Anti-human PVRIG antibody and application thereof

Abstract

The invention relates to the technical field of biology, and discloses an anti-human PVRIG antibody and application thereof. The anti-human PVRIG antibody disclosed by the invention is specifically combined with a human PVRIG antigen and can be used for tumor treatment.

Description

[0001] Priority declaration

[0002] This application claims priority to the Chinese invention patent application with application number 202311579153.1, filing date November 23, 2023, and invention name “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-related2, 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 the activation of T cells. On the other hand, after binding to the ligand, it will inhibit the activity of T cells through the intracellular inhibitory motif.

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

[0006] PVRL2 is mainly expressed on tumor cells, macrophages and activated dendritic cells (also known as DC cells). Its expression is also tumor tissue specific. Analysis of tumor tissues and normal tissues of different tumor patients found that the expression of PVRL2 on lymphocytes infiltrating tumor tissues was significantly higher than that in peripheral normal tissues of the same patient. High expression of PVRL2 is also associated with poor prognosis of tumor patients. Summary of the invention

[0007] The present disclosure provides an anti-human PVRIG antibody.

[0008] 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 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. 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;

[0009] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0010] 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;

[0011] 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;

[0012] 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;

[0013] 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;

[0014] 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;

[0015] 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;

[0016] 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;

[0017] 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;

[0018] 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;

[0019] 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;

[0020] 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

[0021] 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.

[0022] In some embodiments, for the anti-human PVRIG antibody as described in any one of the above, 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;

[0023] 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.

[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 IMGT numbering system.

[0025] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0026] 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 SEQID NO.30;

[0027] 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 SEQID NO.36;

[0028] 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;

[0029] 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 SEQID NO.45;

[0030] 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 SEQID NO.51;

[0031] 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 SEQID NO.51;

[0032] 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;

[0033] 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;

[0034] 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;

[0035] 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 SEQID NO.73;

[0036] 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 SEQID NO.78; or

[0037] 1. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.79, the HCDR2 comprises the amino acid sequence of SEQ ID NO.80, and the HCDR3 comprises the amino acid sequence of SEQ ID NO.81; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.82, the LCDR2 comprises the amino acid sequence of SEQ ID NO.83, and the LCDR3 comprises the amino acid sequence of SEQID NO.68.

[0038] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0039] 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

[0040] 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.

[0041] 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.

[0042] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0043] 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).

[0044] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0045] 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).

[0046] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, wherein

[0047] (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;

[0048] (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;

[0049] (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;

[0050] (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;

[0051] (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;

[0052] (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;

[0053] (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;

[0054] (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;

[0055] (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;

[0056] (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;

[0057] (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;

[0058] (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;

[0059] (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;

[0060] (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;

[0061] (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;

[0062] (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;

[0063] (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;

[0064] (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;

[0065] (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;

[0066] (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

[0067] (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.

[0068] 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

[0069] (a) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.2;

[0070] (b) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.4;

[0071] (c) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.6;

[0072] (d) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.8;

[0073] (e) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.10;

[0074] (f) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.12;

[0075] (g) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.14;

[0076] (h) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.16;

[0077] (i) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.18;

[0078] (j) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.20;

[0079] (k) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.22;

[0080] (l) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.24;

[0081] (m) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.99;

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

[0083] (o) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.105;

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

[0085] (q) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.105;

[0086] (r) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.99;

[0087] (s) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.105;

[0088] (t) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.99; or

[0089] (u) the heavy chain variable region has at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with 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 with SEQ ID NO.105.

[0090] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, (a) the antibody includes 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 includes 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 includes 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.

[0091] In some embodiments, the anti-human PVRIG antibody as described in any of the above items comprises a constant region.

[0092] In some embodiments, the anti-human PVRIG antibody as described in any 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;

[0093] 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;

[0094] In some embodiments, in 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.

[0095] 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.

[0096] In some embodiments, the invention discloses an antibody that competes with any of the above anti-human PVRIG antibodies for binding to human PVRIG, or an antibody that binds to the same epitope as any of the above anti-human PVRIG antibodies.

[0097] 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 properties:

[0098] 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;

[0099] 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;

[0100] 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

[0101] D. The anti-human PVRIG antibody can inhibit the binding activity of PVRIG and PVRL2.

[0102] The present disclosure also provides a multispecific antibody, the 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.

[0103] The present disclosure also provides an antibody conjugate, which comprises the anti-human PVRIG antibody described in any one 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 carrier 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 fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels; in some embodiments, the label is colloidal gold.

[0104] The present disclosure also provides an isolated nucleic acid encoding the anti-human PVRIG antibody described in any one of the preceding items.

[0105] The present disclosure also provides a cell containing the nucleic acid described in any one of the preceding items.

[0106] The present disclosure also provides a pharmaceutical composition comprising: the anti-human PVRIG antibody, bispecific antibody, antibody conjugate, cell, or nucleic acid described in any one of the preceding items, and in some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0107] The present disclosure also provides the use of the anti-human PVRIG antibody, bispecific antibody, antibody conjugate, cell, nucleic acid, or pharmaceutical composition described in any one of the preceding items 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. In some embodiments, the diseases associated with high human PVRIG expression are tumors. In some embodiments, the tumors are 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.

