Anti-PVRIG / anti-TIGIT bispecific antibodies and applications

Abstract

The present invention provides a bispecific antibody capable of specifically binding to both PVRIG and TIGIT. The bispecific antibody can regulate the function of immune cells and can be used as a drug to treat diseases associated with immune abnormalities, such as tumors.

Description

[Technical Field]

[0001] The present invention relates to the field of antibodies, and in particular to anti-PVRIG / anti-TIGIT bispecific antibodies. [Background technology]

[0002] The basis of immunotherapy is the manipulation and / or modulation of the immune system, including both innate and adaptive immune responses. The goal of immunotherapy is to treat disease by controlling the immune response to "foreign agents" (e.g., pathogens or tumor cells). The immune system is a highly complex system composed of many cell types with complex and sophisticated systems for regulating interactions and responses. The concept of cancer immunosurveillance is based on the theory that the immune system can recognize tumor cells, initiate an immune response, and further inhibit tumor development and / or progression. However, it is clear that many cancer cells develop mechanisms to evade the immune system, allowing tumors to grow uninhibited. Cancer / tumor immunotherapy focuses on the development of new and novel antagonists and / or antagonists that can activate and / or activate the immune system to achieve more effective antitumor responses, enhance tumor cell killing, and / or inhibit tumor growth.

[0003] PVRIG is expressed in NK cells and T cells and shares some similarities with other known immune checkpoint receptors. Identification and methods for demonstrating that PVRIG is an immune checkpoint receptor are discussed in WO 2016 / 134333, which is expressly incorporated herein by reference. When PVRIG binds to its ligand (PVRL2), it triggers an inhibitory signal that acts to dampen the NK cell and T cell immune response against target cells (i.e., similar to PD-1 / PD-L1). Blocking PVRL2 binding to PVRIG blocks this inhibitory signal from PVRIG, thereby modulating the NK cell and T cell immune response. The use of PVRIG antibodies that block binding to PVRL2 is a therapeutic approach to enhance cancer cell killing by NK cells and T cells. Blocking antibodies have been generated that bind to PVRIG and block its binding to its ligand, PVRL2.

[0004] Similarly, TIGIT is another target of interest, and binding to its homologous ligand PVR has been shown to directly inhibit NK cell and T cell cytotoxicity via its intracellular ITIM domain. Knockout of the TIGIT gene or antibodies blocking the TIGIT / PVR interaction have been shown to enhance NK cell killing in vitro or exacerbate autoimmune diseases in vivo. In addition to its direct effects on T cells and NK cells, TIGIT can increase the production of anti-inflammatory cytokines (e.g., IL-10) by inducing PVR-mediated signaling in dendritic cells or tumor cells. Notably, TIGIT expression is closely linked to that of another important co-inhibitory receptor, PD-1. TIGIT and PD-1 are co-expressed on many human and murine tumor-infiltrating lymphocytes (TILs).

[0005] Both TIGIT and PVRIG belong to the DNAM superfamily and have been shown to be co-expressed in many tumor-infiltrating lymphocytes and exert immunosuppressive effects. Tumor-infiltrating effector T cells co-expressing TIGIT, PVRIG, and PD-1 are believed to be the most important effector T cells in the infiltrating T cell population. Therefore, bispecific antibodies targeting PVRIG and TIGIT simultaneously offer potential synergistic effects and are an attractive therapeutic approach for use in single-antibody therapy. Such bispecific antibodies, which simultaneously target two immune checkpoint receptors, can also potentially synergize with conventional anti-PD-1 / L-1 antibody therapy, playing an important role in providing new therapeutic avenues for cancer treatment. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the potential synergistic effects of bispecific antibodies, the present invention is particularly proposed to improve the immunosuppressive effect and solve the problem of reduced response efficiency of immune checkpoint inhibitors.

[0007] The present invention provides anti-PVRIG / anti-TIGIT antibodies, nucleic acids encoding them, methods for producing the antibodies, pharmaceutical compositions comprising said antibodies, and related uses of the pharmaceutical compositions for treating tumors. [Means for solving the problem]

[0008] In a first aspect, the present invention provides an anti-PVRIG / anti-TIGIT bispecific antibody, comprising: (a) a first antigen-binding moiety comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL form an anti-TIGIT antigen-binding domain, wherein the TIGIT VH comprises HCDR1, HCDR2, and HCDR3 of the VH set forth in any one of SEQ ID NOs: 72 or 87, and the TIGIT VL comprises LCDR1, LCDR2, and LCDR3 of the VL set forth in any one of SEQ ID NOs: 68 or 91; (b) a second antigen-binding portion comprising a VHH that specifically binds to PVRIG, wherein the VHH comprises CDR1, CDR2 and CDR3 of an array set forth in any one of SEQ ID NOs: 200 or 211.

[0009] In some embodiments, (a) the HCDR1 of the first antigen-binding portion comprises the sequence set forth in any one of SEQ ID NOs: 21 or 33, the HCDR2 comprises the sequence set forth in any one of SEQ ID NOs: 22 or 34, and the HCDR3 comprises the sequence set forth in any one of SEQ ID NOs: 23 or 35; (b) the LCDR1 of the first antigen-binding portion comprises a sequence set forth in any one of SEQ ID NOs: 18 or 96, the LCDR2 comprises a sequence set forth in any one of SEQ ID NOs: 19 or 31, and the LCDR3 comprises a sequence set forth in any one of SEQ ID NOs: 20 or 32; (c) CDR1 of the second antigen-binding portion comprises the sequence set forth in any one of SEQ ID NOs: 168 or 147, CDR2 comprises the sequence set forth in any one of SEQ ID NOs: 207 or 148, and CDR3 comprises the sequence set forth in any one of SEQ ID NOs: 208 or 149.

[0010] In some embodiments, the first antigen-binding portion comprises: (1) the sequences whose SEQ ID NOs are 21, 22, 23, 18, 19, and 20, respectively; or (2) SEQ ID NOs: 33, 34, 35, 96, 31, and 32, respectively; or (3) A sequence having at least 90% identity with the sequence shown in (1) to (2) above, or having one, two, three or more amino acid insertions, deletions and / or substitutions in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, preferably wherein the substitutions are conservative amino acid substitutions.

[0011] In some embodiments, the second antigen-binding portion comprises: (1) the sequences of SEQ ID NOs: 168, 207, and 208, respectively; or (2) the sequences of SEQ ID NOs: 147, 148, and 149, respectively; or (3) The CDR1, CDR2, and CDR3 of a sequence having at least 90% identity with the sequence shown in (1) to (2) above, or having one, two, three, or more amino acid insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions.

[0012] In some embodiments, the VH of the first antigen-binding portion comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72 or 87, and the VL of the first antigen-binding portion comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 68 or 91.

[0013] In some embodiments, the second antigen-binding portion comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 200 or 211.

[0014] In some embodiments, the first antigen-binding portion is a full-length antibody comprising two heavy chains and two light chains, and the C-terminus of the second antigen-binding portion is fused to the N-terminus of at least one heavy chain of the first antigen-binding portion.

[0015] In some embodiments, the heavy chain fusion polypeptide comprises, from N- to C-terminus, PVRIG VHH-(G4S)4 Linker-TIGIT VH-CH1-hinge-CH2-CH3, and the light chain polypeptide comprises, from N- to C-terminus, TIGIT VL-CL.

[0016] In some embodiments, the heavy chain fusion polypeptide comprises a sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 227, 229, 231, or 233, and the light chain polypeptide comprises a sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 226, 228, 230, or 232.

[0017] In some embodiments, the bispecific antibody is a humanized antibody.

[0018] In some embodiments, the bispecific antibody specifically binds to human, monkey PRVIG, or TIGIT protein, preferably has a KD of greater than 1.00E-7M for binding to human or monkey TIGIT and a KD of greater than 1.00E-8M for binding to human or monkey PRVIG, and more preferably can simultaneously bind to TIGIT and PVRIG.

[0019] In another aspect, the present invention provides an antibody or antigen-binding fragment that specifically binds to TIGIT, which comprises: (1) A heavy chain variable region (VH) comprising three complementarity determining regions (HCDRs), i.e., HCDR1, HCDR2, and HCDR3, wherein, when numbered according to the Kabat numbering system, HCDR1 comprises the amino acid sequence set forth in SEQ ID NOs: 21, 27, 33, 39, HCDR2 comprises the amino acid sequence set forth in SEQ ID NOs: 22, 28, 34, 40, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NOs: 23, 29, 35, 41; and when numbered according to the IMGT numbering system, HCDR1 comprises the amino acid sequence set forth in SEQ ID NOs: 45, 51, 57, 63, HCDR2 comprises SEQ ID NOs: 46, 52, 58, 64, and HCDR3 comprises SEQ ID NOs: 47, 53, 59, 65; and (2) A light chain variable region (VL) comprising three complementarity determining regions (LCDRs) designated LCDR1, LCDR2, and LCDR3, wherein, when numbered according to the Kabat numbering system, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NOs: 18, 24, 30, 36, 93, 94, 95, 96, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NOs: 19, 25, 31, 37, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NOs: 20, 26, 32, 38; and when numbered according to the IMGT numbering system, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NOs: 42, 48, 54, 60, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NOs: 43, 49, 55, 61, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NOs: 44, 50, 56, 62.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises: (1) the sequences of SEQ ID NOs: 18, 19, 20, 21, 22, and 23, respectively; or (2) the sequences of SEQ ID NOs: 24, 25, 26, 27, 28, and 29, respectively; or (3) SEQ ID NOs: 30, 31, 32, 33, 34, and 35, respectively; or (4) SEQ ID NOs: 36, 37, 38, 39, 40, and 41, respectively; or (5) SEQ ID NOs: 42, 43, 44, 45, 46, and 47, respectively; or (6) SEQ ID NOs: 48, 49, 50, 51, 52, and 53, respectively; or (7) SEQ ID NOs: 54, 55, 56, 57, 58, and 59, respectively; or (8) SEQ ID NOs: 60, 61, 62, 63, 64, and 65, respectively; or (9) SEQ ID NOs: 93, 31, 32, 33, 34, and 35, respectively; or (10) SEQ ID NOs: 94, 31, 32, 33, 34, and 35, respectively; or (11) SEQ ID NOs: 95, 31, 32, 33, 34, and 35, respectively; or (12) SEQ ID NOs: 96, 31, 32, 33, 34, and 35, respectively; or (13) A sequence having at least 80% identity with the sequence shown in (1) to (12) above, or comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, and having one, two, three, or more amino acid insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions.

[0021] In some embodiments, the antibody or antigen-binding fragment comprises: (1) a heavy chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 10, 11, 12, 13, 69, 70, 71, 72, 81, 82, 83, 84, 85, 87, 101, 102, or 103; or / and (2) a light chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 14, 15, 16, 17, 66, 67, 68, 78, 79, 80, 86, 88, 89, 90, 91, 98, 99, or 100.

