194] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably in this disclosure. Unless otherwise specifically defined in this disclosure, the term "cynomolgus monkey" or "cynomolgus monkey" refers to cynomolgus monkeys (Macaca fascicularis). In certain embodiments, the subject is a human.

[0195] "Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

[0196] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.

[0197] The term "treatment" or "treat" refers to a clinical intervention that attempts to alter the natural course of the individual being treated and can be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the development of the disease or slow the progression of the disease.

[0198] The terms "administering" or "administering" as applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous agent with the animal, human, subject, cell, tissue, organ or biological fluid.

[0199] The term "effective amount" generally refers to an amount sufficient to reduce the severity or frequency of symptoms, eliminate symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring, improve the amount of damage (such as lung disease) associated therewith caused by the disease state. In certain embodiments, an effective amount can be a therapeutically effective amount or a prophylactic effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "prophylactic effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete treatment or prophylactic effect may not occur after administering a dose, but may occur after administering a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more doses. A "therapeutically effective amount" and a "prophylactically effective amount" may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent to elicit a desired response in the individual.

[0200] The terms "cancer" and "tumor" refer to diseases in mammals characterized by unregulated cell growth. Tumors include benign and malignant tumors. Early-stage tumors refer to cancers that are non-invasive or metastatic, or classified as stage 0, stage I, or stage II cancer. In some embodiments, the tumor is a tumor that expresses PVRIG. Examples of cancer include, but are not limited to, lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and hematologic cancer.

[0201] In some embodiments, the present disclosure uses conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are well known in the art, to prepare or detect the antibodies of the present disclosure. Related techniques are disclosed in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), which are incorporated herein by reference.

[0202] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0203] Preparation of some materials used in Examples 1-9 of the present disclosure:

[0204] 1) The target genes encoding human PVRIG (UniProt accession number: Q6DKI7) and monkey PVRIG (UniProt accession number: A0A2K5WVV8) were cloned into plasmids containing His, human Fc, or mouse Fc, respectively, and then transfected into HEK293 cells for transient expression to obtain hPVRIG-mFc (abbreviated as R2248), hPVRIG-hFc (abbreviated as R2249), hPVRIG-His (abbreviated as R2250), and cynoPVRIG-His (abbreviated as R2251);

[0205] 2) The target gene encoding human PVRIG (Q6DKI7) was cloned into the plvx lentiviral plasmid, and the lentivirus was then packaged and titered to infect 293T cells for membrane protein expression, thereby obtaining a cell line overexpressing hPVRIG: 293T-hPVRIG.

[0206] 3) preparing the hIgG4 subtype positive antibody R2243 according to the sequence of SEQ ID No. 81 in International Patent Publication No. WO2021180205A1;

[0207] 4) preparing the mIgG1 subtype positive antibody R2247 according to the sequence of "Antibody 46" in U.S. Patent No. US11279758B2;

[0208] 5) The following proteins were purchased: hPVRL2-His (Cat. No. C440, Novoprotein), hPVRL2-mFc2a (Cat. No. C11A, Novoprotein), and Mouse PVRIG-His (Cat. No. PVG-M52H7, Acro);

[0209] 6) The PVRIG reporter gene system was purchased (Cat. No. CBP74120+CBP74121, Nanjing Kebai).

[0210] Example 1. Preparation of anti-human PVRIG antibodies

[0211] Balb / c or SJL mice were immunized weekly using hPVRIG-mFc (R2248) and hPVRIG-hFc (R2249) as immunogens. Balb / c mice were immunized five times, and SJL mice were immunized four times. After immunization, orbital blood was collected to detect binding of the antibody to hPVRIG-His (R2250) by ELISA, and binding of the antibody to PVRIG reporter cells was detected by FACS. Three mice with good binding activity were screened and given a booster immunization.

[0212] Then, the spleen of the mouse was taken, and the spleen cells were separated and mixed with mouse myeloma cells at a ratio of 1:1 at a ratio of 5E6 / plate. The cells were fused under the action of an electrofusion instrument to obtain hybridomas.