[0108] In some embodiments, the present disclosure provides a method for treating a disease, the method comprising the step of administering a therapeutically effective amount of the anti-human PVRIG antibody, bispecific antibody, antibody conjugate, cell, nucleic acid, or pharmaceutical composition described in any one of the preceding items to a subject in need thereof. In some embodiments, the disease is a disease associated with high human PVRIG expression. In some embodiments, the diseases associated with high human PVRIG expression are tumors. In some embodiments, the tumors are 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.

[0109] In another aspect, the present disclosure also provides an anti-human PVRIG antibody, multispecific antibody, antibody conjugate, cell, nucleic acid or pharmaceutical composition as described in any of the above for use as a drug. In some embodiments, the drug is used to treat a disease. In some embodiments, the drug 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 hematological cancer.

[0110] 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.

[0111] The present disclosure also provides a method for detecting or measuring human PVRIG, which comprises the step of detecting or measuring human PVRIG using the anti-human PVRIG antibody or antibody conjugate as described in any of the preceding items.

[0112] 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 to detect or measure human PVRIG.

[0113] 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 cells as described in any of the preceding items, and then isolating and purifying to obtain the anti-human PVRIG antibody.

[0114] 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

[0115] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0116] Figure 1: ELISA test results of the anti-human PVRIG antibodies (CG004-01, CG004-02, CG004-04, CG004-05, CG004-06, CG004-07) disclosed herein binding to hPVRIG;

[0117] Figure 2 : ELISA test results of the anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14) disclosed herein binding to hPVRIG;

[0118] Figure 3 : A graph showing the results of FACS detection of the binding of the disclosed anti-human PVRIG antibodies (CG004-01, CG004-02, CG004-04, CG004-05, CG004-07, R2247) to hPVRIG;

[0119] Figure 4 : A graph showing the results of FACS detection 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;

[0120] Figure 5 : A graph showing the results of an experiment in which the anti-human PVRIG antibodies (CG004-01, CG004-02, CG004-04, CG004-05, CG004-06, CG004-07, and R2247) disclosed herein block the binding of human PVRIG to its ligand;

[0121] Figure 6 : A graph showing the results of an experiment in which the anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, and R2247) disclosed herein block the binding of human PVRIG to its ligand;

[0122] Figure 7 : A graph showing the results of the binding experiment between the anti-human PVRIG antibodies (CG004-01, CG004-04, CG004-05, CG004-06, CG004-07, R2247) disclosed herein and cynoPVRIG;

[0123] Figure 8 : A graph showing the results of an experiment on the binding of the anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, R2247) disclosed herein to cynoPVRIG;

[0124] Fig. 9 : A graph showing the results of the binding experiment between the anti-human PVRIG antibodies (CG004-01, CG004-04, CG004-05, CG004-06, CG004-07, R2247) disclosed herein and mouse PVRIG;

[0125] Fig.10 : A graph showing the results of the binding experiment between the anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, R2247) disclosed herein and mouse PVRIG;

[0126] Fig.11 : This 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;

[0127] Fig.12 : is a graph showing the activity results of the anti-human PVRIG antibody disclosed in the present invention in activating T cells;

[0128] Fig.13 : It is a graph showing the results of the binding experiment between the disclosed anti-human PVRIG humanized antibodies (R3942-R3950) and 293T-hPVRIG cells;

[0129] Fig.14 : A graph showing the results of the binding experiment between the disclosed anti-human PVRIG humanized antibodies (R3942-R3950) and 293T-cynoPVRIG cells. DETAILED DESCRIPTION

[0130] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention is clearly and completely described below, but the description and embodiments should not be construed as limiting the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified in 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 ordinary technicians in the field to which the present disclosure belongs.

[0131] The terms "a / an", "a", "said", "above", and "aforementioned" in the present disclosure 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 are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. The term "plurality" means at least two, such as 2, 3, etc., unless otherwise clearly and specifically indicated otherwise.

[0132] Unless the context clearly indicates otherwise, in the specification and claims of the present disclosure, the words "include" or "have" should be understood as "include" rather than the exclusive or exhaustive meaning; that is, the meaning of "include but not limited to". For example, "include A" means "include A, but not limited to A".

[0133] "About" means within an acceptable error range for a 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 otherwise expressly stated in the examples or elsewhere in the specification, "about" means within one standard deviation, or up to a range of 5%, as is customary in the art.

[0134] "Optional", "optionally", "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, that is, it includes occasions where the event or circumstance occurs or does not occur, unless the context clearly indicates otherwise.

[0135] The term "and / or" refers to each of the two specific features or a combination thereof, which includes or excludes another specific feature. For example, "A and / or B" means including any of the following situations: A and B, A or B, A (alone) and B (alone); "A, B and / or C" means covering 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).

[0136] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0137] 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 the naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and modified amino acids thereof, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Common natural amino acids are, for example: 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 refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group), 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 refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to that of a naturally occurring amino acid.

[0138] The term "antibody" in the present disclosure is used in the broadest sense and covers 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. .

[0139] "Native antibodies" are naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain of an IgG antibody has a heavy chain variable region (VH, also called a variable heavy domain or a 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 N to C-terminus, each light chain has a light chain variable region (VL, also called a variable light domain, or a 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).

[0140] The terms "full length antibody", "intact antibody" and "whole antibody" in the present disclosure are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure, or an antibody having a heavy chain including an Fc region.

[0141] 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 a purity greater than 90% or 99%, which can be purified 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).

