WO2025140497A1 - Pvrig binding protein, bispecific antibody, and use - Google Patents

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 application thereof Technical Field

The present invention belongs to the field of biomedicine, and in particular relates to PVRIG binding protein, bispecific antibody and application thereof.

Background Art

Most T and NK cell co-inhibitory receptors are members of the immunoglobulin (Ig) superfamily. When they bind to the corresponding ligands expressed on antigen presenting cells or tumor cells, they weaken cell activation through protein tyrosine phosphatases (such as SHIP-1 and SHP-2), ultimately leading to reduced effector function and inhibiting gene expression. Receptor blockers are able to 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 the activity of immune cells and improve anti-tumor efficacy.

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

Summary of the invention

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

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):

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

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

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

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 NO:8-12.

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

In some embodiments, the single domain antibody comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in any one of SEQ ID NOs: 13-17, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 13-17.

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, anti-TIGIT antibodies described in patent CN108290946A, such as 1A4, 1A5, 1D3, 4A3, 10A7 and 4.1D3, anti-TIGIT antibodies described in patent CN108137691A, such as VSIG9#1 and 258-cs1#4, anti-TIGIT antibodies described in patent CN108290936A, such as 14D7 and 26B10, 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.

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.

In some embodiments, the peptide linker comprises glycine and serine. In some embodiments, the amino acid sequence of the peptide linker is ( _GmS ) _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 , and 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.

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):

(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;

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

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

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

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

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

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.

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.

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

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 with 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.

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 with 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.

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 with the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:24. the light chain variable region of the second antigen binding portion comprises the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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.

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

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

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

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

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

In some embodiments, the N-terminus of the antibody heavy chain is connected to the C-terminus of the single-domain antibody through 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 through a peptide linker. In some embodiments, the second antigen binding moiety is connected to the first antigen binding moiety through 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 through 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 through 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 through a peptide linker.

In some embodiments, the peptide linker comprises glycine and serine. In some embodiments, the amino acid sequence of the peptide linker is ( _GmS ) _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 , and 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.

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

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

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

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

(c) HCDR3 comprising an amino acid sequence as 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 as shown in any one of SEQ ID NOs: 8-12;

The second antigen binding portion comprises one or more amino acid sequences of (d)-(i):

(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;

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

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

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

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

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

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; HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:8-12. In some embodiments, the second antigen-binding portion comprises VH and VL; wherein the VH comprises HCDR1, HCDR2 and HCDR3; wherein 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.

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 NO: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.

In some embodiments, the first antigen binding moiety is a single domain antibody. In some embodiments, the single domain antibody comprises an amino acid sequence as 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 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 to an amino acid sequence as 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 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 with 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. sequence; and/or the light chain variable region of the second antigen binding portion comprises the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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.

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 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: 13-17, 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: 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 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: 13-17. : The light chain variable region of the second antigen binding portion comprises an amino acid sequence as shown in SEQ ID NO:25, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO:25, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO:25, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence as shown in SEQ ID NO:25.

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 NO: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.

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

In some embodiments, the second antigen binding moiety 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 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 shown in 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 with the amino acid sequence shown in SEQ ID NO:31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:31.

In some embodiments, the 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.

In some embodiments, the peptide linker comprises glycine and serine. In some embodiments, the amino acid sequence of the peptide linker is ( _GmS ) _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 , and 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.

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

The first antigen binding portion is a single domain antibody, which comprises an amino acid sequence as 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 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;

The second antigen binding portion is an anti-TIGIT antibody, the heavy chain of which comprises the amino acid sequence shown in 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 shown in 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 shown in SEQ ID NO:29 or 30. The light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:31, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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;

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

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

In some embodiments, the 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 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.

In another aspect, the present invention provides a bispecific antibody or antigen-binding fragment, comprising 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, 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 with the amino acid sequence shown in any one of SEQ ID NOs:41-44, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence shown in any one of SEQ ID NOs: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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:31.

In some embodiments, the 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 NO:41-44; and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:31.

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

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

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

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

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

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 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 to the amino acid sequence set forth in any one of SEQ ID NOs: 13-17. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13-17.

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.

In some embodiments, the PVRIG binding protein is a fusion protein and further comprises an Fc fragment. In some embodiments, the Fc fragment is an Fc fragment of an immunoglobulin or a variant thereof, such as an Fc fragment of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof. In some embodiments, the Fc fragment comprises an amino acid sequence as set forth in 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 as set forth in SEQ ID NO: 34 or 35, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence as set forth in SEQ ID NO: 34 or 35.

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 ( _GmS ) _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 , and 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.

In some embodiments, the PVRIG binding protein comprises the amino acid sequence shown in any one of SEQ ID NOs:36-40, 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 with the amino acid sequence shown in any one of SEQ ID NOs:36-40, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence shown in any one of SEQ ID NOs:36-40.

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

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

Table 1 Amino acid sequence

In another aspect, the present invention provides a nucleic acid encoding the above-mentioned 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.

