PVRIG binding proteins, bispecific antibodies and uses thereof

Abstract

The invention relates to PVRIG binding proteins, bispecific antibodies and uses thereof. The bispecific antibody or antigen binding fragment of the present invention comprises a first antigen binding moiety capable of binding to PVRIG and / or a second antigen binding moiety capable of binding to TIGIT. The PVRIG binding proteins or bispecific antibodies of the invention can be used for treating or preventing diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to PVRIG-binding proteins, bispecific antibodies and their applications. Background Art

[0002] Most T- and NK-cell coinhibitory receptors are members of the immunoglobulin (Ig) superfamily. When they bind to their corresponding ligands expressed on antigen-presenting cells or tumor cells, they attenuate cell activation through protein tyrosine phosphatases (such as SHIP-1, SHP-2), ultimately leading to reduced effector function and the expression of inhibitory genes. Receptor blockers are capable of specifically recognizing certain receptors and binding to them to prevent their corresponding ligands from binding. In cancer treatment, blocking the binding of coinhibitory receptors to their ligands with receptor blockers helps to activate the activity of immune cells and improve the anti-tumor efficacy.

[0003] PVRIG is a coinhibitory receptor belonging to the poliovirus receptor (PVR) family, also known as CD112R, and is usually expressed on natural killer (NK) cells and CD8+ T cells. Human PVRIG is a 36-kD single-pass transmembrane protein, containing a transmembrane region, a long intracellular domain, and an extracellular immunoglobulin variable (IgV) domain. Since CD226 and PVRIG have the same binding site on CD112, CD226 competes with the inhibitory immune checkpoint PVRIG for binding to CD112 to promote T-cell activation. Therefore, PVRIG can significantly inhibit the interaction of CD112 / CD226 in T cells. Summary of the Invention

[0004] The purpose of the present invention is to provide PVRIG-binding proteins, bispecific antibodies or antigen-binding fragments and their applications.

[0005] On the one hand, the present invention provides a bispecific antibody or antigen-binding fragment, wherein the bispecific antibody or antigen-binding fragment comprises a first antigen-binding portion capable of binding to PVRIG; the first antigen-binding portion comprises one or more amino acid sequences of (a)-(c):

[0006] (a) HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 6;

[0007] (b) HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 7;

[0008] (c) HCDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO: 8-12, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in any one of SEQ ID NO: 8-12.

[0009] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region (VH), the VH comprising HCDR1, HCDR2, and HCDR3; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7; and HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 8-12.

[0010] In some embodiments, the first antigen-binding portion is a single-domain antibody.

[0011] In some embodiments, the single-domain antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 13-17, or an amino acid sequence having at least 80% identity as compared with the amino acid sequence shown in any one of SEQ ID NO: 13-17, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in any one of SEQ ID NO: 13-17.

[0012] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a second antigen-binding portion capable of binding to other antigens. In some embodiments, the other antigen is TIGIT. In some embodiments, the second antigen-binding portion is an anti-TIGIT antibody or antigen-binding fragment. In some embodiments, the anti-TIGIT antibody is selected from Tiragolumab, Ociperlimab, the anti-TIGIT antibodies described in Patent CN108290946A, such as 1A4, 1A5, 1D3, 4A3, 10A7, and 4.1D3, the anti-TIGIT antibodies described in Patent CN108137691A, such as VSIG9#1 and 258-cs1#4, the anti-TIGIT antibodies described in Patent CN108290936A, such as 14D7 and 26B10, the anti-TIGIT antibodies described in Patent CN109071620A, such as 313R11, 313R12, 313R14, 313R19, and 313R20, the anti-TIGIT antibodies described in Patent CN107207594A, such as 14B2, 13E6, 6F9, 11G11, 10C9, 16F6, 11C9, 10D7, 20G6, 24E8, 24G1, 27F1, 15A6, 4E4, 13D1, 9B11, 10B8, 22G2, 19H2, 8C8, 17G4, 25E7, 26D8, and 16A8, the anti-TIGIT antibodies described in Patent CN107148430A, such as 14A6, 28H5, and 31C6, and the anti-TIGIT antibodies described in US2013 / 0251720, such as 10A7 and 1F4.

[0013] In some embodiments, the second antigen-binding portion is linked to the first antigen-binding portion by a peptide linker. In some embodiments, the second antigen-binding portion is an anti-TIGIT antibody, and the N-terminus or C-terminus of the antibody heavy chain is linked to the first antigen-binding portion by a peptide linker. In some embodiments, the N-terminus of the antibody heavy chain is linked to the C-terminus of the single-domain antibody by a peptide linker. In some embodiments, the C-terminus of the antibody heavy chain is linked to the N-terminus of the single-domain antibody by a peptide linker.

[0014] In some embodiments, the peptide linker comprises glycine and serine. In some embodiments, the amino acid sequence of the peptide linker is (G m S) n , where each m is independently 2, 3, 4, 5, or 6, and n is independently 1, 2, 3, 4, 5, or 6. In some embodiments, the amino acid sequence of the peptide linker is (GGGGS) n, n independently is 1, 2, 3, 4, 5 or 6. In some embodiments, the peptide linker is (GGGGS)2, as shown in SEQ ID NO:32. In some embodiments, the peptide linker is (GGGGS)3, as shown in SEQ ID NO:33.

[0015] On the other hand, the present invention provides a bispecific antibody or antigen-binding fragment, the bispecific antibody or antigen-binding fragment comprising a second antigen-binding portion capable of binding to TIGIT; the second antigen-binding portion comprises one or more of the amino acid sequences in (d)-(i):

[0016] (d) HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:18, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:18;

[0017] (e) HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:19;

[0018] (f) HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:20, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:20;

[0019] (g) LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:21;

[0020] (h) LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:22;

[0021] (i) LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:23.

[0022] In some embodiments, the second antigen-binding portion comprises a heavy-chain variable region (VH) that comprises HCDR1, HCDR2, and HCDR3; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:18; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:20.

[0023] In some embodiments, the second antigen-binding portion comprises a light-chain variable region (VL) that comprises LCDR1, LCDR2, and LCDR3; wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:21; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:22; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:23.

[0024] In some embodiments, the second antigen-binding portion comprises VH and VL; the VH comprises HCDR1, HCDR2, and HCDR3; the VL comprises LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:18; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:20; LCDR1 comprises the amino acid sequence shown in SEQ ID NO:21; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:22; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:23.

[0025] In some embodiments, the heavy-chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:24.

[0026] In some embodiments, the light-chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:25.

[0027] In some embodiments, the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 24; the light chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 25.

[0028] In some embodiments, the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 25.

[0029] In some embodiments, the second antigen-binding portion further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 26 or 27, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 26 or 27, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 26 or 27. In some embodiments, the light chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the heavy chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 26 or 27, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 26 or 27, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 26 or 27; the light chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the heavy chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 26 or 27, and the light chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 28.

[0030] In some embodiments, the second antigen-binding portion is an antibody, and the antibody comprises a heavy chain and a light chain. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 29 or 30, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 29 or 30, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 29 or 30. In some embodiments, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 31. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 29 or 30, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 29 or 30, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 29 or 30; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 31. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 29, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31.

[0031] In some embodiments, the bispecific antibody or antigen-binding fragment comprises a first antigen-binding portion capable of binding to another antigen. In some embodiments, the other antigen is PVRIG.

[0032] In some embodiments, the first antigen-binding portion is a single-domain antibody.

[0033] In some embodiments, the N-terminus of the antibody heavy chain is linked to the C-terminus of the single-domain antibody via a peptide linker. In some embodiments, the C-terminus of the antibody heavy chain is linked to the N-terminus of the single-domain antibody via a peptide linker. In some embodiments, the second antigen-binding portion is linked to the first antigen-binding portion via a peptide linker. In some embodiments, the second antigen-binding portion is an antibody, and the N-terminus or C-terminus of the antibody heavy chain is linked to the first antigen-binding portion via a peptide linker. In some embodiments, the N-terminus of the antibody heavy chain is linked to the C-terminus of the single-domain antibody via a peptide linker. In some embodiments, the C-terminus of the antibody heavy chain is linked to the N-terminus of the single-domain antibody via a peptide linker.

[0034] In some embodiments, the peptide linker comprises glycine and serine. In some embodiments, the amino acid sequence of the peptide linker is (G m S) n , where each m is independently 2, 3, 4, 5, or 6, and n is independently 1, 2, 3, 4, 5, or 6. In some embodiments, the amino acid sequence of the peptide linker is (GGGGS) n , where n is independently 1, 2, 3, 4, 5, or 6. In some embodiments, the peptide linker is (GGGGS)2, as shown in SEQ ID NO:32. In some embodiments, the peptide linker is (GGGGS)3, as shown in SEQ ID NO:33.