[0142] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in antibody binding to an antigen. The heavy chain variable region (VH) and light chain variable region (VL) of a natural antibody, each variable region comprises four conserved framework regions (FR) and three hypervariable regions (also called complementarity determining regions, HVR or CDR).

[0143] 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 in the variable region. VH contains three CDR regions: HCDR1, HCDR2 and HCDR3; VL contains three CDR regions: LCDR1, LCDR2 and LCDR3. Each VH and VL is usually 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.

[0144] The term "light chain" includes a light chain variable region (VL) and a light chain constant region (CL). VL is at the amino terminus of the light chain, and CL is at the carboxyl amino terminus of the light chain. The light chain may be a kappa chain or a lambda chain, etc.

[0145] The term "heavy chain" includes a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain variable region is at the amino terminus of the heavy chain, and the heavy chain constant region is 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 may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgD, IgM, and IgE, etc.

[0146] 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, and has a relatively conserved amino acid sequence compared to VH and VL.

[0147] 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" is composed of VH and VL; "Fab'" is a Fab fragment containing part of the hinge region; "F(ab')2" contains two F(ab')2 domains connected by disulfide bonds in the hinge region. ab' fragment; "scFab" is a polypeptide consisting of VH, CH1, VL, CL and a linker, wherein the antibody domain 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 connected to the head 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 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 bond-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.

[0148] The term "Fc region" refers to the C-terminal region of an antibody heavy chain, including the native antibody Fc region 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, the deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or the deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). In some embodiments, a composition of intact antibodies may include a population of antibodies lacking the K447 residue and / or the G446+K447 residues. In some embodiments, a composition of intact antibodies may include a population of antibodies without the deletion of the K447 residue and / or the G446+K447 residues. In some embodiments, a composition of intact antibodies has a population of antibodies that is a mixture of antibodies with and without the K447 residue and / or the G446+K447 residues. The Fc regions for the antibodies described in this disclosure include the Fc regions of human IgG1, IgG2 (IgG2a, IgG2b), IgG3, and IgG4. Unless otherwise specified in this disclosure, the amino acid residues in the Fc region or constant region are numbered 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.

[0149] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from one particular species and the remaining portion of the heavy chain and / or light chain is derived from other species.

[0150] 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 the non-human antibody and replacing the remaining portion of the antibody with their human counterparts (i.e., the framework regions of the heavy chain variable region of a human antibody, the framework regions of the light chain variable region of a human antibody, and the human antibody constant regions).

[0151] The terms "human antibody", "human-derived antibody", "fully human antibody", "completely human antibody" can be used interchangeably and refer to an antibody in which the variable region and constant region sequences are human sequences. Human antibodies include antibodies having human-derived genes but with altered residues such as cysteine or glycosylation sites. Human antibodies can be antibodies recombinantly produced in non-human cells. Human antibodies include antibodies obtained from transgenic mice with human immunoglobulin heavy and light chain loci.

[0152] 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 ligand (e.g., an antigen). Unless otherwise specified, as disclosed herein, "affinity" refers to internal binding affinity, which reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its ligand Y is usually expressed as a dissociation constant (KD). Affinity can be measured by conventional methods known in the art. The term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to a dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka), usually expressed as a molar concentration (M). The KD value of an antibody can be measured using methods well known in the art. The method for measuring the KD of an antibody includes using a biosensor system such as a system to measure surface plasmon resonance, or measuring affinity in a solution by a solution equilibrium titration method (SET).

[0153] The term "affinity matured" antibody refers to an antibody that has one or more amino acid residue changes in one or more CDRs of the antibody, resulting 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 affinity 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).

[0154] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an Fc region of amino acid sequence mutation). 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.

[0155] 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 natural mutations that may be present in small amounts). In contrast, polyclonal antibodies typically contain 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, they can be prepared by the hybridoma method (Kohler et al., (1975) Nature 256:495), they can be prepared by recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567), they can be prepared 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 monoclonal antibodies can also be prepared using transgenic animals containing all or part of the human immunoglobulin loci (see Presta (2005) J. Allergy Clin. Immunol. 116:731).

[0156] 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).

[0157] The term "epitope" refers to an area (area or region) on an antigen that can specifically bind to an antibody. An epitope is usually composed of an antigenic determinant of a special chemical group with a certain structure. An epitope can be formed by continuous amino acid residues (linear epitope) or by non-continuous amino acid residues (conformational epitope), such as non-continuous amino acid residues that are close to each other in space 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 structural 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 known in the art, including, but not limited to, for example, 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)).

[0158] 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. In order to determine whether a test antibody binds to the same epitope as a reference antibody, it can be tested under saturation conditions 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 saturation 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 saturation binding of the reference antibody, then the test antibody can bind to the same epitope as the reference antibody. To confirm whether the test antibody binds to the same epitope or is merely hindered from binding for steric reasons, 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 to determine that 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 (e.g., 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold amount) of an antibody 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.

[0159] The term "specific binding" or "specifically binds" refers to non-random binding between two molecules, for example, an antibody binds to its corresponding 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, about 1×10 -9 M or smaller, about 1×10 -10 M or smaller, about 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 non-specific antigen (e.g., BSA, casein). The KD value can be measured using methods known in the art, such as by Surface plasmon resonance assay measurement.An antibody that specifically binds 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 (Callithrixjacchus) (common marmoset, marmoset).

[0160] 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.