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

Table 2 Nucleotide sequences

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

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

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

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

On the other hand, the present invention provides use of the above-mentioned bispecific antibody or antigen-binding fragment, the above-mentioned PVRIG binding protein, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned cell or the above-mentioned pharmaceutical composition in treating or preventing a disease.

On the other hand, the present invention provides use of the above-mentioned bispecific antibody or antigen-binding fragment, the above-mentioned PVRIG binding protein, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned cell or the above-mentioned pharmaceutical composition in the preparation of a drug for treating or preventing a disease.

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

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, e.g., 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, urethral cancer, squamous cell carcinoma and Merkel cell carcinoma. In some embodiments, the infection is selected from viral infection, bacterial infection, fungal infection and parasitic infection.

In some embodiments, the present invention relates to a kit or a product comprising the above-mentioned bispecific antibody or antigen-binding fragment, the above-mentioned PVRIG binding protein, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned cell or the above-mentioned pharmaceutical composition.

The PVRIG binding protein or 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

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

FIG2 is a binding curve of the antibody to cells overexpressing TIGIT.

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

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

FIG5 is a graph showing the binding curve of antibody or VHH-Fc fusion protein blocking the binding of ligand PVRL2 to cell surface PVRIG.

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

FIG. 7 shows that antibodies or VHH-Fc fusion proteins block the cellular activity of PVRL2-PVRIG interaction.

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

FIG. 9 shows the activity of antibodies or fusion proteins in activating NK cells.

DETAILED DESCRIPTION

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.

the term

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.

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

As used herein, the terms "comprising" or "including" mean that the antibody, composition, method, etc. include the listed elements, such as components or steps, but do not exclude others. "Essentially consisting of" means that the antibody, composition, method, etc. exclude other elements that have a fundamental effect on the characteristics of the combination, but do not exclude elements that have no essential effect on the antibody, composition, method, etc. "Consisting of" means excluding elements not specifically listed.

"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. Therefore, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to two or more amino acid chains, and "polypeptide" can be used to replace any of the above terms, or used interchangeably with any of the above terms. "Polypeptide" is also intended to refer to products modified after the expression of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage or non-natural amino acid modifications. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a specified nucleic acid sequence, and it may be produced in any manner including chemical synthesis.

Those of ordinary skill in the art will appreciate that the CDR region of an antibody is responsible for the binding specificity of the antibody to an antigen. In the case of known antibody heavy chain and light chain variable region sequences, there are currently several methods for determining the CDR region of an antibody, including Kabat, IMGT, Chothia and AbM numbering systems. However, the application of the definition of the CDR of an antibody or its variant will be within the scope of the term defined and used herein. If the variable region amino acid sequence of the given antibody is given, then those skilled in the art can determine which residues are included in a specific CDR usually, without relying on any experimental data outside the sequence itself.

"Antibody", "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and 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 biological activity of binding to an antigen. Antibodies and antigen-binding fragments include but are not limited to the complementarity determining region (CDR) of a heavy chain or light chain or its ligand-binding portion, a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain constant region (CH), a light chain constant region (CL), a framework region (FR) or any portion thereof, or at least a portion of a binding protein. The CDR region includes the CDR region (LCDR1-3) of the light chain and the CDR region (HCDR1-3) of the heavy chain. The heavy chain constant region (CH) includes the CH1 domain, the hinge (e.g., the upper, middle and/or lower hinge region) domain, the CH2 domain, and the CH3 domain; the crystallizable segment (Fc fragment) is equivalent to the CH2 and CH3 domains. Antibodies and antigen-binding fragments can specifically recognize and bind to polypeptides or polypeptide complexes of one or more (such as two) antigens. Antibodies or antigen-binding fragments that specifically recognize and bind to multiple (such as two) antigens can be referred to as multispecific (such as bispecific) antibodies or antigen-binding fragments.

A complete antibody includes 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 some subclasses (e.g., γ1-γ4). The nature of the chain determines the "type" of the antibody, which is IgG, IgM, IgA, IgD or IgE, respectively. 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 includes two heavy chains or and 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.

"Antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen recognized by the intact antibody. "Antibody fragments" include aptamers, spiegelmers, and bivalent antibodies. "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.

"Fab" generally refers to the part of a conventional antibody (such as IgG) that binds to an antigen, including the heavy chain variable region VH, light chain variable region VL, heavy chain constant region domain CH1, and light chain constant region CL of the antibody. In conventional antibodies, the C-terminus of VH is linked to the N-terminus of CH1 to form a heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form a 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 a heavy chain. In some embodiments, "Fab" also refers to a variant structure of Fab. For example, in some embodiments, the C-terminus of VH is linked to the N-terminus of CL to form a 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 some 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.

"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 to 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, for example, as described in U.S. Patent No. 5,892,019.

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

"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.

"Homology" or "identity" refers to the sequence similarity between two peptides or between two nucleic acids. Homology or identity can be determined by comparing the positions in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then 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.