[0035] On the other hand, the present invention provides a bispecific antibody or antigen-binding fragment, which comprises a first antigen-binding portion capable of binding to PVRIG, and a second antigen-binding portion capable of binding to TIGIT; wherein

[0036] the first antigen-binding portion comprises one or more amino acid sequences of (a)-(c):

[0037] (a) HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO:6;

[0038] (b) HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO:7;

[0039] (c) HCDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs:8-12, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in any one of SEQ ID NOs:8-12;

[0040] the second antigen-binding portion comprises one or more amino acid sequences of (d)-(i):

[0041] (d) HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:18, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO:18;

[0042] (e) HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 19;

[0043] (f) HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 20;

[0044] (g) LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 21;

[0045] (h) LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 22;

[0046] (i) LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 23.

[0047] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region (VH), and the VH comprises HCDR1, HCDR2, and HCDR3; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7; HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 8-12. In some embodiments, the second antigen-binding portion comprises VH and VL; the VH comprises HCDR1, HCDR2, and HCDR3; the VL comprises LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 22; LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 23.

[0048] In some embodiments, the first antigen-binding portion comprises a heavy-chain variable region (VH), the VH comprising HCDR1, HCDR2, and HCDR3; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:6; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:7; HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:8-12; the second antigen-binding portion comprises VH and VL; the VH comprises HCDR1, HCDR2, and HCDR3; the VL comprises LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:18; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:20; LCDR1 comprises the amino acid sequence shown in SEQ ID NO:21; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:22; LCDR3 comprises the amino acid sequence shown in SEQ ID NO:23.

[0049] In some embodiments, the first antigen-binding portion is a single-domain antibody. In some embodiments, the single-domain antibody comprises the amino acid sequence shown in any one of SEQ ID NOs:13-17, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs:13-17, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs:13-17. In some embodiments, the heavy-chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:24; and / or the light-chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:25.

[0050] In some embodiments, the first antigen-binding portion is a single-domain antibody, comprising the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 13-17; the heavy-chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 24; the light-chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 25.

[0051] In some embodiments, the first antigen-binding portion is a single-domain antibody, comprising the amino acid sequence shown in any one of SEQ ID NOs: 13-17; the heavy-chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 24; the light-chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 25.

[0052] In some embodiments, the second antigen-binding portion further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 26 or 27, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 26 or 27, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 26 or 27. In some embodiments, the light chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the heavy chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 26 or 27, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 26 or 27, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 26 or 27; the light chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the heavy chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 26 or 27, and the light chain constant region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 28.

[0053] In some embodiments, the second antigen-binding portion is an antibody, and the antibody comprises a heavy chain and a light chain. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 29 or 30, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 29 or 30, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 29 or 30. In some embodiments, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 31.

[0054] In some embodiments, the second antigen-binding portion is linked to the first antigen-binding portion via a peptide linker. In some embodiments, the second antigen-binding portion is an antibody, and the N-terminus or C-terminus of the antibody heavy chain is linked to the first antigen-binding portion via a peptide linker. In some embodiments, the N-terminus of the antibody heavy chain is linked to the C-terminus of a single-domain antibody via a peptide linker. In some embodiments, the C-terminus of the antibody heavy chain is linked to the N-terminus of a single-domain antibody via a peptide linker.

[0055] In some embodiments, the peptide linker comprises glycine and serine. In some embodiments, the amino acid sequence of the peptide linker is (G m S) n , where each m is independently 2, 3, 4, 5, or 6, and n is independently 1, 2, 3, 4, 5, or 6. In some embodiments, the amino acid sequence of the peptide linker is (GGGGS) n , where n is independently 1, 2, 3, 4, 5, or 6. In some embodiments, the peptide linker is (GGGGS)2, as shown in SEQ ID NO:32. In some embodiments, the peptide linker is (GGGGS)3, as shown in SEQ ID NO:33.

[0056] On the other hand, the present invention provides a bispecific antibody or antigen-binding fragment, the bispecific antibody or antigen-binding fragment comprising a first antigen-binding portion capable of binding to PVRIG, and a second antigen-binding portion capable of binding to TIGIT; wherein

[0057] the first antigen-binding portion is a single-domain antibody, the single-domain antibody comprising the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 13-17;

[0058] The second antigen-binding portion is an anti-TIGIT antibody, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 29 or 30, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 29 or 30, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 29 or 30; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 31;

[0059] The N-terminus or C-terminus of the anti-TIGIT antibody heavy chain is linked to the single-domain antibody via a peptide linker;

[0060] The amino acid sequence of the peptide linker is (GGGGS) n , where n is independently 1, 2, 3, 4, 5 or 6.

[0061] In some embodiments, the N-terminus of the antibody heavy chain is linked to the C-terminus of the single-domain antibody via a peptide linker. In some embodiments, the C-terminus of the antibody heavy chain is linked to the N-terminus of the single-domain antibody via a peptide linker.

[0062] In some embodiments, the peptide linker is (GGGGS)2, as shown in SEQ ID NO: 32. In some embodiments, the peptide linker is (GGGGS)3, as shown in SEQ ID NO: 33.

[0063] On the other hand, the present invention provides a bispecific antibody or antigen-binding fragment, the bispecific antibody or antigen-binding fragment comprising a first polypeptide and a second polypeptide; wherein

[0064] The first polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 41-44, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in any one of SEQ ID NO: 41-44, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 41-44; and / or

[0065] The second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 31.

[0066] In some embodiments, the bispecific antibody or antigen-binding fragment comprises a first polypeptide and a second polypeptide; the first polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 41-44; the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 31.

[0067] On the other hand, the present invention provides a PVRIG-binding protein, which comprises a heavy-chain variable region, and the heavy-chain variable region comprises one or more amino acid sequences in (a)-(c):

[0068] (a) HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 6;

[0069] (b) HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 7;

[0070] (c) HCDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 8-12, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in any one of SEQ ID NOs: 8-12.

[0071] In some embodiments, the heavy-chain variable region comprises HCDR1, HCDR2 and HCDR3; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7; HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 8-12.

[0072] In some embodiments, the heavy-chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having at least 80% identity as compared with the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having one or more conservative amino acid substitutions as compared with the amino acid sequence shown in any one of SEQ ID NOs: 13-17. In some embodiments, the heavy-chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-17.

[0073] In some embodiments, the PVRIG-binding protein is a single-domain antibody, a heavy-chain antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, a Fab, a Fab', an F(ab')2, an F(ab)2, an Fv or an scFv.

[0074] In some embodiments, the PVRIG-binding protein is a fusion protein and further comprises an Fc fragment. In some embodiments, the Fc fragment is an Fc fragment of an immunoglobulin or a variant thereof, such as an Fc fragment of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof. In some embodiments, the Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 34 or 35, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 34 or 35, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 34 or 35.

[0075] In some embodiments, the C-terminus of the heavy chain variable region is linked to the N-terminus of the Fc fragment via a peptide linker. In some embodiments, the amino acid sequence of the peptide linker is (G m S) n , where each m is independently 2, 3, 4, 5 or 6 and n is independently 1, 2, 3, 4, 5 or 6. In some embodiments, the amino acid sequence of the peptide linker is (GGGGS) n , where n is independently 1, 2, 3, 4, 5 or 6. In some embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 32 or 33.

[0076] In some embodiments, the PVRIG-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 36-40, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in any one of SEQ ID NO: 36-40, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 36-40.

[0077] In some embodiments, the PVRIG-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 36-40.

[0078] Exemplary amino acid sequences of the present invention are shown in Table 1.

[0079] Table 1 Amino Acid Sequences

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] On the other hand, the present invention provides a nucleic acid encoding the above-mentioned bispecific antibody or antigen-binding fragment or PVRIG-binding protein or a part thereof. In some embodiments, the nucleic acid is an isolated nucleic acid.

[0087] Exemplary nucleic acid sequences of the present invention are shown in Table 2.

[0088] Table 2 Nucleic Acid Sequences

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] On the other hand, the present invention provides an expression vector comprising the above-mentioned nucleic acid.

[0096] On the other hand, the present invention provides a host cell comprising the above-mentioned nucleic acid, or the above-mentioned expression vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the cell is a CHO cell, a HEK cell (such as HEK293F cell), a BHK cell, a Cos1 cell, a Cos7 cell, a CV1 cell or a murine L cell.

[0097] On the other hand, the present invention provides a method for preparing the bispecific antibody or antigen-binding fragment or PVRIG-binding protein described herein, comprising culturing the above-mentioned host cell in a medium to produce the bispecific antibody or antigen-binding fragment or PVRIG-binding protein. In some embodiments, the method further comprises recovering the bispecific antibody or antigen-binding fragment or PVRIG-binding protein from the host cell or the medium.

[0098] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned bispecific antibody or antigen-binding fragment, the above-mentioned PVRIG-binding protein, the above-mentioned nucleic acid, the above-mentioned expression vector or the above-mentioned cell, and a pharmaceutically acceptable excipient.

[0099] On the other hand, the present invention provides the use of the above-mentioned bispecific antibody or antigen-binding fragment, the above-mentioned PVRIG-binding protein, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned cell or the above-mentioned pharmaceutical composition in the treatment or prevention of diseases.

