Anti-human PVRIG antibodies and uses thereof

Abstract

The present disclosure relates to the field of biotechnology, and discloses an anti-human PVRIG antibody and application thereof. The anti-human PVRIG antibody disclosed by the present disclosure specifically binds to a human PVRIG antigen, and can be used for tumor treatment.

Description

[0001] Priority Statement

[0002] This application claims priority to Chinese Patent Application No. 202311579153.1, filed on November 23, 2023, entitled "Anti-human PVRIG Antibody and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biotechnology, and more specifically, to an anti-human PVRIG antibody and its applications. Background Technology

[0004] PVRIG (poliovirus receptor-related immunoglobulin domain-containing, also known as CD112R) is a novel inhibitory receptor with PVRL2 (poliovirus receptor-related2, also known as CD112) as its ligand. On the one hand, PVRIG can compete with the activating receptor CD226 for binding to the ligand PVRL2 with higher affinity, thereby weakening T cell activation. On the other hand, after binding to the ligand, it will inhibit T cell activity through intracellular inhibitory motifs.

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

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

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

[0008] In some embodiments, this disclosure discloses an anti-human PVRIG antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID Nos. 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 a ... No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 99 or 105 have LCDR1, LCDR2 and LCDR3 in the variable regions with at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity;

[0009] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, wherein,

[0010] A. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 1, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 2;

[0011] B. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 3, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 4;

[0012] C. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 5, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 6;

[0013] D. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 7, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 8;

[0014] E. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 9, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 10;

[0015] F. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 11, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 12;

[0016] G. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 13, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 14;

[0017] H. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 15, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 16;

[0018] I. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 17, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 18;

[0019] J. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 19, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 20;

[0020] K. The heavy chain variable region of the antibody includes HCDR1, HCDR2, and HCDR3 as described in SEQ ID No. 21, and the light chain variable region of the antibody includes LCDR1, LCDR2, and LCDR3 as described in SEQ ID No. 22; or

[0021] L. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 23, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 24.

[0022] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, 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.

[0023] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering system.

[0024] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the IMGT numbering system.

[0025] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, wherein,

[0026] a. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO.25, HCDR2 contains the amino acid sequence of SEQ ID NO.26, and HCDR3 contains the amino acid sequence of SEQ ID NO.27; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO.28, LCDR2 contains the amino acid sequence of SEQ ID NO.29, and LCDR3 contains the amino acid sequence of SEQ ID NO.30;

[0027] b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO.31, HCDR2 contains the amino acid sequence of SEQ ID NO.32, and HCDR3 contains the amino acid sequence of SEQ ID NO.33; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO.34, LCDR2 contains the amino acid sequence of SEQ ID NO.35, and LCDR3 contains the amino acid sequence of SEQ ID NO.36;

[0028] c. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO.37, HCDR2 contains the amino acid sequence of SEQ ID NO.38, and HCDR3 contains the amino acid sequence of SEQ ID NO.39; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO.34, LCDR2 contains the amino acid sequence of SEQ ID NO.35, and LCDR3 contains the amino acid sequence of SEQ ID NO.36;

[0029] d. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO.40, HCDR2 contains the amino acid sequence of SEQ ID NO.41, and HCDR3 contains the amino acid sequence of SEQ ID NO.42; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO.43, LCDR2 contains the amino acid sequence of SEQ ID NO.44, and LCDR3 contains the amino acid sequence of SEQ ID NO.45;

[0030] e. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO.46, HCDR2 contains the amino acid sequence of SEQ ID NO.47, and HCDR3 contains the amino acid sequence of SEQ ID NO.48; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO.49, LCDR2 contains the amino acid sequence of SEQ ID NO.50, and LCDR3 contains the amino acid sequence of SEQ ID NO.51;

[0031] f. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO.46, HCDR2 contains the amino acid sequence of SEQ ID NO.52, and HCDR3 contains the amino acid sequence of SEQ ID NO.48; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO.49, LCDR2 contains the amino acid sequence of SEQ ID NO.50, and LCDR3 contains the amino acid sequence of SEQ ID NO.51;

[0032] g. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO. 53, HCDR2 contains the amino acid sequence of SEQ ID NO. 54, and HCDR3 contains the amino acid sequence of SEQ ID NO. 55; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO. 49, LCDR2 contains the amino acid sequence of SEQ ID NO. 56, and LCDR3 contains the amino acid sequence of SEQ ID NO. 57;

[0033] h. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO. 58, HCDR2 contains the amino acid sequence of SEQ ID NO. 59, and HCDR3 contains the amino acid sequence of SEQ ID NO. 60; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO. 49, LCDR2 contains the amino acid sequence of SEQ ID NO. 61, and LCDR3 contains the amino acid sequence of SEQ ID NO. 62;

[0034] i. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO. 63, HCDR2 contains the amino acid sequence of SEQ ID NO. 64, and HCDR3 contains the amino acid sequence of SEQ ID NO. 65; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO. 66, LCDR2 contains the amino acid sequence of SEQ ID NO. 67, and LCDR3 contains the amino acid sequence of SEQ ID NO. 68;

[0035] j. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO.69, HCDR2 contains the amino acid sequence of SEQ ID NO.70, and HCDR3 contains the amino acid sequence of SEQ ID NO.71; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO.72, LCDR2 contains the amino acid sequence of SEQ ID NO.29, and LCDR3 contains the amino acid sequence of SEQ ID NO.73;

[0036] k. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO. 74, HCDR2 contains the amino acid sequence of SEQ ID NO. 75, and HCDR3 contains the amino acid sequence of SEQ ID NO. 76; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO. 28, LCDR2 contains the amino acid sequence of SEQ ID NO. 77, and LCDR3 contains the amino acid sequence of SEQ ID NO. 78; or

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

[0038] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, 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 implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0042] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, wherein,

[0043] The heavy chain variable region of the antibody includes an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 100-104, and / or the light chain variable region includes an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 99, or 105.

[0044] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, 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 with 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 with SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 99, or 105.

[0046] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, 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 contains the amino acid sequence of SEQ ID NO.7, and the light chain variable region contains 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 contains the amino acid sequence of SEQ ID NO.23, and the light chain variable region contains the amino acid sequence of SEQ ID NO.24;

[0059] (m) The heavy chain variable region contains the amino acid sequence of SEQ ID NO.104, and the light chain variable region contains the amino acid sequence of SEQ ID NO.99;

[0060] (n) The heavy chain variable region contains the amino acid sequence of SEQ ID NO.100, and the light chain variable region contains 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 contains the amino acid sequence of SEQ ID NO.101, and the light chain variable region contains 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 contains the amino acid sequence of SEQ ID NO.102, and the light chain variable region contains 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 contains the amino acid sequence of SEQ ID NO.103, and the light chain variable region contains the amino acid sequence of SEQ ID NO.105.

[0068] In some embodiments, such as the anti-human PVRIG antibody described in any of the preceding embodiments, 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 preceding embodiments, (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 implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, the antibody contains a constant region.

[0092] In some embodiments, such as the anti-human PVRIG antibody described in any of the preceding embodiments, the heavy chain constant region of the antibody is selected from the heavy chain constant regions 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 implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, the species origin of the constant region is mouse or human;

[0094] In some embodiments, such as the anti-human PVRIG antibody described in any of the preceding embodiments, 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, such as the anti-human PVRIG antibody described in any of the preceding embodiments, the antibody is a full-length antibody or any antigen-binding fragment selected from F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv, and scFv.