"Polynucleotide" and "nucleic acid" are used interchangeably. A nucleic acid or polynucleotide (or polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98% or 99%) of "identity or sequence identity" with another sequence means that when the sequences are aligned, that percentage of bases (or amino acids) in the two sequences being compared are the same. The alignment and the identity percentage or sequence identity can be determined visually or using software programs known in the art, such as the software programs 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; expect=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.

"Conservative amino acid substitutions" are those in which an amino acid residue is replaced by an amino acid residue with a similar side chain. 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). Therefore, non-essential amino acid residues of immunoglobulin polypeptides are preferably replaced by other amino acid residues from the same side chain family. In other embodiments, a string of amino acids may be replaced by a structurally similar string of amino acids that differ in sequence and/or in the composition of the side chain family.

In some embodiments, the conservative amino acid substitution is preferably a substitution 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.

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.

The PVRIG binding proteins or bispecific antibodies provided by the present invention 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 by known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, etc. Any of the numerous chemical modifications can be performed by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

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

"Pharmaceutically acceptable" refers to substances approved by government regulatory agencies or listed in recognized pharmacopoeias for use in animals, especially in humans. In addition, "pharmaceutically acceptable excipients" generally refer to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid, etc.

"Excipient" refers to a diluent, adjuvant, excipient or carrier that can be applied to a patient together 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, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifier, or 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 formulations, etc. The composition can be formulated into suppositories with traditional adhesives and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions will contain clinically effective doses of antibodies or antigen binding fragments or fusion proteins, preferably in purified form, together with an appropriate amount of adjuvants, to provide a dosage form suitable for the patient. The preparation should be suitable for administration mode. The parent preparation can be packaged in an ampoule, a disposable syringe, or a multi-dose vial made of glass or plastic.

The PVRIG binding protein or bispecific antibody (including antibodies, antigen binding fragments or fusion proteins, etc.) of the present invention includes neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, salts formed with anions derived from, for example, hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed with cations derived from, for example, sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. The PVRIG binding protein or bispecific antibody described herein may 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 the isoelectric point of the protein, the protein is electrically neutral. The protein described herein may exist in the form of a positive ion, such as when the pH is below the isoelectric point, the protein molecule as a whole exhibits a positive charge. The protein described herein may exist in the form of a negative ion, such as when the pH is above the isoelectric point, the protein molecule as a whole exhibits a negative charge.

The effective dose and treatment regimen for treating a particular patient will depend on various factors, including the specific PVRIG binding protein or bispecific antibody or derivative used, the patient's age and weight, general health, sex and diet, as well as the time of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors are determined by medical care personnel, including those of ordinary skill in the art. The dosage used 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 present invention is administered to the patient at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight each time. In some embodiments, the administration is once a week, every two weeks, every three weeks, or every four weeks.

The antibodies provided by the present invention can be derived from any animal, such as mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, horse or chicken antibodies. In other embodiments, the variable region can be of condricthoid origin (e.g., from sharks).

"Treatment" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent, slow down, improve or stop undesirable physiological changes or disorders, 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 extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, alleviation, reduction or disappearance of the disease state (whether partial or complete), extension of the expected survival period compared to not receiving treatment, etc. Patients in need of treatment include patients who already have a disease or disorder, patients who are susceptible to a disease or disorder, or patients who need to prevent the disease or disorder, and patients who can or are expected to benefit from the administration of the antibodies or pharmaceutical compositions provided by the present invention for detection, diagnostic procedures and/or treatment.

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

Preparation of antibodies or fusion proteins

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.

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 known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. Hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary content, are incorporated herein by reference.

In some embodiments, the DNA encoding the antibody or fusion protein can be designed and synthesized according to the amino acid sequence of the antibody or fusion protein described herein in a conventional manner, placed in an expression vector, and then transfected into a host cell, and the transfected host cell is cultured in a culture medium to produce a monoclonal antibody or fusion protein. In some embodiments, the expression vector includes at least one promoter element, a protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the insertion sequence. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoters of cytomegalovirus, and promoters of other cells 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.

The gene sequence of antibody or fusion protein can be inserted into expression vector by standard methods (for example, complementary restriction sites on connection gene sequence and carrier, or if restriction sites are not present, then flat end connection).Before inserting gene sequence, expression vector can already carry antibody constant region sequence.For example, a method for converting antibody-related VH and VL sequences into full-length antibody gene is to insert them into expression vectors encoding 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 signal peptide that promotes host cell secretion of antibody or fusion protein.Or, gene sequence can be cloned into the carrier of signal peptide that promotes host cell secretion of antibody or fusion protein, so that 3' end of gene sequence encoding signal peptide is connected in frame with 5' end of 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 (i.e., a signal peptide from a non-immunoglobulin protein), for example, MEFGLSLVFLVLILKGVQC (SEQ ID NO:56).