[0100] On the other hand, the present invention provides the use of the above-mentioned bispecific antibody or antigen-binding fragment, the above-mentioned PVRIG-binding protein, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned cell or the above-mentioned pharmaceutical composition in the preparation of a medicament for the treatment or prevention of diseases.

[0101] On the other hand, the present invention provides a method for treating or preventing diseases, comprising administering to a patient in need a therapeutically effective amount of the above-mentioned bispecific antibody or antigen-binding fragment, the above-mentioned PVRIG-binding protein, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned cell or the above-mentioned pharmaceutical composition.

[0102] In some embodiments, the disease is a proliferative disease or an infection. In some embodiments, the disease is a tumor. In some embodiments, the tumor is a benign tumor or cancer. In some embodiments, the tumor is selected from prostate cancer, liver cancer, colorectal cancer, ovarian cancer, uterine cancer (such as endometrial cancer), breast cancer (such as triple-negative breast cancer), pancreatic cancer, gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, bladder cancer, urothelial cancer, lung cancer (such as small cell lung cancer and non-small cell lung cancer), melanoma, non-melanoma skin cancer (such as squamous and basal cell carcinoma), glioma, kidney cancer, lymphoma (such as non-Hodgkin lymphoma, for example diffuse large B-cell lymphoma; Hodgkin lymphoma), leukemia (such as acute myeloid leukemia, T-cell acute lymphoblastic leukemia), testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, high MSI cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, myelodysplastic syndrome, urethral cancer, squamous cell carcinoma and Merkel cell carcinoma. In some embodiments, the infection is selected from viral infection, bacterial infection, fungal infection and parasitic infection.

[0103] In some embodiments, the present invention relates to a kit or article, which comprises the above-mentioned bispecific antibody or antigen-binding fragment, the above-mentioned PVRIG-binding protein, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned cell or the above-mentioned pharmaceutical composition.

[0104] The PVRIG-binding protein or bispecific antibody or antigen-binding fragment of the present invention can be used for the treatment or prevention of various diseases, such as tumors or infections, and can also be used for the diagnosis and prognosis of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] FIG. 1 exemplifies two structures of the bispecific antibody of the present invention; Figure 1AThe structure of bispecific antibody Bi-VH1 Figure 1B The structure of bispecific antibody Bi-VH3

[0106] Figure 2 The binding curve of the antibody to cells overexpressing TIGIT

[0107] Figure 3 The binding curve of the antibody or VHH-Fc fusion protein to cells overexpressing PVRIG

[0108] Figure 4 The binding curve of the antibody or fusion protein to cells overexpressing PVRIG

[0109] Figure 5 The binding curve of the antibody or VHH-Fc fusion protein blocking the binding of ligand PVRL2 to cell surface PVRIG

[0110] Figure 6 Showing the cell activity of the antibody or VHH-Fc fusion protein blocking the PVRL2-PVRIG interaction

[0111] Figure 7 Showing the cell activity of the antibody or VHH-Fc fusion protein blocking the PVRL2-PVRIG interaction

[0112] Figure 8 Showing the cell activity of the antibody blocking the PVR-TIGIT interaction

[0113] Figure 9 Showing the activity of the antibody or fusion protein activating NK cells Detailed implementation methods

[0114] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs

[0115] The term

[0116] It should be noted that the term "a" entity refers to one or more of such entities. For example, "an antibody" should be understood as one or more antibodies. Therefore, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein

[0117] "About" refers to the conventional error range of the corresponding value that is readily known to those skilled in the relevant technical field. In some embodiments, "about" as used herein refers to the described value and its range of ±10%, ±5%, or ±1%

[0118] As used herein, the term "comprising" or "including" means that an antibody, composition, method, etc. includes the recited elements, such as components or steps, but does not exclude others. "Consisting essentially of" means that an antibody, composition, method, etc. excludes other elements that have a fundamental impact on the characteristics of the combination, but does not exclude elements that have no essential impact on the antibody, composition, method, etc. "Consisting of" means excluding elements not specifically recited.

[0119] The term "polypeptide" is intended to cover both the singular "polypeptide" and the plural "polypeptides", and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids, and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to two or more amino acid chains, and "polypeptide" can be used in place of any of the above terms, or used interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to the products of post-expression modification of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. Polypeptides can be derived from natural biological sources or produced by recombinant techniques, but they do not have to be translated from a specified nucleic acid sequence, and they may be produced in any manner, including chemical synthesis.

[0120] Those of ordinary skill in the art will understand that the CDR regions of an antibody are responsible for the binding specificity of the antibody to an antigen. Given the sequences of the variable regions of the heavy and light chains of a known antibody, there are currently several methods for determining the CDR regions of the antibody, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, the application of each definition of the CDR of an antibody or its variant will be within the scope of the terms defined and used herein. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can generally determine which residues are included in a particular CDR without relying on any experimental data outside of the sequence itself.

[0121] "Antibody" and "antigen-binding fragment" refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any of its antigen-binding fragments or its single chain. Thus, the term "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, the complementarity-determining regions (CDRs) of the heavy or light chain or their ligand-binding portions, the variable region of the heavy chain (VH), the variable region of the light chain (VL), the constant region of the heavy chain (CH), the constant region of the light chain (CL), the framework regions (FRs), or any portion thereof, or at least a portion of a binding protein. The CDR regions include the CDR regions of the light chain (LCDR1-3) and the CDR regions of the heavy chain (HCDR1-3). The constant region of the heavy chain (CH) includes the CH1 domain, the hinge (e.g., upper, middle, and / or lower hinge regions) domain, the CH2 domain, and the CH3 domain; the crystallizable fragment (Fc fragment) corresponds to the CH2 and CH3 domains. Antibodies and antigen-binding fragments can specifically recognize and bind to a polypeptide or polypeptide complex of one or more (e.g., two) antigens. Antibodies or antigen-binding fragments that specifically recognize and bind to multiple (e.g., two) antigens can be referred to as multispecific (e.g., bispecific) antibodies or antigen-binding fragments.

[0122] A complete antibody includes a heavy chain or and a light chain. The classes of heavy chains include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and there are also some subclasses (e.g., γ1-γ4). The nature of this chain determines the "class" of the antibody to be IgG, IgM, IgA, IgD, or IgE, respectively. Antibody subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and the functional specificities conferred are also known. All antibody classes are within the scope of protection disclosed in the present invention. In some embodiments, an antibody includes two heavy chains or and two light chains, and these four chains are connected by disulfide bonds in a "Y" configuration, where the light chain starts from the "mouth" of the "Y" and continues through the variable region surrounding the heavy chain.

[0123] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab’)2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen recognized by the complete antibody. The term "antibody fragment" includes aptamers, mirror image isomers, and diabodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

[0124] The term "Fab" generally refers to the antigen-binding portion of a conventional antibody (e.g., IgG), including the variable heavy chain region VH, the variable light chain region VL, the constant heavy chain domain CH1, and the constant light chain domain CL of the antibody. In a conventional antibody, the C-terminus of VH is linked to the N-terminus of CH1 to form the heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form the light chain, and the C-terminus of CH1 is further linked to the hinge region and other constant domain regions of the heavy chain to form the heavy chain. In some embodiments, "Fab" also refers to a variant structure of Fab. For example, in certain embodiments, the C-terminus of VH is linked to the N-terminus of CL to form one polypeptide chain, and the C-terminus of VL is linked to the N-terminus of CH1 to form another polypeptide chain, forming the structure of Fab (cross VH / VL); in certain embodiments, the CH1 of Fab is not linked to the hinge region, but rather the C-terminus of CL is linked to the hinge region of the heavy chain, forming the structure of Fab (cross Fd / LC).

[0125] "Single-chain antibody", "single-chain variable fragment", or "scFv" refers to a fusion protein of the variable heavy chain region (VH) and the variable light chain region (VL) of an immunoglobulin. In some aspects, these regions are linked by a peptide linker of 10 to about 25 amino acids. The peptide linker can be rich in glycine to increase flexibility and rich in serine or threonine to increase solubility, and can link the N-terminus of VH and the C-terminus of VL, and vice versa. Although the constant regions are removed and a linker is introduced, the protein retains the specificity of the original immunoglobulin. scFv molecules are generally known in the art and are described, for example, in U.S. Patent 5,892,019.

[0126] The term "single-domain antibody" or "sdAb" refers to an antigen-binding fragment that contains only a single antibody variable region. A single sdAb can bind to an antigen without the need to pair with a corresponding CDR-containing polypeptide. The variable heavy chain region may also be referred to herein as "VHH". Some VHHs are also called nanobodies. VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this article, a VHH that can bind to PVRIG may be referred to as an anti-PVRIG VHH fragment.

[0127] "Bispecific antibody" refers to an antibody that has two antigen-binding sites, and the two antigen-binding sites can be different epitopes of the same antigen or different epitopes of different antigens.

