Pvrig binding protein, bispecific antibody, and use

Abstract

Provided are a PVRIG binding protein, a bispecific antibody, and a use. The bispecific antibody or antigen-binding fragment 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 protein or bispecific antibody can be used for treating or preventing diseases.

Description

PVRIG binding protein, bispecific antibody and its application Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to PVRIG binding protein, bispecific antibodies and applications thereof. Background Art

[0002] Most T and NK cell co-inhibitory 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 and SHP-2), ultimately leading to reduced effector function and suppression of gene expression. Receptor blockers are drugs that specifically recognize and bind to certain receptors, preventing their corresponding ligands from binding to them. In tumor treatment, blocking the binding of co-inhibitory receptors to their ligands through receptor blockers helps activate immune cell activity and enhance anti-tumor efficacy.

[0003] PVRIG is a co-inhibitory receptor belonging to the poliovirus receptor (PVR) family, also known as CD112R, and is typically expressed on natural killer (NK) cells and CD8+ T cells. Human PVRIG is a 36kD single-pass transmembrane protein containing a transmembrane region, a long intracellular domain, and an extracellular immunoglobulin variable (IgV) domain. Because 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 CD112 / CD226 interaction in T cells. Summary of the Invention

[0004] The object of the present invention is to provide a PVRIG binding protein, a bispecific antibody or an antigen-binding fragment and uses thereof.

[0005] In one aspect, the present invention provides a bispecific antibody or antigen-binding fragment, comprising 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 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 6;

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

[0008] (c) HCDR3 comprising 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 compared to the amino acid sequence shown in any one of SEQ ID NOs: 8-12.

[0009] In some embodiments, the first antigen binding portion comprises a heavy chain variable region (VH), wherein 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; and HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:8-12.

[0010] In some embodiments, the first antigen binding moiety 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 NOs: 13-17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared 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.

[0012] In some embodiments, the bispecific antibody or antigen-binding fragment 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, 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, anti-TIGIT antibodies described in patent CN107148430A, such as 14A6, 28H5 and 31C6, anti-TIGIT antibodies described in patent US2013 / 0251720, such as 10A7 and 1F4.

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

[0015] In another aspect, the present invention provides a 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 amino acid sequences of (d)-(i):

[0016] (d) HCDR1 comprising 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 comprising the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 19;

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

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

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

[0021] (i) LCDR3 comprising 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), wherein the VH 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), wherein the VL 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% 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.

[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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% 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.

[0027] In some embodiments, the heavy chain variable region of the second antigen binding portion comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 24; and the light chain variable region of the second antigen binding portion comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 25. An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:25, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:25.

[0028] In some embodiments, the heavy chain variable region of the second antigen binding moiety comprises the amino acid sequence shown in SEQ ID NO:24, and the light chain variable region of the second antigen binding moiety 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 set forth in SEQ ID NO: 26 or 27, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or an amino acid sequence having one or more conservative amino acid substitutions thereto. 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% 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 of SEQ ID NO: 26 or 27, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26 or 27, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 26 or 27; and the light chain constant region of the second antigen binding portion comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26 or 27. An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the heavy chain constant region of the second antigen binding moiety comprises the amino acid sequence of SEQ ID NO: 26 or 27, and the light chain constant region of the second antigen binding moiety comprises the amino acid sequence of SEQ ID NO: 28.

[0030] In some embodiments, the second antigen-binding portion is an antibody comprising a heavy chain and a light chain. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 29 or 30, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth 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 set forth in SEQ ID NO: 29 or 30. In some embodiments, the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO:31. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to the amino acid sequence of 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 of SEQ ID NO: 29 or 30; the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to the amino acid sequence of SEQ ID NO: 29 or 30; An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 29 or 30, and the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 31.

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

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

[0033] In some embodiments, the N-terminus of the antibody heavy chain is connected 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 connected to the N-terminus of the single-domain antibody via a peptide linker. In some embodiments, the second antigen-binding portion is connected 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 connected to the first antigen-binding portion via a peptide linker. In some embodiments, the N-terminus of the antibody heavy chain is connected 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 connected 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 , wherein 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 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] In another aspect, the present invention provides a 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

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

[0037] (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 6;

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

[0039] (c) HCDR3 comprising 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 compared to 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 comprising 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;

[0042] (e) HCDR2 comprising the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 19;

[0043] (f) HCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 20;

[0044] (g) LCDR1 comprising the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 21;

[0045] (h) LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 22;

[0046] (i) LCDR3 comprising 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.

[0047] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region (VH), wherein the VH comprises HCDR1, HCDR2, and HCDR3; wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 6; HCDR2 comprises the amino acid sequence of SEQ ID NO: 7; and HCDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 8-12. In some embodiments, the second antigen-binding portion comprises a VH and a VL; wherein the VH comprises HCDR1, HCDR2, and HCDR3; and the VL comprises LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 18; HCDR2 comprises the amino acid sequence of SEQ ID NO: 19; HCDR3 comprises the amino acid sequence of SEQ ID NO: 20; LCDR1 comprises the amino acid sequence of SEQ ID NO: 21; LCDR2 comprises the amino acid sequence of SEQ ID NO: 22; and LCDR3 comprises the amino acid sequence of 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 set forth in any one of SEQ ID NOs: 13-17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth 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 set forth 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 of SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 24; and / or the light chain variable region of the second antigen binding portion comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24, An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:25, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:25.

[0050] In some embodiments, the first antigen-binding portion is a single domain antibody comprising the amino acid sequence of any one of SEQ ID NOs: 13-17, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 13-17, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NOs: 13-17; the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 13-17; : The light chain variable region of the second antigen binding portion comprises the amino acid sequence of SEQ ID NO:25, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:24, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO:24; and the light chain variable region of the second antigen binding portion comprises the amino acid sequence of SEQ ID NO:25, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:25, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: The amino acid sequence shown in NO:25 has one or more conservative amino acid substitutions compared to the amino acid sequence.

[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; and 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 set forth in SEQ ID NO: 26 or 27, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or an amino acid sequence having one or more conservative amino acid substitutions thereto. 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% 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 of SEQ ID NO: 26 or 27, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26 or 27, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 26 or 27; and the light chain constant region of the second antigen binding portion comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26 or 27. An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the heavy chain constant region of the second antigen binding moiety comprises the amino acid sequence of SEQ ID NO: 26 or 27, and the light chain constant region of the second antigen binding moiety comprises the amino acid sequence of SEQ ID NO: 28.

[0053] In some embodiments, the second antigen-binding portion is an antibody comprising a heavy chain and a light chain. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 29 or 30, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth 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 set forth in SEQ ID NO: 29 or 30. In some embodiments, the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO:31.