[0213] The hybridoma supernatant was collected, and the binding of the antibody to hPVRIG-His (R2250) was detected by ELISA. The binding of the antibody to PVRIG reporter gene cells was detected by FACS. The blocking activity of the antibody on PVRIG receptor ligand (R2249+hPVRL2-His) was detected by ELISA. The binding of the antibody to cyno-PVRIG-His (R2251) was detected by ELISA. Positive clones were screened for subcloning. 14 positive clones were selected and expanded, and the supernatant was collected. Finally, the antibodies in the supernatant were purified to obtain 14 mouse anti-human PVRIG antibodies. The sequence information of the antibodies is shown in Tables 1 and 2 below:

[0214] Table 1. Anti-human PVRIG antibody variable region sequence list

[0215] Table 2. Anti-human PVRIG antibody CDR sequence list

[0216] Note: The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the anti-human PVRIG antibody are identified according to the Kabat numbering system.

[0217] In addition, the antibody constant regions: the heavy chain constant region of CG004-01, CG004-2, CG004-05, CG004-06, CG004-07, CG004-09, CG004-11, and CG004-14 antibodies is the mouse IgG2a heavy chain constant region, and the light chain constant region is the mouse κ chain constant region; the heavy chain constant region of CG004-04, CG004-12, and CG004-13 is the mouse IgG2b heavy chain constant region, and the light chain constant region is the mouse κ chain constant region; the heavy chain constant region of CG004-10 is the mouse IgG1 heavy chain constant region, and the light chain constant region is the mouse κ chain constant region.

[0218] Example 2. ELISA detection of binding activity of anti-human PVRIG antibody to hPVRIG

[0219] ELISA binding assay protocol: Dilute human PVRIG (R2250) to 1 μg / mL (in 50 mM CB) and coat the plate overnight at 100 μL / well. Wash the plate three times the next morning and pat dry. Add 200 μL / well of blocking buffer (1% BSA) and block at room temperature for 1 hour. Wash the plate once, pat dry, and add 100 μL / well of a 3-fold serial dilution of the test antibody. Incubate at room temperature for 1 hour. Wash the plate five times, pat dry, and add 100 μL / well of anti-mouse Fc-HRP (1:10,000) and incubate at room temperature for 40 minutes. Wash the plate six times, pat dry, and add 50 μL / well of HRP substrates A and B, respectively, and incubate at room temperature for 20 minutes. Add 100 μL / well of stop buffer and read the plate at 450 nm.

[0220] The experimental results are shown in Figures 1 and 2 , which indicate that the anti-human PVRIG antibodies disclosed herein can specifically bind to human PVRIG and have excellent antigen-binding activity.

[0221] Example 3. FACS detection of binding activity of anti-human PVRIG antibody to hPVRIG

[0222] FACS combined experimental method: cell counting, count the PVRIG reporter gene cells to be tested, centrifuge at 300g for 5 minutes, resuspend in FCM buffer, adjust the cell density to 4E+06 cells / mL, and add 2E+05 cells / well of each cell line to a 96-well V-shaped plate; dilute the sample (anti-human PVRIG antibody and R2247 as positive controls) 3-fold with buffer (3% BSA + 1× PBS); add the sample and cells to a 96-well V-shaped plate (sample and cells are added at 50μL / well respectively) and incubate at 4℃ in the dark for 30 minutes; wash: centrifuge (300g / 5min), discard the supernatant, add 200μL FCM buffer to wash once, centrifuge (300g / 5min), discard the supernatant; add secondary antibody (APC anti-mouse IgG Fc, 1:500 dilution) was transferred to a 96-well V-plate (100 μL / well) and incubated at 4°C in the dark for 30 min. Washing: Centrifugation (300 g / 5 min), discard the supernatant, add 200 μL FCM buffer, wash once, centrifuge (300 g / 5 min), and discard the supernatant. Microplate: Resuspend in 100 μL 1× PBS and analyze.

[0223] The experimental results are shown in Figures 3 and 4 , which indicate that the anti-human PVRIG antibody disclosed herein has better antigen binding activity than the positive control antibody R2247.