[0128] "Homology" or "identity" refers to the sequence similarity between two peptides or two nucleic acids. Homology or identity can be determined by comparing the positions in each sequence that can be aligned. When the positions in the sequences being compared are occupied by the same base or amino acid, the molecules are homologous or identical at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0129] The terms "polynucleotide" and "nucleic acid" are used interchangeably. A nucleic acid or polynucleotide (or polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98% or 99%) of "identity or sequence identity" with another sequence means that when the sequences are aligned, that percentage of bases (or amino acids) in the two sequences being compared are the same. The alignment and the percentage of identity or sequence identity can be determined using visual inspection or software programs known in the art, such as the software programs described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignment is preferably performed using default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both of which use the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGHSCORE; Databases = non-redundant; GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides having the specified percentage of identity and encoding polypeptides having the same or similar biological activity.

[0130] "At least 80% identity" means about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range (including the endpoints) between any two of these values or any value therein.

[0131] "Conservative amino acid substitutions" are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, non-essential amino acid residues of an immunoglobulin polypeptide are preferably replaced with other amino acid residues from the same side chain family. In other embodiments, a string of amino acids may be replaced with a structurally similar string of amino acids that differs in sequence and / or in the composition of the side chain family.

[0132] In some embodiments, the conservative amino acid substitutions are preferably those in which one amino acid within one of the following groups (a)-(e) is replaced with another amino acid residue within the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0133] Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln, or Glu; Met is replaced by Leu, Tyr, or Ile; Phe is replaced by Met, Leu, or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp; and / or Phe is replaced by Val, Ile, or Leu.

[0134] The PVRIG-binding protein or bispecific antibody disclosed by the present invention includes modified derivatives, that is, modified by covalent connection of any type of molecule to the PVRIG-binding protein or bispecific antibody, wherein the covalent connection does not prevent the PVRIG-binding protein or bispecific antibody from binding to the epitope. The PVRIG-binding protein or bispecific antibody can be glycosylated, acetylated, polyethylene glycolated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytically cleaved, linked to a cell ligand or other protein, etc. Any one of numerous chemical modifications can be carried out by the prior art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

[0135] In some embodiments, the PVRIG-binding protein or bispecific antibody can be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biologic response modifier, an agent, or PEG.

[0136] The term "pharmaceutically acceptable" refers to substances approved by a government regulatory agency or listed in a recognized pharmacopoeia for use in animals, particularly in humans. In addition, "pharmaceutically acceptable excipients" generally refer to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid, etc.

[0137] The term "excipient" refers to a diluent, adjuvant, excipient or vehicle that can be administered to a patient together with an active ingredient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal or vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Aqueous solutions of salts, aqueous solutions of glucose and aqueous solutions of glycerol can also be used as liquid carriers, especially for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition may also contain a small amount of wetting agent or emulsifying agent, or pH buffer. Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid, chelating agents, and agents for adjusting tonicity such as dextrose are also foreseeable. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, etc. The composition can be formulated into suppositories with conventional binders and carriers such as triglycerides. Oral preparations can include standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions will contain a clinically effective dose of an antibody or antigen-binding fragment or fusion protein, preferably in purified form, together with a suitable amount of excipients to provide a dosage form suitable for the patient. The preparation should be suitable for the mode of administration. The parenteral preparation can be encapsulated in an ampoule, a disposable syringe or a multi-dose vial made of glass or plastic.

[0138] The PVRIG-binding protein or bispecific antibody (including antibody, antigen-binding fragment or fusion protein, etc.) of the present invention includes neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, salts formed with anions derived from, for example, hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-aminoethanol, histidine, procaine, etc. The PVRIG-binding protein or bispecific antibody described herein can be neutral, that is, substantially without a net charge, such as when the pH of the composition containing the PVRIG-binding protein or bispecific antibody is at the isoelectric point of the protein, the protein is electrically neutral. The protein described herein can exist in a positively charged form, such as when the pH is below the isoelectric point, the protein molecule as a whole shows a positive charge. The protein described herein can exist in a negatively charged form, such as when the pH is above the isoelectric point, the protein molecule as a whole shows a negative charge.

[0139] The effective dose and treatment regimen for treating a particular patient will depend on various factors, including the specific PVRIG-binding protein or bispecific antibody or derivative used, the patient's age and weight, general health, gender and diet, as well as the time of administration, excretion frequency, drug combination, and the severity of the particular disease being treated. These factors are judged by medical care personnel including those within the scope of ordinary skill in the art. The dose used can be determined by pharmacological and pharmacokinetic principles well known in the art. In some embodiments, the dose of the PVRIG-binding protein or bispecific antibody of the present invention administered to a patient is from 0.01 mg / kg to 100 mg / kg of the patient's body weight per administration. In some embodiments, it is administered once a week, once every two weeks, once every three weeks, or once every four weeks.

[0140] The antibodies disclosed in the present invention can be derived from any animal, such as a mammal. Preferably, the antibody is a human, murine, donkey, rabbit, goat, camel, llama, horse, or chicken-derived antibody. In another embodiment, the variable region can be of chondricthoid origin (e.g., from a shark).

[0141] "Treatment" refers to therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent, slow down, improve, or stop adverse physiological changes or disorders, such as the progression of a disease, including but not limited to the following whether detectable or not, remission of symptoms, reduction in the degree of the disease, stabilization of the disease state (i.e., not getting worse), delay or slowdown in the progression of the disease, improvement in the disease state, mitigation, alleviation, or disappearance (whether partial or complete), extension of the survival period compared to that expected without treatment, etc. Patients in need of treatment include those who already have a disease or disorder, those who are prone to having a disease or disorder, or those who need to prevent the disease or disorder, and who can or are expected to benefit from the administration of the antibodies or pharmaceutical compositions disclosed in the present invention for detection, diagnostic procedures, and / or treatment.

[0142] "Patient" refers to any mammal in need of diagnosis, prevention, prognosis, or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, etc.

[0143] Preparation of Antibody or Fusion Protein

[0144] There are various methods known in the art for preparing antibodies or fusion proteins, such as hybridoma technology, recombinant DNA technology, transgenic mouse technology, and phage display libraries, etc.

[0145] Antibodies or fusion proteins can be prepared by using conventional recombinant DNA techniques. Vectors and cell lines for producing antibodies or fusion proteins can be selected, constructed, and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. Hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, the entire content including supplementary content of which is incorporated herein by reference.

[0146] In some embodiments, DNA encoding an antibody or fusion protein can be designed and synthesized according to the amino acid sequence of the antibody or fusion protein described herein by conventional methods, inserted into an expression vector, and then transfected into a host cell. The transfected host cell is cultured in a medium to produce a monoclonal antibody or fusion protein. In some embodiments, the expression vector includes at least one promoter element, a protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the inserted sequence. High-efficiency transcription can be obtained through the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and other cell promoters such as the actin promoter can also be applied. Suitable expression vectors can include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), pSVL, pMSG, pRSVcat, pSV2dhfr, pBC12MI, pCS2, and pCHO1.0, etc. Commonly used mammalian host cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, murine L cells, and CHO cells, etc.

[0147] The gene sequence of an antibody or fusion protein can be inserted into an expression vector by standard methods (e.g., ligating the gene sequence to complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are present). Before inserting the gene sequence, the expression vector may already carry the antibody constant region sequence. For example, one method of converting antibody-related VH and VL sequences into a full-length antibody gene is to insert them separately into an expression vector that already encodes the heavy chain constant region and the light chain constant region, such that the VH sequence is operably linked to the CH sequence in the vector, and the VL sequence is operably linked to the CL sequence in the vector. Alternatively, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody or fusion protein by the host cell. Or, the gene sequence can be cloned into a vector encoding a signal peptide that promotes the secretion of the antibody or fusion protein by the host cell, such that the 3' end of the gene sequence encoding the signal peptide is in-frame with the 5' end of the gene sequence encoding the antibody (heavy chain and / or light chain) or fusion protein. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein), e.g., MEFGLSLVFLVLILKGVQC (SEQ ID NO:56).

[0148] In some embodiments, the expression vector needs to contain a selection marker. Common selection markers include selection genes such as dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, etc., to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into a host cell lacking the above genes, and after culturing in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein. The obtained antibody or fusion protein can be separated or purified by conventional technical means, such as protein A-agarose gel, ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, or affinity chromatography, etc.

[0149] Examples

[0150] The technical solutions of the present invention are further illustrated by the following specific examples. The specific examples do not represent a limitation on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0151] Example 1: Preparation of PVRIG recombinant protein and anti-PVRIG antibodies SF35 and H4

[0152] The DNA sequence of the PVRIG recombinant protein PVRIG-his (such as SEQ ID NO:1) was inserted into an expression plasmid to obtain a recombinant plasmid. Then, the above plasmid was transiently transfected into HEK293 cells by polyetherimide (PEI). After culturing, the supernatant was collected and the PVRIG-his protein sample was purified.