[0022] In some embodiments, the antibody or antigen-binding fragment comprises: (1) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 10 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 14; or (2) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 15; or (3) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 12 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, or (4) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, or (5) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 69 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 66, 67, or 68; or (6) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 70 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 66, 67, or 68; or (7) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 71 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 66, 67, or 68; or (8) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 72 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 66, 67, or 68; or (9) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 81, 82, 83, 84, or 85, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 78; or (10) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 81, 82, 83, 84, or 85, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 79; or (11) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 81, 82, 83, 84, or 85, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 80; or (12) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 87 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 86, 88, 89, 90, or 91; or (13) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 101 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 98, 99, or 100; or (14) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 102 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 98, 99, or 100; (15) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 103 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 98, 99, or 100; or A sequence having at least 80% identity with the sequence shown in (1) to (15) above or having up to 20 mutations, wherein the mutations may be selected from insertions, deletions, and / or substitutions, and preferably the substitutions are conservative amino acid substitutions.

[0023] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region, when numbered in natural order relative to the VH set forth in SEQ ID NO: 10, has at least one mutation selected from the group consisting of S30T, G44K, W47Y, I48M, V67I, or V71R, preferably has at least S30T and V71R mutations, more preferably has at least S30T, G44K, and V71R mutations, more preferably has at least S30T, G44K, I48M, V67I, and V71R mutations, more preferably has at least S30T, G44K, W47Y, and V71R mutations, or having, when numbered in natural order relative to the VH set forth in SEQ ID NO: 11, at least a mutation selected from the group consisting of T28A, R72A, T74K or A76S, preferably having at least T28A, R72A, T74K and A76S mutations; or having, when numbered in natural order relative to the VH shown in SEQ ID NO: 12, at least mutations selected from the group consisting of I29M, S30T, G44K, W47Y, I48M, V67I or V71R, preferably having at least S30T and V71R mutations, more preferably having at least I29M, S30T and V71R mutations, more preferably having at least I29M, S30T, G44K and V71R mutations, more preferably having at least I29M, S30T, G44K, I48M, V67I and V71R mutations, more preferably having at least I29M, S30T, G44K, W47Y and V71R mutations; Alternatively, when numbered in natural order compared to the VH shown in SEQ ID NO: 13, it has at least mutations selected from the group consisting of R44G, R72V, T74K, S75L or A76S, preferably it has at least R72V and T74K mutations, more preferably it has at least R72V, T74K, S75L and A76S mutations, more preferably it has at least R44G, R72V, T74K, S75L and A76S mutations.

[0024] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region, when numbered in natural order relative to the VL set forth in SEQ ID NO: 14, has at least a mutation selected from the group consisting of L37Q, P43S, or L47M, preferably has at least an L47M mutation, more preferably has at least an L37Q and an L47M mutation, more preferably has at least an P43S and an L47M mutation; or when numbered in natural order relative to the VL shown in SEQ ID NO: 15, has at least a mutation selected from the group consisting of N31Q, N31T, N31D, G32A, Q38H or P43S, preferably has at least Q38H and P43S mutations, more preferably has at least N31Q, Q38H and P43S mutations, more preferably has at least N31T, Q38H and P43S mutations, more preferably has at least N31D, Q38H and P43S mutations, more preferably has at least G32A, Q38H and P43S mutations; or having, when numbered in natural order relative to the VL shown in SEQ ID NO: 16, at least a mutation selected from the group consisting of L37Q, P43S or Q45K, preferably having at least L37Q and Q45K mutations, more preferably having at least a P43S mutation; Alternatively, when numbered in natural order compared to the VL shown in SEQ ID NO: 17, it has at least a mutation selected from the group consisting of A43S, P43S or I48V, preferably it has at least the A43S mutation, more preferably it has at least the A43S and I48V mutations, more preferably it has at least the P43S and I48V mutations.

[0025] In some embodiments, the antibody or antigen-binding fragment specifically binds to human or monkey TIGIT protein, and preferably has a KD of greater than 1.00E-8M for binding to human or monkey TIGIT.

[0026] In some embodiments, the antibody or antigen-binding fragment is a murine antibody, a humanized antibody, a fully human antibody, or a chimeric antibody.

[0027] In some embodiments, the antibody or antigen-binding fragment is chosen from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a single-specific antibody, a multispecific molecule (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a complete antibody, a fragment of a complete antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, a fully human antibody, Fab, Fab', Fab'-SH, F(ab'), Fd, Fv, scFv, a diabody, or a single domain antibody.

[0028] In another aspect, the present invention provides a Nanobody or antigen-binding fragment that specifically binds to PVRIG, comprising HCDR1, HCDR2 and HCDR3 of a VH set forth in any one of SEQ ID NOs: 107 to 119, 198 to 204, 211 to 216 and 219 to 225.

[0029] In some embodiments, the HCDR1, HCDR2 and HCDR3 of the Nanobody or antigen-binding fragment are determined according to the IMGT numbering system, e.g., selected from Table 21, and the HCDR1, HCDR2 and HCDR3 are determined according to the Kabat numbering system, e.g., selected from Table 22 or Table 29.

[0030] In some embodiments, the Nanobody or antigen-binding fragment has HCDRs 1 to 3 of the VH of SEQ ID NO: 107, which have the sequences set forth in SEQ ID NOs: 120 to 122 or SEQ ID NOs: 159 to 161 according to the IMGT or Kabat numbering system; HCDRs 1 to 3 of VH shown in SEQ ID NO: 108 have the sequences shown in SEQ ID NOs: 123 to 125 or 162 to 164 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 109 have the sequences shown in SEQ ID NOs: 126 to 128 or 165 to 167 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 110 have the sequences shown in SEQ ID NOs: 129 to 131 or 168 to 170 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 111 have the sequences shown in SEQ ID NOs: 132 to 134 and 171 to 173 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 112 have the sequences shown in SEQ ID NOs: 135 to 137 or 174 to 176 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 113 have the sequences shown in SEQ ID NOs: 138 to 140 or 177 to 179 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 114 have the sequences shown in SEQ ID NOs: 141 to 143 or 180 to 182 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 115 have the sequences shown in SEQ ID NOs: 144 to 146 or 183 to 185 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 116 have the sequences shown in SEQ ID NOs: 147 to 149 or 186 to 188 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 117 have the sequences shown in SEQ ID NOs: 150 to 152 or 189 to 191 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 118 have the sequences shown in SEQ ID NOs: 153 to 155 or 192 to 194 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 119 have the sequences shown in SEQ ID NOs: 156 to 158 or 195 to 197 according to the IMGT or Kabat numbering system, HCDRs 1 to 3 of VH shown in SEQ ID NO: 198 have the sequences shown in SEQ ID NOs: 168 to 170 according to the Kabat numbering system; HCDRs 1 to 3 of VH shown in SEQ ID NO: 199 have the sequences shown in SEQ ID NOs: 168, 207 and 170 according to the Kabat numbering system; HCDRs 1 to 3 of VH shown in SEQ ID NO: 200 have the sequences shown in SEQ ID NOs: 168, 207 and 208 according to the Kabat numbering system; HCDRs 1 to 3 of VH shown in SEQ ID NO: 201 have the sequences shown in SEQ ID NOs: 168, 207 and 209 according to the Kabat numbering system; HCDRs 1 to 3 of VH shown in SEQ ID NO: 202 have the sequences shown in SEQ ID NOs: 168, 169 and 208 according to the Kabat numbering system; HCDR1 to HCDR3 of VH shown in SEQ ID NOs: 203 and 204 have the sequences shown in SEQ ID NOs: 168, 210 and 208 according to the Kabat numbering system; HCDRs 1 to 3 of VHs shown in SEQ ID NOs: 211 to 215 have the sequences shown in SEQ ID NOs: 147 to 149 according to the IMGT numbering system; HCDRs 1 to 3 of VH shown in SEQ ID NO: 216 have the sequences shown in SEQ ID NOs: 147, 148 and 218 according to the IMGT numbering system; HCDRs 1 to 3 of VH shown in SEQ ID NOs: 219 to 225 have the sequences shown in SEQ ID NOs: 156 to 158 according to the IMGT numbering system.

[0031] In some embodiments, the Nanobody or antigen-binding fragment comprises a CDR sequence that has at least 80% identity with the HCDR1, HCDR2 and HCDR3 or that has one, two, three or more amino acid insertions, deletions and / or substitutions, preferably wherein the substitutions are conservative amino acid substitutions.

[0032] In some embodiments, the Nanobody or antigen-binding fragment comprises a VH as set forth in any one of SEQ ID NOs: 107-119, 198-204, 211-216, 219-225, or a sequence having at least 80% identity to a VH as set forth in any one of SEQ ID NOs: 107-119, 198-204, 211-216, 219-225, or having up to 20 mutations, which may be selected from insertions, deletions and / or substitutions, and preferably, the substitutions are conservative amino acid substitutions.

[0033] In some embodiments, the Nanobody or antigen-binding fragment comprises a sequence which, when numbered in natural order relative to the VH set forth in SEQ ID NO: 110, has at least a mutation selected from the group consisting of A97V, K98E, N54D, N108S, S110A, G55A or S75T, more preferably has at least A97V and K98E mutations, more preferably has at least A97V, K98E and N54D mutations, more preferably has at least A97V, K98E, N54D and N108S mutations, more preferably has at least A97V, K98E, N54D and S110A mutations, more preferably has at least A97V, K98E and N108S mutations, more preferably has at least A97V, K98E, G55A and N108S mutations, more preferably has at least S75T, A97V, K98E, G55A and N108S mutations; Alternatively, when numbered in natural order compared to the VH set forth in SEQ ID NO: 116, it has at least mutations selected from the group consisting of S35T, V37F, G44E, L45R, W47F, N50T, L79V, V61S, D62H, T122I or M123Q, more preferably has at least V37F, G44E, L45R, W47F and N50T mutations, more preferably has at least S35T, V37F, G44E, L45R, W47F and N50T mutations, more preferably at least S35T, V37F, G44E, L45R, W47F, N50T and L79V mutations, more preferably at least S35T, V37F, G44E, L45R, W47F, N50T, V61S and D62H mutations, more preferably at least S35T, V37F, G44E, L45R, W47F, N50T, T122I and M123Q mutations; Alternatively, when numbered in natural order compared to the VH set forth in SEQ ID NO: 119, it has at least mutations selected from the group consisting of S35G, V37Y, G44D, L45R, W47L, N50T, Y58K, Y59I, D72G, N73D, Y79S, L78V or Y94F, more preferably it has at least S35G, V37Y, G44D, L45R, W47L and N50T mutations, more preferably it has at least S35G, V37Y, G44D, L45R, W47L, N50T and Y58K mutations, more preferably it has at least S35G, V37Y, G44D, L45R, W47L, N50T, Y58K, D72G and N73D mutations, more preferably at least S35G, V37Y, G44D, L45R, W47L, N50T, Y58K, D72G, N73D and Y79S mutations, more preferably at least S35G, V37Y, G44D, L45R, W47L, N50T, Y58K, D72G, N73D and L78V mutations, more preferably at least S35G, V37Y, G44D, L45R, W47L, N50T, Y58K, Y59I, D72G and N73D mutations, more preferably at least S35G, V37Y, G44D, L45R, W47L, N50T, Y58K, D72G, N73D and Y94F mutations.

[0034] In some embodiments, the Nanobody or antigen-binding fragment is (1) a chimeric Nanobody or fragment thereof, (2) a humanized Nanobody or fragment thereof, or (3) a fully human Nanobody or fragment thereof.