[0054] In some embodiments, the second antigen binding moiety is connected to the first antigen binding moiety via a peptide linker. In some embodiments, the second antigen binding moiety is an antibody, and the N-terminus or C-terminus of the antibody heavy chain is connected to the first antigen binding moiety via a peptide linker. In some embodiments, the N-terminus of the antibody heavy chain is connected 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 connected to the N-terminus of the 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 , wherein 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 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] In another aspect, the present invention provides a 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 comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 13-17, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 13-17, or an amino acid sequence having one or more conservative amino acid substitutions compared to an amino acid sequence as set forth in any one of SEQ ID NOs: 13-17;

[0058] The second antigen-binding portion is an anti-TIGIT antibody, the heavy chain of which comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 29 or 30; the light chain of which comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29 or 30. an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO:31;

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

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

[0061] In some embodiments, the N-terminus of the antibody heavy chain is connected 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 connected 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] In another aspect, the present invention provides a bispecific antibody or antigen-binding fragment, comprising a first polypeptide and a second polypeptide;

[0064] The first polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-44, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of any one of SEQ ID NOs: 41-44, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NOs: 41-44; and / or

[0065] The second polypeptide comprises the amino acid sequence of SEQ ID NO:31, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of 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; and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:31.

[0067] In another aspect, the present invention provides a PVRIG binding protein comprising a heavy chain variable region comprising one or more amino acid sequences of (a) to (c):

[0068] (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 6;

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

[0070] (c) HCDR3 comprising 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 compared to 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; and 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 set forth in any one of SEQ ID NOs: 13-17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having one or more conservative amino acid substitutions thereto. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth 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', a F(ab')2, a F(ab)2, a Fv, or a 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 immunoglobulin Fc fragment or a variant thereof, such as a human IgG1, IgG2, IgG3, or IgG4 Fc fragment or a variant thereof. In some embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 34 or 35, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of 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 of SEQ ID NO: 34 or 35.

[0075] In some embodiments, the C-terminus of the heavy chain variable region is connected 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 , wherein 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 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 of any one of SEQ ID NOs: 36-40, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 36-40, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NOs: 36-40.

[0077] In some embodiments, the PVRIG binding protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 36-40.

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

[0079] Table 1 Amino acid sequence

[0080] In another aspect, the present invention provides a nucleic acid encoding the aforementioned bispecific antibody or antigen-binding fragment or PVRIG binding protein or a portion thereof. In some embodiments, the nucleic acid is an isolated nucleic acid.

[0081] The exemplary nucleic acid sequences of the present invention are shown in Table 2.

[0082] Table 2 Nucleotide sequences

[0083] In another aspect, the present invention provides an expression vector comprising the above-mentioned nucleic acid.

[0084] In another aspect, the present invention provides a host cell comprising the nucleic acid or expression vector described above. 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 a HEK293F cell), a BHK cell, a Cos1 cell, a Cos7 cell, a CV1 cell, or a mouse L cell.

[0085] In another aspect, the present invention provides a method for producing the bispecific antibody, antigen-binding fragment, or PVRIG-binding protein described herein, comprising culturing the host cells described above in a culture medium to produce the bispecific antibody, antigen-binding fragment, or PVRIG-binding protein. In some embodiments, the method further comprises recovering the bispecific antibody, antigen-binding fragment, or PVRIG-binding protein from the host cells or the culture medium.

[0086] On the other hand, the present invention provides a pharmaceutical composition comprising the aforementioned bispecific antibody or antigen-binding fragment, the aforementioned PVRIG-binding protein, the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned cell, and pharmaceutically acceptable excipients.

[0087] In another aspect, the present invention provides uses of the aforementioned bispecific antibody or antigen-binding fragment, the aforementioned PVRIG-binding protein, the aforementioned nucleic acid, the aforementioned expression vector, the aforementioned cell, or the aforementioned pharmaceutical composition in treating or preventing a disease.

[0088] In another aspect, the present invention provides use of the aforementioned bispecific antibody or antigen-binding fragment, the aforementioned PVRIG-binding protein, the aforementioned nucleic acid, the aforementioned expression vector, the aforementioned cell, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating or preventing a disease.

[0089] In another aspect, the present invention provides a method for treating or preventing a disease, comprising administering a therapeutically effective amount of the aforementioned bispecific antibody or antigen-binding fragment, the aforementioned PVRIG-binding protein, the aforementioned nucleic acid, the aforementioned expression vector, the aforementioned cell, or the aforementioned pharmaceutical composition to a patient in need thereof.

[0090] 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 a 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 tumors, adult T-cell leukemia / lymphoma, myelodysplastic syndrome, urinary tract 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.

[0091] In some embodiments, the present invention relates to a kit or article of manufacture comprising the aforementioned bispecific antibody or antigen-binding fragment, the aforementioned PVRIG-binding protein, the aforementioned nucleic acid, the aforementioned expression vector, the aforementioned cell, or the aforementioned pharmaceutical composition.

[0092] The PVRIG binding protein, bispecific antibody, or antigen-binding fragment of the present invention can be used to treat or prevent 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

[0093] FIG1 illustrates two structures of the bispecific antibodies of the present invention; FIG1A shows the structure of the bispecific antibody Bi-VH1, and FIG1B shows the structure of the bispecific antibody Bi-VH3.

[0094] Figure 2 shows the binding curve of the antibody to cells overexpressing TIGIT.

[0095] FIG3 shows the binding curves of antibodies or VHH-Fc fusion proteins and cells overexpressing PVRIG.

[0096] FIG4 shows the binding curves of antibodies or fusion proteins and cells overexpressing PVRIG.

[0097] FIG5 shows the binding curve of antibody or VHH-Fc fusion protein blocking the binding of ligand PVRL2 to cell surface PVRIG.

[0098] FIG6 shows that antibodies or VHH-Fc fusion proteins block the cellular activity of PVRL2-PVRIG interaction.

[0099] FIG7 shows that antibodies or VHH-Fc fusion proteins block the cellular activity of PVRL2-PVRIG interaction.

[0100] FIG8 shows cellular activity of antibodies blocking PVR-TIGIT interaction.

[0101] FIG9 shows the activity of antibodies or fusion proteins in activating NK cells. DETAILED DESCRIPTION

[0102] Unless defined otherwise, 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.

[0103] the term

[0104] It should be noted that the term "a" entity refers to one or more of that entity, e.g., "an antibody" should be understood as one or more antibodies, and thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0105] "About" refers to the normal error range of the corresponding numerical value that is easily known to those skilled in the relevant art. In some embodiments, "about" mentioned herein refers to the described numerical value and its ±10%, ±5% or ±1% range.

[0106] As used herein, the terms "comprising" or "including" mean that the antibody, composition, or method, etc. includes the listed elements, such as components or steps, but does not exclude others. "Essentially consisting of" means that the antibody, composition, or method, etc. excludes other elements that have a fundamental effect on the characteristics of the combination, but does not exclude elements that do not substantially affect the antibody, composition, or method, etc. "Consisting of" means excluding elements not specifically listed.