[0153] The amino acid sequence of PVRIG-his is as follows:

[0154] TPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPE RGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPP SSDPGLSAPPTPAPILRADGGGGSHHHHHHHH(SEQ ID NO:1)

[0155] The amino acid sequences of the heavy and light chains of antibody SF35 and antibody H4 are shown in Table 3. The nucleic acid sequences of the antibody heavy and light chains were cloned into expression plasmids respectively, then transfected into CHO-K1 cells. After culturing, the supernatant was collected and the antibody was purified.

[0156] Table 3 Amino acid sequences of antibody SF35 and antibody H4

[0157]

[0158]

[0159] Example 2: Preparation of anti-PVRIG / TIGIT bispecific antibody and VHH-Fc fusion protein

[0160] Two structures of anti-PVRIG / TIGIT bispecific antibodies were constructed and named respectively as (1) bispecific antibody Bi-VH1, the schematic diagram of whose structure is as Figure 1A shown; (2) bispecific antibody Bi-VH3, the schematic diagram of whose structure is as Figure 1B shown; The structural feature of bispecific antibody Bi-VH1 is that the VHH fragment of anti-PVRIG is linked to the N-terminus of the heavy chain of anti-TIGIT antibody, such as bispecific antibodies Bi-VH1-94Y and Bi-VH1-94W; while the structural feature of bispecific antibody Bi-VH3 is that the VHH fragment of anti-PVRIG is linked to the C-terminus of the heavy chain of anti-TIGIT antibody, such as bispecific antibodies Bi-VH3-94Y and Bi-VH3-94W. The VHH-Fc fusion protein contains the following structures in sequence from the N-terminus to the C-terminus: the VHH fragment of anti-PVRIG, a peptide linker and an Fc fragment.

[0161] The relevant sequences of the antibody or VHH-Fc fusion protein are shown in Table 1-2 and Table 4-7; among them, the VHH-Fc fusion protein N1-94Y-Fc contains two identical sequences (as shown in SEQ ID NO: 36), and its nucleic acid sequence is as shown in SEQ ID NO: 46; the VHH-Fc fusion protein N1-94W-Fc contains two identical sequences (as shown in SEQ ID NO: 37), and its nucleic acid sequence is as shown in SEQ ID NO: 47; the VHH-Fc fusion protein 32 contains two identical sequences (as shown in SEQ ID NO: 38), and its nucleic acid sequence is as shown in SEQ ID NO: 48; the VHH-Fc fusion protein 34 contains two identical sequences (as shown in SEQ ID NO: 39), and its nucleic acid sequence is as shown in SEQ ID NO: 49; the VHH-Fc fusion protein 37 contains two identical sequences (as shown in SEQ ID NO: 40), and its nucleic acid sequence is as shown in SEQ ID NO: 50.

[0162] The bispecific antibody Bi-VH1-94Y contains two identical first polypeptides (shown as SEQ ID NO: 41) and two identical second polypeptides (shown as SEQ ID NO: 31). The first polypeptide consists of a VHH fragment against PVRIG (shown as SEQ ID NO: 13), a peptide linker L1 (GGGGSGGGGS, shown as SEQ ID NO: 32), and an anti-TIGIT heavy chain sequence (shown as SEQ ID NO: 29). The second polypeptide consists of an anti-TIGIT light chain sequence (shown as SEQ ID NO: 31). The nucleic acid sequence of its first polypeptide is shown as SEQ ID NO: 51, and the nucleic acid sequence of the second polypeptide is shown as SEQ ID NO: 45. The bispecific antibody Bi-VH1-94W contains two identical first polypeptides (shown as SEQ ID NO: 42) and two identical second polypeptides (shown as SEQ ID NO: 31). The first polypeptide consists of a VHH fragment against PVRIG (shown as SEQ ID NO: 14), a peptide linker L1 (GGGGSGGGGS, shown as SEQ ID NO: 32), and an anti-TIGIT heavy chain sequence (shown as SEQ ID NO: 29). The second polypeptide consists of an anti-TIGIT light chain sequence (shown as SEQ ID NO: 31). The nucleic acid sequence of its first polypeptide is shown as SEQ ID NO: 52, and the nucleic acid sequence of the second polypeptide is shown as SEQ ID NO: 45. The bispecific antibody Bi-VH3-94Y contains two identical first polypeptides (shown as SEQ ID NO: 43) and two identical second polypeptides (shown as SEQ ID NO: 31). The first polypeptide consists of an anti-TIGIT heavy chain sequence (shown as SEQ ID NO: 30), a peptide linker L2 (GGGGSGGGGSGGGGS, shown as SEQ ID NO: 33), and a VHH fragment against PVRIG (shown as SEQ ID NO: 13). The second polypeptide consists of an anti-TIGIT light chain sequence (shown as SEQ ID NO: 31). The nucleic acid sequence of its first polypeptide is shown as SEQ ID NO: 53, and the nucleic acid sequence of the second polypeptide is shown as SEQ ID NO: 45.The bispecific antibody Bi-VH3-94W contains two identical first polypeptides (as shown in SEQ ID NO: 44) and two identical second polypeptides (as shown in SEQ ID NO: 31). The first polypeptide consists of an anti-TIGIT heavy chain sequence (as shown in SEQ ID NO: 30), a peptide linker L2 (GGGGSGGGGSGGGGS, as shown in SEQ ID NO: 33), and a VHH fragment against PVRIG (as shown in SEQ ID NO: 14). The second polypeptide consists of an anti-TIGIT light chain sequence (as shown in SEQ ID NO: 31). The nucleic acid sequence of the first polypeptide is as shown in SEQ ID NO: 54, and the nucleic acid sequence of the second polypeptide is as shown in SEQ ID NO: 45.;

[0163] Table 4 Sequences of VHH Fragments Against PVRIG and Their CDRs

[0164]

[0165]

[0166] Table 5 Sequences of VHH-Fc Fusion Proteins and Their Compositions

[0167]

[0168] The nucleic acid sequences of the first and second polypeptides of the bispecific antibody were respectively cloned into expression plasmids, then transiently transfected into HEK293F cells. After cultivation, the supernatant was collected and the bispecific antibody was purified.

[0169] The nucleic acid sequence of the VHH-Fc fusion protein was cloned into an expression plasmid, then transiently transfected into HEK293F cells. After cultivation, the supernatant was collected and the VHH-Fc fusion protein was purified.

[0170] Example 3: Determination of the Dissociation Constants of Anti-PVRIG / TIGIT Bispecific Antibody and VHH-Fc Fusion Protein Using Biacore

[0171] The affinity of antibodies and VHH-Fc fusion proteins for PVRIG-his and TIGIT-his proteins was detected using a Biacore T200. Antibodies or VHH-Fc fusion proteins were conjugated to the surface of a CM5 chip, and then PVRIG-his or TIGIT-his was captured using the chip. Proteins of different concentrations of PVRIG-his or TIGIT-his were injected into the CM5 chip. As shown in Table 6, VHH-Fc fusion proteins 32, 34, and 37 showed strong affinity for the PVRIG-his protein, with KD values of 0.65 nM, 0.89 nM, and 0.96 nM, respectively. As shown in Table 7, the bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y, and Bi-VH3-94W had high affinity for binding PVRIG-his, with KD values of 3.05 nM, 2.35 nM, 5.64 nM, and 5.83 nM, respectively, which were comparable to the affinity of the VHH-Fc fusion proteins N1-94W-Fc and N1-94Y-Fc. As shown in Table 8, the bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y, and Bi-VH3-94W had high affinity for binding TIGIT-his, with KD values of 4.99 nM, 5.58 nM, 3.91 nM, and 3.70 nM, respectively, which were comparable to the affinity of the parental antibody h10D8OF (the sequence of its heavy chain is shown in SEQ ID NO:29, and the sequence of its light chain is shown in SEQ ID NO:31).

[0172] Table 6 Affinity data of antibodies or VHH-Fc fusion proteins for PVRIG-his (prepared in Example 1)

[0173] Sample Ka (1 / Ms) Kd (1 / s) KD (M) VHH-Fc Fusion Protein 32 9.47E+05 6.17E-04 6.52E-10 VHH-Fc Fusion Protein 34 7.36E+05 6.57E-04 8.91E-10 VHH-Fc Fusion Protein 37 4.79E+05 4.60E-04 9.60E-10 SF35 8.17E+05 2.99E-04 3.66E-10

[0174] Table 7 Affinity data of antibodies or VHH-Fc fusion proteins for PVRIG-his (purchased from ACRO, catalog number PVG-H52H5)

[0175]

[0176] Table 8 Affinity data of antibodies for TIGIT-his (purchased from ACRO, catalog number TIT-H52H5)

[0177] Sample Ka (1 / Ms) Kd (1 / s) KD (M) Bi-VH1-94Y 4.75E+05 2.37E-03 4.99E-09 Bi-VH3-94Y 5.16E+05 2.02E-03 3.91E-09 Bi-VH1-94W 4.01E+05 2.24E-03 5.58E-09 Bi-VH3-94W 5.37E+05 1.98E-03 3.70E-09 h10D8OF 4.61E+05 3.50E-03 7.60E-09

[0178] Example 4: Binding experiment of anti-PVRIG / TIGIT bispecific antibody to cells overexpressing TIGIT

[0179] Take appropriate amounts of bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y, and Bi-VH3-94W, as well as the parental anti-TIGIT antibody h10D8OF. Dilute them to an initial concentration of 200 nM in a 96-well V-bottom plate with 1×PBS and perform 3-fold serial dilutions with 1×PBS to obtain a total of 9 serial dilution concentrations.