[0035] In some embodiments, the Nanobody or antigen-binding fragment antibody may or may not comprise a heavy chain constant region; optionally, the antibody heavy chain constant region may be selected from human, alpaca, mouse, rat, rabbit, or sheep; optionally, the antibody heavy chain constant region may be selected from IgG, IgM, IgA, IgE, or IgD; the IgG may be selected from IgG1, IgG2, IgG3, or IgG4; optionally, the heavy chain constant region may be selected from an Fc region, a CH3 region, or a complete heavy chain constant region; preferably, the heavy chain constant region is a human Fc region; and preferably, the Nanobody or antigen-binding fragment is a heavy chain antibody.

[0036] In another aspect, the anti-PVRIG / anti-TIGIT bispecific antibody, the antibody or antigen-binding fragment that specifically binds to TIGIT, or the nanobody or antigen-binding fragment that specifically binds to PVRIG described in the present invention is further conjugated to a therapeutic agent or tracer, preferably the therapeutic agent is selected from a drug, a toxin, a radioisotope, a chemotherapeutic agent or an immunomodulatory agent, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer.

[0037] In another aspect, the present invention provides a multispecific molecule comprising the anti-PVRIG / anti-TIGIT bispecific antibody, the antibody or antigen-binding fragment that specifically binds to TIGIT, or the nanobody or antigen-binding fragment that specifically binds to PVRIG, wherein preferably the multispecific molecule may be bispecific, trispecific or tetraspecific, more preferably the multispecific molecule may be bivalent, tetravalent or hexavalent.

[0038] In some embodiments, the multispecific molecule is a tandem scFv, a bifunctional antibody (Db), a single-chain bifunctional antibody (scDb), a dual affinity retargeting (DART) antibody, a F(ab')2, a dual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dock-and-lock (DNL) antibody, a chemically cross-linked antibody, a heteropolymer nanobody, or a heteroconjugate antibody.

[0039] In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment that specifically binds to TIGIT, or a nanobody or antigen-binding fragment that specifically binds to PVRIG.

[0040] In another aspect, the present invention provides an immune effector cell that expresses the chimeric antigen receptor or comprises a nucleic acid fragment encoding the chimeric antigen receptor, wherein the immune effector cell is preferably selected from a T cell, a natural killer cell (NK cell), a natural killer T cell (NKT cell), a double negative T cell (DNT cell), a monocyte, a macrophage, a dendritic cell, or a mast cell, preferably the T cell is selected from a cytotoxic T cell, a regulatory T cell, or a helper T cell, and preferably the immune effector cell is an autoimmune effector cell or an allogeneic immune effector cell.

[0041] In another aspect, the present invention provides an isolated nucleic acid fragment encoding any one of the above bispecific antibodies, any one of the above antibodies or antigen-binding fragments that specifically bind to TIGIT, any one of the above nanobodies or antigen-binding fragments that specifically bind to PVRIG, any one of the above multispecific molecules, or any one of the above chimeric antigen receptors.

[0042] In another aspect, the present invention provides a vector comprising the nucleic acid fragment.

[0043] In another aspect, the present invention provides a host cell comprising the vector as described above, preferably wherein the cell is a prokaryotic or eukaryotic cell, such as a bacterial (E. coli), fungal (yeast), insect cell or mammalian cell (CHO cell line or 293T cell line).

[0044] In another aspect, the present invention provides a method for producing any one of the above bispecific antibodies, any one of the above antibodies or antigen-binding fragments that specifically bind to any one of the above TIGIT, any one of the above nanobodies or antigen-binding fragments that specifically bind to any one of the above PVRIG, or any one of the above multispecific molecules, the method comprising culturing the host cells as described above and isolating the antibody or molecule expressed in the cells.

[0045] In another aspect, the present invention provides a method of producing the immune effector cells, comprising introducing a nucleic acid fragment encoding any one of the CARs described above into the immune effector cells, and optionally further comprising initiating expression of any one of the CARs described above by the immune effector cells.

[0046] In another aspect, the present invention provides a pharmaceutical composition comprising any one of the above bispecific antibodies, any one of the above antibodies or antigen-binding fragments that specifically bind to TIGIT, any one of the above nanobodies or antigen-binding fragments that specifically bind to PVRIG, any one of the above multispecific molecules, any one of the above immune effector cells, nucleic acid fragments, vectors, host cells, or products produced by the above methods, and a pharmaceutically acceptable vector.

[0047] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, preferably, the additional therapeutic agent is an anti-tumor agent, more preferably, the anti-tumor agent is a PD-1 axis binding antagonist.

[0048] In another aspect, there is provided a use of any one of the above bispecific antibodies disclosed by the present invention, any one of the above antibodies or antigen-binding fragments that specifically bind to TIGIT, any one of the above nanobodies or antigen-binding fragments that specifically bind to PVRIG, any one of the above multispecific molecules, any one of the above immune effector cells, nucleic acid fragments, vectors, host cells, products produced by the above methods, or pharmaceutical compositions in the manufacture of a medicament for treating cancer or an infectious disease, wherein the cancer is selected from solid tumors and hematological tumors, preferably The present invention further provides a use wherein the tumor is selected from the group consisting of leukemia, multiple myeloma, lymphoma, myelodysplastic syndrome, prostate cancer, liver cancer, colorectal cancer, anal cancer, ovarian cancer, endometrial cancer, cervical cancer, abdominal cancer, breast cancer, pancreatic cancer, gastric cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer, glioma, kidney cancer, mesothelioma, esophageal cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, sarcoma, glioblastoma, thymic cancer, fungal granuloma, Merkel cell carcinoma, microsatellite instability-high cancer, and KRAS mutant tumors.

[0049] In some embodiments, the drug is used in combination with an additional therapeutic agent or surgery, wherein the additional therapeutic agent or surgery is selected from radiation therapy, chemotherapy, an oncolytic agent, a cytotoxic agent, a cytokine, surgery, an immunostimulatory antibody, an immunomodulatory agent, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, or a cellular immunotherapy.

[0050] In some embodiments, the additional therapeutic agent is administered before or after the drug, or simultaneously with the drug.

[0051] In some embodiments, the agent is used in combination with a PD-1 axis binding antagonist.

[0052] In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. Preferably, the PD-1 binding antagonist is an anti-PD-1 antibody. More preferably, the PD-1 binding antagonist is MDX. 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108, preferably the PD-L1 binding antagonist is an anti-PD-L1 antibody, and more preferably the PD-L1 binding antagonist is selected from the group consisting of MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), Tecentriq, and MSB0010718C (avelumab), preferably the PD-L2 binding antagonist is an anti-PD-L2 antibody, and more preferably the PD-L2 binding antagonist is an immunoadhesin.

[0053] In another aspect, the present invention provides a method for treating cancer or an infectious disease, comprising administering to a patient in need thereof an effective amount of any one of the above bispecific antibodies, any one of the above antibodies or antigen-binding fragments that specifically bind to TIGIT, any one of the above nanobodies or antigen-binding fragments that specifically bind to PVRIG, any one of the above multispecific molecules, any one of the above immune effector cells, nucleic acid fragments, vectors, host cells, products produced by the above methods, or pharmaceutical compositions, wherein the cancer is selected from solid tumors and hematological tumors, and or wherein the tumor is selected from leukemia, multiple myeloma, lymphoma, myelodysplastic syndrome, prostate cancer, liver cancer, colorectal cancer, anal cancer, ovarian cancer, endometrial cancer, cervical cancer, abdominal cancer, breast cancer, pancreatic cancer, gastric cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer, glioma, kidney cancer, mesothelioma, esophageal cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, sarcoma, glioblastoma, thymic carcinoma, fungal granuloma, Merkel cell carcinoma, microsatellite instability-high cancer, and KRAS mutant tumors.

[0054] In some embodiments, the method further comprises administering to a patient in need thereof an effective amount of a PD-1 axis binding antagonist, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist; preferably, the PD-1 binding antagonist is an anti-PD-1 antibody; and more preferably, the PD-1 binding antagonist is MDX. 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108, preferably the PD-L1 binding antagonist is an anti-PD-L1 antibody, and more preferably the PD-L1 binding antagonist is selected from the group consisting of MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), Tecentriq, and MSB0010718C (avelumab), preferably the PD-L2 binding antagonist is an anti-PD-L2 antibody, and more preferably the PD-L2 binding antagonist is an immunoadhesin.

[0055] In another aspect, the present invention further provides any one of the above bispecific antibodies, any one of the above antibodies or antigen-binding fragments that specifically bind to TIGIT, any one of the above nanobodies or antigen-binding fragments that specifically bind to PVRIG, any one of the above multispecific molecules, any one of the above immune effector cells, nucleic acid fragments, vectors, host cells, products produced by the above methods, or pharmaceutical compositions for use in preventing or treating cancer or an infectious disease, wherein the cancer is selected from solid tumors and hematological tumors, preferably wherein the tumor is selected from leukemia, multiple myeloma, lymphoma, myelodysplastic syndrome, prostate cancer, liver cancer, colorectal cancer, anal cancer, ovarian cancer, endometrial cancer, cervical cancer, abdominal cancer, breast cancer, pancreatic cancer, gastric cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer, glioma, kidney cancer, mesothelioma, esophageal cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, sarcoma, glioblastoma, thymic carcinoma, fungal granuloma, Merkel cell carcinoma, microsatellite instability-high cancer, and KRAS mutant tumors. [Effects of the Invention]

[0056] The anti-PVRIG×TIGIT humanized bispecific antibody of the present invention can specifically target tumor cells and effectively mediate killing of tumor cell lines, exhibiting excellent tumor-inhibiting effects while also having good safety characteristics.

[0057] Definitions and explanations of terms Unless otherwise defined herein, scientific and technical terms associated with the present invention shall have the meanings that are understood by those skilled in the art.

[0058] Further, unless otherwise stated herein, singular terms herein shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "such" include plural referents unless the context clearly dictates otherwise.

[0059] The terms "comprise," "include," and "have" are used interchangeably herein and are intended to indicate the comprehensiveness of the method, meaning that the method may contain elements other than those listed. At the same time, it should be understood that the descriptions "comprise," "include," and "have" used herein also provide a method "consisting of." For example, a "composition comprising A and B" should be understood as a technical solution in which both a composition consisting of A and B and a composition further containing other components in addition to A and B are included in the scope of the aforementioned "composition."

[0060] The term "and / or" as used herein includes the meaning of "and", "or" and "all or any combination of the elements linked by the term to which it belongs".

[0061] The terms "T cell immunoreceptor with Ig and ITIM domains," "TIGIT," "TIGIT antigen," "Vstm3," and "WUCAM" are used interchangeably and include various mammalian isotypes, such as human Tigit, orthologs of human Tigit, and analogs that contain at least one epitope within Tigit and analogs that share at least one epitope with TIGIT. The amino acid sequence of TIGIT (e.g., human TIGIT) and the nucleotide sequence encoding it are known in the art.

[0062] The terms "PVRIG" or "PVRIG protein," as used herein, may optionally include any such protein or variant, complex, or fragment thereof, including, but not limited to, the known or wild-type PVRIG described herein, as well as any naturally occurring splice variant, amino acid variant, or isoform, particularly the ECD fragment of PVRIG. "Anti-PVRIG antibodies" (including antigen-binding fragments) that bind to PVRIG and prevent activation by PVRL2 (e.g., most commonly by blocking the interaction of PVRIG with PVLR2) enhance the activation of T cells and / or NK cells and are used to treat diseases such as cancer and pathogen infections.