[0107] "Polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). "Polypeptide" refers to any single chain or multiple chains of two or more amino acids and does not refer to the specific length of the product. Thus, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and "polypeptide" may be used in place of or interchangeably with any of the foregoing terms. "Polypeptide" is also intended to refer to products of polypeptides that have been modified after expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but it need not be translated from a specified nucleic acid sequence and may be produced by any means, including chemical synthesis.

[0108] Those of ordinary skill in the art will appreciate that the CDR district of antibody is responsible for the binding specificity of antibody to antigen. In the case of known antibody heavy chain and light chain variable region sequences, there are currently several methods for determining antibody CDR district, including Kabat, IMGT, Chothia and AbM numbering systems. However, the application of the definition of the CDR of each antibody or its variant will fall within the scope of the term defined and used herein. If the variable region amino acid sequence of the given antibody is given, those skilled in the art can determine which residues are included in the specific CDR, and do not rely on any experimental data outside the sequence itself.

[0109] "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, any antigen-binding fragment thereof, or a single chain thereof. Therefore, "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule that has the 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 heavy chain variable region (VH), the light chain variable region (VL), the heavy chain constant region (CH), the light chain constant region (CL), the framework regions (FRs), or any portion thereof, or at least a portion of a binding protein. CDR regions include the light chain CDR regions (LCDR1-3) and the heavy chain CDR regions (HCDR1-3). The heavy chain constant region (CH) includes the CH1 domain, the hinge (e.g., the upper, middle, and / or lower hinge regions), the CH2 domain, and the CH3 domain; the crystallizable segment (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.

[0110] A complete antibody comprises a heavy chain and / or a light chain. The categories of heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), among which there are also some subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, namely IgG, IgM, IgA, IgD or IgE. Antibody subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been fully characterized and the functional specificity conferred is also known. All antibody types are within the scope of protection of the present invention. In some embodiments, the antibody comprises two heavy chains or two light chains, and these four chains are connected in a "Y" configuration by disulfide bonds, wherein the light chain starts from the "Y" mouth and continues through the variable region to surround the heavy chain.

[0111] "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 their structure, antibody fragments bind to the same antigen recognized by the intact antibody. "Antibody fragments" include aptamers, Spiegelmers, and diabodies. "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.

[0112] "Fab" generally refers to the antigen-binding portion of a conventional antibody (e.g., IgG), comprising the antibody's heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region domain (CH1), and light chain constant region (CL). In conventional antibodies, 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 region domains of the heavy chain to form the heavy chain. In some embodiments, "Fab" also refers to variant structures 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 a Fab (cross VH / VL) structure. In certain embodiments, the CH1 of Fab is not linked to the hinge region, but the C-terminus of CL is linked to the hinge region of the heavy chain, forming a Fab (cross Fd / LC) structure.

[0113] "Single-chain antibody", "single-chain variable fragment" or "scFv" refers to a fusion protein of the heavy chain variable region (VH) and the light chain variable region (VL) of an immunoglobulin. In some aspects, these regions are connected with a peptide linker of 10 to about 25 amino acids. The peptide linker can be rich in glycine to increase flexibility, as well as rich in serine or threonine to increase solubility, and can connect the N-terminus of VH and the C-terminus of VL, or vice versa. Although the protein has been stripped of the constant region and a linker has been introduced, it retains the specificity of the original immunoglobulin. scFv molecules are generally known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0114] A "single-domain antibody" or "sdAb" refers to an antigen-binding fragment comprising only a single antibody variable region. An sdAb alone is capable of binding to an antigen without being paired with a corresponding CDR-containing polypeptide. The heavy chain variable region may also be referred to herein as a "VHH." Some VHHs are also referred to as nanobodies. From N-terminus to C-terminus, a VHH has the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Herein, a VHH that can bind to PVRIG may be referred to as an anti-PVRIG VHH fragment.

[0115] "Bispecific antibody" refers to an antibody that has two antigen-binding sites, which may be different epitopes of the same antigen or different epitopes of different antigens.

[0116] "Homology" or "identity" refers to the sequence similarity between two peptides or between two nucleic acids. Homology or identity can be determined by comparing alignable positions in each sequence. When a position in the compared sequences is 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.

[0117] "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" to 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 percent identity or sequence identity can be determined visually or using software programs known in the art, such as those described in Ausubel et al., eds. (2007), in Current Protocols in Molecular Biology. Preferably, the alignment is performed using the default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expectation=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above specified percentage identities and encode polypeptides having the same or similar biological activity.

[0118] "Conservative amino acid substitutions" are substitutions of amino acid residues with similar side chains. Families of amino acid residues with 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues of immunoglobulin polypeptides are preferably substituted with other amino acid residues from the same side chain family. In other embodiments, a string of amino acids may be substituted with a string of structurally similar amino acids that differ in sequence and / or in the composition of the side chain family.

[0119] In some embodiments, the conservative amino acid substitutions are preferably substitutions in which an amino acid within the following groups (a)-(e) is substituted by another amino acid residue within the same group: (a) small aliphatic, non-polar 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, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0120] 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.

[0121] The PVRIG binding proteins or bispecific antibodies provided herein include modified derivatives, i.e., modified by covalent attachment of any type of molecule to the PVRIG binding protein or bispecific antibody, wherein the covalent attachment 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, pegylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications can be performed using conventional techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0122] In some embodiments, the PVRIG binding protein or bispecific antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0123] "Pharmaceutically acceptable" refers to substances approved by government regulatory agencies or listed in recognized pharmacopeias for use in animals, especially 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 auxiliary agent.

[0124] "Excipient" refers to a diluent, adjuvant, vehicle, or carrier that can be administered to a patient along with the active ingredient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions, aqueous glucose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like. If necessary, the composition can also contain a small amount of a wetting agent or emulsifier, or a pH buffer. Antibacterial agents such as benzyl alcohol or methyl parahydroxybenzoate, antioxidants such as ascorbic acid, chelating agents, and agents for regulating tension such as dextrose are also foreseeable. These compositions can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release preparation etc.Said composition can be formulated into suppository with traditional adhesive and carrier such as triglyceride.Oral preparation can comprise standard carrier, for example pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate etc.Such composition will contain the antibody or antigen-binding fragment or fusion protein of clinical effective dose, preferably in the form after purification, together with the adjuvant of suitable amount, to provide the administration form that is suitable for patient.Said preparation should be applicable to administration mode.Parent preparation can be encapsulated in ampoule bottle, disposable syringe or the multiple dose bottle made of glass or plastic.

[0125] The PVRIG-binding proteins or bispecific antibodies (including antibodies, antigen-binding fragments, or fusion proteins) of the present invention include 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, and tartaric acid, and salts formed with cations derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, and procaine. The PVRIG-binding proteins or bispecific antibodies described herein can be neutral, i.e., substantially free of 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 proteins described herein can exist in a positive ionic form, such as when the pH is below the isoelectric point, the protein molecule exhibits a positive charge overall. The proteins described herein can exist in a negative ionic form, such as when the pH is above the isoelectric point, the protein molecule exhibits a negative charge overall.