[0096] In some embodiments, the invention discloses antibodies that competitively bind to human PVRIG, or antibodies that bind to the same epitope as the anti-human PVRIG antibodies described in any of the preceding claims.

[0097] In some embodiments, such as the anti-human PVRIG antibody described in any of the preceding embodiments, said 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 this 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 this 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 this application; and / or

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

[0102] This disclosure also provides a multispecific antibody comprising the anti-human PVRIG antibody described in any of the preceding embodiments. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a trispecific antibody.

[0103] This disclosure also provides an antibody-drug conjugate comprising the anti-human PVRIG antibody described in any of the preceding embodiments. In some embodiments, the antibody-drug conjugate further comprises a therapeutic agent or imaging agent conjugated to the antibody; in some embodiments, the antibody-drug conjugate further comprises biotin or a biotin derivative conjugated to the antibody; in some embodiments, the antibody-drug conjugate further comprises a solid-phase support conjugated to the antibody; in some embodiments, the antibody-drug 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] This disclosure also provides an isolated nucleic acid encoding the anti-human PVRIG antibody described in any of the preceding claims.

[0105] This disclosure also provides a cell containing the nucleic acid described in any of the preceding claims.

[0106] This disclosure also provides a pharmaceutical composition comprising: an anti-human PVRIG antibody as described in any of the preceding claims, a multispecific antibody, an antibody-drug conjugate, a cell, or a nucleic acid. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0107] This disclosure also provides the use of the anti-human PVRIG antibody, multispecific antibody, antibody-drug conjugate, cell, nucleic acid, or pharmaceutical composition described in any of the preceding claims 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 human PVRIG-associated disease is a tumor. In some embodiments, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, and hematologic malignancies.

[0108] In some embodiments, this disclosure provides a method of treating a disease, the method comprising administering to a subject in need a therapeutically effective amount of any of the preceding anti-human PVRIG antibodies, multispecific antibodies, antibody-drug conjugates, cells, nucleic acids, or pharmaceutical compositions. In some embodiments, the disease is a human PVRIG-associated disease. In some embodiments, the human PVRIG-associated disease is a tumor. In some embodiments, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, and hematologic malignancies.

[0109] In another aspect, this disclosure also provides anti-human PVRIG antibodies, multispecific antibodies, antibody-drug conjugates, cells, nucleic acids, or pharmaceutical compositions as described in any of the foregoing embodiments for use as medicaments. In some embodiments, the medicament is used to treat a disease. In some embodiments, the medicament is used to detect human PVRIG expression or to treat diseases associated with high human PVRIG expression. In some embodiments, the human PVRIG high expression-associated disease is a tumor. In some embodiments, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, and hematologic malignancies.

[0110] In some embodiments, the treatment described in any of the preceding embodiments further includes administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent.

[0111] This disclosure also provides a method for detecting or determining human PVRIG, the method comprising the step of detecting or determining human PVRIG using an anti-human PVRIG antibody or antibody-drug conjugate as described in any of the preceding claims.

[0112] This disclosure also provides a kit comprising an anti-human PVRIG antibody or antibody-drug conjugate as described in any of the preceding claims; in some embodiments, the kit is used to detect or determine human PVRIG.

[0113] This disclosure also provides a method for preparing an anti-human PVRIG antibody as described in the preceding claim, comprising the steps of culturing cells as described in the preceding claim, and then isolating and purifying to obtain the anti-human PVRIG antibody.

[0114] The anti-human PVRIG antibody disclosed herein can specifically bind to human PVRIG; in some embodiments, the anti-human PVRIG antibody can specifically bind to mouse PVRIG and / or monkey PVRIG; the anti-human PVRIG antibody can inhibit the binding activity of PVRIG to PVRL2; and / or the anti-human PVRIG antibody can inhibit tumor growth. Attached Figure Description

[0115] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

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

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

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

[0119] Figure 4 : This is a graph showing the FACS detection results of the binding of the disclosed anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, R2247) to hPVRIG;

[0120] Figure 5 : This is a diagram showing the experimental results of blocking the binding of human PVRIG to its ligands by the anti-human PVRIG antibodies (CG004-01, CG004-02, CG004-04, CG004-05, CG004-06, CG004-07, R2247) disclosed in this publication;

[0121] Figure 6 : This is a diagram showing the experimental results of blocking the binding of human PVRIG to its ligands by the anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, R2247) disclosed in this publication;

[0122] Figure 7 : This is a figure showing the experimental results of the binding of the disclosed anti-human PVRIG antibodies (CG004-01, CG004-04, CG004-05, CG004-06, CG004-07, R2247) to cynoPVRIG;

[0123] Figure 8 : This is a graph showing the experimental results of the binding of the disclosed anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, R2247) to cynoPVRIG;

[0124] Figure 9 : This is a figure showing the experimental results of binding of the disclosed anti-human PVRIG antibodies (CG004-01, CG004-04, CG004-05, CG004-06, CG004-07, R2247) to mouse PVRIG;

[0125] Figure 10 : This is a figure showing the experimental results of binding of the disclosed anti-human PVRIG antibodies (CG004-09, CG004-10, CG004-11, CG004-12, CG004-13, CG004-14, R2247) to mouse PVRIG;

[0126] Figure 11 : This is a diagram showing the experimental results of the anti-human PVRIG antibody inhibiting the interaction between PVRIG and PVRL2 reporter genes disclosed in this publication;

[0127] Figure 12 : This is a diagram showing the activity results of the anti-human PVRIG antibody activating T cells disclosed in this publication;

[0128] Figure 13 This is a diagram showing the results of the binding experiment between the humanized anti-human PVRIG antibody (R3942-R3950) and 293T-hPVRIG cells.

[0129] Figure 14 Figure : This is a diagram showing the results of the binding experiment between the humanized anti-human PVRIG antibody (R3942-R3950) disclosed in this publication and 293T-cynoPVRIG cells. Detailed Implementation

[0130] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. However, the description and embodiments should not be construed as limiting the scope of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Unless expressly defined in this disclosure, the technical and scientific terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0131] In this disclosure, the terms "a / an," "an," "the," "the," and "the foregoing" include plural references unless the context clearly indicates otherwise. For example, "an antibody" means one or more antibodies. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. The term "multiple" means at least two, such as 2, 3, etc., unless otherwise explicitly stated otherwise.

[0132] Unless the context clearly indicates otherwise, the words “comprising” or “having” in this disclosure and in the claims should be understood as meaning “including” rather than “exclusive” or “exhaustive”; that is, meaning “including but not limited to”. For example, “comprising A” means “including A, but not limited to A”.

[0133] "About" means within an acceptable range of error 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 measurement, result, or implementation, unless otherwise expressly stated in the embodiments or elsewhere in the specification, "about" means within one standard deviation, or up to 5%, according to convention in the art.

[0134] "Optional," "optionally," "maybe," or "optionally" means that the event or circumstance described below may but not necessarily occur; that is, it includes situations where the event or circumstance may or may not occur, unless the context clearly indicates otherwise.

[0135] The term "and / or" refers to each of two specific features or a combination thereof, which may or may not contain the other specific feature. For example, "A and / or B" means including any of the following: A and B, A or B, A (alone) and B (alone); "A, B and / or C" means covering any of the following: 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 and single-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 mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0138] The term "antibody" in this disclosure is used in the broadest sense and encompasses a wide variety of 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 called antigen-binding portions or antibody fragments), etc., as long as they exhibit the desired antigen-binding activity.