[0224] Example 4. Anti-human PVRIG antibody blocking activity experiment

[0225] The ELISA method was used to evaluate the blocking activity of anti-human PVRIG antibodies on the binding of hPVRIG-hFc to its ligand.

[0226] Experimental Methods: Antigen Coating: Dilute hPVRIG-hFc (R2249) with coating buffer to a concentration of 1 μg / mL. Add 100 μL / well to a 96-well ELISA plate. Coat two plates. Gently shake to mix, seal with sealing film, and incubate at 2-8°C overnight. Washing: Remove the plate, discard the supernatant, and pat dry on absorbent paper. Add PBST wash buffer (200 μL / well), discard the supernatant, and pat dry on absorbent paper. Repeat washing three times. Blocking: Add 1% BSA blocking buffer (200 μL / well) and incubate at 37°C for 1 hour. Dilute the candidate antibody and positive antibody R2247 three-fold in 1X BSA solution. Transfer 70 μL of the diluted antibody to a 96-well microtiter plate and incubate at 37°C for 60 min. Then, add 70 μL / well of the diluted 0.5 μg / mL nectin-2-his ligand protein and incubate at 37°C for 60 min. Wash: Remove the microtiter plate, discard the supernatant, and pat dry on absorbent paper. Add PBST wash buffer (200 μL / well), discard the supernatant, and pat dry on absorbent paper. Repeat the wash three times. Incubate with enzyme-linked secondary antibody: Add 100 μL / well of HRP-anti-his secondary antibody (1:10,000) diluted in 1% BSA (PBST) to columns 1-12 of the microtiter plate and incubate at 37°C for 40 min. Washing: Remove the plate, discard the supernatant, and pat dry on absorbent paper. Add 200 μL / well of PBST wash buffer, discard the supernatant, and pat dry on absorbent paper. Repeat washing three times. Color development: First add 50 μL / well of Solution A followed by 50 μL / well of Solution B, incubate at room temperature in the dark for 10 minutes. Stop and read: Add 100 μL / well of stop buffer to the plate and immediately read the OD450 absorbance on a microplate reader.

[0227] The experimental results are shown in Figures 5 and 6 , which indicate that the anti-human PVRIG antibody disclosed herein can effectively block the binding of human PVRIG to its ligand.

[0228] Example 5. Cross-binding experiment of anti-human PVRIG antibody and mPVRIG / cynoPVRIG

[0229] Experimental Methods: The day before the experiment, dilute cynoPVRIG (R2251) or mouse PVRIG (Acro) protein to 1 μg / mL and add 100 μL / well to a 96-well microtiter plate. Coat two microtiter plates, gently shake to mix, seal with sealing film, and incubate at 2-8°C overnight. Wash: Remove the microtiter plate, discard the supernatant, and pat dry on absorbent paper. Add 200 μL / well of PBST wash buffer, discard the supernatant, and pat dry on absorbent paper. Repeat the wash three times. Block: Add 200 μL / well of 1% BSA blocking buffer and incubate at 37°C for 1 hour. Dilute the candidate antibody and positive antibody R2247 three-fold in a 1X BSA solution. Transfer 100 μL of the diluted antibody to a 96-well microtiter plate and incubate at 37°C for 60 minutes. Washing: Remove the plate, discard the supernatant, and pat dry on absorbent paper. Add PBST wash buffer (200 μL / well), discard the supernatant, and pat dry on absorbent paper. Repeat washing three times. Enzyme-linked secondary antibody incubation: Dilute goat anti-mouse Fc enzyme-linked secondary antibody (1:15,000) in 1% BSA (PBST) and apply 100 μL / well to columns 1-12 of the plate. Incubate at 37°C for 40 min. Washing: Remove the plate, discard the supernatant, and pat dry on absorbent paper. Add PBST wash buffer (200 μL / well), discard the supernatant, and pat dry on absorbent paper. Repeat washing three times. Color development: Mix Solution A and Solution B in a 1:1 ratio and add 100 μL / well to the plate. Incubate at room temperature in the dark for 10 min. Stop and read the plate: Add stop solution to the ELISA plate, 100 μL / well, and immediately read the OD450 absorbance on a ELISA reader.