[0126] The effective dosage and treatment regimen for treating a particular patient will depend on various factors, including the specific PVRIG-binding protein, bispecific antibody, or derivative used, the patient's age, weight, general health, sex, and diet, as well as the timing of administration, frequency of excretion, drug combination, and the severity of the specific condition being treated. These factors are within the discretion of a healthcare provider, who is within the skill of the art. The dosage employed can be determined by pharmacological and pharmacokinetic principles well known in the art. In some embodiments, the PVRIG-binding protein or bispecific antibody of the invention is administered to a patient at a dose of 0.01 mg / kg to 100 mg / kg of the patient's body weight per dose. In some embodiments, administration is weekly, biweekly, three-weekly, or four-weekly.

[0127] The antibodies provided herein can be derived from any animal, such as a mammal. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, horse, or chicken. In other embodiments, the variable region can be of condricthoid origin (e.g., from shark).

[0128] "Treatment" refers to both therapeutic treatment and preventative or prophylactic measures, the purpose of which is to prevent, slow, ameliorate or halt an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following results, whether detectable or undetectable, relief of symptoms, reduction in the severity of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, alleviation, reduction or elimination of the disease state (whether partial or complete), and prolongation of life expectancy compared to that expected in the absence of treatment. Patients in need of treatment include those already suffering from a condition or disorder, those susceptible to a condition or disorder, or those in need of prevention of the condition or disorder, and those who can or are expected to benefit from the administration of the antibodies or pharmaceutical compositions provided herein for detection, diagnostic procedures and / or treatment.

[0129] "Patient" refers to any mammal in need of diagnosis, prevention, prognosis or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, etc.

[0130] Preparation of antibodies or fusion proteins

[0131] Various methods for preparing antibodies or fusion proteins are known in the art, such as hybridoma technology, recombinant DNA technology, transgenic mouse technology, and phage display library methods.

[0132] Antibodies or fusion proteins can be prepared using conventional recombinant DNA techniques. Vectors and cell lines that produce 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, DL Hacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary content, are incorporated herein by reference.

[0133] In some embodiments, DNA encoding the antibody or fusion protein can be designed and synthesized according to conventional methods based on the amino acid sequence of the antibody or fusion protein described herein, inserted into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in culture medium to produce the monoclonal antibody or fusion protein. In some embodiments, the expression vector comprises 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 either side of the inserted sequence. Efficient transcription can be achieved using the early and late promoters of SV40, and the early promoters of long terminal repeats from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus. Other cellular promoters, such as the actin promoter, can also be used. Suitable expression vectors may 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, mouse L cells, and CHO cells, etc.

[0134] The gene sequence of antibody or fusion protein can be inserted into expression vector by standard methods (for example, connecting complementary restriction sites on gene sequence and carrier, or if there is no restriction site, then flat end connection).Before inserting gene sequence, expression vector can have carried antibody constant region sequence.For example, a kind of method that the VH and VL sequence relevant to antibody is converted to full-length antibody gene is to insert them into expression vector having encoded heavy chain constant region and light chain constant region respectively, so that VH sequence is operably connected to CH sequence in carrier, and VL sequence is operably connected to CL sequence in carrier.Or, recombinant expression vector can encode the signal peptide that promotes host cell secretion antibody or fusion protein.Or, gene sequence can be cloned into the carrier of the signal peptide that promotes host cell secretion antibody or fusion protein, so that the 3 ' end of the gene sequence encoding signal peptide is connected in frame with the 5 ' end of the gene sequence encoding antibody (heavy chain and / or light chain) or fusion protein. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein), for example, MEFGLSLVFLVLILKGVQC (SEQ ID NO: 56).

[0135] In some embodiments, the expression vector must contain a selection marker. Common selection markers include genes for dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and other selection markers to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking these genes. After culture in a selective medium, the successfully transfected cells grow in large quantities and produce the desired target protein. The resulting antibody or fusion protein can be isolated or purified using conventional techniques, such as protein A-Sepharose, ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, or affinity chromatography.

[0136] Example

[0137] The following is a detailed description of the technical solution of the present invention, which does not limit the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0138] Example 1: Preparation of PVRIG recombinant protein and anti-PVRIG antibodies SF35 and H4

[0139] The DNA sequence of the PVRIG recombinant protein PVRIG-his (shown in SEQ ID NO: 1) was inserted into an expression plasmid to generate a recombinant plasmid. The plasmid was then transiently transfected into HEK293 cells using polyetherimide (PEI). After culture, the supernatant was collected and purified to obtain a PVRIG-his protein sample.

[0140] The amino acid sequence of PVRIG-his is as follows:

[0141] The amino acid sequences of the heavy and light chains of antibodies SF35 and H4 are shown in Table 3. The nucleic acid sequences of the heavy and light chains of the antibodies were cloned into expression plasmids, respectively, and then transformed into CHO-K1 cells. After culture, the supernatant was collected and purified to obtain the antibodies.

[0142] Table 3 Amino acid sequences of antibody SF35 and antibody H4

[0143] Example 2: Preparation of anti-PVRIG / TIGIT bispecific antibodies and VHH-Fc fusion proteins

[0144] Two types of anti-PVRIG / TIGIT bispecific antibodies were constructed, designated as (1) bispecific antibody Bi-VH1, whose schematic diagram is shown in Figure 1A; and (2) bispecific antibody Bi-VH3, whose schematic diagram is shown in Figure 1B. The structural feature of the bispecific antibody Bi-VH1 is that the anti-PVRIG VHH fragment is connected to the N-terminus of the heavy chain of the anti-TIGIT antibody, such as the bispecific antibodies Bi-VH1-94Y and Bi-VH1-94W; while the structural feature of the bispecific antibody Bi-VH3 is that the anti-PVRIG VHH fragment is connected to the C-terminus of the heavy chain of the anti-TIGIT antibody, such as the bispecific antibodies Bi-VH3-94Y and Bi-VH3-94W. The VHH-Fc fusion protein comprises the following structures from the N-terminus to the C-terminus: the anti-PVRIG VHH fragment, a peptide linker, and an Fc fragment.

[0145] Relevant sequences of antibodies or VHH-Fc fusion proteins are shown in Tables 1-2 and 4-7; wherein, VHH-Fc fusion protein N1-94Y-Fc contains two identical sequences (as shown in SEQ ID NO:36), and its nucleic acid sequence is shown in SEQ ID NO:46; VHH-Fc fusion protein N1-94W-Fc contains two identical sequences (as shown in SEQ ID NO:37), and its nucleic acid sequence is shown in SEQ ID NO:47; VHH-Fc fusion protein 32 contains two identical sequences (as shown in SEQ ID NO:38), and its nucleic acid sequence is shown in SEQ ID NO:48; VHH-Fc fusion protein 34 contains two identical sequences (as shown in SEQ ID NO:39), and its nucleic acid sequence is shown in SEQ ID NO:49; VHH-Fc fusion protein 37 contains two identical sequences (as shown in SEQ ID NO:40), and its nucleic acid sequence is shown in SEQ ID NO:50.