In some embodiments, the expression vector needs to contain a screening marker, and common screening markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other screening genes, so as to facilitate the screening and separation of successfully transfected cells. The constructed plasmid is transfected into host cells without the above genes, and after culturing in a selective medium, the successfully transfected cells grow in large quantities and produce the desired target protein. The obtained antibody or fusion protein can be separated or purified by conventional technical means, such as protein A-agarose gel, ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis or affinity chromatography.

Example

The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

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

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

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

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

Table 3 Amino acid sequences of antibody SF35 and antibody H4

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

Two structures of anti-PVRIG/TIGIT bispecific antibodies were constructed, named as (1) bispecific antibody Bi-VH1, whose schematic diagram is shown in FIG1A ; (2) bispecific antibody Bi-VH3, whose schematic diagram is shown in FIG1B ; 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; and 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, the peptide linker and the Fc fragment.

The relevant sequences of antibodies or VHH-Fc fusion proteins are shown in Tables 1-2 and 4-7; among them, 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.

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 is composed 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), and the second polypeptide is composed of an anti-TIGIT light chain sequence (as shown in SEQ ID NO:31), the nucleic acid sequence of the first polypeptide is as shown in SEQ ID NO:51, and the nucleic acid sequence of the second polypeptide is as shown in SEQ ID NO:45; The heterologous 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 is composed 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), and the second polypeptide is composed of an anti-TIGIT light chain sequence (as shown in SEQ ID NO:31), the nucleic acid sequence of the first polypeptide is as shown in SEQ ID NO:52, and the nucleic acid sequence of the second polypeptide is as 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 is composed 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 is composed of an anti-TIGIT light chain sequence (as shown in SEQ ID NO:31), the nucleic acid sequence of the first polypeptide is as shown in SEQ ID NO:53, and the nucleic acid sequence of the second polypeptide is as shown in SEQ ID NO:45; The heterologous antibody Bi-VH3-94W contains two first polypeptides with identical sequences (as shown in SEQ ID NO:44) and two second polypeptides with identical sequences (as shown in SEQ ID NO:31), the first polypeptide is composed 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), and the second polypeptide is composed 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.

Table 4 Sequences and CDRs of VHH fragments against PVRIG

Table 5 Sequence and composition of VHH-Fc fusion protein

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

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

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

Biacore T200 was used to detect the affinity of antibodies and VHH-Fc fusion proteins to PVRIG-his and TIGIT-his proteins. Antibodies or VHH-Fc fusion proteins were coupled to the surface of the CM5 chip, and then the chip was used to capture PVRIG-his or TIGIT-his, respectively. Different 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 showed strong affinity to PVRIG-his protein, with KD values of 0.65nM, 0.89nM, and 0.96nM, respectively. As shown in Table 7, the bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y and Bi-VH3-94W bind 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 affinities, with KD values of 4.99nM, 5.58nM, 3.91nM and 3.70nM, respectively, which are comparable to the affinities 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).

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

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

Table 8 Affinity data of antibodies and TIGIT-his (purchased from ACRO, catalog number TIT-H52H5)

Example 4: Binding experiment of anti-PVRIG/TIGIT bispecific antibody and cells overexpressing TIGIT

Take appropriate amounts of bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y and Bi-VH3-94W, and parent anti-TIGIT antibody h10D8OF, dilute them to 200 nM with 1× PBS as the starting concentration in a 96-well V-shaped plate, and perform 3-fold gradient dilution with 1× PBS to obtain a total of 9 serial dilution concentrations.

Take Jurkat-TIGIT cells with good growth status (transfect the human full-length TIGIT gene (SEQ ID NO: 55) into the 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, resuspend the cells with 1×PBS to a density of 1×10 ⁷ cells/mL, add 50μL/well to a 96-well V-shaped plate, at this time the number of cells is 5×10 ⁵ cells/well, add 50μL of the corresponding concentration of antibody sample to each well and mix well, at this time the maximum final concentration of the antibody is 100nM. Place the 96-well V-shaped plate in a 4°C refrigerator and incubate for 60min. Then centrifuge the 96-well V-shaped plate to remove the supernatant, wash it with 1×PBS and remove the supernatant for the next incubation. Anti-FC PE (purchased from BioLegend, catalog number 366903) was diluted with 1×PBS at a ratio of 1:500. 100 μL of diluted anti-FC PE was added to each well except the blank well to resuspend the cells, and placed in a 4°C refrigerator in the dark for 30 minutes. After incubation, the cells were washed with 1×PBS and finally resuspended with 1×PBS at a volume of 150 μL per well. The cells were collected by flow cytometry, and the fluorescent antibodies bound to the cell surface were detected to obtain the mean fluorescence intensity (MFI) value of the original data. The final results were fitted with a four-parameter model with antibody concentration as the horizontal axis and MFI as the vertical axis, and the results are shown in Figure 2.

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

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 with EC ₅₀ values of 0.23 nM, 0.23 nM, 0.23 nM and 0.12 nM, respectively, which were similar to the binding ability of the parent anti-TIGIT antibody h10D8OF to TIGIT expressed on the cell surface (EC ₅₀ = 0.13 nM).