[0230] The experimental results are shown in Figures 7-10. The experimental results show that the anti-human PVRIG antibodies disclosed herein can bind to cynoPVRIG and mouse PVRIG, and are superior to the positive antibody R2247.

[0231] Example 6. Reporter gene activity detection

[0232] PVRIG (CD112R) is a regulatory protein expressed on T cells. When it binds to its ligand PVRL2 (CD112, Nectin-2), expressed on dendritic cells (DCs) or tumor cells, it can inhibit T cell immune activation. The luciferase reporter (Luc) system uses luciferin as a substrate to detect the activity of firefly luciferase. Luciferase catalyzes the oxidation of luciferin to oxyluciferin, which emits bioluminescence. Therefore, a reporter gene activity assay with a narrow detection window was designed using PVRIG Effector Reporter Cells and CD112 / TCR Activator / CHO Cells to verify whether candidate antibodies have the ability to inhibit the interaction between PVRIG and PVRL2.

[0233] Experimental Methods: Count cells, centrifuge at 300g for 5 minutes, discard the supernatant, wash once with F12K + 10% FBS medium, resuspend in F12K + 10% FBS medium, adjust the cell density to 2E+05 cells / mL, and add 2E+04 cells / well (100 μL / well). Incubate overnight in a CO2 incubator. The next morning, dilute the sample (using 1640 μg of 10% FBS medium). Count the cells, centrifuge at 300 g for 5 minutes, discard the supernatant, wash once with 1640+10% FBS medium, and resuspend in 1640+10% FBS medium to adjust the PVRIG cell density to 1.6E+06 cells / mL. Discard the F12K+10% FBS medium and add 50 μL of antibody diluent to each well of a 96-well white cell culture plate according to the experimental design. Add 8E+04 PVRIG cells / well (50 μL / well) and add a volume of 1640+10% FBS medium to the corresponding wells to bring the total volume to 100 μL. Incubate the cell culture plate in a CO2 incubator for 6 hours. Thaw Bright-Lumi™ Firefly Luciferase Assay Reagent in advance and equilibrate to room temperature. Remove the cell culture plate and equilibrate at room temperature for 10 minutes (no more than 30 minutes). Add 100 μL of Bright-Lumi™ Firefly Luciferase Assay Reagent to each well and incubate at room temperature for 5 minutes. Perform chemiluminescence detection on a flow cytometer.

[0234] The experimental results are shown in FIG11 , which indicate that the anti-human PVRIG antibody disclosed herein can inhibit the interaction between PVRIG and PVRL2.

[0235] Example 7. Preparation and evaluation of humanized PVRIG mouse-derived conventional antibodies

[0236] The hIgG4 (S228P / F234A / L235A / K447A) subtype chimeric antibody R3014 was prepared using the murine antibody sequence of CG004-1 (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO. 9, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO. 10).

[0237] Three-dimensional homology modeling was performed on the selected sequences, and acceptable structures were determined using methods such as Pull-Draw. The antibody sequences were compared with the GermLine database to obtain a human germline template (IGKV4-1*01) with high homology. The CDRs were then grafted into the corresponding humanized template. The grafted antibodies were subjected to further three-dimensional structural modeling and analysis. Residues that were buried, directly interacted with the CDR regions, and significantly influenced the conformation of the variable region were reverse-mutated. Chemically labile amino acid residues in the CDR regions were also optimized to generate a series of humanized antibody sequences. A series of humanized molecules of the hIgG4 (S228P / F234A / L235A / K447A) subtype were generated: R3942-R3950. The amino acid sequences of the heavy chain variable regions of antibodies R3942-R3950 are shown in Table 3, and the amino acid sequences of the light chain variable regions are shown in Table 4. The combinations of heavy and light chain variable regions are shown in Table 5.