[0063] As used herein, the terms "anti-PVRIG / anti-TIGIT antibody," "bispecific PVRIG / TIGIT antibody," and "anti-PVRIG / anti-TIGIT bispecific antibody" are used interchangeably, and the anti-PVRIG / anti-TIGIT bispecific antibody of the present invention specifically binds to human TIGIT, preferably the ECD of human TIGIT, and PVRIG, more preferably the ECD of human PVRIG.

[0064] As used herein, the term "specifically binds" means that an antigen-binding molecule (e.g., an antibody) specifically binds to an antigen and a substantially identical antigen with high affinity, but does not bind to unrelated antigens with high affinity. Affinity is generally reflected by the equilibrium dissociation constant (KD), where a relatively low KD indicates a relatively high affinity. Taking antibodies as an example, high affinity is generally about 1×10 -7 M or less, approximately 1 x 10 -8 M or less, approximately 1 x 10 -9 M or less, approximately 1 x 10 -10 M or less, 1 x 10 -11 M or less or 1×10 -12This means that the equilibrium dissociation constant KD is equal to or less than M. The formula for calculating KD is KD=Kd / Ka, where Kd represents the dissociation rate and Ka represents the binding rate. The equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0065] The term "antigen-binding molecule" as used herein is used in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplary antigen-binding molecules include, but are not limited to, antibodies or antibody mimetics. An "antibody mimetic" refers to an organic compound or domain that can specifically bind to an antigen but is unrelated to the antibody structure. Exemplary antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0066] The term "antibody" as used herein is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contain sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region and are therefore capable of specifically binding to an antigen. The term "antibody" as used herein encompasses a variety of forms and structures, so long as they exhibit the desired antigen-binding activity. The term "antibody" as used herein encompasses alternative protein scaffolds or artificial scaffolds with grafted complementarity-determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (including the introduction of mutations that stabilize the three-dimensional structure of the antibody) as well as fully synthetic scaffolds, such as biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may further include non-antibody derived scaffolds, such as scaffold proteins amenable to CDR grafting known in the art, including, but not limited to, tenascin, fibronectin, peptide aptamers, and the like.

[0067] As used herein, the term "antibody" includes typical "four-chain antibodies" belonging to immunoglobulins consisting of two heavy chains (HC) and two light chains (LC). The heavy chain, from N- to C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. If the full-length antibody is an IgE isotype, the heavy chain refers to a polypeptide chain that optionally further contains a heavy chain constant region CH4 domain. The light chain refers to a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) from N- to C-terminus, with the heavy and light chains linked by disulfide bonds to form a "Y"-shaped structure. The immunoglobulin heavy chain constant regions differ in amino acid composition and sequence, resulting in different antigenicities. Thus, "immunoglobulins" herein can be classified into five classes, namely, immunoglobulin isotypes also called IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are divided into κ chains and λ chains based on differences in the constant region. Each Ig in the five classes may have a κ chain or a λ chain.

[0068] "Antibody" as used herein also includes antibodies without light chains, such as heavy-chain antibodies (HCAbs) originating from camelids, e.g., dromedary (Camelus dromedarius), Bactrian camel (Camelus bactrianus), glama (Lama glama), guanicoe (Lama guanicoe), and alpaca (Vicugna pacos), and immunoglobulin neoantigen receptors (IgNARs) found in cartilaginous fish, e.g., sharks.

[0069] As used herein, the term "heavy chain antibody" refers to an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen-binding domain and CH2 and CH3 constant domains, where the CH1 domain is absent.

[0070] As used herein, the term "nanobody" refers to the smallest functional antigen-binding fragment of a naturally occurring heavy-chain antibody devoid of light chains present in vivo in camelids, whose variable regions are cloned to yield a single-domain antibody consisting only of the heavy-chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody).

[0071] As used herein, the terms "nanobody" and "single domain antibody" (sdAb) have the same meaning and are used interchangeably, and refer to the smallest fully functional antigen-binding fragment obtained by cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region. Generally, a naturally occurring heavy chain antibody lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is further cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0072] Further discussion of "heavy chain antibodies" and "nanobodies" can be found in Hamers-Casterman et al., Nature. 1993, 363, 446-8, and in the review by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001), as well as WO94 / 04678, WO95 / 04079, WO96 / 34103, WO94 / 25591, WO99 / 37681, WO00 / 40968, WO00 / 43507, WO00 / 65057, WO01 / 40310, WO01 / 44301, EP1134231, WO02 / 48193, WO97 / 49805, WO01 / 21817, WO03 / 035694, WO03 / 054016, WO03 / 055527, WO03 / 05 Reference can be made to the above mentioned patent applications mentioned as general background art, such as WO0531, WO01 / 90190, WO03 / 025020, WO04 / 041867, WO04 / 041862, WO04 / 041865, WO04 / 041863, WO04 / 062551, WO05 / 044858, WO06 / 40153, WO06 / 079372, WO06 / 122786, WO06 / 122787 and WO06 / 122825, and other prior art mentioned in these applications.

[0073] An "antibody" herein may be derived from any animal, including, but not limited to, a human or a non-human animal, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, grama, guanicoea, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0074] As used herein, "antibody" includes, but is not limited to, a monoclonal antibody, a polyclonal antibody, a single-specific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a complete antibody, a fragment of a complete antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0075] The term "monoclonal antibody" herein refers to an antibody obtained from an essentially homogeneous antibody population; i.e., except for possible variants (e.g., naturally occurring variants or variants present in minor amounts during production of the preparation), each antibody comprising the population is identical and / or binds to the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. The use of the modifier "monoclonal" herein should not be construed as requiring production of the antibody or antigen-binding molecule by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology, and methods using genetically modified animals containing all or part of the human immunoglobulin loci, as well as other methods known in the art.

[0076] As used herein, the term "monospecific" refers to having one or more binding sites, each of which binds to the same epitope of the same antigen.

[0077] As used herein, the term "multispecific" refers to having at least two antigen-binding sites, each of which binds to a different epitope on the same antigen or a different epitope on a different antigen. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes that an antibody / antigen-binding molecule can bind.

[0078] The term "valency" as used herein refers to the presence of a given number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0079] The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably herein and refer to having a structure essentially similar to that of a natural antibody.

[0080] The terms "antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of a complete antibody, but include only a part or a variant of a complete antibody, which part or variant has the ability to bind to an antigen. The term "antigen-binding fragment" or "antibody fragment" as used herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabodies, and single domain antibodies.

[0081] Papain digestion of a complete antibody produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment containing the VL domain and constant domain (CL) of the light chain, and the VH domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by containing one or more cysteines from the antibody hinge region and additional small residues at the carboxy terminus of the heavy chain CH1 domain. Fab'-SH is a Fab' fragment in which the cysteine residues in the constant domains bear a free thiol group. Pepsin treatment produces an F(ab')2 fragment containing two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0082] The term "Fd" herein refers to an antibody consisting of a VH and CH1 domain. The term "Fv" herein refers to an antibody fragment consisting of a single arm VL and VH domain. An Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. Generally, the six CDRs are considered to confer antigen-binding specificity to the antibody. However, even a single variable region (e.g., an Fd fragment, containing only three antigen-specific CDRs) can recognize and bind to an antigen, although the affinity may be lower than that of the complete binding site.

[0083] The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH domains are linked via a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeated GGGGS amino acid sequence or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, as may variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56; and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may be present between the VH and VL of an scFv, forming a disulfide-linked Fv (dsFv).

[0084] As used herein, the term "diabody" refers to a diabody whose VH and VL domains are expressed on a single polypeptide chain, but because a linker that is too short to allow pairing between the two domains on the same chain is used, the domains pair with potential domains on another chain to generate two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0085] As used herein, the term "naked antibody" refers to an antibody that is not conjugated to a therapeutic agent or tracer, and the term "conjugated antibody" refers to an antibody that is conjugated to a therapeutic agent or tracer, preferably, the therapeutic agent may be selected from a drug, a toxin, a radioisotope, a chemotherapeutic agent, or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, and a photosensitizer.

[0086] The term "chimeric antibody" as used herein refers to an antibody in which a portion of its light and / or heavy chains is derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass) and another portion of its light and / or heavy chains is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), but which retains binding activity for a target antigen (USP 4,816,567 to Cabilly et al., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). For example, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions are derived from a first antibody (e.g., a murine antibody) but the heavy and light chain constant regions are derived from a second antibody (e.g., a human antibody) (e.g., a human-mouse chimeric antibody).

[0087] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to enhance sequence homology with human antibodies. Generally speaking, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune responses, etc.

[0088] The term "fully human antibody" herein refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. A fully human antibody herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutagenesis in vitro or introduced by somatic mutation in vivo). However, the term "fully human antibody" herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

[0089] The term "variable region" as used herein refers to the region of an antibody's heavy or light chain that binds to an antigen, and "heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The heavy and light chain variable domains of a natural antibody (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity. As used herein, the terms "complementarity-determining region" and "CDR" are used interchangeably and generally refer to the hypervariable region (HVR) of a heavy chain variable region (VH) or a light chain variable region (VL). These regions are also called complementarity-determining regions because their spatial structure allows them to form precise complementarity with an antigen epitope. The CDR of a heavy chain variable region is abbreviated as HCDR, and the CDR of a light chain variable region is abbreviated as LCDR. The terms "framework region" or "FR region" are also used interchangeably and refer to amino acid residues other than CDRs in the heavy chain variable region or light chain variable region of an antibody. A typical antibody variable region generally consists of four FR regions and three CDR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0090] Further description of CDRs can be found in Kabat et al., J. Biol. Chem., 252:6609-6616 (1977), Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991), Chothia et al., J. Mol. Biol., 196:901-917 (1987), Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997), MacCallum et al., J. Mol. Biol., 262:732-745 (1996), Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008), Lefranc MPet See, e.g., Hönegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). The term "CDR" as used herein may be designated and defined by methods known in the art, including, but not limited to, the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and tool sites that may be used include, but are not limited to, the AbRSA site (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis site (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT site (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlapping and subsets of amino acid residues defined in different ways.

[0091] The term "Kabat numbering system" as used herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0092] The term "IMGT numbering system" as used herein generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol.27:55-77, 2003.

[0093] As used herein, the term "heavy chain constant region" refers to the carboxy-terminal portion of an antibody heavy chain, which is not directly involved in binding an antibody to an antigen but exhibits effector functions such as interaction with Fc receptors. This means that the amino acid sequence is more conserved than that of the antibody variable domain. The "heavy chain constant region" includes at least the CH1 domain, hinge region, CH2 domain, and CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes "full-length heavy chain constant regions" and "heavy chain constant region fragments," the former having a structure essentially similar to that of a native antibody constant region, and the latter containing only a "portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of the CH1 domain, hinge region, CH2 domain, and CH3 domain. If the antibody is an IgE antibody, it also contains the CH4 domain, but if the antibody is a heavy chain antibody, it does not contain the CH1 domain. For example, a typical "heavy chain constant region fragment" may be selected from the CH1, Fc, or CH3 domain.

[0094] The term "light chain constant region" as used herein refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in the binding of the antibody to an antigen, and said light chain constant region may be selected from a constant kappa domain or a constant lambda domain.