[0180] Take Jurkat-TIGIT cells in good growth state (the full-length human TIGIT gene (SEQ ID NO: 55) was transfected into the Jurkat cell line (ATCC, Clone E6-1, TIB-152 TM ), and the resulting cell line that could stably express human TIGIT was named Jurkat-TIGIT cells), centrifuge to discard the supernatant, resuspend the cells with 1×PBS to a density of 1×10 7 cells / mL, and add 50 μL / well to a 96-well V-bottom plate. At this time, the number of cells is 5×10 5 cells / well. Add 50 μL of the antibody sample at the corresponding concentration to each well and mix well. At this time, the maximum final concentration of the antibody is 100 nM. Place the 96-well V-bottom plate in a 4°C refrigerator and incubate for 60 min. Then centrifuge the 96-well V-bottom plate to remove the supernatant, wash it with 1×PBS and remove the supernatant for the next incubation. Dilute anti-FC PE (purchased from BioLegend, catalog number 366903) with 1×PBS at a ratio of 1:500. Add 100 μL of the diluted anti-FC PE to resuspend the cells in each well except the blank well, and place it in a 4°C refrigerator in the dark and incubate for 30 min. After incubation, wash with 1×PBS, and finally resuspend with 1×PBS at a volume of 150 μL / well. Collect the cells with a flow cytometer to detect the fluorescent antibody bound to the cell surface, and obtain the original data of the mean fluorescence intensity (MFI) value. The final result is plotted with the antibody concentration on the x-axis and the MFI on the y-axis using a four-parameter model for curve fitting, and the result is as Figure 2 shown.

[0181] The full-length human TIGIT gene sequence is as follows:

[0182] Atgcgctggtgtctgctgctgatttgggcccagggactgagacaggctcctctggcttcaggaatgatgaccggcaccatcgagaccaccggaaacatcagcgccgagaagggaggaagcatcatcctccagtgccacctgagtagcacaaccgcacaggtcacccaggtcaattgggagcagcaggaccagctgctggccatttgcaacgccgatctgggttggcacatctctcctagcttcaaggacagagtggccccaggaccaggactgggactgacactgcagagtctgaccgtgaacgacaccggcgagtacttctgcatctaccacacctacccagacggcacctacacaggacggatcttcctggaggtgctggagtctagcgtggcagagcacggagccagattccagatccctctgctgggagctatggcagctacactggtcgtgatctgcaccgcagtgatcgtggtcgtggctctgacacggaagaagaaggccctgagaatccacagcgtggagggagacctgagaagaaagagcgccggacaggaggagtggtctcctagcgctccttctcctccaggctcttgtgtgcaggcagaagcagctccagcaggtctctgcggagaacagagaggagaggattgcgccgagctgcacgactacttcaacgtgctgagctaccggagcctgggcaattgcagcttcttcaccgagaccggatga(SEQ ID NO:55)

[0183] The results showed that the bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y and Bi-VH3-94W could all bind to Jurkat-TIGIT cells, and the EC 50 values were 0.23 nM, 0.23 nM, 0.23 nM and 0.12 nM, respectively. The binding ability to TIGIT expressed on the cell surface by the parental anti-TIGIT antibody h10D8OF (EC 50 = 0.13 nM) was similar.

[0184] Example 5: Binding experiment of anti-PVRIG / TIGIT bispecific antibody and cells overexpressing PVRIG

[0185] Take appropriate amounts of bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y, and Bi-VH3-94W, as well as VHH-Fc fusion proteins N1-94W-Fc, N1-94Y-Fc, 32, 34, and 37, and antibodies SF35, hIgG1 (Beijing Sino Biological Inc., Catalog No.: MA14OC2903). Dilute them to an initial concentration of 200 nM in a 96-well V-bottom plate with 1×PBS and perform 3-fold serial dilutions with 1×PBS to obtain a total of 9 serial dilution concentrations.

[0186] Transfer the cDNA of PVRIG (purchased from Beijing Sino Biological Inc., Catalog No. HG28312-UT) into Jurkat-NFAT cells (Jurkat-NFAT cells construction: electrotransfer the plasmid containing the NFAT luciferase reporter gene into Jurkat cells) to obtain Jurkat-NFAT cells overexpressing PVRIG, named Jurkat-NFAT-PVRIG. Take Jurkat-NFAT-PVRIG cells in good growth state, centrifuge to discard the supernatant, resuspend the cells with 1×PBS to a density of 1×10 7 cells / mL, and add 50 μL / well to a 96-well V-bottom plate (Corning, Catalog No.: 3897). At this time, the number of cells is 5×10 5 cells / well. Add 50 μL of the corresponding concentration of antibody or VHH-Fc fusion protein sample to each well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 100 nM. Place the 96-well V-bottom plate in a 4°C refrigerator and incubate for 60 min. After incubation, centrifuge the 96-well V-bottom plate to remove the supernatant, wash with 1×PBS and then remove the supernatant for the next incubation. Dilute anti-FC PE (purchased from BioLegend, Catalog No. 366903) with 1×PBS at a ratio of 1:500. Add 100 μL of the diluted anti-FC PE to resuspend the cells in each well except the blank well, and place it in a 4°C refrigerator for dark incubation for 30 min. After incubation, wash with 1×PBS, and finally resuspend with 1×PBS at a volume of 150 μL / well. Collect the cells with a flow cytometer to detect the fluorescent antibody bound to the cell surface and obtain the original data MFI value. The final results are plotted with the antibody or VHH-Fc fusion protein concentration on the x-axis and MFI on the y-axis using a four-parameter model for curve fitting. The results are shown in Tables 9, Figure 3 and Figure 4 as shown.

[0187] Table 9 Cellular-level Affinity of Antibodies or VHH-Fc Fusion Proteins

[0188] Sample <![CDATA[EC 50 (nM)]]> Relative Activity (%) * VHH-Fc Fusion Protein 32 0.67 136.16 VHH-Fc Fusion Protein 34 0.95 96.09 VHH-Fc Fusion Protein 37 0.87 105.21 SF35 0.91 100.00 hIgG1 NA NA

[0189] * Relative Activity % = {EC 50 (Sample) - EC 50 (SF35)} / EC 50 (SF35) × 100%

[0190] From Table 9 and Figure 3 the results, it can be seen that VHH-Fc fusion proteins 32, 34, and 37 can all bind to human PVRIG highly expressed on the surface of Jurkat-NFAT-PVRIG cells with high affinity. The EC 50 of VHH-Fc fusion protein 32 is 0.67 nM, the EC 50 of VHH-Fc fusion protein 34 is 0.95 nM, the EC 50 of VHH-Fc fusion protein 37 is 0.87 nM. The binding activities of VHH-Fc fusion proteins 32 and 37 are superior to those of antibody SF35 (EC 50 = 0.91 nM).

[0191] From Figure 4 the results, it can be seen that bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y, and Bi-VH3-94W can all significantly bind to Jurkat-NFAT-PVRIG cells, and their EC 50 are 0.26 nM, 0.32 nM, 1.72 nM, and 2.33 nM respectively. The binding abilities of bispecific antibodies Bi-VH1-94Y and Bi-VH1-94W to PVRIG expressed on the cell surface are comparable to those of VHH-Fc fusion proteins N1-94W-Fc (EC 50 = 0.34 nM) and N1-94Y-Fc (EC 50 = 0.40 nM).

[0192] Example 6: Experiment on the Blocking of the Binding of Ligand PVRL2 to PVRIG on the Cell Surface by VHH-Fc Fusion Proteins

[0193] Take appropriate amounts of VHH-Fc fusion proteins 32, 34, 37, as well as antibodies SF35, H4, and hIgG1 (Beijing Protein Innovation Co., Ltd., product number: MA14OC2903), and dilute them to an initial concentration of 600 nM with 1×PBS, and perform 4-fold serial dilutions with 1×PBS to obtain a total of 10 serial dilution concentrations.

[0194] Take Jurkat-NFAT-PVRIG cells in good growth condition, centrifuge to discard the supernatant, and resuspend the cells with 1×PBS to a density of 1×10 7 cells / mL. Add the cells to a 96-well V-bottom plate (Corning, catalog number: 3897) at 50 μL / well. At this time, the number of cells is 5×10 5 cells / well. Add 50 μL of the antibody or VHH-Fc fusion protein at the corresponding concentration to each well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 300 nM. Place the 96-well V-bottom plate in a 4°C refrigerator and incubate for 10 min. During the incubation of the antibody, take an appropriate amount of the ligand PVRL2-mFc-bio (purchased from Acro, catalog number CD2-H82A3) and dilute it to 600 nM with 1×PBS. After the incubation is completed, take out the 96-well V-bottom plate, quickly add 50 μL of the ligand PVRL2-mFc dilution to each well and pipette to mix well. At this time, the final concentration of the ligand is 200 nM, and the maximum final concentration of the antibody or VHH-Fc fusion protein is 200 nM. Place the 96-well V-bottom plate in a 4°C refrigerator and continue to incubate for 60 min.