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

Take appropriate amounts of bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y and Bi-VH3-94W, VHH-Fc fusion proteins N1-94W-Fc, N1-94Y-Fc, 32, 34 and 37, and antibodies SF35 and hIgG1 (Beijing Sino Biological Technology Co., Ltd., Cat. No. MA14OC2903). Dilute to 200 nM with 1× PBS as the starting concentration in a 96-well V-shaped plate, and perform 3-fold gradient dilution with 1× PBS to obtain a total of 9 serial dilution concentrations.

The cDNA of PVRIG (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 were taken, centrifuged and the supernatant was discarded, and the cells were resuspended in 1×PBS to a density of 1×10 ⁷ cells/mL, and added to a 96-well V-shaped plate (Corning, catalog number: 3897) at a rate of 50 μL/well. At this time, the number of cells was 5×10 ⁵ cells/well. 50 μL of the corresponding concentration of antibody or VHH-Fc fusion protein sample was added to each well and mixed. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein was 100 nM. The 96-well V-shaped plate was placed in a 4°C refrigerator for incubation for 60 minutes. After the incubation was completed, the 96-well V-shaped plate was centrifuged to remove the supernatant, and then washed with 1×PBS and the supernatant was removed for the next incubation. Anti-FC PE (purchased from BioLegend, catalog number 366903) was diluted with 1×PBS at a ratio of 1:500, and 100μL of diluted anti-FC PE was added to each well except the blank well to resuspend the cells, and placed in a 4°C refrigerator in the dark for 30 minutes. After the incubation was completed, it was washed with 1×PBS and finally resuspended with 1×PBS at a volume of 150μL per well. The cells were collected by flow cytometry, and the fluorescent antibodies bound to the cell surface were detected to obtain the original data MFI value. The final results were fitted with a four-parameter model with the antibody or VHH-Fc fusion protein concentration as the horizontal axis and MFI as the vertical axis. The results are shown in Table 9, Figure 3 and Figure 4.

Table 9 Cellular affinity of antibodies or VHH-Fc fusion proteins

*Relative activity % = {EC ₅₀ (sample) - EC ₅₀ (SF35)} / EC ₅₀ (SF35) × 100%

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 _EC50 of VHH-Fc fusion protein 32 is 0.67nM, the _EC50 of VHH-Fc fusion protein 34 is 0.95nM, and the _EC50 of VHH-Fc fusion protein 37 is 0.87nM. The binding activity of VHH-Fc fusion proteins 32 and 37 is better than that of antibody SF35 ( _EC50 = 0.91nM).

As shown in the results of 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 significantly, with EC ₅₀ values of 0.26 nM, 0.32 nM, 1.72 nM and 2.33 nM, respectively. The bispecific antibodies Bi-VH1-94Y and Bi-VH1-94W have comparable binding abilities to PVRIG expressed on the cell surface as the VHH-Fc fusion proteins N1-94W-Fc (EC ₅₀ = 0.34 nM) and N1-94Y-Fc (EC ₅₀ = 0.40 nM).

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

Take appropriate amount of VHH-Fc fusion proteins 32, 34, 37 and antibodies SF35, H4, hIgG1 (Beijing Sino Biological Technology Co., Ltd., Catalog No.: MA14OC2903) and dilute to 600 nM with 1×PBS as the starting concentration, and perform 4-fold gradient dilution with 1×PBS to obtain 10 serial dilution concentrations.

Take Jurkat-NFAT-PVRIG cells with good growth status, centrifuge and discard the supernatant, resuspend the cells with 1×PBS to a density of 1×10 ⁷ cells/mL, add 50μL/well to a 96-well V-shaped plate (Corning, Catalog No.: 3897), at this time the number of cells is 5×10 ⁵ cells/well, add 50μL of the corresponding concentration of antibody or VHH-Fc fusion protein to each well and mix well, at this time the maximum final concentration of antibody or VHH-Fc fusion protein is 300nM. Place the 96-well V-shaped plate in a 4℃ refrigerator and incubate for 10min. While waiting for the antibody incubation, take an appropriate amount of ligand PVRL2-mFc-bio (purchased from Acro, Catalog No. CD2-H82A3) and dilute it to 600nM with 1×PBS. After the incubation is completed, take out the 96-well V-shaped plate, add the ligand PVRL2-mFc dilution as soon as possible according to the volume of 50 μL per well and mix it with a pipette. At this time, the final concentration of the ligand is 200 nM, and the maximum final concentration of the antibody or VHH-Fc fusion protein is 200 nM. Place the 96-well V-shaped plate in a 4°C refrigerator and continue incubation for 60 minutes.

After the incubation is completed, the 96-well V-shaped plate is centrifuged to remove the supernatant, and then washed with 1×PBS to remove the supernatant for the next incubation. Streptavidin-phycoerythrin (SA-PE; BioLegend, Cat. No. 740452) is diluted with 1×PBS at a ratio of 1:800, and 100 μL of diluted SA-PE is added to each well except the blank well to resuspend the cells, and incubated in a 4°C refrigerator away from light for 30 minutes.