[0095] The term "Fc," as used herein, refers to the carboxy-terminal portion of an antibody obtained by papain hydrolysis of a whole antibody and typically contains the CH3 and CH2 domains of the antibody. Fc regions include, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxy-terminus thereof. Because the C-terminal lysine of the Fc region (based on residue 447 of the Kabat numbering system) can be removed, for example, during antibody production or purification or by recombinant manipulation of a nucleic acid encoding an antibody heavy chain, the Fc region may or may not include Lys447.

[0096] As used herein, the term "conservative amino acids" generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, rigidity, etc.). Illustratively, amino acids in each of the following groups are conserved amino acid residues, and substitutions of amino acid residues within the groups are conservative amino acid substitutions.

[0097] By way of example, the following six groups are considered to be illustrative of amino acids that are conservatively substituted for one another: 1) Alanine (A), serine (S), threonine (T), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), Histidine (H), 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0098] As used herein, the term "identity" can be calculated to determine the percentage "identity" of two amino acid sequences or two nucleic acid sequences by aligning the sequences for optimal comparison purposes (e.g., for optimal alignment, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at this position.

[0099] The percentage identity between two sequences varies depending on the identical positions shared by the sequences, taking into account the number of gaps that need to be introduced and the length of each gap to optimally align the two sequences.

[0100] A mathematical algorithm can be used to compare the sequences and calculate the percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm integrated into the GCG software package GAP program (available at www.gcg.com) determines the percent identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. As another example, the GAP program of the GCG software package (available at www.gcg.com) determines the percent identity between two nucleotide sequences using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (which should be used unless otherwise stated) is the Blossum62 scoring matrix, which uses 12 gap penalty points, 4 gap extension penalty points and 5 transcoding gap penalty points.

[0101] The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) as implemented in the ALIGN program (version 2.0) using a PAM120 weighted coset table, a gap length penalty point of 12, and a gap penalty point of 4.

[0102] Additionally or alternatively, the nucleic acid and protein sequences described herein can be further used as "query sequences" to perform searches against a common database, e.g., to identify other family member sequences or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention, BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12. To obtain amino acid sequences homologous to the protein molecules of the present invention, BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3. To obtain gapped alignment results for comparison purposes, gapped BLAST can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0103] The term "chimeric antigen receptor (CAR)" herein refers to an artificial cell surface receptor that is engineered for expression on immune effector cells and specifically binds to an antigen, and includes at least (1) an extracellular antigen-binding domain, such as the variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR to the immune effector cell, and (3) an intracellular signaling domain. CARs can use the extracellular antigen-binding domain to redirect T cells and other immune effector cells to a selected target, such as cancer cells, in an MHC-unrestricted manner.

[0104] The term "immunostimulatory antibody" as used herein refers to 1) anti-CTLA4 mAb (e.g., ipilimumab, tremelimumab), anti-PD-1 (nivolumab BMS-936558 / MDX-1106 / ONO-4538, CT-011, lambrozilumab MK-3475, MEDI-0680 (AMP-514), PDR001, REGN2810, BGB-108), anti-PDL-1 antagonists (BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A, MSB0010718C, YW243.55.S70), anti-LAG-3 (e.g., IMP-321), and anti-TIM-3 1) antagonistic antibodies that target inhibitory immune checkpoints, including anti-BTLA, anti-B7-H4, anti-B7-H3, and anti-VISTA; and 2) agonistic antibodies that enhance immunostimulatory proteins, including anti-CD40 mAbs (e.g., CP-870, 893, lucatumumab, dacizumab), anti-CD137 mAbs (e.g., BMS-663513, urelumab, PF-05082566), anti-OX40 mAbs (e.g., anti-OX40), anti-GITR mAbs (e.g., TRX518), anti-CD27 mAbs (e.g., CDX-1127), and anti-ICOS mAbs. "Immunostimulatory antibodies" can promote antitumor immunity by directly modulating immune function, i.e., by blocking other inhibitory targets or by enhancing immunostimulatory proteins.

[0105] The term "immunomodulatory agent" as used herein may refer, for example, to thymosin alpha 1. Principle: Thymosin alpha 1 (Tα1) is a naturally occurring thymopeptide that acts as an endogenous regulator of the innate and adaptive immune systems. It is used worldwide to treat diseases associated with immune dysfunction, including viral infections such as hepatitis B and C, and certain cancers, and to enhance vaccines. In particular, recent advances in immunomodulatory research have demonstrated the beneficial effects of Tα1 treatment in sepsis patients (Wu et al., Critical Care 2013, 17:R8).

[0106] The term "nucleic acid" as used herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are generally represented by a sequence of bases, which thereby represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is generally represented 5' to 3'. As used herein, the term "nucleic acid molecule" encompasses deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers of two or more of these molecules. Nucleic acid molecules may be linear or circular. Furthermore, the term "nucleic acid molecule" includes both sense and antisense strands, and both single- and double-stranded forms. The nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for directly expressing antibodies of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, to enhance the stability of the RNA vector and / or the expression of the encoded molecule, mRNA can be chemically modified, thereby injecting the mRNA into a subject for in vivo antibody production (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1). An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from a component of its natural environment.An isolated nucleic acid includes a nucleic acid molecule contained in a cell that generally contains the nucleic acid molecule, but in which the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0107] The term "vector," as used herein, refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0108] As used herein, the term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," and include the original transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. As used herein, progeny include mutants that have the same function or biological activity as screened or selected for in the original transformed cell.

[0109] The term "pharmaceutical composition" as used herein refers to a formulation that is present in an effective form that allows for the biological activity of the active ingredient contained therein and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the pharmaceutical composition is administered.

[0110] The term "treatment," as used herein, refers to surgical or therapeutic treatment aimed at preventing or alleviating (reducing) undesirable physiological changes or pathologies in a subject, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, a stable disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Subjects in need of treatment include those already suffering from a disease or disorder, as well as those susceptible to a disease or disorder, or those in whom a disease or disorder is to be prevented. References to terms such as slowing, alleviating, reducing, mitigating, and remission also include conditions such as resolution, elimination, and non-occurrence.

[0111] The term "subject" herein refers to a living organism being treated for a particular disease or condition described in the present invention. Examples of subjects and patients include mammals being treated for a disease or condition, such as humans, primates (e.g., monkeys), or non-primate mammals.

[0112] The term "effective amount" herein refers to the amount of a therapeutic agent that, when administered to a cell, tissue, or subject alone or in combination with another therapeutic agent, is effective to prevent or ameliorate the symptoms of a disease or the progression of that disease. "Effective amount" also refers to the amount of a compound sufficient to ameliorate a symptom, e.g., treat, cure, prevent, or ameliorate an associated medical condition, or treat, cure, prevent, or ameliorate an increased rate of such a condition. When an active ingredient is administered alone to an individual, the therapeutically effective dose is the amount of that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined dose of the active ingredients that produces a therapeutic effect, regardless of combined, sequential, or simultaneous administration.

[0113] The term "cancer," as used herein, refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes benign and malignant cancers. The term "tumor" or "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor," when referred to herein, are not mutually exclusive.

[0114] The term "EC50" herein refers to the half-maximal effective concentration, including the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents 50% of the antibody concentration at which the maximum effect is observed and can be measured by methods known in the art.

[0115] The term "G4S linker peptide" as used herein refers to a G-S combination of glycine (G) and serine (S) used to link multiple proteins to form a fusion protein. A commonly used G-S combination is (GGGGS)n, where the length of the linker sequence is changed by changing the size of n. At the same time, glycine and serine can also be combined to produce different linker sequences. For example, the (G4S)4 Linker used in the present invention has the G-S combination of GGGGS. [Brief explanation of the drawings]