[0238] Table 3 Humanized PVRIG mouse antibody heavy chain sequence

[0239] Table 4 Humanized PVRIG mouse antibody light chain sequence

[0240] Table 5 Combinations of heavy chain variable regions and light chain variable regions of humanized PVRIG mouse antibody

[0241] Example 8. Evaluation of reporter gene activity of humanized PVRIG mouse antibody

[0242] The activity of the humanized PVRIG murine antibodies R3942-R3950 was evaluated using a reporter gene assay, using the same method as in Example 6. The results are shown in Figure 12 . R3014 is the parent molecule, and R3942-R3950 are the humanized molecules. Of the humanized molecules, R3944 exhibited superior reporter gene activity compared to the parent R3014.

[0243] Example 9. Binding activity of humanized PVRIG nanobodies to 293T-hPVRIG cells and 293T-cynoPVRIG cells

[0244] The binding activity of humanized PVRIG nanobodies R3942-R3950 to 293T-hPVRIG cells and 293T-cynoPVRIG cells was evaluated using the same method as in Example 5. As shown in Figures 13 and 14, the binding activity of humanized molecule R3944 to 293-hPVRIG cells was close to that of the parent R3014.

[0245] The foregoing description is merely a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure. Industrial Applicability

[0246] The anti-human PVRIG antibodies disclosed in the present disclosure can specifically bind to human PVRIG; in some embodiments, the anti-human PVRIG antibodies can specifically bind to mouse PVRIG and / or monkey PVRIG; the anti-human PVRIG antibodies can inhibit the binding activity of PVRIG to PVRL2; and / or the anti-human PVRIG antibodies can inhibit tumor growth.

Claims

1. An anti-human PVRIG antibody, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in a variable region having at least 90% sequence identity with SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 100, 101, 102, 103 or 104, and / or the light chain variable region comprising LCDR1, LCDR2 and LCDR3 in a variable region having at least 90% sequence identity with SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 99 or 105.