[0161] In some embodiments, the anti-human PVRIG antibody as described in any of the above items, the antibody comprises 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 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. ID 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, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in the variable region having at least 95% sequence identity with 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 with 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, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in the variable region having at least 98% sequence identity with 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 with 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, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in the variable region having at least 100% sequence identity with 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 with SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24.

[0162] 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.

[0163] 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 Kabat numbering system, wherein

[0164] a. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.25, the amino acid sequence of HCDR2 is SEQ ID NO.26, and the amino acid sequence of HCDR3 is SEQ ID NO.27; 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.29, and the amino acid sequence of LCDR3 is SEQ ID NO.30;

[0165] b. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.31, the amino acid sequence of HCDR2 is SEQ ID NO.32, and the amino acid sequence of HCDR3 is SEQ ID NO.33; 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;

[0166] 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;

[0167] 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;

[0168] 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;

[0169] f. 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.52, 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] 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;

[0171] 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;

[0172] i. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.63, the amino acid sequence of HCDR2 is SEQ ID NO.64, and the amino acid sequence of HCDR3 is SEQ ID NO.65; and the amino acid sequence of LCDR1 of the light chain variable region is SEQ ID NO.66, the amino acid sequence of LCDR2 is SEQ ID NO.67, and the amino acid sequence of LCDR3 is SEQ ID NO.68;

[0173] j. the amino acid sequence of HCDR1 of the heavy chain variable region is SEQ ID NO.69, the amino acid sequence of HCDR2 is SEQ ID NO.70, and the amino acid sequence of HCDR3 is SEQ ID NO.71; and the amino acid sequence of LCDR1 of the light chain variable region is SEQ ID NO.72, the amino acid sequence of LCDR2 is SEQ ID NO.29, and the amino acid sequence of LCDR3 is SEQ ID NO.73;

[0174] 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

[0175] 1. The HCDR1 of the heavy chain variable region is an amino acid sequence such as SEQ ID NO.79, the HCDR2 is an amino acid sequence such as SEQ ID NO.80, and the HCDR3 is an amino acid sequence such as SEQ ID NO.81; and the LCDR1 of the light chain variable region is an amino acid sequence such as SEQ ID NO.82, the LCDR2 is an amino acid sequence such as SEQ ID NO.83, and the LCDR3 is an amino acid sequence such as SEQ ID NO.68.

[0176] 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 includes the amino acid sequence of GNTFSDSY (SEQ ID NO.84), HCDR2 includes the amino acid sequence of INPYNGDT (SEQ ID NO.85), and HCDR3 includes the amino acid sequence of ARSNWDVGNCFDY (SEQ ID NO.86); and the LCDR1 of the light chain variable region includes the amino acid sequence of QDISNY (SEQ ID NO.87), LCDR2 includes the amino acid sequence of YTS (SEQ ID NO.88), and LCDR3 includes the amino acid sequence of SEQ ID NO.30.

[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 Chothia numbering system; in some embodiments, the HCDR1 of the heavy chain variable region includes the amino acid sequence of GNTFSDS (SEQ ID NO.89), HCDR2 includes the amino acid sequence of NPYN (SEQ ID NO.90), and HCDR3 includes 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 SEQID 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.

[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 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 (SEQID 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).

[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 AbM numbering system; in some embodiments, the HCDR1 of the heavy chain variable region includes the amino acid sequence of GNTFSDSYMN (SEQ ID NO.97), HCDR2 includes the amino acid sequence of IINPYNGDTY (SEQ ID NO.98), and HCDR3 includes the amino acid sequence of SEQ ID NO.27; and the LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO.28, LCDR2 includes the amino acid sequence of SEQ ID NO.29, and LCDR3 includes the amino acid sequence of SEQ ID NO.30.

[0180] The binding of the anti-human PVRIG antibody provided by the present disclosure to human PVRIG can also be expressed as a "half-maximal effective concentration" (EC50) value. Generally, the smaller the EC50, the better the affinity, indicating that the antibody can bind to the antigen at a lower concentration. The EC50 value can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometric fluorescence sorting technology, and other binding assays).

[0181] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in the present disclosure to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or connections, and nucleic acids can be synthetic, naturally occurring, and non-naturally occurring, such as non-natural nucleic acids that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to reference nucleotides. 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 the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which normally contain the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or at a chromosomal location different from its natural chromosomal location. "Nucleic acid encoding anti-human PVRIG antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (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 in 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 replaced by mixed bases 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).

[0182] 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.

[0183] The terms "identity" and "sequence identity" refer to the degree (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 obtain the optimal alignment of the two sequences, gaps may be introduced, if necessary, to obtain the maximum percentage of sequence identity. The percentage of amino acid sequence identity can be determined in various ways known in the art, such as software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA, which are well known in the art. Those 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 comparison sequence.