[0146] The bispecific antibody Bi-VH1-94Y contains two first polypeptides with identical sequences (as shown in SEQ ID NO:41) and two second polypeptides with identical sequences (as shown in SEQ ID NO:31). The first polypeptide consists of an anti-PVRIG VHH fragment (as shown in SEQ ID NO:13), a peptide linker L1 (GGGGSGGGGS, as shown in SEQ ID NO:32), and an anti-TIGIT heavy chain sequence (as shown in SEQ ID NO:29). 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 shown in SEQ ID NO:51, and the nucleic acid sequence of the second polypeptide is shown in SEQ ID NO:45. The bispecific antibody Bi-VH1-94W contains two first polypeptides with identical sequences (as shown in SEQ ID NO:42) and two second polypeptides with identical sequences (as shown in SEQ ID NO:31). The first polypeptide consists of an anti-PVRIG VHH fragment (as shown in SEQ ID NO:14), a peptide linker L1 (GGGGSGGGGS, as shown in SEQ ID NO:32) and an anti-TIGIT heavy chain sequence (as shown in SEQ ID NO:29), 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 shown in SEQ ID NO:52, and the nucleic acid sequence of the second polypeptide is shown in SEQ ID NO:45; the bispecific antibody Bi-VH3-94Y contains two first polypeptides with identical sequences (as shown in SEQ ID NO:43) and two second polypeptides with identical sequences (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 an anti-PVRIG VHH fragment (as shown in SEQ ID NO:13), 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 shown in SEQ ID NO:53, and the nucleic acid sequence of the second polypeptide is shown in 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 an anti-PVRIG VHH fragment (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 shown in SEQ ID NO:54, and the nucleic acid sequence of the second polypeptide is shown in SEQ ID NO:45.

[0147] Table 4 Sequences and CDRs of VHH fragments against PVRIG

[0148] Table 5 Sequence and composition of VHH-Fc fusion protein

[0149] The nucleic acid sequences of the first polypeptide and the second polypeptide of the bispecific antibody were cloned into expression plasmids respectively, and then transiently transfected into HEK293F cells. After culture, the supernatant was collected and purified to obtain the bispecific antibody.

[0150] The nucleic acid sequence of the VHH-Fc fusion protein was cloned into an expression plasmid, and then transiently transfected into HEK293F cells. After culture, the supernatant was collected and purified to obtain the VHH-Fc fusion protein.

[0151] Example 3: Determination of the dissociation constants of anti-PVRIG / TIGIT bispecific antibodies and VHH-Fc fusion proteins using Biacore

[0152] The affinity of antibodies and VHH-Fc fusion proteins for PVRIG-his and TIGIT-his proteins was measured using Biacore T200. Antibodies or VHH-Fc fusion proteins were coupled to the surface of a CM5 chip, and PVRIG-his or TIGIT-his, respectively, were captured using the chip. Various concentrations of PVRIG-his or TIGIT-his proteins were injected into the CM5 chip. As shown in Table 6, VHH-Fc fusion proteins 32, 34, and 37 exhibited strong affinity for PVRIG-his, 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 bound to PVRIG-his with high affinity, with KD values ​​of 3.05 nM, 2.35 nM, 5.64 nM and 5.83 nM, respectively, which are comparable to the affinities 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 bound to TIGIT-his with high affinity, with KD values ​​of 4.99nM, 5.58nM, 3.91nM and 3.70nM, respectively, which are comparable to the affinity of the parent 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).

[0153] Table 6 Affinity data of antibodies or VHH-Fc fusion proteins and PVRIG-his (prepared in Example 1)

[0154] Table 7 Affinity data of antibodies or VHH-Fc fusion proteins and PVRIG-his (purchased from ACRO, product number PVG-H52H5)

[0155] Table 8 Affinity data of antibodies to TIGIT-his (purchased from ACRO, product number TIT-H52H5)

[0156] Example 4: Binding experiment of anti-PVRIG / TIGIT bispecific antibody to cells overexpressing TIGIT

[0157] 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, were diluted to a starting concentration of 200 nM with 1× PBS in a 96-well V-plate. Three-fold serial dilutions were then performed with 1× PBS to obtain a total of nine serial dilution concentrations.

[0158] Jurkat-TIGIT cells with good growth status (human full-length TIGIT gene (SEQ ID NO: 55) was transfected into Jurkat cell line (ATCC, Clone E6-1, TIB-152 TM ) to obtain a cell line that can stably express human TIGIT, named Jurkat-TIGIT cells), centrifuge and discard the supernatant, and resuspend the cells in 1× PBS to a density of 1×10 7 cells / mL, add 50 μL / well to a 96-well V-shaped plate, at which point the cell number is 5×10 5 Cells were measured at 4°C / well. 50 μL of the corresponding antibody sample was added to each well and mixed thoroughly, achieving a maximum final antibody concentration of 100 nM. The 96-well V-shaped plate was incubated in a 4°C refrigerator for 60 min. The plate was then centrifuged, the supernatant removed, and the cells washed with 1× PBS and removed for the next incubation. Anti-FC PE (BioLegend, Cat. No. 366903) was diluted 1:500 in 1× PBS. 100 μL of the diluted anti-FC PE was added to each well, except for the blank well, to resuspend the cells. The cells were incubated in a 4°C refrigerator protected from light for 30 min. After incubation, the cells were washed with 1× PBS and resuspended in 150 μL of 1× PBS per well. Cells were collected using a flow cytometer and the fluorescent antibody bound to the cell surface was detected. The mean fluorescence intensity (MFI) values ​​were calculated. The results were fitted using a four-parameter model with antibody concentration as the horizontal axis and MFI as the vertical axis. The results are shown in Figure 2.

[0159] The full-length human TIGIT gene sequence is as follows:

[0160] The results showed that the bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y and Bi-VH3-94W could bind to Jurkat-TIGIT cells, and EC 50 The binding capacity of the parent anti-TIGIT antibody h10D8OF to TIGIT expressed on the cell surface (EC 50 =0.13 nM).

[0161] Example 5: Binding experiment of anti-PVRIG / TIGIT bispecific antibody and cells overexpressing PVRIG

[0162] 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 and hIgG1 (Beijing Sino Biological Science and Technology Co., Ltd., Cat. No. MA14OC2903) were diluted to a starting concentration of 200 nM with 1× PBS in a 96-well V-shaped plate. Three-fold serial dilutions were then performed with 1× PBS to obtain a total of nine serial dilution concentrations.