2. The anti-human PVRIG antibody according to claim 1, wherein The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system; Optionally, A. The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.2; B. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.3, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.4; C. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.5, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.6; D. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.7, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.8; E. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.9, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.10; F. The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 11, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 12; G. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 13, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 14; H. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 15, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 16; I. The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 17, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 18; J. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.19, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.20; K. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.21, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.22; or L. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.23, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.24; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat numbering system; Optionally, the anti-human PVRIG antibody, wherein, a. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.25, HCDR2 comprises the amino acid sequence of SEQ ID NO.26, and HCDR3 comprises the amino acid sequence of SEQ ID NO.27; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.28, LCDR2 comprises the amino acid sequence of SEQ ID NO.29, and LCDR3 comprises the amino acid sequence of SEQ ID NO.30; b. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.31, HCDR2 comprises the amino acid sequence of SEQ ID NO.32, and HCDR3 comprises the amino acid sequence of SEQ ID NO.33; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34, LCDR2 comprises the amino acid sequence of SEQ ID NO.35, and LCDR3 comprises the amino acid sequence of SEQ ID NO.36; c. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.37, HCDR2 comprises the amino acid sequence of SEQ ID NO.38, and HCDR3 comprises the amino acid sequence of SEQ ID NO.39; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34, LCDR2 comprises the amino acid sequence of SEQ ID NO.35, and LCDR3 comprises the amino acid sequence of SEQ ID NO.36; d. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.40, HCDR2 comprises the amino acid sequence of SEQ ID NO.41, and HCDR3 comprises the amino acid sequence of SEQ ID NO.42; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.43, LCDR2 comprises the amino acid sequence of SEQ ID NO.44, and LCDR3 comprises the amino acid sequence of SEQ ID NO.45; e. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.46, HCDR2 comprises the amino acid sequence of SEQ ID NO.47, and HCDR3 comprises the amino acid sequence of SEQ ID NO.48; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.49, LCDR2 comprises the amino acid sequence of SEQ ID NO.50, and LCDR3 comprises the amino acid sequence of SEQ ID NO.51; f. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.46, HCDR2 comprises the amino acid sequence of SEQ ID NO.52, and HCDR3 comprises the amino acid sequence of SEQ ID NO.48; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.49, LCDR2 comprises the amino acid sequence of SEQ ID NO.50, and LCDR3 comprises the amino acid sequence of SEQ ID NO.51; g. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.53, HCDR2 comprises the amino acid sequence of SEQ ID NO.54, and HCDR3 comprises the amino acid sequence of SEQ ID NO.55; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.49, LCDR2 comprises the amino acid sequence of SEQ ID NO.56, and LCDR3 comprises the amino acid sequence of SEQ ID NO.57; h. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.58, HCDR2 comprises the amino acid sequence of SEQ ID NO.59, and HCDR3 comprises the amino acid sequence of SEQ ID NO.60; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.49, LCDR2 comprises the amino acid sequence of SEQ ID NO.61, and LCDR3 comprises the amino acid sequence of SEQ ID NO.62; i. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.63, HCDR2 comprises the amino acid sequence of SEQ ID NO.64, and HCDR3 comprises the amino acid sequence of SEQ ID NO.65; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.66, LCDR2 comprises the amino acid sequence of SEQ ID NO.67, and LCDR3 comprises the amino acid sequence of SEQ ID NO.68; j. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.69, HCDR2 comprises the amino acid sequence of SEQ ID NO.70, and HCDR3 comprises the amino acid sequence of SEQ ID NO.71; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.72, LCDR2 comprises the amino acid sequence of SEQ ID NO.29, and LCDR3 comprises the amino acid sequence of SEQ ID NO.73; k. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.74, HCDR2 comprises the amino acid sequence of SEQ ID NO.75, and HCDR3 comprises the amino acid sequence of SEQ ID NO.76; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.28, LCDR2 comprises the amino acid sequence of SEQ ID NO.77, and LCDR3 comprises the amino acid sequence of SEQ ID NO.78; or 1. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.79, HCDR2 comprises the amino acid sequence of SEQ ID NO.80, and HCDR3 comprises the amino acid sequence of SEQ ID NO.81; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.82, LCDR2 comprises the amino acid sequence of SEQ ID NO.83, and LCDR3 comprises the amino acid sequence of SEQ ID NO.68; Optionally, A. the anti-human PVRIG antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, respectively; or B. The anti-human PVRIG antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO.33, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, respectively.

3. The anti-human PVRIG antibody according to claim 1 or 2, wherein the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

4. The anti-human PVRIG antibody according to any one of claims 1 to 3, wherein The heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 100, 101, 102, 103 or 104, and / or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 99 or 105; Optionally, (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.2; (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.4; (c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.6; (d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.8; (e) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.10; (f) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.12; (g) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.14; (h) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.15, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.16; (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.17, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.18; (j) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.19, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.20; (k) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.21, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.22; (l) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.23, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.24; (m) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.104, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.99; (n) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.100, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.99; (o) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.100, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.105; (p) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.101, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.99; (q) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.101, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.105; (r) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.102, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.99; (s) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.102, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.105; (t) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.103, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.99; or (u) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.103, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.

105.

5. An anti-human PVRIG antibody, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.1, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.2; (b) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.3, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.4; (c) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.5, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.6; (d) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.7, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.8; (e) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.9, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.10; (f) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.11, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.12; (g) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.13, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.14; (h) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.15, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.16; (i) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.17, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.18; (j) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.19, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.20; (k) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.21, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.22; (l) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.23, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.24; (m) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.104, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.99 (n) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.100, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.99; (o) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.100, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.105; (p) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.101, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.99; (q) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.101, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.105; (r) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.102, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.99; (s) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.102, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.105; or (t) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.103, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.99; (u) the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.103, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.105; alternatively, (a) the antibody comprises a heavy chain variable region as shown in SEQ ID NO.1, and a light chain variable region as shown in SEQ ID NO.2; (b) the antibody comprises a heavy chain variable region as shown in SEQ ID NO.3, and a light chain variable region as shown in SEQ ID NO.4; or (c) The antibody comprises a heavy chain variable region as shown in SEQ ID NO.101, and a light chain variable region as shown in SEQ ID NO.