[0184] 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 coupling an anti-human PVRIG antibody to a therapeutic agent or an imaging agent. In an optional embodiment, the antibody conjugate is formed by coupling 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 harmful effect on the growth or proliferation of a cell (such as maytansine, 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 coupled to the antibody. In an optional embodiment, the antibody conjugate also includes a marker coupled to the antibody. In an optional embodiment, the above-mentioned marker refers to a class of substances with 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 radioactive isotope, 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 kind of marker is used, it belongs to the protection scope of the present disclosure. In an optional embodiment, the fluorescent dye is 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, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 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), pre-chlorophyll protein (preCP), etc.).In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase. In alternative embodiments, the radioisotope 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 Ru, 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 optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, lophan and its derivatives, and peroxyoxalate and its derivatives. In an optional 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 optional embodiment, the colloid includes but is not limited to colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex. In an optional embodiment, the colloidal metal includes but is not limited to colloidal gold or colloidal silver. In an optional embodiment, the colloidal metal is colloidal gold. In an optional embodiment, the antibody conjugate may also include a solid phase carrier coupled to the antibody. In the antibody conjugate, the antibody is coupled to a solid phase carrier. In an optional 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, microplates, glass, capillaries, nylon and nitrocellulose membranes. In an alternative embodiment, the solid phase carrier is a nitrocellulose membrane.

[0185] The term "linker", "Linker" or "connector" refers to a connecting unit connecting two polypeptide fragments, which usually has a certain flexibility, and the use of the linker will 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.

[0186] The term "conservatively modified variants" or "conservative substitutions" of amino acids refers to the use of other amino acids with similar characteristics (e.g., charge, side chain size, hydrophilicity / hydrophobicity, backbone morphology and rigidity, etc.) to replace the amino acids in the sequence, but does not change 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 change the biological activity (see, for example, 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 nucleic acids refers to nucleic acids that encode the same or substantially the same amino acid sequence, and also includes nucleic acids of 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 changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Each 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 usually the only codon for methionine; and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule.

[0187] 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 (YAC), bacterial artificial chromosomes (BAC), or artificial chromosomes (PAC) derived from P1, bacteriophages such as lambda phages or M13 phages, and animal viruses, etc. In some embodiments, a vector is a "plasmid," which is a circular double-stranded DNA loop to which additional DNA segments can be connected. In some embodiments, a vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), and a DNA segment can be connected to a viral genome. A vector may contain a variety of elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene, a vector may also contain a replication initiation site, and a vector may also include components that assist it in entering a cell, including, but not limited to, viral particles, liposomes, or protein shells. A vector may 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 include at least one selection marker.

[0188] 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 (together with the host cell) the expression of one or more coding regions operably linked thereto. An expression vector may include, but is not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of a coding region operably linked thereto.

[0189] The term "operably linked" refers to a connection relationship in which the components referred to by the term allow them to perform their inherent functions under suitable conditions. For example, a control sequence "operably linked" to a protein coding sequence refers to a sequence that is linked to it and achieves the expression of the protein coding sequence under conditions compatible with the transcriptional activity of the control sequence.

[0190] 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 considering the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Mutant offspring with 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 (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Hansenula polymorpha. polymorpha), Kluyveromyces sp., 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, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa.

[0191] The term "pharmaceutical composition" refers to a mixture containing one or more drugs (eg, antibodies described herein) and other components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0192] The term "pharmaceutically acceptable carrier" refers to a component 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.

[0193] 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), sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably in the present disclosure. Unless explicitly defined in the present disclosure, the term "cynomolgus" or "cynomolgus" refers to cynomolgus (Macaca fascicularis). In certain embodiments, the subject is a human.

[0194] "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.

[0195] 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, excretions, 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 removed tissues, organ cultures, or cell cultures.

[0196] 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. The 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 formation of the disease or slow the progression of the disease.

[0197] The term "administering" or "administering" as it applies to an animal, a human, an experimental subject, a cell, a tissue, an organ or a biological fluid, refers to the contact of an exogenous drug with the animal, a human, a subject, a cell, a tissue, an organ or a biological fluid.

[0198] 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, and improve the amount of damage (e.g., lung disease) associated with the disease state. In some embodiments, an effective amount may be a therapeutically effective amount or a preventive 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 "preventive 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 preventive effect may not occur after administering a dose, but may occur after administering a series of doses. Thus, a therapeutically or preventive effective amount may be administered in one or more administrations. 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 to elicit a desired response in the individual.

[0199] The terms "cancer" and "tumor" refer to diseases characterized by unregulated cell growth in mammals. Tumors include benign tumors and malignant tumors, and early 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, and 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 hematological cancer.

[0200] 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 have been disclosed in the literature, such as Molecular Cloning: A Laboratory Manual, Second 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 (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Chain Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), which are incorporated herein by reference.

[0201] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0202] Preparation of some materials used in Examples 1-9 of the present disclosure:

[0203] 1) The target genes encoding human PVRIG (UniProt registration number: Q6DKI7) and monkey PVRIG (UniProt registration number: A0A2K5WVV8) were cloned into plasmids containing His, human Fc or mouse Fc, and then transfected into HEK293 cells of the transient expression system for transient expression, thereby obtaining hPVRIG-mFc (abbreviated as R2248), hPVRIG-hFc (abbreviated as R2249), hPVRIG-His (abbreviated as R2250), and cyno PVRIG-His (abbreviated as R2251);

[0204] 2) The target gene encoding human PVRIG (Q6DKI7) was cloned into the plvx lentiviral plasmid, and then the lentivirus was packaged and the titer was tested, and 293T cells were infected for membrane protein expression to obtain a cell line overexpressing hPVRIG: 293T-hPVRIG;

[0205] 3) preparing a positive antibody R2243 of the hIgG4 subtype according to the sequence of SEQ ID No. 81 in International Patent Publication No. WO2021180205A1;

[0206] 4) preparing mIgG1 subtype positive antibody R2247 according to the sequence of "Antibody 46" in U.S. Pat. No. US11279758B2;

[0207] 5) The following proteins were purchased: hPVRL2-His (Cat. No. C440, Novoprotein), hPVRL2-mFc2a (Cat. No. C11A, Novoprotein), Mouse PVRIG-His (Cat. No. PVG-M52H7, Acro);

[0208] 6) The PVRIG reporter gene system was purchased (Cat. No. CBP74120+CBP74121, Nanjing Kebai).