Cells were collected by flow cytometry, and fluorescent antibodies bound to the cell surface were detected to obtain the original data MFI value. The final result was obtained by using the antibody or VHH-Fc fusion protein concentration as the horizontal axis and MFI as the vertical axis to perform curve fitting using a four-parameter model, and the results are shown in FIG5 .

As can be seen from the results in Figure 5, VHH-Fc fusion proteins 32, 34 and 37 can all block the binding of the ligand PVRL2 to human PVRIG expressed on the surface of Jurkat-NFAT-PVRIG cells. The _IC50 of VHH-Fc fusion protein 32 is 24.48nM, the _IC50 of VHH-Fc fusion protein 34 is 1.67nM, and the _IC50 of VHH-Fc fusion protein 37 is 1.01nM. The blocking abilities of VHH-Fc fusion proteins 34 and 37 are comparable to those of antibody SF35 ( _IC50 = 0.56).

Example 7: PVRIG cell activity experiment of bispecific antibodies

1) Take appropriate amount of VHH-Fc fusion proteins 32, 34, 37 and antibodies SF35, H4, hIgG1 (Beijing Yiqiao Shenzhou Technology Co., Ltd., Catalog No.: MA14OC2903) and dilute to 600nM as the starting concentration with detection diluent (add 55mL FBS (ExCell Biology, Catalog No.: FSP500) to 500mL RPMI 1640 culture medium (Gibco, Catalog No.: 11875093) as detection diluent), and perform 3-fold gradient dilution with detection diluent to obtain 10 serial dilution concentrations; take a 96-well white plate and add 50μL volume to each well for later use.

The cDNA of PVRL2 (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number HG10005-UT) was transferred into CHO-OKT3 cells (for the construction method, see patent CN111748580A) to obtain CHO-OKT3 cells overexpressing PVRL2, named CHO-OKT3-PVRL2. Take Jurkat-NFAT-PVRIG cells and CHO-OKT3-PVRL2 cells with good growth status, centrifuge and discard the supernatant, resuspend them in the detection diluent, and count them with a cell counter. According to the cell density, Jurkat-NFAT-PVRIG cells are diluted with the detection diluent to make the cell density 4×10 ⁶ cells/mL. Take out the 96-well white plate with 50 μL/well antibody or VHH-Fc fusion protein, add 50 μL Jurkat-NFAT-PVRIG cells to each well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 300nM.

Place the 96-well white plate in a 37°C CO ₂ constant temperature incubator and incubate for 10 minutes, then remove the 96-well white plate. Dilute 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. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 200 nM. Place the 96-well white plate in a CO ₂ constant temperature incubator and incubate at 37°C for 6 hours. Take out the Bio-Lite ^TM Luciferase Assay System colorimetric solution from the -20°C refrigerator in advance and dissolve it at room temperature. After incubation, remove the 96-well white plate from the incubator, equilibrate at room temperature for about 8 minutes, then add 70 μL of colorimetric solution to each well, place it at room temperature away from light for 5 minutes, and read the plate as soon as possible.

Use the Luminescence detection module on the SpectraMax multi-function microplate reader, set PMT and Optics to 500, read the relative light unit (RLU), and shake before reading the plate. With the antibody or VHH-Fc fusion protein concentration as the horizontal axis and the relative light unit as the vertical axis, the sample was curve-fitted using the four-parameter model, and the four-parameter curve was obtained as shown in Figure 6.

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. The EC ₅₀ of VHH-Fc fusion protein 32 was 140.9 nM, the EC ₅₀ of VHH-Fc fusion protein 34 was 124.8 nM, and the EC ₅₀ of VHH-Fc fusion protein 37 was 10.0 nM.

2) Take appropriate amount of bispecific antibodies Bi-VH1-94Y, Bi-VH1-94W, Bi-VH3-94Y, Bi-VH3-94W, VHH-Fc fusion proteins N1-94Y-Fc and N1-94W-Fc and dilute them to 600 nM with RPMI 1640 medium as the starting concentration, and perform 5-fold gradient dilution with RPMI 1640 medium to obtain 8 serial dilution concentrations in total; take a 96-well white plate and add 100 μL volume to each well for later use.

Take Jurkat-NFAT-PVRIG cells and CHO-OKT3-PVRL2 cells that are in good growth condition, centrifuge and discard the supernatant, resuspend them in RPMI 1640 medium containing 10% FBS (fetal bovine serum), and count them using a cell counter. Dilute Jurkat-NFAT-PVRIG cells with RPMI 1640 medium containing 10% FBS to a cell density of 4×10 ⁶ cells/mL. Take out 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 well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 400 nM.