[0139] "Natural antibodies" are naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N to the C-terminus, each heavy chain of an IgG antibody has a heavy chain variable region (VH, also called a variable heavy domain or heavy chain variable region), followed by a heavy chain constant region (CH, also called a constant heavy domain), which includes three constant domains (CH1, CH2, and CH3); similarly, from the N to the C-terminus, each light chain has a light chain variable region (VL, also called a variable light domain or light chain variable region), followed by a light chain constant region (CL, also called a constant light domain). Based on whether an antibody contains α, δ, ε, γ, and μ heavy chains, antibodies can be classified into five isotypes: IgA, IgD, IgE, IgG, and IgM. Isotype antibodies can be further divided into different subtypes. For example, the IgG isotype includes four subtypes: IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), and IgG4 (γ4 heavy chain). The IgA isotype is divided into two subtypes: IgA1 (α1 heavy chain) and IgA2 (α2 heavy chain).

[0140] The terms "full-length antibody," "intact antibody," and "all antibody" used in this disclosure are used interchangeably to refer to antibodies having a structure substantially similar to that of natural antibodies, or antibodies containing a heavy chain with an Fc region.

[0141] "Separated" antibodies are antibodies that have been separated from components of their natural environment. In some embodiments, antibodies can be purified to a purity greater than 90% or 99%, and can be purified and determined by methods such as 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 in the heavy or light chain of an antibody that is involved in antibody binding to the antigen. Natural antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), each of which contains four conserved framework regions (FRs) and three hypervariable regions (also known as complementarity-determining regions, HVRs, or CDRs).

[0143] The terms "complementarity-determining region," "hypervariant region," or "CDR" refer to the main region within the variable domain that facilitates antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. A VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; a VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL typically consists of three CDRs and four FRs arranged in the following order (from the amino terminus to the carboxyl terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid sequence boundaries of CDRs can be determined using methods well known to those skilled in the art, such as the "Kabat" numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering system, the "ABM" numbering system, the "contact" numbering system (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering system (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 the variable region (VL) and the constant region (CL) of the light chain. The VL is located at the amino terminus of the light chain, and the CL is located at the carboxylamino terminus of the light chain. The light chain can be a κ chain or a λ chain, etc.

[0145] The term "heavy chain" includes the variable region (VH) and the constant region (CH). The variable region is located at the amino terminus of the heavy chain, while the constant region is located at the carboxyl-amino terminus. The constant region of the IgG antibody heavy chain includes three constant region domains: CH1, CH2, and CH3. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgD, IgM, and IgE, etc.

[0146] The term "constant region" or "constant domain" refers to the carboxyl-terminal portion of the light and heavy chains that does not directly participate in the binding of antibodies to antigens. Compared to VH and VL, the constant region has a relatively conserved amino acid sequence.

[0147] The term "antigen-binding fragment" refers to a portion containing a complete antibody, but unlike the complete antibody, this portion specifically binds to the antigen bound by the complete antibody. 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 (e.g., scFv), bispecific antibodies, linear antibodies, and multispecific antibodies formed from antigen-binding fragments. Specifically, "Fab" is a monovalent fragment composed of VL, VH, CL, and CH1 domains; "Fv" consists of VH and VL; "Fab'" is a Fab fragment containing a portion of the hinge region; and "F(ab')2" contains two F1 fragments connected by disulfide bonds in the hinge region. The bivalent fragment of the ab' fragment; "scFab" is a polypeptide composed 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 containing a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are connected by peptide linkages. The head linker enables expression as a single-chain polypeptide, and scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFv may have VL and VH variable regions in any order, such as the N-terminus to C-terminus of the scFv polypeptide, which may contain: a) VL-linker-VH or b) VH-linker-VL; “dsFv” is a disulfide-stabilized Fv fragment; (dsFv)2 is a dimerized dsFv; “Fab'-SH” is a cysteine ​​residue in the hinge region of the Fab' fragment carrying a free thiol group.

[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 the human IgG heavy chain extends from an amino acid residue at position Cys226 to its carboxyl terminus. In some embodiments, the Fc region of the human IgG heavy chain extends from Pro230 to its carboxyl terminus. The C-terminus of the Fc region can also be, for example, lacking the C-terminal lysine residue (residue 447 according to the EU numbering system) or lacking both the C-terminal glycine and lysine residues (residues 446 and 447 according to the EU numbering system). In some embodiments, the composition of intact antibodies may include a population of antibodies lacking the K447 residue and / or G446+K447 residue. In some embodiments, the composition of intact antibodies may include a population of antibodies without the lack of the K447 residue and / or G446+K447 residue. In some embodiments, the composition of intact antibodies has a population of antibodies containing and without a mixture of K447 residues and / or G446+K447 residues. The Fc region used in the antibodies described in this disclosure includes 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 edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0149] The term "chimeric" refers to antibodies in which a portion of the heavy and / or light chains is derived from a particular species, while the remainder of the heavy and / or light chains is derived from other species.

[0150] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in the human body. For example, this can be achieved by retaining the CDR region of a non-human antibody and replacing the rest of the antibody with its human counterpart (i.e., the framework region of the heavy chain variable region, the framework region of the light chain variable region, and the constant region of the human antibody).

[0151] The terms "human antibody," "human-derived antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. Human antibodies include antibodies derived from human genes but with altered cysteine ​​or glycosylation sites. Human antibodies can be recombinant antibodies produced in non-human cells. Human antibodies include antibodies obtained from transgenic mice containing human immunoglobulin heavy and light chain loci.

[0152] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its bound ligand (e.g., an antigen). Unless otherwise specified, as in this disclosure, "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y is typically expressed as a dissociation constant (KD). Affinity can be measured using conventional methods known in the art. The terms "kassoc" or "ka" refer to the association rate of a particular antibody-antigen interaction, and the terms "kdis" or "kd" refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka), and is typically expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. Methods for determining antibody KD include measuring surface plasmon resonance using a biosensing system, such as a system, or measuring affinity in solution by solution equilibrium titration (SET).

[0153] The term "affinity-matured" antibody refers to an antibody with one or more amino acid residue alterations in one or more CDRs, resulting in improved affinity 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) describes affinity maturation via VH and VL domain shuffling. The following literature records the random mutagenesis of CDR and / or framework residues: 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 biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with a mutated amino acid sequence). 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 group of antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence (except for the possible small number of naturally occurring mutations). In contrast, polyclonal antibodies typically contain different antibodies with variable domains having different amino acid sequences, and they are usually specific to different epitopes and / or different antigens. "Monoclonal" indicates an antibody obtained from a substantially homogeneous group of antibodies. Monoclonal antibodies can be produced by methods known in the art, for example, by hybridoma methods (Kohler et al., (1975) Nature 256:495), by recombinant DNA methods (see, for example, US Patent No. 4,816,567), by isolation methods from phage antibody libraries (Clackson et al., (1991) Nature 352:624-628 and Marks et al., (1991) J. Mol. Biol. 222:581-597), and also by 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 antigen-binding proteins (such as antibodies). An antigen may have one or more epitopes that interact with different antigen-binding proteins (such as antibodies).