[0163] PVRIG cDNA (purchased from Beijing Sino Biological Shenzhou Technology Co., Ltd., catalog number HG28312-UT) was transferred into Jurkat-NFAT cells (Construction of Jurkat-NFAT cells: Plasmid containing NFAT luciferase reporter gene was electroporated into Jurkat cells) to obtain Jurkat-NFAT cells overexpressing PVRIG, named Jurkat-NFAT-PVRIG. Jurkat-NFAT-PVRIG cells with good growth status were taken, centrifuged and the supernatant was discarded. The cells were resuspended in 1×PBS to a density of 1×10 7 cells / mL, and 50 μL / well was added to a 96-well V-shaped plate (Corning, Cat. No. 3897). At this time, the number of cells was 5×10 5 Cells were plated at 4°C / well. 50 μL of the corresponding antibody or VHH-Fc fusion protein sample was added to each well and mixed. The maximum final concentration of the antibody or VHH-Fc fusion protein was 100 nM. The 96-well V-shaped plate was incubated at 4°C for 60 min. After incubation, the plate was centrifuged, the supernatant removed, and the cells were washed with 1× PBS and removed for the next incubation. Anti-FC PE (BioLegend, Cat. No. 366903) was diluted 1:500 in 1× PBS. 100 μL of the diluted anti-FC PE was added to each well, except for the blank well, and the cells were resuspended in a 4°C refrigerator protected from light for 30 min. After incubation, the cells were washed with 1× PBS and resuspended in 150 μL of 1× PBS per well. Cells were collected by flow cytometry and detected for cell surface-bound fluorescent antibodies to obtain raw data MFI values. The final results were obtained by curve fitting using a four-parameter model with the antibody or VHH-Fc fusion protein concentration as the horizontal axis and the MFI as the vertical axis. The results are shown in Table 9, Figures 3 and 4.

[0164] Table 9 Cellular affinity of antibodies or VHH-Fc fusion proteins *Relative activity % = {EC 50 (Sample)-EC 50 (SF35)} / EC 50 (SF35)×100%

[0165] From the results in Table 9 and Figure 3, it can be seen that VHH-Fc fusion proteins 32, 34 and 37 can bind to human PVRIG highly expressed on the surface of Jurkat-NFAT-PVRIG cells with high affinity. The EC of VHH-Fc fusion protein 32 is 50 The EC of VHH-Fc fusion protein 34 was 0.67 nM. 50 The EC of VHH-Fc fusion protein 37 was 0.95 nM. 50 The binding activity of VHH-Fc fusion proteins 32 and 37 was better than that of antibody SF35 (EC 50 =0.91nM).

[0166] As shown in Figure 4, the bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y and Bi-VH3-94W can all bind to Jurkat-NFAT-PVRIG cells. 50 The bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W and VHH-Fc fusion protein N1-94W-Fc (EC 50 =0.34 nM) and N1-94Y-Fc (EC 50 =0.40 nM) had comparable binding ability to PVRIG expressed on the cell surface.

[0167] Example 6: Experiment on the blocking effect of VHH-Fc fusion protein on the binding of ligand PVRL2 to cell surface PVRIG

[0168] Appropriate amounts of VHH-Fc fusion proteins 32, 34, and 37 and antibodies SF35, H4, and hIgG1 (Beijing Sino Biological Technology Co., Ltd., catalog number: MA14OC2903) were taken and diluted to 600 nM with 1× PBS as the starting concentration. 4-fold serial dilutions were performed with 1× PBS to obtain a total of 10 serial dilution concentrations.

[0169] Take Jurkat-NFAT-PVRIG cells that are in good growth condition, centrifuge and discard the supernatant, and resuspend the cells in 1×PBS to a density of 1×10 7 cells / mL, and 50 μL / well was added to a 96-well V-shaped plate (Corning, Cat. No. 3897). At this time, the number of cells was 5×10 5Cells / well, add 50 μL of the corresponding concentration of antibody or VHH-Fc fusion protein to each well and mix thoroughly. The maximum final concentration of the antibody or VHH-Fc fusion protein is 300 nM. Incubate the 96-well V-shaped plate in a 4°C refrigerator for 10 min. While waiting for the antibody incubation, dilute an appropriate amount of the ligand PVRL2-mFc-bio (purchased from Acro, Cat. No. CD2-H82A3) to 600 nM in 1× PBS. After incubation is complete, remove the 96-well V-shaped plate and quickly add 50 μL of the ligand PVRL2-mFc dilution to each well. Mix thoroughly by pipetting. 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. Incubate the 96-well V-shaped plate in a 4°C refrigerator for a further 60 min.

[0170] After incubation, the 96-well V-shaped plate was centrifuged and the supernatant removed. The cells were washed with 1× PBS and the supernatant removed before the next incubation. Streptavidin-phycoerythrin (SA-PE; BioLegend, Cat. No. 740452) was diluted 1:800 in 1× PBS. 100 μL of the diluted SA-PE was added to each well, except for the blank well, to resuspend the cells. The cells were incubated in a dark refrigerator at 4°C for 30 min.

[0171] Cells were collected using a flow cytometer and fluorescent antibodies bound to the cell surface were detected to obtain raw data MFI values. The final results were fitted using a four-parameter model with antibody or VHH-Fc fusion protein concentration as the horizontal axis and MFI as the vertical axis. The results are shown in Figure 5.

[0172] As shown in Figure 5, VHH-Fc fusion proteins 32, 34, and 37 can all block the binding of ligand PVRL2 to human PVRIG expressed on the surface of Jurkat-NFAT-PVRIG cells. The IC 50 The IC of VHH-Fc fusion protein 34 was 24.48 nM. 50 The IC of VHH-Fc fusion protein 37 was 1.67 nM. 50 The blocking ability of VHH-Fc fusion proteins 34 and 37 was comparable to that of antibody SF35 (IC 50 =0.56) is equivalent.

[0173] Example 7: PVRIG cell activity assay of bispecific antibodies

[0174] 1) Take appropriate amounts of VHH-Fc fusion proteins 32, 34, and 37, as well as antibodies SF35, H4, and hIgG1 (Beijing Sino Biological Science Co., Ltd., Catalog No. MA14OC2903) and dilute them to a starting concentration of 600 nM using assay diluent (add 55 mL of FBS (ExCell Biology, Catalog No. FSP500) to 500 mL of RPMI 1640 medium (Gibco, Catalog No. 11875093) as assay diluent). Perform a 3-fold serial dilution with assay diluent to obtain 10 serial dilution concentrations. Add 50 μL per well of a 96-well white plate and set aside.

[0175] PVRL2 cDNA (purchased from Beijing Sino-Bio Technologies Co., Ltd., catalog number HG10005-UT) was transformed into CHO-OKT3 cells (for the construction method, see patent CN111748580A) to obtain CHO-OKT3 cells overexpressing PVRL2, named CHO-OKT3-PVRL2. Jurkat-NFAT-PVRIG cells and CHO-OKT3-PVRL2 cells in good growth condition were centrifuged, the supernatant discarded, and the cells were resuspended in assay diluent and counted using a cell counter. Based on the cell density, Jurkat-NFAT-PVRIG cells were diluted with assay diluent to a cell density of 4×10 6 Remove the 96-well white plate to which 50 μL / well of antibody or VHH-Fc fusion protein has been added, add 50 μL of Jurkat-NFAT-PVRIG cells to each well, and mix thoroughly. The maximum final concentration of the antibody or VHH-Fc fusion protein is now 300 nM.