[0195] After the incubation is completed, centrifuge the 96-well V-bottom plate to remove the supernatant, wash it with 1×PBS and then remove the supernatant for the next incubation. Dilute streptavidin-phycoerythrin (SA-PE; BioLegend, catalog number 740452) with 1×PBS at a ratio of 1:800. Add 100 μL of the diluted SA-PE to resuspend the cells in each well except the blank wells, and place them in a 4°C refrigerator in the dark for 30 min.

[0196] Collect the cells with a flow cytometer, detect the fluorescent antibody bound to the cell surface, and obtain the original data MFI value. Finally, the results are plotted with the concentration of the antibody or VHH-Fc fusion protein on the x-axis and MFI on the y-axis using a four-parameter model for curve fitting. The results are as Figure 5 shown.

[0197] From Figure 5 the results, it can be seen that VHH-Fc fusion proteins 32, 34, and 37 can all block the binding of the ligand PVRL2 to human PVRIG expressed on the surface of Jurkat-NFAT-PVRIG cells. The IC 50 of VHH-Fc fusion protein 32 is 24.48 nM, the IC 50 of VHH-Fc fusion protein 34 is 1.67 nM, the IC 50 of VHH-Fc fusion protein 37 is 1.01 nM. The blocking abilities of VHH-Fc fusion proteins 34 and 37 are comparable to those of the antibody SF35 (IC 50 = 0.56).

[0198] Example 7: PVRIG cell activity experiment of bispecific antibody

[0199] 1) Take appropriate amounts of VHH-Fc fusion proteins 32, 34, 37, and antibodies SF35, H4, hIgG1 (Beijing Protein Innovation Co., Ltd., catalog number: MA14OC2903), and dilute them to an initial concentration of 600 nM with the detection dilution solution (add 55 mL of FBS (ExCell Biology, catalog number: FSP500) to 500 mL of RPMI 1640 medium (Gibco, catalog number: 11875093) as the detection dilution solution), and perform 3-fold serial dilutions with the detection dilution solution to obtain a total of 10 serial dilution concentrations; take a 96-well white plate and add 50 μL per well for standby.

[0200] Transfer the cDNA of PVRL2 (purchased from Beijing Protein Innovation Co., Ltd., catalog number HG10005-UT) into CHO-OKT3 cells (the construction method can be referred to in Patent CN111748580A) to obtain CHO-OKT3 cells overexpressing PVRL2, named CHO-OKT3-PVRL2. Take Jurkat-NFAT-PVRIG cells and CHO-OKT3-PVRL2 cells in good growth state, centrifuge to discard the supernatant, resuspend with the detection dilution solution, and count with a cell counter. According to the cell density, dilute Jurkat-NFAT-PVRIG cells with the detection dilution solution to make the cell density 4×10 6 cells / mL. Take out the 96-well white plate with 50 μL / well of antibody or VHH-Fc fusion protein added, add 50 μL of Jurkat-NFAT-PVRIG cells to each well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 300 nM.

[0201] Place the 96-well white plate in a 37°C CO2 incubator and incubate for 10 min, then take out the 96-well white plate. Dilute CHO-OKT3-PVRL2 cells with the detection dilution solution to make the cell density 2×10 6 cells / mL. Add CHO-OKT3-PVRL2 cells at a volume of 50 μL per well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 200 nM. Place the 96-well white plate in a CO2 incubator at 37°C and incubate for 6 h. Take out the Bio-Lite TM Luciferase Assay System chromogenic solution from the -20°C refrigerator in advance and dissolve it at room temperature. After incubation, take out the 96-well white plate from the incubator, equilibrate at room temperature for about 8 min, then add 70 μL of chromogenic solution to each well, place it in the dark at room temperature for 5 min, and read the plate as soon as possible.

[0202] Using the Luminescence detection module on the SpectraMax multi-functional microplate reader, set PMT and Optics to 500, read the relative light units (RLU), and shake the plate before reading. With the concentration of antibody or VHH-Fc fusion protein as the abscissa and the relative light units as the ordinate, perform curve fitting on the samples using a four-parameter model to obtain the four-parameter curve as Figure 6 shown.

[0203] The results showed that VHH-Fc fusion proteins 32, 34, and 37 were all able to effectively block the inhibitory signal of PVRL2-PVRIG, thereby activating the NFAT signaling pathway in Jurkat. The EC 50 of VHH-Fc fusion protein 32 was 140.9 nM, the EC 50 of VHH-Fc fusion protein 34 was 124.8 nM, and the EC 50 of VHH-Fc fusion protein 37 was 10.0 nM.

[0204] 2) Take appropriate amounts of bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y, Bi-VH3-94W, VHH-Fc fusion proteins N1-94Y-Fc and N1-94W-Fc and dilute them to 600 nM as the starting concentration with RPMI 1640 medium, and perform 5-fold serial dilutions with RPMI1640 medium to obtain a total of 8 series of dilution concentrations; take a 96-well white plate and add 100 μL per well for standby.

[0205] Take Jurkat-NFAT-PVRIG cells and CHO-OKT3-PVRL2 cells in good growth state, centrifuge to discard the supernatant, resuspend them with RPMI 1640 medium containing 10% FBS (fetal bovine serum), and count them using a cell counter. Jurkat-NFAT-PVRIG cells were diluted with RPMI 1640 medium containing 10% FBS to make the cell density 4×10 6 cells / mL. Take out the 96-well white plate that has been added with 100 μL / well of antibody or VHH-Fc fusion protein, add 50 μL of Jurkat-NFAT-PVRIG cells to each well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 400 nM.

[0206] CHO-OKT3-PVRL2 cells were diluted with RPMI 1640 medium containing 10% FBS to make the cell density 2×10 6cells / mL. Take out the 96-well white plate containing the antibody and Jurkat-NFAT-PVRIG cells, add CHO-OKT3-PVRL2 cells at a volume of 50 μL per well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 300 nM. Place the white plate in a CO2 incubator and incubate at 37 °C for 6 h.

[0207] Take out Bio-Lite from the -20 °C refrigerator in advance TM Luciferase Assay System chromogenic solution and dissolve it at room temperature. Take out the white plate from the incubator, equilibrate it at room temperature for about 8 min, then add 70 μL of the chromogenic solution to each well, place it in the dark at room temperature for 5 min, and read the plate as soon as possible. Use the Luminescence detection module on the SpectraMax multifunctional microplate reader, set PMT and Optics to 500, read the relative light units (RLU), and shake before reading the plate. With the concentration of the antibody or VHH-Fc fusion protein as the abscissa and the relative light units as the ordinate, perform curve fitting on the samples with a four-parameter model to obtain the four-parameter curve as Figure 7 shown.

[0208] The results show that both the bispecific antibody and the VHH-Fc fusion protein can effectively block the interaction between the PVRIG antigen expressed on the surface of Jurkat-NFAT-PVRIG cells and the highly expressed PVRL2 on the surface of CHO-OKT3-PVRL2 cells, and promote the activation of T cells. The EC 50 of Bi-VH1-94Y for activating T cells is 35.34 nM, Bi-VH1-94W is 51.27 nM, Bi-VH3-94Y is 67.15 nM, which is comparable to the blocking effects of the VHH-Fc fusion proteins N1-94W-Fc (EC 50 = 21.79 nM) and N1-94Y-Fc (EC 50 = 56.60 nM).

[0209] Example 8: Activity experiment of bispecific antibody and TIGIT cells

[0210] Take appropriate amounts of the bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y, and Bi-VH3-94W and dilute them to 700 nM as the starting concentration with RPMI1640 medium. Dilute the antibody and RPMI 1640 medium in a 1:4 multiple dilution to obtain a total of 9 series of dilution concentrations.

[0211] Take CHO-K1-CD155 cells in good growth condition (the construction method refers to the construction of the sAPC cell line in Example 1 of Patent CN111748580A), centrifuge to discard the supernatant, resuspend the cells with RPMI 1640 medium containing 10% FBS, count the cells, and prepare a cell suspension with a density of 1×10 5 cells / mL. Add 100 μL / well (at this time, the number of cells is 1×10 4 cells / well) to a 96-well white plate, supplement sterile water to the side wells, and place it in a 5% CO2, 37 °C incubator overnight.

[0212] Take Jurkat-TIGIT cells in good growth condition, centrifuge to discard the supernatant, resuspend the cells with RPMI 1640 medium containing 10% FBS, count the cells, and prepare a cell suspension with a density of 5.0×10 6 cells / mL for later use. Take out the 96-well white plate with CHO-K1-CD155 cells from the incubator, discard the supernatant, and then add 40 μL / well of antibody dilution solution and 40 μL / well of Jurkat-TIGIT cell suspension (at this time, the number of cells is 2×10 5 cells / well) to columns 2-11, rows B-H of the 96-well white plate. At this time, the maximum final concentration of the antibody is 350 nM. Supplement sterile water to the side wells. Place the cell plate in a 5% CO2, 37 °C incubator and incubate for 6 h.