[0157] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody. Epitopes are typically composed of antigenic determinants consisting of specific chemical groups with a defined structure. Epitopes can be formed from consecutive amino acid residues (linear epitopes) or from discontinuous amino acid residues (conformal epitopes), such as spatially close discontinuous amino acid residues formed due to antigen folding. In some embodiments, an 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. Epitopes 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 assays (e.g., nuclear magnetic resonance spectroscopy). Antibodies that bind to specific epitopes (i.e., those that bind to the same epitope) can be obtained using methods known in the art, including but not limited to, alanine scanning, Western 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] "Antibody that binds to the same epitope as the reference antibody" or "antibody that competes with the reference antibody for binding" refers to an antibody that, in a competitive assay, blocks the binding of the reference antibody to the antigen by 50% or more, or whose binding to the antigen is blocked by the reference antibody by 50% or more. To determine whether a test antibody binds to the same epitope as the reference antibody, the ability of the reference antibody to bind to the antigen can be tested under saturation conditions. For example, after removing excess reference antibody, the ability of the test antibody to bind to the antigen can be evaluated. If the test antibody can 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 cannot 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 an antibody to be tested binds to the same epitope or is only hindered from binding by steric reasons, a conventional assay (e.g., peptide mutation and detection using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative method known in the art) can be used. If, in both settings (i.e., both antibodies are used as saturating antibodies), only the saturating antibody can bind to the antigen, it can be concluded that the antibody to be tested and the reference antibody compete for binding to the antigen. In some embodiments, in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 50 (1990) 1495-1502), if an excess (e.g., 1, 5, 10, 20, or 100 times the amount) of one 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, the two antibodies are considered to bind to the same or overlapping epitopes.

[0159] The term "specific binding" or "specifically bound" refers to a non-random binding between two molecules. For example, an antibody binds to the antigen or epitope it corresponds to with a higher affinity than it binds to other antigens or epitopes. Typically, antibodies bind with an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or about 1×10 -12 The equilibrium dissociation constant (KD) of an antibody (M or less) binds to an antigen or an epitope within the antigen. In some embodiments, the KD of antibody binding to an antigen is 10%, 1%, or less of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). The KD value can be measured using methods known in the art, such as by... Surface plasmon resonance assay. Antibodies that specifically bind to antigens or epitopes within antigens may exhibit cross-reactivity with other related antigens, for example, cross-reactivity with corresponding antigens from other species (such as humans or monkeys, such as cynomolgus (Cyno cyno) and chimpanzee (Chimpanzee)) or common marmoset (Callithrix jacchus (Marmoset)).

[0160] The terms "anti-human PVRIG antibody" and "PVRIG-binding antibody" refer to antibodies capable of binding to PVRIG with sufficient affinity. In one embodiment, the anti-human PVRIG antibody binds to non-PVRIG proteins to a degree less than about 10%, 1%, or less of its binding to PVRIG, said binding can be achieved through... Measured by surface plasmon resonance assay. In some embodiments, the antibody binding to PVRIG has the following dissociation constant (KD): < 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 conserved PVRIG epitopes from PVRIGs of different species.

[0161] In some embodiments, such as the anti-human PVRIG antibody described in any of the preceding claims, 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 a variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 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 variable regions of IDNo. ​​2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity. In some embodiments, the present disclosure discloses an anti-human PVRIG antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of 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 of 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 present disclosure discloses an anti-human PVRIG antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of 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 of 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 present disclosure discloses an anti-human PVRIG antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of 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 of 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, as described in any of the preceding anti-human PVRIG antibodies, 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, as described in any of the preceding anti-human PVRIG antibodies, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering system.

[0163] In some implementations, such as the anti-human PVRIG antibody described in any of the preceding embodiments, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering system, wherein

[0164] a. The amino acid sequences of HCDR1 (e.g., SEQ ID NO. 25), HCDR2 (e.g., SEQ ID NO. 26), and HCDR3 (e.g., SEQ ID NO. 27) of the heavy chain variable region; and the amino acid sequences of LCDR1 (e.g., SEQ ID NO. 28), LCDR2 (e.g., SEQ ID NO. 29), and LCDR3 (e.g., SEQ ID NO. 30) of the light chain variable region;

[0165] b. The amino acid sequences of HCDR1 (SEQ ID NO. 31), HCDR2 (SEQ ID NO. 32), and HCDR3 (SEQ ID NO. 33) of the heavy chain variable region; and the amino acid sequences of LCDR1 (SEQ ID NO. 34), LCDR2 (SEQ ID NO. 35), and LCDR3 (SEQ ID NO. 36) of the light chain variable region;

[0166] c. The amino acid sequences of HCDR1 (e.g., SEQ ID NO. 37), HCDR2 (e.g., SEQ ID NO. 38), and HCDR3 (e.g., SEQ ID NO. 39) of the heavy chain variable region; and the amino acid sequences of LCDR1 (e.g., SEQ ID NO. 34), LCDR2 (e.g., SEQ ID NO. 35), and LCDR3 (e.g., SEQ ID NO. 36) of the light chain variable region;

[0167] d. The amino acid sequences of HCDR1 (SEQ ID NO. 40), HCDR2 (SEQ ID NO. 41), and HCDR3 (SEQ ID NO. 42) of the heavy chain variable region; and the amino acid sequences of LCDR1 (SEQ ID NO. 43), LCDR2 (SEQ ID NO. 44), and LCDR3 (SEQ ID NO. 45) of the light chain variable region;

[0168] e. The amino acid sequences of HCDR1 (e.g., SEQ ID NO. 46), HCDR2 (e.g., SEQ ID NO. 47), and HCDR3 (e.g., SEQ ID NO. 48) of the heavy chain variable region; and the amino acid sequences of LCDR1 (e.g., SEQ ID NO. 49), LCDR2 (e.g., SEQ ID NO. 50), and LCDR3 (e.g., SEQ ID NO. 51) of the light chain variable region;

[0169] f. The amino acid sequences of HCDR1 (e.g., SEQ ID NO. 46), HCDR2 (e.g., SEQ ID NO. 52), and HCDR3 (e.g., SEQ ID NO. 48) of the heavy chain variable region; and the amino acid sequences of LCDR1 (e.g., SEQ ID NO. 49), LCDR2 (e.g., SEQ ID NO. 50), and LCDR3 (e.g., SEQ ID NO. 51) of the light chain variable region;

[0170] g. The amino acid sequences of HCDR1 (e.g., SEQ ID NO. 53), HCDR2 (e.g., SEQ ID NO. 54), and HCDR3 (e.g., SEQ ID NO. 55) of the heavy chain variable region; and the amino acid sequences of LCDR1 (e.g., SEQ ID NO. 49), LCDR2 (e.g., SEQ ID NO. 56), and LCDR3 (e.g., SEQ ID NO. 57) of the light chain variable region;

[0171] h. The amino acid sequences of HCDR1 (e.g., SEQ ID NO. 58), HCDR2 (e.g., SEQ ID NO. 59), and HCDR3 (e.g., SEQ ID NO. 60) of the heavy chain variable region; and the amino acid sequences of LCDR1 (e.g., SEQ ID NO. 49), LCDR2 (e.g., SEQ ID NO. 61), and LCDR3 (e.g., SEQ ID NO. 62) of the light chain variable region;

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

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

[0174] k. The amino acid sequences of HCDR1 (e.g., SEQ ID NO. 74), HCDR2 (e.g., SEQ ID NO. 75), and HCDR3 (e.g., SEQ ID NO. 76) of the heavy chain variable region; and the amino acid sequences of LCDR1 (e.g., SEQ ID NO. 28), LCDR2 (e.g., SEQ ID NO. 77), and LCDR3 (e.g., SEQ ID NO. 78) of the light chain variable region; or

[0175] l. The heavy chain variable region HCDR1 as in SEQ ID NO. 79, HCDR2 as in SEQ ID NO. 80, and HCDR3 as in SEQ ID NO. 81; and the light chain variable region LCDR1 as in SEQ ID NO. 82, LCDR2 as in SEQ ID NO. 83, and LCDR3 as in SEQ ID NO. 68.