[0116] [Figure 1] 1 shows the binding activity of anti-TIGIT human-mouse chimeric antibodies to human TIGIT ECD-mFc fusion protein. [Figure 2] 1 shows the binding activity of anti-TIGIT human-mouse chimeric antibodies to cynomolgus monkey TIGIT ECD-mFc fusion protein. [Figure 3] 1 shows the binding activity of an anti-TIGIT human-mouse chimeric antibody to CHO-K1 cells highly expressing human TIGIT. [Figure 4]1 shows the binding activity of anti-TIGIT human-mouse chimeric antibodies to CHO-K1 cells that moderately express human TIGIT. [Figure 5] 1 shows the binding activity of an anti-TIGIT human-mouse chimeric antibody to CHO-K1 human TIGIT low-expressing cell line. [Figure 6] 1 shows the binding activity of anti-TIGIT human-mouse chimeric antibodies to CHO-K1 cynomolgus monkey TIGIT cells. [Figure 7] 1 shows the effect of anti-TIGIT human-mouse chimeric antibody in blocking the interaction between Bio-CD155-His and CHO-K1 TIGIT. [Figure 8] 1 shows the effect of anti-TIGIT human-mouse chimeric antibodies in blocking the interaction between TIGIT ECD-mFc and CHO-K1 CD155. [Figure 9] FACS detection of the expression levels of PVRIG and TIGIT on NK cells derived from different donors (donor-010 and donor-050) and PVR and PVRL2 on the tumor cell line WIDR. (A) The black open peaks indicate the expression of PVRIG / TIGIT on NK cells, and the gray solid peaks indicate the isotype controls corresponding to the two detection antibodies. (B) The black open peaks indicate the expression of PVR / PVRL2 on WIDR cells, and the gray solid peaks indicate the isotype controls corresponding to the two detection antibodies. [Figure 10] This figure shows the effect of co-incubating WIDR cells and NK cells with anti-TIGIT human-mouse chimeric antibodies on NK cell degranulation (CD107a). The abscissa represents the test antibody concentration, and the ordinate represents the percentage of CD107a-positive cells. RG6058-hIgG1 is the positive control antibody, and anti-HEL-hIgG1 is the negative isotype control antibody. [Figure 11] Figure 1 shows the effect of anti-TIGIT human-mouse chimeric antibodies on the killing activity of NK cells against WIDR target cells. The abscissa represents the concentration of the test antibody, the ordinate represents the target cell mortality rate, RG6058-hIgG1 is a positive control antibody, and anti-HEL-hIgG1 is a negative isotype control antibody. [Figure 12] This figure shows the effect of anti-TIGIT human-mouse chimeric antibody on antigen-specific CD8 T cell functional activity using a CMV antigen-recall assay. (A) Percentage of CD8 T cells in PBMCs from 128 CMV IgG-positive donors after 11 days of CMV pp65 (495-503) induction, percentage of CMV pp65-specific CD8 T cells, and FMO control. (B) Expression of PVRIG, TIGIT, and PD-1 in CMV pp65-specific CD8 T cells. (C) Expression of PVRL2 and PVR in Colo205. (D) IFN-γ secretion levels in cell supernatants after 18 hours of plate co-incubation. The positive control was RG6058-hIgG1, and the negative control was no treatment (no drug treatment). The percentages on the bar graphs represent the percentage increase in IFN-γ secretion by the test antibody compared to the no-treatment group. [Figure 13] 1 shows the binding activity of humanized anti-TIGIT antibodies to human TIGIT ECD-mFc fusion protein. [Figure 14] 1 shows the binding activity of humanized anti-TIGIT antibodies to cynomolgus monkey TIGIT ECD-mFc fusion protein. [Figure 15] 1 shows the binding activity of humanized anti-TIGIT antibodies to CHO-K1 cell lines that highly express human TIGIT. [Figure 16] 1 shows the binding activity of humanized anti-TIGIT antibodies to CHO-K1 human TIGIT moderately expressing cell line. [Figure 17] 1 shows the binding activity of humanized anti-TIGIT antibodies to CHO-K1 human TIGIT low-expressing cell line. [Figure 18] 1 shows the binding activity of humanized anti-TIGIT antibodies to CHO-K1 cynomolgus monkey TIGIT cells. [Figure 19] 1 shows the effect of blocking the interaction of anti-TIGIT humanized antibody Bio-CD155-His with CHO-K1 human TIGIT. [Figure 20]1 shows the effect of anti-TIGIT humanized antibodies in blocking the interaction between TIGIT ECD-mFc and CHO-K1 CD155. [Figure 21] 1 shows the effect of anti-TIGIT humanized antibodies in blocking the interaction between TIGIT ECD-mFc and CHO-K1 CD112. [Figure 22] 1 shows the binding activity of humanized anti-TIGIT antibodies to human PBMCs. [Figure 23] Figure 1 shows the effect of humanized anti-TIGIT antibodies on the killing activity of NK cells against WIDR target cells. In the figure, the abscissa represents the test antibody concentration, and the ordinate represents the target cell mortality rate. TIGIT-CHI-002, TIGIT-CHI-005, TIGIT-CHI-006, and TIGIT-CHI-070 are pre-humanized chimeric antibodies. RG6058-hIgG1 is a positive control antibody, and anti-HA HcAb-hIgG1 is a negative isotype control antibody. [Figure 24] This figure shows the effect of anti-TIGIT humanized antibodies on antigen-specific CD8 T cell functional activity using a CMV antigen-recall assay. (A) Percentage of CD8 T cells in PBMCs from CMV IgG-positive donor 622 after 11 days of CMV pp65 (495-503) induction, percentage of CMV pp65-specific CD8 T cells, and FMO control. (B) Expression of PVRIG, TIGIT, PD-1, and CD226 in CMV pp65-specific CD8 T cells. (C) IFN-γ secretion levels in cell supernatants after 18 hours of plate co-incubation. Positive controls are RG6058-hIgG1 and TIGIT-CHI-002, and the negative control is no treatment (no drug treatment). The percentages on the bars represent the percentage increase in IFN-γ secretion by the test antibody compared to the no-treatment group. [Figure 25] This is the detection of human TIGIT by FACS in the human TIGIT-overexpressing cell line CHO-K1. [Figure 26A]Binding ability of PVRIG test antibodies to human PVRIG recombinant protein. The figure shows the binding ability of test antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, and A118 to human PVRIG protein. Among them, COM701-hIgG1, COM701-hIgG4, and SRF813-hIgG1 were positive controls for this experiment, and anti-HA HcAb-hIgG1 and anti-CD38 HcAb-hIgG1 were negative controls for this experiment. [Figure 26B] Binding ability of test PVRIG antibodies to cynomolgus monkey PVRIG recombinant protein. The figure shows the binding ability of test antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, and A118 to cynomolgus monkey PVRIG protein. Among them, COM701-hIgG1, COM701-hIgG4, and SRF813-hIgG1 were positive controls for this experiment, and anti-HA HcAb-hIgG1, anti-CD38 HcAb-hIgG1, and anti-Fluorescein-hIgG1 were negative controls for this experiment. [Figure 27A] Binding activity of test PVRIG antibodies to human PVRIG on Flpin CHO-PVRIG cells. The figure shows the binding ability of test antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, and A118 to human PVRIG on Flpin CHO-PVRIG cells. Among them, COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies, and anti-CD38 HcAb-hIgG1 is a negative isotype control antibody. [Figure 27B]Binding activity of test PVRIG antibodies to cynomolgus monkey PVRIG expressed on FlpinCHO-cyno PVRIG cells. The figure shows the binding ability of test antibodies PVRIG-A11, A15, A30, A50, A60, A105, A117, and A118 to cynomolgus monkey PVRIG expressed on FlpinCHO-cyno PVRIG cells. Among them, COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies, and anti-CD38 HcAb-hIgG1 is a negative isotype control antibody. [Figure 28] Blockade of the interaction between human PVRIG and human PVRL2 recombinant protein by PVRIG test antibodies. The figure shows the effect of test antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, and A118 in blocking the binding of PVRIG to PVRL2, of which COM701-hIgG4 and SRF813-hIgG1 were positive controls, and anti-HA HcAb-hIgG1 and anti-CD38 HcAb-hIgG1 were negative controls. [Figure 29] Blockade of CHO-K1-CD112 cell binding to human PVRIG-mFc protein by PVRIG test antibodies. The figure shows the ability of test antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, and A118 to compete for CHO-K1-CD112 cell binding to human PVRIG-mFc protein. Among them, COM701-hIgG4 and SRF813-hIgG1 are positive control antibodies, and anti-CD38 HcAb-hIgG1 is a negative isotype control antibody. [Figure 30] Cell surface PVR and PVRL2 expression levels in the tumor cell line Reh. In the figure, the black hollow peaks represent the expression of PVR / PVRL2 on the Reh cell surface, and the gray solid peaks represent the isotype control corresponding to the detection antibody. [Figure 31]The effect of the PVRIG test antibody on NK cell degranulation. Figure A shows the effect of test antibodies PVRIG-A11, A15, and A30 on NK cell CD107a expression when NK cells (donor-010) were incubated with target cells Reh. Figure B shows the effect of test antibodies PVRIG-A60, A75, A43, and A35 on NK cell CD107a expression when NK cells (donor-010) were incubated with target cells WIDR. Figure C shows the effect of test antibodies PVRIG-A104, A105, A118, A113, and A117 on NK cell CD107a expression when NK cells (donor-050) were incubated with target cells WIDR. The abscissa represents the concentration of the test antibody, and the ordinate represents the percentage of strongly CD107a-positive cells. COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies, and anti-HA HcAb-hIgG1 is a negative isotype control antibody. [Figure 32] Effect of PVRIG test antibodies on NK cell killing of target cells. Panel A shows the stimulatory effect of test antibodies PVRIG-A11, A15, and A30 on NK cell (donor-010) killing of WIDR target cells at a concentration of 6.87 nM. Each test antibody was statistically significant compared to the negative isotype control anti-HA HcAb-hIgG1 (**p<0.01, ***p<0.001, ****p<0.0001, One-Way ANOVA Analysis). Figure B shows the stimulatory effect of test antibody PVRIG-A50 on NK cell (donor-010) killing of target WIDR cells at different concentrations. Figure C shows the stimulatory effect of test antibodies PVRIG-A60, A75, A35, A43, A104, A105, A113, A117, and A118 on NK cell (donor-050) killing of target WIDR cells at different concentrations. In panels B and C, the abscissa represents the concentration of the test antibody, and the ordinate represents the mortality of target cells. COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies, and anti-HA HcAb-hIgG1 is a negative isotype control antibody. [Figure 33]The functional improvement of antigen-specific CD8 T cells was demonstrated by a CMV antigen-recall assay. Figure A shows the percentage of CD8 and CMV pp65-specific CD8 T cells in PBMCs from CMV IgG-positive donor 021 after 11 days of CMV pp65 (495-503) induction. Figure B shows the expression of PVRIG, TIGIT, PD-1, and CD226 in CMV pp65-specific CD8 T cells (donor 021). Figure C shows the expression of PVRL2, PVR, and HLA-A2 in colo205 T cells. Figure D shows the IFN-γ secretion level in cell supernatants after 18 hours of plate co-incubation. In this experimental system, the positive controls were COM701-hIgG4 and SRF813-hIgG1, and the negative control was no treatment (no drug treatment). The final antibody concentration was 70 nM. Compared to the no-treatment group, the secretion of IFN-γ in the cell supernatant was significantly increased after the treatment with antibodies PVRIG-A15, A30, A60, 75, 105, 117, and 118 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA Analysis). [Figure 34A] Binding ability of PVRIG humanized antibodies to human PVRIG recombinant protein. The figure shows the binding activity of the test humanized molecules and their control parental antibodies PVRIG-A50, A105, and A118 to human PVRIG protein, respectively, of which anti-HA HcAb-hIgG1, anti-CD38 HcAb-hIgG1, and anti-Fluorescein-hIgG1 were negative controls for the experiment. [Figure 34B]Binding ability of PVRIG humanized antibodies to cynomolgus monkey PVRIG recombinant protein. The figure shows the binding activity of the test humanized molecules and their control parental antibodies PVRIG-A50, A105, and A118 to cynomolgus monkey PVRIG protein, respectively, of which anti-HA HcAb-hIgG1, anti-CD38 HcAb-hIgG1, and anti-Fluorescein-hIgG1 were negative controls for the experiment. [Figure 35A] Binding activity of humanized PVRIG antibodies to human PVRIG surfaced on FlpinCHO-PVRIG cells. The figures show the binding ability of the test humanized molecules and their control parental antibodies, PVRIG-A50, A105, and A118, to human PVRIG surfaced on FlpinCHO-PVRIG cells, respectively. COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies, and anti-CD38 HcAb-hIgG1 and anti-Fluorescein-hIgG1 are negative isotype control antibodies. [Figure 35B] Binding of humanized PVRIG antibodies to cynomolgus monkey PVRIG surfaced on FlpinCHO-cyno PVRIG cells. The figures show the binding ability of the test humanized molecules and their control parental antibodies, PVRIG-A50, A105, and A118, to cynomolgus monkey PVRIG surfaced on FlpinCHO-cyno PVRIG cells, respectively. COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies, and anti-CD38 HcAb-hIgG1 and anti-Fluorescein-hIgG1 are negative isotype control antibodies. [Figure 36] Blockade of human PVRIG binding to human PVRL2 by humanized PVRIG antibodies. The figure shows the blocking effect of test humanized molecules and their control parental antibodies PVRIG-A50, A105, and A118 on binding to human PVRIG protein and human PVRL2 protein, respectively. Anti-HA HcAb-hIgG1 and anti-Fluorescein-hIgG1 were negative controls for this experiment. [Figure 37]Figure 1 shows the blocking of CHO-K1-CD112 cell binding to human PVRIG-mFc protein by humanized PVRIG antibodies. The figure shows the competitive ability of the test humanized molecules and their control parental antibodies, PVRIG-A50, A105, and A118, to CHO-K1-CD112 cell binding to human PVRIG-mFc protein. COM701-hIgG4 and SRF813-hIgG1 are positive control antibodies, and anti-CD38 HcAb-hIgG1 and anti-Fluorescein-hIgG1 are negative isotype control antibodies. [Figure 38] Effect of humanized PVRIG antibody on NK cell killing. The potentiation of NK cell killing of WIDR target cells by the test humanized molecule and its control parental antibodies, PVRIG-A50 (A), A118 (B), and A105 (C), at different concentrations was shown. In the figure, the abscissa represents the test antibody concentration, and the ordinate represents the target cell mortality rate. COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies, and anti-HA HcAb-hIgG1 and anti-Fluorescein-hIgG1 are negative isotype control antibodies. [Figure 39]This figure shows the effect of humanized PVRIG antibody on improving the function of antigen-specific CD8 T cells, as determined by a CMV antigen-recall assay. The level of IFN-γ secretion in cell supernatants after 18 hours of plate co-incubation was measured. In this experimental system, the positive control was the parental PVRIG antibody before humanization, and the negative control was no treatment (no drug treatment). The final antibody concentration was 70 nM in both cases. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. no treatment, one-way ANOVA analysis. The effects of the four humanized antibodies of PVRIG-A50 (PVRIG-A50-H1a, H1b, H1d, H2a) were not significantly different from PVRIG-A50 (One-way ANOVA Analysis), the effects of the two humanized antibodies of PVRIG-A118 (PVRIG-A118-H3, H5) were not significantly different from PVRIG-A118, the effect of PVRIG-A105-H2 was significantly lower than that of PVRIG-A105 (*p<0.05, One-way ANOVA Analysis), and the remaining two humanized antibodies (PVRIG-A105-H1, H3) were not significantly different from PVRIG-A105 (One-way ANOVA Analysis). [Figure 40] FIG. 1 shows the composition and structure of four humanized bispecific antibodies. [Figure 41] ELISA detection of binding of four humanized biantibodies to human PVRIG-ECD-mFc protein. [Figure 42] ELISA detection of binding of four humanized biantibodies to cynomolgus monkey PVRIG-ECD-mFc protein. [Figure 43] ELISA detection of binding of four humanized biantibodies to human TIGIT-ECD-mFc protein. [Figure 44] ELISA detection of binding of four humanized biantibodies to cynomolgus monkey TIGIT-ECD-mFc protein. [Figure 45]FACS detection of the binding activity of four humanized biantibodies to FlpinCHO-human PVRIG cells. [Figure 46] FACS detection of the binding activity of four humanized dual antibodies to FlpinCHO-cynomolgus PVRIG cells. [Figure 47] This is FACS detection of the binding activity of four humanized double antibodies to the CHO-K1 cell line highly expressing human TIGIT. [Figure 48] Binding activity of four humanized double antibodies against CHO-K1-human TIGIT moderately expressing cell line was detected by FACS. [Figure 49] This is FACS detection of the binding activity of four humanized double antibodies to the CHO-K1-human TIGIT low-expressing cell line. [Figure 50] Binding activity of four humanized dual antibodies against the CHO-K1-cynomolgus TIGIT cell line detected by FACS. [Figure 51] Four humanized biantibodies block the binding of PVRIG protein to PVRL2 protein as detected by HTRF. [Figure 52] Four humanized biantibodies block the binding activity of PVRIG-ECD-mFc to CHO-K1-CD112 as detected by FACS. [Figure 53] ELISA detection of the blocking activity of TIGIT-ECD-mFc to CHO-K1-CD155 by four humanized biantibodies. [Figure 54] Four humanized dual antibodies block the binding activity of Bio-CD155-His protein to CHO-K1 human TIGIT, as detected by FACS. [Figure 55] Binding activity of four humanized biantibodies to human PBMCs detected by FACS. [Figure 56]Figure 1 shows BIAcore detection of co-binding of humanized bispecific antibodies against PVRIG and TIGIT. Binding curves of LC-BsAb-002 (A) and LC-BsAb-006 (B) humanized bispecific antibodies against human TIGIT protein and human PVRIG protein, and antibody-antigen binding curves for sequential injections of human TIGIT and human PVRIG proteins, respectively. The black triangles indicate the injection time points of the corresponding proteins. [Figure 57] The functional enhancement effect of the anti-PVRIG x TIGIT humanized bispecific antibody on NK cells was detected by NK cell degranulation assay. NK cell degranulation assay protocol (A), FACS detection of the expression levels of PVRIG and TIGIT on NK cells, and PVR and PVRL2 on WIDR cells (B), the effects of the humanized bispecific antibodies LC-BsAb-002 and LC-BsAb-006 on the expression levels of CD107a on NK cells (EC50, AUC, target cells WIDR, C), and the effect of the humanized bispecific antibody LC-BsAb-002 on the expression levels of CD107a on NK cells (target cells TF-1, D). [Figure 58] NK cell killing assays demonstrate the anti-PVRIG x TIGIT humanized bispecific antibody-mediated NK cell killing of WIDR cells. Expression levels of PVRIG and TIGIT on NK cells from different donors (Donors 050, 831, and 715) (A), expression levels of PVRIG and PVRL2 on target WIDR cells (B), NK cell cytotoxicity assay protocol (C), NK cell-mediated killing of WIDR cells by LC-BsAb-002 and LC-BsAb-006 from three different donors (EC50, AUC, D), and NK cell-mediated killing of TF-1 cells by LC-BsAb-002 (E). [Figure 59] Direct killing of human Treg cells mediated by the anti-PVRIG x TIGIT humanized bispecific antibody was detected by NK cell ADCC experiments. NK cell-mediated ADCC killing experiment protocol (A), PVRIG and TIGIT expression levels on human Treg cells (B), and comparison of ADCC killing of human Tregs by different IgG Fc antibodies LC-BsAb-002 and LC-BsAb-006 (EC50, AUC, C). [Figure 60] ADCP activity against human Treg cells mediated by anti-PVRIG x TIGIT humanized bispecific antibody. [Figure 61] The functional enhancement effect of the anti-PVRIG x TIGIT humanized bispecific antibody on antigen-specific CD8 T cells was detected by a CMV antigen-recall assay. (A) CMV antigen-recall experimental protocol. (B) Expression levels of PVRIG, TIGIT, and PD-1 on pp65-specific CD8 T cells. (C) Expression levels of PVRL2, PVR, and PD-L1 on Colo205 cells after IFN-γ pretreatment. (D) IFN-γ secretion levels in cell supernatants after 18 hours of plate co-incubation. Controls in this experimental system were the anti-TIGIT antibodies RG6058, TIGIT-002-H4L3, and TIGIT-005-H2L1d, and the anti-PVRIG antibodies COM701 and PVRIG-A50-H1b. The negative control was no treatment (C). IFN-γ secretion levels in cell supernatants after 18 hours of plate-coincubation with the humanized biantibody, single-agent combinations, and their respective combinations with anti-PD-L1 antibodies were measured. Controls in this experimental system were the anti-TIGIT antibodies RG6058, TIGIT-002-H4L3, and TIGIT-005-H2L1d, the anti-PVRIG antibodies COM701 and PVRIG-A50-H1b, and the anti-PD-L1 antibody Tecentriq. The negative control was no treatment (D). [Figure 62]CMV antigen-recall assay demonstrates the functional enhancement of antigen-specific CD8 T cells after the combination of the anti-PVRIG x TIGIT humanized bispecific antibody and Tecentriq. Expression levels of PVRIG, TIGIT, and PD-1 on pp65-specific CD8 T cells, and expression levels of PVRL2, PVR, and PD-L1 on Colo205 cells after IFN-γ pretreatment (A). IFN-γ secretion levels in cell supernatants after 18 hours of plate-coincubation with the humanized bispecific antibody, alone, and in combination with the respective anti-PD-L1 antibodies. Controls in this experimental system include the anti-TIGIT antibody RG6058, the anti-PVRIG antibody COM701, and the anti-PD-L1 antibody Tecentriq (B). [Figure 63] 1 shows the tumor growth curves of each test group in the A375 hPBMC humanized animal model. [Figure 64] 1 shows the tumor growth curves of a single mouse in each test group in the A375 hPBMC humanized animal model. [Figure 65] 1 shows changes in mouse body weight after administration of each test group in the A375 hPBMC humanized animal model. [Figure 66] Figure 1 shows tumor growth curves for each test group of bispecific antibody alone and in combination with Tecentriq at different doses in an A375 hPBMC humanized animal model. [Figure 67] Figure 1 shows the tumor growth curves of a single mouse in each test group treated with different doses of the bispecific antibody alone and in combination with Tecentriq in an A375 hPBMC humanized animal model. [Figure 68] Figure 1 shows changes in mouse body weight after administration of different doses of bispecific antibody alone and in combination with Tecentriq in each test group in an A375 hPBMC humanized animal model. DETAILED DESCRIPTION OF THE INVENTION