[0209] Example 1. Preparation of anti-human PVRIG antibody

[0210] Balb / c or SJL mice were immunized once a week using hPVRIG-mFc (R2248) and hPVRIG-hFc (R2249) as immunogens. Balb / c mice were immunized 5 times in total, and SJL mice were immunized 4 times. After the immunization, orbital blood was collected to detect the binding of the antibody to hPVRIG-His (R2250) by ELISA binding, and the binding of the antibody to PVRIG reporter gene cells was detected by FACS. The 3 mice with good binding activity were screened out and immunized once more.

[0211] Then, the spleen of the mouse was taken, spleen cells were separated, and mixed with mouse myeloma cells at a ratio of 1:1 at a ratio of 5E6 / plate, and fused under the action of an electric fusion instrument to obtain hybridomas;

[0212] The hybridoma supernatant was taken, 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 binding blocking activity of the antibody to PVRIG receptor ligand (R2249+hPVRL2-His) was detected by ELISA, and the binding of the antibody to cyno-PVRIG-His (R2251) was detected by ELISA. The positive clones were screened for subcloning, 14 positive clones were selected and expanded, the supernatant was collected, and 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:

[0213] Table 1. Anti-human PVRIG antibody variable region sequence list

[0214]

[0215]

[0216]

[0217] Table 2. Anti-human PVRIG antibody CDR sequence list

[0218]

[0219]

[0220] Note: The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the anti-human PVRIG antibody are identified according to the Kabat numbering system.

[0221] 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 a mouse IgG2a heavy chain constant region, and the light chain constant region is a mouse κ chain constant region; the heavy chain constant region of CG004-04, CG004-12, and CG004-13 is a mouse IgG2b heavy chain constant region, and the light chain constant region is a mouse κ chain constant region; the heavy chain constant region of CG004-10 is a mouse IgG1 heavy chain constant region, and the light chain constant region is a mouse κ chain constant region.

[0222] Example 2. ELISA detection of binding activity of anti-human PVRIG antibody to hPVRIG

[0223] ELISA binding assay method: dilute human PVRIG (R2250) to 1 μg / mL (diluent: 50 mM CB), and coat the plate at 100 μL / well overnight; wash 3 times the next morning and pat dry. Add 200 μL / well blocking solution (1% BSA) and block at room temperature for 1 hour; wash the strips once, pat dry, dilute the antibody to be tested 3 times, add 100 μL / well, incubate at room temperature for 1 hour; wash the strips 5 times, pat dry, dilute anti mouse Fc-HRP (1:10000), add 100 μL / well, incubate at room temperature for 40 minutes; wash the strips 6 times, pat dry, add HRP substrate A solution and B solution, add 50 μL / well respectively, incubate at room temperature for 20 minutes; add 100 μL / well stop solution, and read the plate at 450 nm.

[0224] The experimental results are attached. Figure 1 and 2 The experimental results show that the anti-human PVRIG antibodies disclosed in the present invention can specifically bind to human PVRIG and have good antigen binding activity.

[0225] Example 3. FACS detection of binding activity of anti-human PVRIG antibody to hPVRIG

[0226] FACS combined experimental method: cell counting, count the cells of PVRIG reporter gene to be detected, centrifuge at 300g for 5min, resuspend in FCM buffer, adjust the cell density to 4E+06 / 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 times with buffer (3% BSA+1×PBS); add the sample and cells to a 96-well V-shaped plate (the sample and cells are added at 50μL / well respectively) and incubate at 4℃ in the dark for 30min; 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) to a 96-well V-shaped plate (according to 100 μL / well), incubate at 4°C in the dark for 30 min; wash: centrifuge (300g / 5min), discard the supernatant, add 200 μL FCM buffer to wash once, centrifuge (300g / 5min), discard the supernatant; apply to the machine: add 100 μL 1×PBS to resuspend, and apply to the machine for detection.

[0227] The experimental results are attached. Figure 3 and 4,The experimental results show that compared with the positive control antibody R2247, the anti-human PVRIG antibody disclosed in the present invention has better antigen binding activity.

[0228] Example 4. Anti-human PVRIG antibody blocking activity experiment

[0229] The ELISA method was used to evaluate the blocking activity of anti-human PVRIG antibody on hPVRIG-hFc and its ligand.