[0176] In some embodiments, the anti-human PVRIG antibody as described in any of the preceding embodiments, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the IMGT numbering system; in some embodiments, wherein the heavy chain variable region HCDR1 comprises the amino acid sequence GNTFSDSY (SEQ ID NO. 84), HCDR2 comprises the amino acid sequence INPYNGDT (SEQ ID NO. 85), and HCDR3 comprises the amino acid sequence ARSNWDVGNCFDY (SEQ ID NO. 86); and the light chain variable region LCDR1 comprises the amino acid sequence QDISNY (SEQ ID NO. 87), LCDR2 comprises the amino acid sequence YTS (SEQ ID NO. 88), and LCDR3 comprises the amino acid sequence SEQ ID NO. 30.

[0177] In some embodiments, the anti-human PVRIG antibody as described in any of the preceding embodiments, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Chothia numbering system; in some embodiments, wherein the heavy chain variable region HCDR1 comprises the amino acid sequence of GNTFSDS (SEQ ID NO. 89), HCDR2 comprises the amino acid sequence of NPYN (SEQ ID NO. 90), and HCDR3 comprises the amino acid sequence of SEQ ID NO. 27; and the light chain variable region LCDR1 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.

[0178] In some embodiments, the anti-human PVRIG antibody as described in any of the preceding embodiments, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Contact numbering system; in some embodiments, wherein the heavy chain variable region HCDR1 comprises the amino acid sequence HQGYSLPRT (SEQ ID NO. 91), HCDR2 comprises the amino acid sequence WIGIINPYNGDTY (SEQ ID NO. 92), and HCDR3 comprises the amino acid sequence ARSNWDVGNCFD (SEQ ID NO. 93); and the light chain variable region LCDR1 comprises the amino acid sequence SNYLNWY (SEQ ID NO. 94), LCDR2 comprises the amino acid sequence LLIYYTSRLH (SEQ ID NO. 95), and LCDR3 comprises the amino acid sequence HQGYSLPR (SEQ ID NO. 96).

[0179] In some embodiments, the anti-human PVRIG antibody as described in any of the preceding embodiments, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the AbM numbering system; in some embodiments, wherein the heavy chain variable region HCDR1 comprises the amino acid sequence of GNTFSDSYMN (SEQ ID NO. 97), HCDR2 comprises the amino acid sequence of IINPYNGDTY (SEQ ID NO. 98), and HCDR3 comprises the amino acid sequence of SEQ ID NO. 27; and the light chain variable region LCDR1 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.

[0180] The binding of the anti-human PVRIG antibody provided in this disclosure to human PVRIG can also be expressed as a "half-maximum 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 detection methods known in the art, such as direct or indirect binding detection methods (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometry, and other binding detection methods).

[0181] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this disclosure, referring to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and their polymers. Nucleic acids include those containing known nucleotide analogs or modified backbone residues or links. Nucleic acids can be synthetic, naturally occurring, or non-natural, such as non-natural nucleic acids having similar binding properties to a reference nucleic acid and being metabolized in a manner similar to a reference nucleotide. This includes, but is not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acid (PNA) modified nucleic acids. "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from its components in its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain such molecules but are present outside the chromosome or at a chromosomal location different from their natural chromosomal location. "Nucleic acid encoding anti-human PVRIG antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including one or more nucleic acid molecules in a single or separate vector, and one or more nucleic acid molecules present at one or more locations in the host cell. In some embodiments, the nucleic acid sequence further includes conserved 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 sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of 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 “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to both naturally occurring 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 two sequences are identical at equivalent positions when two sequences are optimally aligned. To obtain optimal alignment of two sequences, gaps may be introduced if necessary to achieve the maximum percentage of sequence identity. The percentage of amino acid sequence identity can be determined using various methods known in the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine appropriate parameters for the aligned sequences, including any algorithms required to achieve maximum alignment of the full length of the compared sequences.

[0184] The terms "antibody conjugate" and "antibody-drug conjugate" refer to antibodies conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents). In some embodiments of this disclosure, an "antibody-drug conjugate" includes a conjugate formed by conjugating an anti-human PVRIG antibody to a therapeutic agent or imaging agent. In optional embodiments, the antibody-drug conjugate is formed by conjugating an anti-human PVRIG antibody to a therapeutic agent via a linker; in optional embodiments, the therapeutic agent is a cytotoxic agent; in optional embodiments, the therapeutic agent may be selected from toxins, chemotherapeutic agents, antibiotics, radioisotopes, and ribolysins. In this disclosure, a "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction; a "toxin" refers to a substance that can have a harmful effect on cell growth or proliferation (e.g., maytansine, calichiomycin, taxanes, vincristine, colchicine, auratestatin, etc.); a "chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer. In optional embodiments, the antibody-drug conjugate further includes biotin or a biotin derivative conjugated to the antibody. In optional embodiments, the antibody conjugate further includes a label conjugated to the antibody. In optional embodiments, the label refers to a substance possessing properties that can be directly observed with the naked eye or detected by instruments, such as luminescence, color development, or radioactivity, which enables qualitative or quantitative detection of the corresponding target analyte. In optional embodiments, the label is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle labels. In practical use, those skilled in the art can select other suitable labels according to detection conditions or actual needs; regardless of the label used, it falls within the scope of protection of this disclosure. In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase. In optional embodiments, the radioactive isotopes include, but are not limited to, those mentioned above. 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 optional embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, luciferin, luteolin and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives. In optional embodiments, the nanoparticle labeling includes, but is not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles. In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, dispersed dyes, dye-labeled microspheres, and latexes. In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold or colloidal silver. In optional embodiments, the colloidal metal is colloidal gold. In optional embodiments, the antibody conjugate may further include a solid-phase support conjugated to the antibody. In the antibody conjugate, the antibody is conjugated to a solid-phase support. In optional embodiments, the solid-phase support is selected from microspheres, plates, and membranes. In optional embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes. In optional embodiments, the solid phase support is a nitrocellulose membrane.

[0185] The terms "linker" or "connector" refer to a linking unit that connects two polypeptide fragments. Linkers are typically flexible, and their use does not cause the loss of the original function of the protein domains. Linkers can be peptide linkers, containing 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, where x is an integer from 1 to 5 and y is an integer from 0 to 6.

[0186] The term "conservatively modified variant" or "conservative substitution" for amino acids refers to replacing an amino acid in a sequence with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophilicity / hydrophobicity, ossicular structure, and rigidity), without altering the protein's biological activity. Those skilled in the art will recognize that, generally, single amino acid substitutions in non-essential regions of a polypeptide typically do not alter 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 variant" or "conservative substitution" for nucleic acids refers to those nucleic acids encoding the same or substantially the same amino acid sequence, including nucleic acids with substantially the same sequence where the nucleic acid does not encode an 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 designates alanine, that codon can be changed to any of the corresponding codons without altering the encoded polypeptide. These types of nucleic acid variations are "silent variations," and they are a type of conserved modification variation. Each nucleic acid sequence encoding a polypeptide in this disclosure contains every possible silent variation of the nucleic acid. Each codon in the nucleic acid (except for AUG—usually the only codon for methionine; and TGG—usually the only codon for tryptophan) can be modified to produce a functionally identical molecule.