[0176] Place the 96-well white plate in a 37°C CO2 constant temperature incubator and incubate for 10 minutes. Take out the 96-well white plate. Dilute the CHO-OKT3-PVRL2 cells with the detection diluent to a cell density of 2×10 6 cells / mL. Add CHO-OKT3-PVRL2 cells at a volume of 50 μL per well and mix well. The maximum final concentration of the antibody or VHH-Fc fusion protein is 200 nM. Place the 96-well white plate in a CO2 constant temperature incubator at 37°C and incubate for 6 hours. Take out the Bio-Lite TM Dissolve the Luciferase Assay System colorimetric solution at room temperature. After incubation, remove the 96-well white plate from the incubator and equilibrate at room temperature for approximately 8 minutes. Then, add 70 μL of colorimetric solution to each well and incubate at room temperature in the dark for 5 minutes. Read the plate as soon as possible.

[0177] Using the Luminescence Detection Module on a SpectraMax Multi-Mode Microplate Reader, set the PMT and Optics to 500, read relative light units (RLU), and shake the plate before reading. A four-parameter model was used to fit the samples, with antibody or VHH-Fc fusion protein concentration as the horizontal axis and relative light units as the vertical axis. The resulting four-parameter curve is shown in Figure 6.

[0178] The results showed that VHH-Fc fusion proteins 32, 34, and 37 could effectively block the inhibitory signal of PVRL2-PVRIG, thereby activating the NFAT signaling pathway of Jurkat. 50 The EC of VHH-Fc fusion protein 34 was 140.9 nM. 50 The EC of VHH-Fc fusion protein 37 was 124.8 nM. 50 is 10.0nM.

[0179] 2) Dilute 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 to a starting concentration of 600 nM using RPMI 1640 medium. Perform a 5-fold serial dilution using RPMI 1640 medium to obtain a total of 8 serial dilution concentrations. Add 100 μL of the solution to each well of a 96-well white plate for later use.

[0180] Jurkat-NFAT-PVRIG cells and CHO-OKT3-PVRL2 cells in good growth condition were taken, centrifuged and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium containing 10% FBS (fetal bovine serum) and counted using a cell counter. Jurkat-NFAT-PVRIG cells were diluted with RPMI 1640 medium containing 10% FBS to a cell density of 4×10 6 Remove the 96-well white plate to which 100 μL / well of antibody or VHH-Fc fusion protein has been added, add 50 μL of Jurkat-NFAT-PVRIG cells to each well, and mix thoroughly. The maximum final concentration of the antibody or VHH-Fc fusion protein is now 400 nM.

[0181] CHO-OKT3-PVRL2 cells were diluted with RPMI 1640 medium containing 10% FBS to a cell density of 2 × 10 6Remove the 96-well white plate containing the antibody and Jurkat-NFAT-PVRIG cells and add 50 μL of CHO-OKT3-PVRL2 cells to each well. Mix thoroughly to achieve a maximum final concentration of 300 nM for the antibody or VHH-Fc fusion protein. Incubate the plate in a CO2 incubator at 37°C for 6 h.

[0182] Take Bio-Lite out of the -20℃ freezer in advance TM Dissolve the Luciferase Assay System colorimetric solution at room temperature. Remove the white plate from the incubator and equilibrate at room temperature for approximately 8 minutes. Then, add 70 μL of colorimetric solution to each well and incubate at room temperature, protected from light, for 5 minutes. Read the plate as soon as possible. Using the Luminescence Detection Module on the SpectraMax Multi-Mode Microplate Reader, set the PMT and Optics to 500 and read the relative light units (RLU). Shake the plate before reading. A four-parameter model was used to fit the sample curve, with the antibody or VHH-Fc fusion protein concentration as the horizontal axis and the relative light units as the vertical axis. The resulting four-parameter curve is shown in Figure 7.

[0183] The results showed that both bispecific antibodies and VHH-Fc fusion proteins could effectively block the interaction between PVRIG antigen expressed on the surface of Jurkat-NFAT-PVRIG cells and PVRL2 highly expressed on the surface of CHO-OKT3-PVRL2 cells, thereby promoting T cell activation. 50 was 35.34 nM, Bi-VH1-94W was 51.27 nM, and Bi-VH3-94Y was 67.15 nM, which was comparable to the VHH-Fc fusion protein N1-94W-Fc (EC 50 =21.79 nM) and N1-94Y-Fc (EC 50 =56.60 nM) had a comparable blocking effect.

[0184] Example 8: Bispecific Antibody and TIGIT Cell Activity Experiment

[0185] An appropriate amount of bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y, and Bi-VH3-94W were diluted to 700 nM with RPMI 1640 medium as the starting concentration. The antibodies were diluted with RPMI 1640 medium at a ratio of 1:4 to obtain a total of 9 serial dilution concentrations.

[0186] Take CHO-K1-CD155 cells with good growth status (the construction method refers to the construction of sAPC cell line in Example 1 of patent CN111748580A) and centrifuge to discard the supernatant. Resuspend the cells in RPMI 1640 medium containing 10% FBS, count the cells, and prepare 1×10 5 cells / mL density of cell suspension, 100 μL / well (the number of cells is 1×10 4 cells / well) were added into a 96-well white plate, sterile water was added to the side wells, and the plate was placed in a 5% CO2, 37°C incubator for overnight incubation.

[0187] Jurkat-TIGIT cells in good growth condition were centrifuged and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium containing 10% FBS, counted, and the cells were prepared into 5.0×10 6 Cell suspension with a density of 2 × 10 cells / mL was prepared for use. The 96-well white plate with CHO-K1-CD155 cells was removed from the incubator, and the supernatant was discarded. Then, 40 μL / well of antibody dilution and 40 μL / well of Jurkat-TIGIT cell suspension (the cell number at this time was 2 × 10 5 cells / well). The maximum final antibody concentration is 350 nM. Sterile water is added to the side wells. Incubate the cell plate in a 5% CO2, 37°C incubator for 6 h.

[0188] Take Bio-Lite out of the -20℃ freezer in advance TM Dissolve the Luciferase Assay System colorimetric solution at room temperature. Remove the 96-well white plate from the incubator and equilibrate at room temperature for approximately 8 minutes. Then, add 60 μL of colorimetric solution to each well. Incubate at room temperature, protected from light, for 5 minutes. Read the plate as soon as possible. Using the Luminescence Detection Module on the SpectraMax Multi-Mode Microplate Reader, set the PMT and Optics to 500 and read the relative light units (RLU). A four-parameter model was used to fit the sample curve, with antibody concentration as the horizontal axis and relative light units as the vertical axis, as shown in Figure 8.