[0230] Experimental method: Antigen coating: dilute hPVRIG-hFc (R2249) with coating solution to a concentration of 1μg / mL, add 100μL / well to a 96-well ELISA plate, coat two ELISA plates, gently shake to mix, seal with sealing film, and place at 2-8℃ overnight. Washing: Take the ELISA plate, discard the supernatant, and pat dry the residual liquid on absorbent paper; add PBST washing solution, 200μL / well, discard the supernatant, pat dry the residual liquid on absorbent paper; repeat washing 3 times. Blocking: Add 1% BSA blocking solution, 200μL / well, incubate at 37℃ for 1h. Dilute the candidate antibody and positive antibody R2247 with 1XBSA solution 3 times, transfer 70μL of the diluted antibody to a 96-well ELISA plate, and incubate at 37℃ for 60min; then add 70μL / well of the diluted 0.5μg / mL Nectin-2-his ligand protein, and incubate at 37℃ for 60min. Wash: Take the ELISA plate, discard the supernatant, and pat dry the residual liquid on absorbent paper; add PBST washing solution, 200μL / well, discard the supernatant, and pat dry the residual liquid on absorbent paper; repeat washing 3 times. Enzyme-labeled secondary antibody incubation: HRP-anti-his secondary antibody (1:10000) diluted with 1% BSA (PBST) is added to the 1st to 12th columns of the ELISA plate, 100μL / well, and incubate at 37℃ for 40min. Washing: Take the ELISA plate, discard the supernatant, and pat dry the residual liquid on absorbent paper; add PBST washing solution, 200μL / well, discard the supernatant, and pat dry the residual liquid on absorbent paper; repeat washing 3 times. Color development: first add 50μL / well of solution A and then add 50μL / well of solution B, incubate at room temperature and avoid light for 10min. Stop and read the plate: Add stop solution to the ELISA plate, 100μL / well, and immediately read the OD450 absorbance value on the ELISA reader.

[0231] The experimental results are attached. Figure 5 and 6 ,The experimental results show that the anti-human PVRIG antibody disclosed in the present invention can effectively block the binding of human PVRIG to its ligand.

[0232] Example 5. Cross-binding experiment of anti-human PVRIG antibody and mPVRIG / cynoPVRIG

[0233] Experimental method: The day before the experiment, cyno PVRIG (R2251) protein or mouse PVRIG (Acro) protein was diluted to 1 μg / mL, 100 μL / well was added to a 96-well ELISA plate, two ELISA plates were coated, gently shaken to mix, sealed with sealing film, and placed at 2-8°C overnight. Washing: Take the ELISA plate, discard the supernatant, and pat dry the residual liquid on absorbent paper; add PBST washing solution, 200 μL / well, discard the supernatant, pat dry the residual liquid on absorbent paper; repeat washing 3 times. Blocking: Add 1% BSA blocking solution, 200 μL / well, incubate at 37°C for 1h. Dilute the candidate antibody and positive antibody R2247 with 1XBSA solution 3 times, transfer 100 μL of the diluted antibody to the 96-well ELISA plate, and incubate at 37°C for 60min. Washing: Take the ELISA plate, discard the supernatant, and pat dry the residual liquid on absorbent paper; add PBST washing solution, 200μL / well, discard the supernatant, and pat dry the residual liquid on absorbent paper; repeat washing 3 times. Enzyme-labeled secondary antibody incubation: Dilute the goat anti-mouse Fc enzyme-labeled secondary antibody (1:15000) with 1% BSA (PBST), add to the 1-12 columns of the ELISA plate, 100μL / well, and incubate at 37℃ for 40min. Washing: Take the ELISA plate, discard the supernatant, and pat dry the residual liquid on absorbent paper; add PBST washing solution, 200μL / well, discard the supernatant, and pat dry the residual liquid on absorbent paper; repeat washing 3 times. Color development: Mix solution A and solution B in a 1:1 ratio, add to the ELISA plate, 100μL / well, and incubate at room temperature in the dark for 10min. Stop and read the plate: Add stop solution to the ELISA plate, 100 μL / well, and immediately read the OD450 absorbance value on a ELISA reader.

[0234] The experimental results are attached. Figure 7-10 ,The experimental results show that the anti-human PVRIG antibodies disclosed in the present invention can bind to cynoPVRIG and mouse PVRIG, and are superior to the positive antibody R2247.

[0235] Example 6. Reporter gene assay activity detection

[0236] PVRIG (CD112R) is a regulatory protein expressed on T cells. When it binds to the ligand PVRL2 (CD112, Nectin-2) expressed on DCs or tumor cells, it can inhibit the immune activation of T cells. Luciferase reporter gene (Luc) is a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase. Luciferase can catalyze the oxidation of luciferin to oxyluciferin, and during the oxidation of luciferin, bioluminescence is emitted. Therefore, by using PVRIG Effector Reporter Cell and CD112 / TCR Activator / CHO Cell, a reporter gene activity detection method with a better detection window is designed to verify whether the candidate antibody has the function of inhibiting the interaction between PVRIG and PVRL2.

[0237] Experimental method: Cell count, take an appropriate amount of CHO cells expressing PVRIG ligand, centrifuge at 300g for 5min, discard the supernatant, wash once with F12K+10% FBS medium, resuspend with F12K+10% FBS medium and adjust the cell density to 2E+05 cells / mL, add 2E+04 cells / well, i.e. 100μL / well; incubate in a carbon dioxide incubator overnight. The next morning, dilute the sample (1640+10% FBS medium for dilution). Count the cells, take an appropriate amount of PVRIG reporter gene cells, centrifuge at 300g for 5min, discard the supernatant, wash once with 1640+10% FBS medium, resuspend with 1640+10% FBS medium and adjust the PVRIG cell density to 1.6E+06 / mL. According to the experimental design, discard the F12K+10% FBS medium in a 96-well white cell culture flat-bottom plate, add 50μL of antibody diluent to each well, add PVRIG cells at 8E+04 / well, i.e. 50μL / well, and add a certain volume of 1640+10% FBS medium to the corresponding wells, so that the total volume of all wells is 100μl. Place the cell culture plate in a CO2 incubator and incubate for 6h. Thaw the Bright-LumiTM firefly luciferase detection reagent in advance and equilibrate to room temperature. Take out the cell culture plate and equilibrate at room temperature for 10min (not more than 30min). Add 100 μL Bright-LumiTM Firefly Luciferase Detection Reagent to each well and incubate at room temperature for 5 min. Perform chemiluminescence detection on a microscope.