[0187] The term "vector" is a delivery vehicle capable of transporting a genetic element (e.g., nucleic acid) linked to it. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. Exemplarily, vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. In some embodiments, the vector is a "plasmid," which is a circular double-stranded DNA loop in which additional DNA segments can be linked. In some embodiments, the vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which DNA segments can be linked to a viral genome. Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain replication initiation sites and may include components that facilitate their entry into cells, including, but not limited to, viral particles, liposomes, or protein coats. Vectors can be expression vectors. In some embodiments, the vector (e.g., an expression vector) contains the nucleic acid sequence of the antibody encoded by this disclosure, a promoter (e.g., SV40, CMV, EF1α), and may also contain at least one selection marker.

[0188] The term "expression vector" or "expression construct" is a vector containing a nucleic acid sequence suitable for transformation of host cells and which directs and / or controls (together with the host cell) the expression of one or more coding regions operatively linked to it. Expression vectors may include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, affect the splicing of coding regions operatively linked to them.

[0189] The term "operably linked" refers to a connection that allows the components referred to by the term to perform their inherent functions under suitable conditions. For example, a control sequence "operably linked" to a protein-coding sequence is a sequence that, when linked to, enables the expression of the protein-coding sequence under conditions compatible with the transcriptional activity of that 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 progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young 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*, *Pichiamembranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. The fungi include *Candida polymorpha*, *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" means a mixture containing one or more drugs (such as the antibodies described in this disclosure) and other components, such as physiological / pharmaceutical carriers and excipients.

[0192] The term "pharmaceuticalally 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 terms "subject" or "individual" include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless specifically indicated, the terms "patient" or "subject" are used interchangeably in this disclosure. Unless explicitly defined in this disclosure, the terms "cynomolgus monkey" or "cynomolgus" refer to the cynomolgus monkey (Macacafascicularis). In some embodiments, the subject is a human.

[0194] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and 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 within the subject's body. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0196] The term "treatment" or "treat" refers to a clinical intervention that attempts to alter the natural processes of the individual being treated, and may be administered for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some embodiments, antibodies of this disclosure are used to delay the onset of disease or slow its progression.

[0197] The term “application” or “giving”, when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug with an animal, human, subject, cell, tissue, organ, or biological fluid.

[0198] The term "effective dose" generally refers to an amount sufficient to reduce the severity or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, or improve damage associated with a disease state (e.g., lung disease). In some embodiments, an effective dose may be a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when administered to a subject, will have a predetermined preventative effect, 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 related symptoms. A complete therapeutic or preventative effect may not occur after a single dose is administered, but may occur after a series of doses. Thus, a therapeutically or preventatively effective dose may be administered in a single or multiple-dose manner. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the therapeutic agent to elicit the desired response in the individual.

[0199] The terms "cancer" and "tumor" refer to diseases in mammals characterized by unregulated cell growth. Tumors include benign and malignant tumors, and early-stage tumors refer to non-invasive or metastatic cancers, or cancers classified as stage 0, stage I, or stage II. In some embodiments, the tumor is a PVRIG-expressing tumor, and examples of cancers include, but are not limited to, lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and hematologic malignancies.

[0200] In some embodiments, the present disclosure uses conventional techniques known in the art, such as cell biology, molecular biology (including recombinant technology), microbiology, biochemistry and immunology, to prepare or detect the antibodies of the present disclosure. The relevant techniques have been disclosed in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.C. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in this paper are: ChainReaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (JEColigan et al., eds., 1991).

[0201] The features and performance of this disclosure will be further described in detail below with reference to embodiments.

[0202] Preparation of some of the materials used in embodiments 1-9 of this disclosure:

[0203] 1) The target genes encoding human PVRIG (UniProt accession number: Q6DKI7) and monkey PVRIG (UniProt accession number: A0A2K5WVV8) were cloned into plasmids containing His, human Fc, or mouse Fc, respectively. They were then transfected into HEK293 cells for transient expression to obtain hPVRIG-mFc (R2248), hPVRIG-hFc (R2249), hPVRIG-His (R2250), and cyno PVRIG-His (R2251).

[0204] 2) The target gene encoding human PVRIG (Q6DKI7) was cloned into the plvx lentiviral plasmid, then the lentivirus was packaged and the titer was detected. The virus was then used to infect 293T cells to express membrane proteins, thereby obtaining a cell line overexpressing hPVRIG: 293T-hPVRIG.

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

[0206] 4) Prepare a positive antibody R2247 for the mIgG1 subtype according to the sequence of “Antibody 46” in US Patent No. US11279758B2;

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

[0208] 6) Purchased a PVRIG reporter gene system (product number CBP74120+CBP74121, Nanjing Kebai).

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

[0210] Balb / c or SJL mice were immunized weekly using hPVRIG-mFc (R2248) and hPVRIG-hFc (R2249) as immunogens. Balb / c mice were immunized a total of 5 times, and SJL mice were immunized 4 times. After immunization, orbital blood was collected for ELISA binding to detect the binding of antibodies to hPVRIG-His (R2250), and the binding of antibodies to PVRIG reporter cells was detected by FACS. Three mice with good binding activity were selected for a booster immunization.

[0211] Then, mouse spleens were taken, spleen cells were separated, and mouse myeloma cells were mixed 1:1 at a rate of 5E6 / plate. The mixtures were then fused under the action of an electrofusion device to obtain hybridomas.

[0212] Hybridoma supernatant was collected. ELISA was used to detect the binding of the antibody to hPVRIG-His (R2250), FACS was used to detect the binding of the antibody to PVRIG reporter cells, ELISA was used to detect the antibody's blocking activity against the PVRIG receptor ligand (R2249+hPVRL2-His), and ELISA was used to detect the binding of the antibody to cyno-PVRIG-His (R2251). Positive clones were screened for subcloning, and 14 positive clones were selected and expanded. The supernatant was collected, and the antibodies in the supernatant were purified to obtain 14 murine anti-human PVRIG antibodies. The sequence information of the antibodies is shown in Tables 1 and 2 below.

[0213] Table 1. Sequence listing of variable regions of anti-human PVRIG antibodies

[0214]

[0215]

[0216]

[0217] Table 2. CDR sequence listing of anti-human PVRIG antibodies

[0218]

[0219]

[0220] Note: The anti-human PVRIG antibodies HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 mentioned above were identified according to the Kabat coding system.

[0221] In addition, the antibody constant regions are as follows: CG004-01, CG004-2, CG004-05, CG004-06, CG004-07, CG004-09, CG004-11, and CG004-14 have a heavy chain constant region corresponding to the mouse IgG2a heavy chain constant region and a light chain constant region corresponding to the mouse κ chain constant region; CG004-04, CG004-12, and CG004-13 have a heavy chain constant region corresponding to the mouse IgG2b heavy chain constant region and a light chain constant region corresponding to the mouse κ chain constant region; and CG004-10 has a heavy chain constant region corresponding to the mouse IgG1 heavy chain constant region and a light chain constant region corresponding to the mouse κ chain constant region.