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

Take out the Bio-Lite ^TM LuciferaseAssay System colorimetric solution from the -20℃ refrigerator in advance and dissolve it at room temperature. Take out the white plate from the incubator and equilibrate it at room temperature for about 8 minutes. Then add 70μL of colorimetric solution to each well, place it at room temperature away from light for 5 minutes, and read the plate as soon as possible. Use the Luminescence detection module on the SpectraMax multi-function microplate reader, set the PMT and Optics to 500, read the relative light units (RLU), and shake before reading the plate. With the antibody or VHH-Fc fusion protein concentration as the horizontal axis and the relative light unit as the vertical axis, the sample was curve-fitted using the four-parameter model, and the four-parameter curve was obtained as shown in Figure 7.

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

Example 8: Bispecific Antibody and TIGIT Cell Activity Experiment

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

CHO-K1-CD155 cells in good growth state (the construction method refers to the construction of sAPC cell line in Example 1 of patent CN111748580A) 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 a cell suspension with a density of 1×10 ⁵ cells/mL. 100 μL/well (the cell number at this time was 1×10 ⁴ cells/well) was added to a 96-well white plate, and sterile water was added to the side wells. The plate was placed in a 5% CO ₂ , 37°C incubator for overnight incubation.

Take Jurkat-TIGIT cells that are growing well, centrifuge and discard the supernatant, resuspend the cells in RPMI 1640 medium containing 10% FBS, count the cells, and prepare a cell suspension with a density of 5.0×10 ⁶ cells/mL for later use. Take out the 96-well white plate that has been plated with CHO-K1-CD155 cells from the incubator, discard the supernatant, and then add 40μL/well of antibody diluent and 40μL/well of Jurkat-TIGIT cell suspension to the 2nd to 11th columns and rows B to H of the 96-well white plate (the number of cells is 2×10 ⁵ cells/well at this time), and the maximum final antibody concentration is 350nM at this time. Add sterile water to the side wells. Place the cell plate in a 5% CO ₂ , 37°C incubator and incubate for 6h.

Take out the Bio-Lite ^TM Luciferase Assay System colorimetric solution from the -20℃ refrigerator in advance and dissolve it at room temperature. Take out the 96-well white plate from the incubator, equilibrate it at room temperature for about 8 minutes, then add 60μL of colorimetric solution to each well, place it at room temperature away from light for 5 minutes, and read the plate as soon as possible. Use the Luminescence detection module on the SpectraMax multi-function microplate reader, set PMT and Optics to 500, and read the relative light units (RLU). With the antibody concentration as the horizontal axis and the relative light unit as the vertical axis, the sample is curve-fitted using the four-parameter model, as shown in Figure 8.

The results showed that the bispecific antibodies Bi-VH1-94Y (EC ₅₀ = 1.42nM), Bi-VH1-94W (EC ₅₀ = 1.68nM), Bi-VH3-94Y (EC ₅₀ = 2.15nM), and Bi-VH3-94W (EC ₅₀ = 1.71nM) could 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.

Example 9: Detection of NK cell activity of bispecific antibodies

Add 55 mL of FBS to 500 mL of RPMI 1640 medium as a diluent for detection. Take appropriate amounts of bispecific antibodies Bi-VH1-94W, Bi-VH3-94W, VHH-Fc fusion protein N1-94W-Fc, and antibody h10D8OF. The sample groups are Bi-VH1-94W and Bi-VH3-94W, and the control groups are N1-94W-Fc, antibody h10D8OF, N1-94W-Fc+antibody h10D8OF (where the added amounts of N1-94W-Fc and antibody h10D8OF are the same as those of N1-94W-Fc and antibody h10D8OF alone), and hIgG1 (Beijing Sino Biological Technology Co., Ltd., Cat. No.: MA14OC2903). The sample group and the control group were diluted to 200 nM as the starting concentration with the detection diluent in a 96-well V-shaped plate, and then 100-fold gradient dilution was performed with the detection diluent to obtain 3 serial dilution concentrations.

Take the PBMC cells in good condition and transfer them to a six-well plate or cell culture flask and culture them at 37°C for 4 hours. Centrifuge and discard the supernatant. Resuspend the cells with RPMI 1640 medium containing 10% FBS to a density of 4×10 ⁶ cells/mL. 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 well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 66.7 nM. Place the 96-well U-shaped plate in a 5% CO ₂ , 37°C incubator and incubate for 4 hours.

Take K562 cells (highly expressing PVRL2 and PVR) with good growth status, centrifuge and discard the supernatant, resuspend the cells in RPMI 1640 medium containing 10% FBS to a density of 1×10 ⁷ cells/mL. Take out the 96-well U-shaped plate that has been incubated for 4 hours, add 50 μL of K562 cells to each well and mix well. At this time, the maximum final concentration of the antibody or VHH-Fc fusion protein is 50 nM. Place the 96-well U-shaped plate in a 5% CO ₂ , 37°C incubator and incubate for 21 hours.