[0189] The results showed that the bispecific antibody Bi-VH1-94Y (EC 50 =1.42nM), Bi-VH1-94W (EC 50 =1.68nM), Bi-VH3-94Y (EC 50 =2.15 nM), Bi-VH3-94W (EC 50=1.71nM), which can effectively block the interaction between TIGIT antigen expressed on the surface of Jurkat-TIGIT cells and CD155 highly expressed on the surface of CHO-K1-CD155 cells, thereby promoting the activation of T cells.

[0190] Example 9: Detection of NK cell activity of bispecific antibodies

[0191] Add 55 mL of FBS to 500 mL of RPMI 1640 medium as the assay diluent. Take appropriate amounts of the bispecific antibodies Bi-VH1-94W and Bi-VH3-94W, the VHH-Fc fusion protein N1-94W-Fc, and the antibody h10D8OF. The sample groups included Bi-VH1-94W and Bi-VH3-94W, while the controls included N1-94W-Fc, the antibody h10D8OF, N1-94W-Fc plus the antibody h10D8OF (the amounts of N1-94W-Fc and the antibody h10D8OF added were the same as for N1-94W-Fc and the antibody h10D8OF alone), and hIgG1 (Beijing Sino Biological Science Co., Ltd., Cat. No. MA14OC2903). The sample group and the control group were diluted to 200 nM as the starting concentration using the detection diluent in a 96-well V-shaped plate, and then 100-fold serial dilutions were performed using the detection diluent to obtain 3 serial dilution concentrations.

[0192] Transfer PBMC cells in good condition to a six-well plate or cell culture flask and culture at 37°C for 4 h. Centrifuge and discard the supernatant. Resuspend the cells in 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-shaped plate (Corning, Cat. No. 7007). At this time, the number of cells is 4×10 5 cells / well, add 50 μL of the corresponding concentration of antibody or VHH-Fc fusion protein sample to each well and mix thoroughly. The maximum final concentration of the antibody or VHH-Fc fusion protein is 66.7 nM. Place the 96-well U-shaped plate in a 5% CO2, 37°C incubator and incubate for 4 hours.

[0193] K562 cells with good growth status (high expression of PVRL2 and PVR) were centrifuged and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium containing 10% FBS to a density of 1×10 7 Remove the 96-well U-shaped plate after 4 hours of incubation, add 50 μL of K562 cells to each well, and mix thoroughly. The maximum final concentration of the antibody or VHH-Fc fusion protein is now 50 nM. Incubate the 96-well U-shaped plate in a 5% CO2, 37°C incubator for 21 hours.

[0194] The 96-well U-shaped plate was centrifuged, the supernatant removed, and the cells were washed with PBS containing 2% FBS and then removed for the next incubation. Anti-CD3 APC (BioLegend, Catalog No. 344812), Anti-NKp46 BV421 (BioLegend, Catalog No. 331914), and Anti-CD137 PE (BioLegend, Catalog No. 309804) were added simultaneously at a 1:500 dilution ratio in PBS containing 2% FBS to prepare the fluorescent secondary antibody dilution solution. 100 μL of the fluorescent secondary antibody dilution solution was added to each well, except for the blank well, and the cells were resuspended in a refrigerator at 4°C in the dark for 25 minutes. After incubation, the 96-well U-shaped plate was centrifuged, the supernatant removed, and the cells were washed with PBS containing 2% FBS. Finally, the cells were resuspended in 150 μL of PBS containing 2% FBS per well. Cells were collected using a flow cytometer and fluorescent antibodies bound to the cell surface were detected to obtain raw data MFI values. The final results were fitted using a four-parameter model with antibody concentration as the horizontal axis and NK cell percentage as the vertical axis, resulting in a dose-dependent binding curve, as shown in Figure 9.

[0195] The results showed that both bispecific antibodies Bi-VH1-94W and Bi-VH3-94W were able to effectively activate NK cells, resulting in the expression of the activation marker CD137 on the NK cell surface at 32.63% and 35.88%, respectively, while the hIgG1 control group had only 22.45%.

Claims

1. A PVRIG-binding protein comprising a heavy chain variable region, said heavy chain variable region comprising 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.

2. The PVRIG-binding protein according to claim 1, wherein 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.

3. The PVRIG-binding protein according to claim 1 or 2, 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 compared 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.

4. The PVRIG-binding protein according to any one of claims 1-3, wherein the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs:13-17.

5. A PVRIG-binding protein comprising the amino acid sequence shown in any one of SEQ ID NOs: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 NOs: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 NOs:36-40.

6. The PVRIG-binding protein according to any one of claims 1-5, which is a single domain antibody.

7. A bispecific antibody or antigen-binding fragment comprising a first antigen-binding portion capable of binding to PVRIG; said first antigen-binding portion comprises the PVRIG-binding protein according to any one of claims 1-6.

8. The bispecific antibody or antigen-binding fragment according to claim 7, comprising a second antigen-binding portion capable of binding to TIGIT, said second antigen-binding portion comprising 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.

9. The bispecific antibody or antigen-binding fragment according to claim 8, 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.

10. The bispecific antibody or antigen-binding fragment according to claim 8 or 9, 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 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 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.

11. The bispecific antibody or antigen-binding fragment according to claim 10, 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.

12. The bispecific antibody or antigen-binding fragment according to any one of claims 8-11, wherein the second antigen-binding portion is linked to the first antigen-binding portion by a peptide linker.

13. The bispecific antibody or antigen-binding fragment according to any one of claims 8-12, wherein 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 by a peptide linker.

14. The bispecific antibody or antigen-binding fragment according to any one of claims 8-12, wherein the N-terminus of the antibody heavy chain is linked to the C-terminus of the first antigen-binding portion by a peptide linker.

15. The bispecific antibody or antigen-binding fragment according to any one of claims 8-12, wherein the C-terminus of the antibody heavy chain is linked to the N-terminus of the first antigen-binding portion by a peptide linker.

16. 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 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.

17. The biological material is: 1) A nucleic acid encoding the PVRIG-binding protein according to any one of claims 1-6 or the bispecific antibody or antigen-binding fragment according to any one of claims 7-16; 2) An expression vector comprising a nucleic acid encoding the PVRIG-binding protein according to any one of claims 1-6 or the bispecific antibody or antigen-binding fragment according to any one of claims 7-16; or 3) A host cell comprising a nucleic acid or expression vector encoding the PVRIG-binding protein according to any one of claims 1-6 or the bispecific antibody or antigen-binding fragment according to any one of claims 7-16.

18. A pharmaceutical composition comprising the PVRIG-binding protein according to any one of claims 1-6 or the bispecific antibody or antigen-binding fragment according to any one of claims 7-16 and a pharmaceutically acceptable excipient.

19. Use of the PVRIG-binding protein according to any one of claims 1-6, the bispecific antibody or antigen-binding fragment according to any one of claims 7-16, the biological material according to claim 17 or the pharmaceutical composition according to claim 18 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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