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

[0223] ELISA assay: Human PVRIG (R2250) was diluted to 1 μg / mL (diluent: 50 mM CB), and 100 μL / well was coated onto the plate and incubated overnight. The next morning, the plate was washed three times and patted dry. 200 μL / well blocking buffer (1% BSA) was added, and the plate was blocked at room temperature for 1 h. The plate was washed once, patted dry, and the antibody was serially diluted 3-fold at 100 μL / well. The plate was incubated at room temperature for 1 h. The plate was washed five times, patted dry, and anti-mouse Fc-HRP (1:10000) was diluted at 100 μL / well. The plate was incubated at room temperature for 40 min. The plate was washed six times, patted dry, and HRP substrate solutions A and B were added at 50 μL / well, respectively. The plate was incubated at room temperature for 20 min. 100 μL / well stop solution was added, and the plate was read at 450 nm.

[0224] The experimental results are attached. Figure 1 and 2 Experimental results show that the anti-human PVRIG antibodies disclosed herein can all specifically bind to human PVRIG and have excellent antigen-binding activity.

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

[0226] FACS combined with experimental methods: Cell counting: PVRIG reporter cells were counted, centrifuged at 300g for 5 min, resuspended in FCM buffer, and the cell density was adjusted to 4E+06 cells / mL. The amount of cells added to each 96-well V-plate was 2E+05 cells / well. The sample was serially diluted 3-fold with buffer (3% BSA + 1×PBS) (anti-human PVRIG antibody and R2247 were positive controls). The sample and cells were added to 96-well V-plates (50 μL of sample and cells per well) and incubated at 4°C in the dark for 30 min. Washing: Centrifuged (300g / 5 min), discarded the supernatant, washed once with 200 μL of FCM buffer, centrifuged (300g / 5 min), discarded the supernatant, and the secondary antibody (APC anti-mouse IgG) was added. Fc (1:500 dilution) into 96-well V-plates (100 μL / well), incubate at 4°C in the dark for 30 min; Wash: Centrifuge (300 g / 5 min), discard supernatant, add 200 μL FCM buffer to wash once, centrifuge (300 g / 5 min), discard supernatant; Analyze: Resuspend in 100 μL 1×PBS, and analyze.

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

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

[0229] The blocking activity of anti-human PVRIG antibodies against hPVRIG-hFc and its ligands was evaluated using an ELISA method.

[0230] Experimental Methods: Antigen Coating: Dilute hPVRIG-hFc(R2249) with coating buffer to a concentration of 1 μg / mL, add 100 μL / well to two wells of a 96-well microplate, gently vortex to mix, seal with sealing film, and incubate overnight at 2-8℃. Washing: Remove the microplate, discard the supernatant, and blot dry with absorbent paper; add 200 μL / well of PBST washing buffer, discard the supernatant, and blot dry with absorbent paper; repeat washing 3 times. Blocking: Add 200 μL / well of 1% BSA blocking buffer and incubate at 37℃ for 1 h. The candidate antibody and positive antibody R2247 were serially diluted 3-fold with 1X BSA solution. 70 μL of the diluted antibody was transferred to a 96-well ELISA plate and incubated at 37°C for 60 min. Then, 70 μL / well of pre-diluted 0.5 μg / mL Nectin-2-his ligand protein was added, and the plate was incubated at 37°C for 60 min. Washing: The ELISA plate was removed, the supernatant was discarded, and the plate was blotted dry on absorbent paper. 200 μL / well of PBST washing buffer was added, the supernatant was discarded, and the plate was blotted dry on absorbent paper. This washing process was repeated 3 times. Secondary antibody incubation: 100 μL / well of HRP-anti-his secondary antibody diluted 1% BSA (PBST) was added to columns 1-12 of the ELISA plate and incubated at 37°C for 40 min. Washing: Discard the supernatant from the microplate and blot dry on absorbent paper. Add 200 μL of PBST washing buffer per well, discard the supernatant, and blot dry on absorbent paper. Repeat washing three times. Development: Add 50 μL of solution A per well, then add 50 μL of solution B per well. Incubate at room temperature in the dark for 10 min. Termination and Reading: Add 100 μL of stop solution per well to the microplate and immediately read the OD450 absorbance using a microplate reader.

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

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

[0233] Experimental Methods: One day prior to the experiment, dilute cyno PVRIG (R2251) protein or mouse PVRIG (Acro) protein to 1 μg / mL and add 100 μL / well to two wells of a 96-well microplate. Gently vortex to mix, seal with sealing film, and incubate overnight at 2-8℃. Washing: Remove the microplate, discard the supernatant, and blot dry with absorbent paper. Add 200 μL / well of PBST washing buffer, discard the supernatant, and blot dry with absorbent paper. Repeat washing three times. Blocking: Add 200 μL / well of 1% BSA blocking buffer and incubate at 37℃ for 1 h. Dilute the candidate antibody and positive antibody R2247 three-fold with 1X BSA solution. Transfer 100 μL of the diluted antibody to each well of a 96-well microplate and incubate at 37℃ for 60 min. Washing: Discard the supernatant from the microplate and blot dry on absorbent paper. Add 200 μL of PBST washing buffer to each well, discard the supernatant, and blot dry on absorbent paper. Repeat washing 3 times. Secondary antibody incubation: Dilute goat anti-mouse Fc enzyme-labeled secondary antibody (1:15000) with 1% BSA (PBST) and add 100 μL to columns 1-12 of the microplate. Incubate at 37°C for 40 min. Washing: Discard the supernatant from the microplate and blot dry on absorbent paper. Add 200 μL of PBST washing buffer to each well, discard the supernatant, and blot dry on absorbent paper. Repeat washing 3 times. Development: Mix solution A and solution B in a 1:1 ratio and add 100 μL to each well of the microplate. Incubate at room temperature in the dark for 10 min. Termination and plate reading: Add stop solution to the microplate, 100 μL / well, and immediately read the OD450 absorbance value using a microplate reader.

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

[0235] Example 6. Reporter gene activity assay

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

[0237] Experimental Methods: Cell counting was performed on CHO cells expressing PVRIG ligand. Cells were centrifuged at 300g for 5 min, the supernatant was discarded, and the cells were washed once with F12K + 10% FBS medium. After resuspending the cells in F12K + 10% FBS medium, the cell density was adjusted to 2E+05 cells / mL. Cells were added at a rate of 2E+04 cells / well (100 μL / well). The cells were incubated overnight in a CO2 incubator. The following morning, the samples were diluted (using 1640 + 10% FBS medium). Cell counting: Take an appropriate amount of PVRIG reporter cells, centrifuge at 300g for 5 min, discard the supernatant, wash once with 1640 + 10% FBS medium, resuspend in 1640 + 10% FBS medium, and adjust the PVRIG cell density to 1.6E+06 cells / mL. According to the experimental design, in a 96-well white blood cell culture plate, aspirate F12K + 10% FBS medium, add 50 μL of antibody dilution to each well, and add PVRIG cells at a rate of 8E+04 cells / well (i.e., 50 μL / well). Add a certain volume of 1640 + 10% FBS medium to the corresponding wells, making the total volume of all wells 100 mL. Incubate the cell culture plate in a CO2 incubator for 6 h. Thaw the Bright-Lumi™ firefly luciferase assay reagent in advance and equilibrate to room temperature. Remove the cell culture plate and equilibrate at room temperature for 10 min (not exceeding 30 min). Add 100 μL of Bright-Lumi™ firefly luciferase assay reagent to each well and incubate at room temperature for 5 min. Then perform chemiluminescence detection.

[0238] The experimental results are attached. Figure 11 Experimental results show that the anti-human PVRIG antibody disclosed in this paper 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 murine antibody sequence of CG004-1 (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.9, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.10).