99.

6. The anti-human PVRIG antibody according to any one of claims 1 to 5, wherein the antibody comprises a constant region; Optionally, the heavy chain constant region of the antibody is selected from the heavy chain constant region of IgG1, IgG2, IgG3 and IgG4, and / or the light chain constant region of the antibody is selected from the κ or λ chain constant region; Optionally, the species origin of the constant region is mouse or human; Optionally, the heavy chain constant region is a mouse IgG2a constant region, a mouse IgG1 constant region, a human IgG1 constant region or a human IgG4 constant region, and / or the light chain constant region is a mouse κ constant region or a human κ constant region.

7. The anti-human PVRIG antibody according to any one of claims 1 to 6, wherein the antibody is a full-length antibody or any antigen-binding fragment selected from F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv and scFv.

8. An antibody that competitively binds to human PVRIG with the anti-human PVRIG antibody according to any one of claims 1 to 7, or an antibody that binds to the same epitope as the anti-human PVRIG antibody according to any one of claims 1 to 7.

9. The anti-human PVRIG antibody according to any one of claims 1 to 8, wherein the anti-human PVRIG antibody has at least one of the following properties: A. The anti-human PVRIG antibody can specifically bind to human PVRIG; optionally, the antibody can bind to human PVRIG protein with an EC50 value of ≤10 nM, wherein: The EC50 value was determined by flow cytometry; B. The anti-human PVRIG antibody can specifically bind to human PVRIG; optionally, the antibody can bind to monkey PVRIG protein with an EC50 value of ≤10 nM, wherein the EC50 value is determined by flow cytometry; C. The anti-human PVRIG antibody can specifically bind to human PVRIG; optionally, the antibody can bind to mouse PVRIG protein with an EC50 value of ≤10 nM, wherein the EC50 value is determined by flow cytometry; D. The anti-human PVRIG antibody can inhibit the binding activity of PVRIG and PVRL2. 10 . A multispecific antibody, comprising the anti-human PVRIG antibody according to any one of claims 1 to 9 .

11. An antibody conjugate, comprising the anti-human PVRIG antibody according to any one of claims 1 to 10.

12. An isolated nucleic acid encoding the anti-human PVRIG antibody of any one of claims 1 to 9 or encoding the multispecific antibody of claim 10.

13. A cell comprising the nucleic acid of claim 12.

14. A pharmaceutical composition comprising the anti-human PVRIG antibody of any one of claims 1 to 9, the multispecific antibody of claim 10, the antibody conjugate of claim 11, the nucleic acid of claim 12, or the cell of claim 13; optionally, further comprising one or more pharmaceutically acceptable carriers, diluents or excipients.

15. Use of the anti-human PVRIG antibody according to any one of claims 1 to 9, the multispecific antibody according to claim 10, the antibody conjugate according to claim 11, the nucleic acid according to claim 12, the cell according to claim 13 or the pharmaceutical composition according to claim 14 in the preparation of a product having at least one of the following uses: detecting human PVRIG expression, treating diseases associated with high human PVRIG expression; Optionally, the disease is a tumor; Optionally, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma and hematological cancer.

16. A method for treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the anti-human PVRIG antibody according to any one of claims 1 to 9, the multispecific antibody according to claim 10, the antibody conjugate according to claim 11, the nucleic acid according to claim 12, the cell according to claim 13 or the pharmaceutical composition according to claim 14; optionally, the disease is a disease associated with high expression of human PVRIG; Optionally, the disease is a tumor; Optionally, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma and hematological cancer.

17. The anti-human PVRIG antibody according to any one of claims 1 to 9, the multispecific antibody according to claim 10, the antibody conjugate according to claim 11, the nucleic acid according to claim 12, the cell according to claim 13 or the pharmaceutical composition according to claim 14 for use as a medicament; optionally, the medicament is used to treat diseases associated with high expression of human PVRIG; Optionally, the disease is a tumor; Optionally, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma and hematological cancer.

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