[0117] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. Unless specific conditions are specified in the examples, general conditions or conditions recommended by the manufacturer are used. Unless the manufacturer of the reagents or equipment used is specified, they may be commercially available general products.

[0118] The embodiments of the present invention are merely illustrative and do not limit the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, and all such modifications and replacements fall within the protection scope of the present invention. [Example]

[0119] Example 1 Production of TIGIT antigen Using human TIGIT protein (NCBI sequence number: XP_024309156.1) and cynomolgus monkey TIGIT protein (NCBI: XP_015300911.1) as templates for the TIGIT of the present disclosure, the amino acid sequences of the antigens and detection proteins of the present disclosure were designed, and different tags were selectively fused based on the TIGIT protein. These were then cloned into the PTT5 vector (Invitrogen), and purified by transient expression in 293 cells or stable expression in CHO cells to obtain the antigens and detection proteins encoding the present disclosure.

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[0125] Example 2 Construction of a CHO-K1 engineered cell line The nucleotide sequences encoding the full-length amino acid sequence of human TIGIT (NCBI: XP_024309156.1), the full-length amino acid sequence of cynomolgus monkey TIGIT (NCBI: XP_015300911.1), the full-length amino acid sequence of human CD155 (NCBI: NP_006496.4), and the full-length amino acid sequence of human CD112 (NCBI: NP_001036189.1) were cloned into the pcDNA3.1 vector (purchased from Clontech) to prepare plasmids. CHO-K1 cell line (purchased from ATCC) was transfected with plasmids (Lipofectamine® 3000 Transfection Kit, purchased from Invitrogen, catalog number: L3000-015) and selectively cultured in DMEM / F12 medium containing 10% (w / w) fetal bovine serum and 10 μg / mL puromycin for two weeks. Monoclonal cells were then seeded into 96-well plates and cultured at 37°C in 5% (v / v) CO2. After approximately two weeks, some monoclonal wells were selected and expanded. Cloning after expansion was performed. Monoclonal cell lines with good growth and high fluorescence intensity were selected and further expanded and cryopreserved in liquid nitrogen.

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[0130] Example 3 Generation of anti-human TIGIT mouse monoclonal antibodies Anti-human TIGIT antibodies were obtained using hybridoma technology. Mice were immunized with human TIGIT-ECD-mFc fusion protein, splenocytes were isolated from the immunized mice, and then electrofusion-fused with mouse myeloid tumor cells. These were then cultured in HAT selective medium, and the culture supernatant was isolated and identified. Clones secreting the target antibody were selected and subcloned, and finally, mouse monoclonal antibodies were obtained through production and purification. The detailed description is as follows:

[0131] A. Animal immunity For the experiment, female SJL white mice, 6-8 weeks old (Shanghai Slux Laboratory Animal Co., Ltd., Animal Production Permit Number: SCXK(Hu)2017-0005) were used. The breeding environment was SPF grade. After purchasing, the mice were acclimatized for one week in a laboratory animal room (Hegen Biotechnology (Shanghai) Co., Ltd.) under a 12 / 12-hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. After acclimatization, the mice were immunized according to the following protocol.