[0213] Take out the Bio-Lite TM Luciferase Assay System chromogenic solution from the -20 °C refrigerator in advance and dissolve it at room temperature. Take out the 96-well white plate from the incubator, equilibrate at room temperature for about 8 min, then add 60 μL of chromogenic solution to each well, place it in the dark at room temperature for 5 min, and read the plate as soon as possible. Use the Luminescence detection module on the SpectraMax multi-functional microplate reader, set PMT and Optics to 500, and read the relative light units (RLU). Use the Four-Parameter model to perform curve fitting on the samples, as Figure 8 shown.

[0214] The results show that the bispecific antibodies Bi-VH1-94Y (EC 50 = 1.42 nM), Bi-VH1-94W (EC 50 = 1.68 nM), Bi-VH3-94Y (EC 50 = 2.15 nM), Bi-VH3-94W (EC 50(= 1.71 nM) can effectively block the interaction between the TIGIT antigen expressed on the surface of Jurkat-TIGIT cells and the highly expressed CD155 on the surface of CHO-K1-CD155 cells, and promote the activation of T cells.

[0215] Example 9: Detection of the NK cell activity of bispecific antibodies

[0216] Add 55 mL of FBS to 500 mL of RPMI 1640 medium as the dilution solution for detection. Take appropriate amounts of bispecific antibodies Bi-VH1-94W, Bi-VH3-94W, VHH-Fc fusion protein N1-94W-Fc, and antibody h10D8OF. The sample groups are Bi-VH1-94W and Bi-VH3-94W, and the control groups are N1-94W-Fc, antibody h10D8OF, N1-94W-Fc + antibody h10D8OF (where the addition amounts of N1-94W-Fc and antibody h10D8OF are the same as the concentrations of N1-94W-Fc and antibody h10D8OF alone), and hIgG1 (Beijing Sino Biological Inc., product number: MA14OC2903). The sample groups and the control groups are respectively diluted to an initial concentration of 200 nM with the dilution solution for detection in a 96-well V-bottom plate, and serially diluted 100-fold with the dilution solution for detection to obtain a total of 3 series of dilution concentrations.

[0217] Transfer PBMC cells in good condition to a six-well plate or cell culture flask and culture them at 37 °C for 4 h. Centrifuge and discard the supernatant, and resuspend the cells with RPMI 1640 medium containing 10% FBS to a density of 4 × 10 6 cells / mL, and add 100 μL / well to a 96-well U-bottom plate (Corning, product number: 7007). At this time, the number of cells is 4 × 10 5 cells / well. Add 50 μL of the antibody or VHH-Fc fusion protein sample at the corresponding concentration to each well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 66.7 nM. Place the 96-well U-bottom plate in a 5% CO2, 37 °C incubator and incubate for 4 h.

[0218] Centrifuge K562 cells (highly expressing PVRL2 and PVR) in good growth condition and discard the supernatant. Resuspend the cells with RPMI 1640 medium containing 10% FBS to a density of 1 × 10 7 cells / mL. Take out the 96-well U-bottom plate that has been incubated for 4 h, add 50 μL of K562 cells to each well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 50 nM. Place the 96-well U-bottom plate in a 5% CO2, 37 °C incubator and incubate for 21 h.

[0219] Centrifuge the 96-well U-bottom plate to remove the supernatant, wash it with PBS containing 2% FBS and then remove the supernatant for the next incubation. Add Anti-CD3 APC (purchased from BioLegend, catalog number 344812), Anti-NKp46 BV421 (purchased from BioLegend, catalog number 331914) and anti-CD137 PE (purchased from BioLegend, catalog number 309804) to the PBS containing 2% FBS at a dilution ratio of 1:500 to obtain the fluorescent secondary antibody dilution. Add 100 μL of the fluorescent secondary antibody dilution to resuspend the cells in each well except the blank well, and incubate in the dark at 4 °C for 25 min. After incubation, centrifuge the 96-well U-bottom plate to remove the supernatant, wash it with PBS containing 2% FBS, and finally resuspend it with PBS containing 2% FBS at a volume of 150 μL per well. Collect the cells with a flow cytometer to detect the fluorescent antibodies bound to the cell surface and obtain the original data MFI value. The final results are plotted with the antibody concentration on the abscissa and the percentage of NK cells on the ordinate using a four-parameter model for curve fitting to obtain an antibody dose-dependent binding curve, as Figure 9 shown.

[0220] The results showed that both bispecific antibodies Bi-VH1-94W and Bi-VH3-94W could effectively activate NK cells, and the expressions of the activation marker CD137 on the surface of NK cells were 32.63% and 35.88% respectively, while the hIgG1 control group was only 22.45%.

Claims

1. A PVRIG-binding protein, which comprises a heavy chain variable region, and the heavy chain variable region comprises one or more of HCDR1 shown in SEQ ID NO: 6, HCDR2 shown in SEQ ID NO: 7, and HCDR3 shown in any one of SEQ ID NOs: 8-12; or, the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 6, HCDR2 shown in SEQ ID NO: 7, and HCDR3 shown in any one of SEQ ID NOs: 8-12.

2. The PVRIG-binding protein according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having one or more conservative amino acid substitutions compared with the amino acid sequence shown in any one of SEQ ID NOs: 13-17; or, the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-17.

3. A PVRIG-binding protein, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 36-40, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 36-40, or an amino acid sequence having one or more conservative amino acid substitutions compared with the amino acid sequence shown in any one of SEQ ID NOs: 36-40.

4. The PVRIG-binding protein according to any one of claims 1-3, which is a single-domain antibody.

5. A bispecific antibody or antigen-binding fragment, which comprises a first antigen-binding portion capable of binding to PVRIG; the first antigen-binding portion comprises the PVRIG-binding protein according to any one of claims 1-4.

6. The bispecific antibody or antigen-binding fragment according to claim 5, which comprises a second antigen-binding portion capable of binding to TIGIT, and the second antigen-binding portion comprises one or more of HCDR1 shown in SEQ ID NO: 18, HCDR2 shown in SEQ ID NO: 19, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 21, LCDR2 shown in SEQ ID NO: 22, and LCDR3 shown in SEQ ID NO:

23.

7. The bispecific antibody or antigen-binding fragment according to claim 6, wherein the second antigen-binding portion comprises HCDR1 shown in SEQ ID NO: 18, HCDR2 shown in SEQ ID NO: 19, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 21, LCDR2 shown in SEQ ID NO: 22, and LCDR3 shown in SEQ ID NO:

23.

8. The bispecific antibody or antigen-binding fragment according to claim 6 or 7, wherein the second antigen-binding portion comprises a heavy chain variable region and a light chain variable region; wherein, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 24; and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

25.

9. The bispecific antibody or antigen-binding fragment according to claim 8, wherein the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region of the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:

25.

10. The bispecific antibody or antigen-binding fragment according to any one of claims 6-9, wherein the second antigen-binding portion is linked to the first antigen-binding portion through a peptide linker; or, the second antigen-binding portion is an antibody, and the N-terminus or C-terminus of the antibody heavy chain is linked to the first antigen-binding portion through a peptide linker; or, the N-terminus of the antibody heavy chain is linked to the C-terminus of the first antigen-binding portion through a peptide linker; or, the C-terminus of the antibody heavy chain is linked to the N-terminus of the first antigen-binding portion through a peptide linker.

11. A bispecific antibody or antigen-binding fragment, comprising a first polypeptide and a second polypeptide; wherein the first polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 41-44, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in any one of SEQ ID NO: 41-44, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 41-44; and / or the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 80% identity compared to the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

31.

12. The biological material is: 1) Nucleic acid encoding the PVRIG-binding protein according to any one of claims 1-4 or the bispecific antibody or antigen-binding fragment according to any one of claims 5-11; 2) An expression vector comprising the nucleic acid encoding the PVRIG-binding protein according to any one of claims 1-4 or the bispecific antibody or antigen-binding fragment according to any one of claims 5-11; or 3) A host cell comprising a nucleic acid or expression vector encoding the PVRIG-binding protein according to any one of claims 1-4 or the bispecific antibody or antigen-binding fragment according to any one of claims 5-11.

13. A pharmaceutical composition comprising the PVRIG-binding protein according to any one of claims 1-4 or the bispecific antibody or antigen-binding fragment according to any one of claims 5-11 and a pharmaceutically acceptable excipient.

14. Use of the PVRIG-binding protein according to any one of claims 1-4, the bispecific antibody or antigen-binding fragment according to any one of claims 5-11, the biological material according to claim 12, or the pharmaceutical composition according to claim 13 in the treatment or prevention of diseases or in the preparation of a medicament for the treatment or prevention of diseases.

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