[0238] The experimental results are attached. Fig.11 ,The experimental results show that the anti-human PVRIG antibody disclosed in the present invention can inhibit the interaction between PVRIG and PVRL2.

[0239] Example 7. Preparation and evaluation of humanized PVRIG mouse-derived conventional antibodies

[0240] The hIgG4 (S228P / F234A / L235A / K447A) subtype chimeric antibody R3014 was prepared using the mouse 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).

[0241] The selected sequence was subjected to three-dimensional homology modeling, and was evaluated as an acceptable structure by methods such as the pull-through diagram. The antibody sequence was compared with the antibody GermLine database to obtain a human germline template (IGKV4-1*01) with high homology. The CDR was transplanted into the corresponding human template. The transplanted antibody was simulated and analyzed again in three dimensions, and the buried residues, residues that directly interacted with the CDR region, and residues that had an important effect on the conformation of the variable region were reversely transformed, and the chemically unstable amino acid residues in the CDR region were optimized to generate a series of humanized antibody sequences. A series of humanized molecules of the hIgG4 (S228P / F234A / L235A / K447A) subtype were prepared: R3942-R3950, the amino acid sequence of the heavy chain variable region of the antibody R3942-R3950 is shown in Table 3, the amino acid sequence of the light chain variable region is shown in Table 4, and the combination of the heavy chain variable region and the light chain variable region is shown in Table 5.

[0242] Table 3 Humanized PVRIG mouse antibody heavy chain sequence

[0243]

[0244] Table 4 Humanized PVRIG mouse antibody light chain sequence

[0245]

[0246] Table 5 Combinations of heavy chain variable regions and light chain variable regions of humanized PVRIG mouse antibody

[0247] serial number Heavy chain Light chain R3942 SEQ ID NO.100 SEQ ID NO.99 R3943 SEQ ID NO.100 SEQ ID NO.105 R3944 SEQ ID NO.101 SEQ ID NO.99 R3945 SEQ ID NO.101 SEQ ID NO.105 R3946 SEQ ID NO.102 SEQ ID NO.99 R3947 SEQ ID NO.102 SEQ ID NO.105 R3948 SEQ ID NO.103 SEQ ID NO.99 R3949 SEQ ID NO.103 SEQ ID NO.105 R3950 SEQ ID NO.104 SEQ ID NO.99

[0248] Example 8. Evaluation of reporter gene activity of humanized PVRIG mouse antibody

[0249] The activity of humanized PVRIG mouse antibodies R3942-R3950 was evaluated by reporter gene method. The evaluation method was the same as Example 6. The results are as follows: Fig.12 As shown. R3014 is the parent molecule, and R3942-R3950 are humanized molecules. Among the humanized molecules, R3944 showed better reporter gene assay activity than the parent R3014.

[0250] Example 9. Binding activity of humanized PVRIG nanobodies to 293T-hPVRIG cells and 293T-cynoPVRIG cells

[0251] The binding activity of humanized PVRIG nanobodies R3942-R3950 to 293T-hPVRIG cells and 293T-cynoPVRIG cells was evaluated using the same evaluation method as in Example 5. Fig.13 and Fig.14 As shown, the binding activity of the humanized molecule R3944 to 293-hPVRIG cells is close to that of the parent R3014.

[0252] The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

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; Optionally, 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; Optionally, (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.

4. The anti-human PVRIG antibody according to any one of claims 1 to 3, 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.

5. The anti-human PVRIG antibody according to any one of claims 1 to 4, wherein the antibody is a full-length antibody or any one antigen-binding fragment selected from F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv and scFv.

6. An antibody that competitively binds to human PVRIG with the anti-human PVRIG antibody according to any one of claims 1 to 5, or an antibody that binds to the same epitope as the anti-human PVRIG antibody according to any one of claims 1 to 5.

7. The anti-human PVRIG antibody according to any one of claims 1 to 6, 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.

8. A biomaterial, characterized in that The biological material includes any one of the following (i) to (v): (i) a multispecific antibody, comprising the anti-human PVRIG antibody according to any one of claims 1 to 7; (ii) an antibody conjugate, the antibody conjugate comprising the anti-human PVRIG antibody according to any one of claims 1 to 7; (iii) an isolated nucleic acid encoding the anti-human PVRIG antibody according to any one of claims 1 to 7 or encoding the multispecific antibody according to (i); (iv) an expression vector comprising the isolated nucleic acid described in (iii); (v) A cell, characterized in that the cell carries the isolated nucleic acid described in (iii) and the expression vector described in (iv).

9. A pharmaceutical composition comprising the anti-human PVRIG antibody according to any one of claims 1 to 7 and the biomaterial according to claim 8; optionally, it further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

10. Use of the anti-human PVRIG antibody according to any one of claims 1 to 7, the biomaterial according to claim 8 or the pharmaceutical composition according to claim 9 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.

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