[0241] Three-dimensional structural homology modeling was performed on the selected sequences, and the structures were determined to be acceptable through methods such as Laplace plots. The antibody sequences were compared with the GermLine antibody database to obtain a human template (IGKV4-1*01) with high homology. The CDR was transplanted into the corresponding human template. The transplanted antibodies were subjected to three-dimensional structural simulation and analysis again. Residues that were embedded, interacted directly with the CDR region, and had a significant impact on the conformation of the variable region were subjected to backcomplex modification. 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 hIgG4 (S228P / F234A / L235A / K447A) isotypes were prepared: R3942-R3950. The amino acid sequences of the heavy chain variable region of antibody R3942-R3950 are shown in Table 3, the amino acid sequences of the light chain variable region are shown in Table 4, and the combinations of the heavy chain variable region and the light chain variable region are shown in Table 5.

[0242] Table 3 Heavy chain sequences of humanized PVRIG mouse antibody

[0243]

[0244] Table 4. Light chain sequence of humanized PVRIG mouse antibody

[0245]

[0246] Table 5. Combination of heavy chain variable regions and light chain variable regions of humanized PVRIG mouse antibodies.

[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 the reporter gene activity of humanized PVRIG mouse antibody

[0249] The activity of humanized PVRIG mouse antibodies R3942-R3950 was evaluated using a reporter gene assay, the same method as in Example 6, and the results are as follows. Figure 12 As shown in the figure, R3014 is the parent molecule, and R3942-R3950 are humanized molecules. Among the humanized molecules, R3944 exhibits superior reporter gene activity compared to the parent molecule R3014.

[0250] Example 9. Binding activity of humanized PVRIG nanobody with 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 methods as in Example 5. Results are as follows: Figure 13 and Figure 14 As shown, the binding activity of the humanized molecule R3944 to 293-hPVRIG cells is close to that of the parental molecule R3014.

[0252] The above description is merely a preferred embodiment of this disclosure and is not intended to limit this disclosure. Various modifications and variations can be made to this disclosure by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.

Claims

1. An anti-human PVRIG antibody, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are identical to those of HCDR1, HCDR2, and HCDR3 in the variable region shown in SEQ ID No. 9, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are identical to those of LCDR1, LCDR2, and LCDR3 in the variable region shown in SEQ ID No. 10, respectively. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are identical to those of HCDR1, HCDR2, and HCDR3 in the variable region shown in SEQ ID No. 11, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are identical to those of LCDR1, LCDR2, and LCDR3 in the variable region shown in SEQ ID No. 12, respectively; or The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are identical to those of HCDR1, HCDR2, and HCDR3 in the variable region shown in SEQ ID No. 13, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are identical to those of LCDR1, LCDR2, and LCDR3 in the variable region shown in SEQ ID No.

14.

2. The anti-human PVRIG antibody according to claim 1, characterized in that, The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat numbering system; The heavy chain variable region of the anti-human PVRIG antibody has the amino acid sequence HCDR1 of SEQ ID NO.46, HCDR2 of SEQ ID NO.47, and HCDR3 of SEQ ID NO.48; and the light chain variable region has the amino acid sequence LCDR1 of SEQ ID NO.49, LCDR2 of SEQ ID NO.50, and LCDR3 of SEQ ID NO.

51. The heavy chain variable region has the amino acid sequence HCDR1 of SEQ ID NO. 46, HCDR2 of SEQ ID NO. 52, and HCDR3 of SEQ ID NO. 48; and the light chain variable region has the amino acid sequence LCDR1 of SEQ ID NO. 49, LCDR2 of SEQ ID NO. 50, and LCDR3 of SEQ ID NO. 51; or The heavy chain variable region has the amino acid sequence HCDR1 of SEQ ID NO. 53, HCDR2 of SEQ ID NO. 54, and HCDR3 of SEQ ID NO. 55; and the light chain variable region has the amino acid sequence LCDR1 of SEQ ID NO. 49, LCDR2 of SEQ ID NO. 56, and LCDR3 of SEQ ID NO.

57.

3. The anti-human PVRIG antibody according to any one of claims 1 to 2, characterized in that, The antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

4. The anti-human PVRIG antibody according to claim 3, characterized in that, The heavy chain variable region is the amino acid sequence of SEQ ID NO. 9, and the light chain variable region is the amino acid sequence of SEQ ID NO. 10; or 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; or 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; or The heavy chain variable region is the amino acid sequence of SEQ ID NO. 104, and the light chain variable region is the amino acid sequence of SEQ ID NO. 99; or The heavy chain variable region is the amino acid sequence of SEQ ID NO. 100, and the light chain variable region is the amino acid sequence of SEQ ID NO. 99; or The heavy chain variable region is the amino acid sequence of SEQ ID NO. 100, and the light chain variable region is the amino acid sequence of SEQ ID NO. 105; or The heavy chain variable region is the amino acid sequence of SEQ ID NO. 101, and the light chain variable region is the amino acid sequence of SEQ ID NO. 99; or The heavy chain variable region is the amino acid sequence of SEQ ID NO. 101, and the light chain variable region is the amino acid sequence of SEQ ID NO. 105; or The heavy chain variable region is the amino acid sequence of SEQ ID NO. 102, and the light chain variable region is the amino acid sequence of SEQ ID NO. 99; or The heavy chain variable region is the amino acid sequence of SEQ ID NO. 102, and the light chain variable region is the amino acid sequence of SEQ ID NO. 105; or The heavy chain variable region is the amino acid sequence of SEQ ID NO. 103, and the light chain variable region is the amino acid sequence of SEQ ID NO. 99; or The heavy chain variable region is the amino acid sequence of SEQ ID NO.103, and the light chain variable region is the amino acid sequence of SEQ ID NO.

105.

5. The anti-human PVRIG antibody according to claim 1, 2 or 4, wherein the antibody comprises a constant region.

6. The anti-human PVRIG antibody according to claim 5, characterized in that, The heavy chain constant region of the antibody is selected from the heavy chain constant regions 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.

7. The anti-human PVRIG antibody according to claim 5, characterized in that, The species origin of the constant region is either rat or human.

8. The anti-human PVRIG antibody according to claim 6, characterized in that, The heavy chain constant region is the mouse IgG2a constant region, mouse IgG1 constant region, human IgG1 constant region or human IgG4 constant region, and / or the light chain constant region is the mouse κ constant region or human κ constant region.

9. The anti-human PVRIG antibody according to claim 1, 2, 4, 6, 7 or 8, characterized in that, The antibody is a full-length antibody or any antigen-binding fragment selected from F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv and scFv.

10. A biomaterial, characterized in that, The biomaterial includes any one of the following (i) to (iv): (i) A multispecific antibody, wherein the multispecific antibody comprises the anti-human PVRIG antibody according to any one of claims 1 to 9; (ii) an isolated nucleic acid, wherein the isolated nucleic acid encodes the anti-human PVRIG antibody according to any one of claims 1 to 9 or encodes the multispecific antibody according to (i); (iii) an expression vector comprising the isolated nucleic acid (ii); (iv) A cell characterized in that the cell carries (ii) the isolated nucleic acid and (iii) the expression vector.

11. A pharmaceutical composition, characterized in that, It comprises the anti-human PVRIG antibody as described in any one of claims 1 to 9 and the biomaterial as described in claim 10.

12. The pharmaceutical composition according to claim 11, characterized in that, It also contains one or more pharmaceutically acceptable carriers.

13. The pharmaceutical composition according to claim 12, characterized in that, The carrier includes excipients.

14. The pharmaceutical composition according to claim 13, characterized in that, The excipients include diluents.

Images