The 96-well U-shaped plate was centrifuged to remove the supernatant, and the supernatant was removed after washing with PBS containing 2% FBS for the next incubation. Anti-CD3 APC (purchased from BioLegend, item number 344812), Anti-NKp46 BV421 (purchased from BioLegend, item number 331914) and anti-CD137 PE (purchased from BioLegend, item number 309804) were added to PBS containing 2% FBS at a dilution ratio of 1:500 to obtain a fluorescent secondary antibody dilution solution. 100 μL of fluorescent secondary antibody dilution solution was added to each well except the blank well to resuspend the cells, and placed in a 4°C refrigerator in the dark for 25 minutes. After the incubation is completed, the 96-well U-shaped plate was centrifuged to remove the supernatant, washed with PBS containing 2% FBS, and finally resuspended with PBS containing 2% FBS according to a volume of 150 μL per well. Cells were collected by flow cytometry, and fluorescent antibodies bound to the cell surface were detected to obtain the raw data MFI value. The final result was obtained by fitting the curve using a four-parameter model with antibody concentration as the horizontal axis and the percentage of NK cells as the vertical axis to obtain the antibody dose-dependent binding curve, as shown in Figure 9.

The results showed that the 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 surface of NK cells at 32.63% and 35.88%, respectively, while the hIgG1 control group was only 22.45%.

Claims (19)

A PVRIG binding protein, comprising a heavy chain variable region, wherein the heavy chain variable region comprises one or more of the HCDR1 shown in SEQ ID NO:6, the HCDR2 shown in SEQ ID NO:7, and the HCDR3 shown in any one of SEQ ID NOs:8-12. The PVRIG binding protein as described in 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. The PVRIG binding protein as described in claim 1 or 2, wherein the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 13-17, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NO: 13-17, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 13-17. The PVRIG binding protein as described in any one of claims 1-3, wherein the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO:13-17. A PVRIG binding protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 36-40, or an amino acid sequence having at least 80% identity with the amino acid sequence as 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 as shown in any one of SEQ ID NOs: 36-40. The PVRIG binding protein according to any one of claims 1 to 5, which is a single domain antibody. A bispecific antibody or antigen-binding fragment, comprising a first antigen-binding portion capable of binding to PVRIG; the first antigen-binding portion comprises the PVRIG binding protein according to any one of claims 1 to 6. The bispecific antibody or antigen-binding fragment as described in claim 7 comprises a second antigen-binding portion capable of binding to TIGIT, wherein the second antigen-binding portion comprises one or more of HCDR1 shown in SEQ ID NO: 18, HCDR2 shown in SEQ ID NO: 19, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 21, LCDR2 shown in SEQ ID NO: 22 and LCDR3 shown in SEQ ID NO: 23. The bispecific antibody or antigen-binding fragment of 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. The bispecific antibody or antigen-binding fragment of 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 with the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having one or more conservative amino acid substitutions with 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 with the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence shown in SEQ ID NO:25. The bispecific antibody or antigen-binding fragment of 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. The bispecific antibody or antigen-binding fragment of any one of claims 8 to 11, wherein the second antigen-binding moiety is linked to the first antigen-binding moiety via a peptide linker. The bispecific antibody or antigen-binding fragment according to any one of claims 8 to 12, wherein 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. The bispecific antibody or antigen-binding fragment of any one of claims 8 to 12, wherein the N-terminus of the antibody heavy chain is connected to the C-terminus of the first antigen-binding moiety via a peptide linker. The bispecific antibody or antigen-binding fragment of any one of claims 8 to 12, wherein the C-terminus of the antibody heavy chain is connected to the N-terminus of the first antigen-binding moiety via a peptide linker. A bispecific antibody or antigen-binding fragment comprising a first polypeptide and a second polypeptide; wherein The first polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NOs:41-44, or an amino acid sequence having at least 80% identity with the amino acid sequence as shown in any one of SEQ ID NOs:41-44, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence as shown in any one of SEQ ID NOs: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 with the amino acid sequence shown in SEQ ID NO:31, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence shown in SEQ ID NO:31. Biological materials, for: 1) A nucleic acid encoding the PVRIG binding protein according to any one of claims 1 to 6 or the bispecific antibody or antigen-binding fragment according to any one of claims 7 to 16; 2) an expression vector comprising a nucleic acid encoding the PVRIG binding protein of any one of claims 1 to 6 or the bispecific antibody or antigen-binding fragment of any one of claims 7 to 16; or 3) A host cell comprising a nucleic acid or an expression vector encoding the PVRIG binding protein according to any one of claims 1 to 6 or the bispecific antibody or antigen-binding fragment according to any one of claims 7 to 16. A pharmaceutical composition comprising the PVRIG binding protein according to any one of claims 1 to 6 or the bispecific antibody or antigen-binding fragment according to any one of claims 7 to 16 and a pharmaceutically acceptable excipient. Use of the PVRIG binding protein of any one of claims 1 to 6, the bispecific antibody or antigen-binding fragment of any one of claims 7 to 16, the biomaterial of claim 17, or the pharmaceutical composition of claim 18 in treating or preventing a disease or in the preparation of a medicament for treating or preventing a disease.