[0132] Mice were immunized with mFc-tagged human TIGIT-ECD (Acro Cat No. TIT-H5253). The immunization protocol was cross-immunized with TiterMax® Gold Adjuvant (Sigma Cat No. T2684) and Thermo Imject® Alum (Thermo Cat No. 77161). The antigen-to-adjuvant (TiterMax® Gold Adjuvant) ratio was 1:1, and the antigen-to-adjuvant (Thermo Imject® Alum) ratio was 2:1. The doses were 50 μg / mouse / injection (priming immunization) and 25 μg / mouse / injection (boosting immunization). After antigen emulsification, immunizations were administered at time points 1, 8, 15, 22, 29, 36, 43, and 50. On day 1, 50 μg / mouse of emulsified antigen was intraperitoneally injected (IP). On day 8, 25 μg / mouse was injected subcutaneously (SC, typically into four sites on the back and groin). Intraperitoneal and subcutaneous injections were alternated every other week, and blood samples were collected on days 20, 27, 34, and 48. Antibody titers in the mouse serum were determined by ELISA. After the sixth to eighth immunization, spleen and lymph node cells from mice with high serum antibody titers were selected and fused. Three days before fusion, shock immunization was performed, and 50 μg / mouse of the antigen solution prepared in saline was injected intraperitoneally (IP).

[0133] BB cell fusion Hybridoma cells were obtained by fusing spleen and lymph node cells with bone marrow tumor SP2 / 0 cells (ATCC® CRL-1581) using an optimized electrofusion (BTX ECM2001+) procedure. The hybridoma cells obtained after fusion were 5 × 10 5The cells were resuspended at a density of 1 / mL in DMEM (Gibco Cat No. 10569044) complete medium containing 20% FBS (Excell Cat No. FND500), 1x HT (Sigma Cat No. H0262-10VL), and 1x NEAA, and seeded at 100 μL / well into a 96-well flat-bottom plate. After incubation at 37°C and 5% CO for 6–7 days, the supernatant was removed and added at 200 μL / well to DMEM complete medium containing HT, 10% FBS, 1x HT (Sigma Cat No. H0137-10VL), and 1x NEAA. After overnight incubation at 37°C and 5% CO, ELISA detection was performed.

[0134] C. Screening of Hybridoma Cells After 7 to 10 days of fusion, the hybridoma cell supernatants were collected and their binding activity to human TIGIT ECD-hFc (internally generated) was detected by ELISA to screen for positive clones. On the second day, the binding activity of the positive cloned supernatants to CHO-K1 human TIGIT (internally constructed), CHO-K1 cynomolgus monkey TIGIT (internally constructed), and the blocking effect of human TIGIT ECD-hFc binding to CHO-K1 CD155 (internally constructed) were detected to screen for target clones and perform subcloning.

[0135] The subclone cells were cultured for 7-10 days and then screened by ELISA. The target hybridoma monoclonal cells were screened and expanded to 24-well plates. After 2-3 days, the culture supernatants were tested for their binding activity to human TIGIT ECD-hFc, CHO-K1 human TIGIT (internal construction), CHO-K1 cynomolgus monkey TIGIT (internal construction), cynomolgus monkey TIGIT ECD-hFc (internal production), and their blocking effect on the interaction between Bio-CD155-His (Yiqiao Shenzhou, 10109-H08H) and CHO-K1 human TIGIT (internal construction), as well as the blocking effect on the interaction between human TIGIT ECD-hFc and CHO-K1 CD155 (internal construction). Target clones were selected, purified, and 116 monoclonal antibodies were obtained.

[0136] D. Identification of Mouse Monoclonal Antibodies The 116 monoclonal antibodies obtained above were identified by ELISA, FACS, BIAcore, etc., and 13 candidate antibodies were obtained: TIGIT-F2-002, TIGIT-F2-005, TIGIT-F2-006, TIGIT-F2-011, TIGIT-F3-034, TIGIT-F4-044, TIGIT-F5-057, TIGIT-F5-067, TIGIT-F5-070, TIGIT-F5-072, TIGIT-F6-084, TIGIT-F6-088, and TIGIT-F6-104.

[0137] (a) Detection of binding activity of anti-TIGIT mouse monoclonal antibody to human TIGIT ECD-hFc and cynomolgus monkey TIGIT ECD-hFc. Sheep anti-human IgG antibody (Jackson, CAT:109-006-098) was diluted to 4 μg / mL in pH 7.4 PBS (Hyclone, CAT#SH30256) and added at 50 μL / well to an ELISA plate (Corning, CAT#9018). The plate was left at 4°C overnight, and the coating solution was shaken off. 250 μL / well of 5% nonfat dry milk (Seiko Seiko, CAT#A600669-0250)-PBS was added. The plate was then incubated at 37°C for 2-4 hours. The plate was then washed three times with 0.05% Tween 20-PBS (Seiko Seiko, CAT#A100777-0500, B548117-0500) in a plate washer (Biotek, CAT#405TUS). 50 μL / well of human TIGIT antibody was added. ECD-hFc (internally constructed, working concentration 30 ng / mL) and cynomolgus monkey TIGIT ECD-hFc (internally constructed, working concentration 30 ng / mL) were added and incubated overnight at 4°C. The plates were washed three times with 0.05% Tween 20-PBS in a plate washer. 50 μL / well of purified mouse monoclonal antibody (diluted to 13 nM with 1% BSA and diluted to 12 concentration points in a 3-fold serial gradient) was added and incubated for 1.5 to 2 hours at 37°C. The plates were washed three times with 0.05% Tween 20-PBS in a plate washer. 1:5000 dilution ratio was diluted with 1% BSA (Seiko Seiko Bio, CAT# A500023- HRP enzyme-conjugated antibody (Jackson, CAT#115-035-003) was diluted in 0.0100% Tween 20-PBS and added to the ELISA plate at 50 μL / well. The plate was then washed three times with 0.05% Tween 20-PBS in a plate washer. 50 μL / well of TMB color development solution (KPL, CAT#52-00-03) was added and incubated at room temperature for 7-10 minutes. 50 μL / well of 1 M HCl was added to stop the reaction. The OD450nm readings were performed using a microplate reader (Biotek, Powerwave HT). The experimental results demonstrated that the anti-TIGIT mouse monoclonal antibody effectively bound to human TIGIT ECD-hFc and cynomolgus monkey TIGIT ECD-hFc compared to the control anti-TIGIT antibody, Roche RG6058.

[0138] The amino acid sequence corresponding to RG6058 is shown below.

[0139] RG6058 VH SEQ ID NO: 234: EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS RG6058 VL SEQ ID NO 235: DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK (b) Detection of binding activity of anti-TIGIT mouse monoclonal antibodies to CHO-K1 human TIGIT and CHO-K1 cynomolgus monkey TIGIT by ELISA Cells were harvested and diluted to a concentration of 5 × 10 in 10% FBS-DMEM / F12 medium (Excell, CAT#FSP500; Gibco, CAT#11330). 5The plate was then adjusted to 1 / mL and added to a 96-well cell culture plate (Corning, CAT#3599) at 100 μL / well. The plate was then incubated overnight at 37°C and 5% CO2. The culture supernatant was then shaken off, and 50 μL / well of cell fixative (Biyuntian, CAT#P0098) was added. The plate was then fixed at room temperature for 1 hour. The plate was then washed once with 0.05% Tween 20-PBS in a plate washer. 250 μL / well of 5% nonfat dry milk in PBS was added, and the plate was then incubated at 37°C for 2-4 hours. The plate was then washed three times with 0.05% Tween 20-PBS in a plate washer. Purified mouse monoclonal antibody dilution solution was added and diluted to 13 nM with 1% BSA. 50 μL / well of the purified antibody was diluted to 13 nM with 1% BSA to give 12 concentration points in a 3-fold gradient. The diluted solution was incubated at 37°C for 1.5-2 h. The plate was then washed three times with 0.05% Tween 20-PBS in a plate washer. HRP enzyme-labeled antibody (Jackson, CAT#115-035-003) was diluted 1:5000 in 1% BSA (Seiko Seiko Bio, CAT#A500023-0100)-PBS and added to the cell plate at 50 μL / well. The plate was then incubated at 37°C for 1 hour. The plate was then washed three times with 0.05% Tween 20-PBS in a plate washer. TMB color development solution (KPL, CAT#52-00-03) was added at 50 μL / well. The plate was then incubated at 37°C for 10 minutes. 1M HCl was added at 50 μL / well to stop the reaction. The OD450nm was then read using a microplate reader (Biotek, Powerwave HT). The experimental results showed that, compared with the control anti-TIGIT antibody Roche RG6058, the purified mouse anti-TIGIT monoclonal antibody could effectively bind to the CHO-K1 human TIGIT high-expressing cell line, the CHO-K1 human TIGIT medium-expressing cell line, the CHO-K1 human TIGIT low-expressing cell line, and the CHO-K1 cynomolgus monkey TIGIT cell line.

[0140] (c) Anti-TIGIT mouse monoclonal antibody blocks the interaction between Bio-CD155-His and CHO-K1 human TIGIT, as detected by FACS. Cells were collected, washed once with PBS (Hyclone, CAT#SH30256), and diluted to 2 × 10 in 1% BSA-PBS. 5The cells were resuspended in 40 μL / well. The antibody was diluted to 210 nM in 1% BSA-PBS and diluted threefold to 12 concentration points. Bio-CD155-His (Yiqiao Shenzhou, 10109-H08H) was diluted to 3 μg / mL in 1% BSA-PBS. 40 μL of the antibody dilution and 40 μL of the Bio-CD155-His dilution were then mixed with 40 μL of the diluted antibody and 40 μL of the diluted Bio-CD155-His solution and incubated at 4°C for 60 minutes. The cells were washed twice with PBS, and 100 μL / well of APC-labeled streptavidin (working dilution 1:1700, Biolegend, 405243) was added. The cells were resuspended and incubated at 4°C for 40 minutes. The cells were washed twice with PBS and resuspended in 1% BSA-PBS at 100 μL / well. The cell samples were then analyzed using a flow cytometer (BD, Canto II). The experimental results showed that both the purified mouse monoclonal antibody and the control antibody RG6058 could effectively block the binding of Bio-CD155-His protein to the CHO-K1-human TIGIT-high-expressing cell line.

[0141] (d) ELISA detection of anti-TIGIT mouse monoclonal antibody inhibiting the interaction between human TIGIT and CHO-K1 CD155. CHO-K1 CD155 cells were harvested and diluted to a concentration of 5 × 10 in 10% FBS-DMEM / F12 medium (Excell, CAT#FSP500, Gibco, CAT#11330). 5The plate was then washed once with 0.05% Tween 20-PBS in a plate washer, and 250 μL / well of 5% nonfat dry milk in PBS was added. The plate was then incubated at 37°C for 2-4 hours. The plate was then washed three times with 0.05% Tween 20-PBS in a plate washer. The plate was then incubated at 37°C for 2-4 hours. The plate was then washed three times with 0.05% Tween 20-PBS in a plate washer, and the mouse monoclonal antibody and human TIGIT The plate was mixed with ECD-hFc (working concentration 30 ng / mL) and incubated for 0.5 hours. 50 μL of the antigen-antibody mixture was then added to the cell plate at a rate of 50 μL / well and incubated at 37°C for 1.5 to 2 hours. The plate was then washed three times with 0.05% Tween 20-PBS in a plate washer. Finally, a 1:5000 dilution of HRP enzyme-labeled antibody (Merck, CAT# A500023-0100) was added to the plate in 1% BSA (Seiko Seiko Bio, CAT# A500023-0100)-PBS. P113P) was diluted and added to the cell plate at 50 μL / well. The plate was incubated at 37°C for 1 hour. After washing three times with 0.05% Tween 20-PBS in a plate washer, 50 μL / well of TMB color development solution (KPL, CAT#52-00-03) was added. The plate was incubated at 37°C for 10 minutes. 50 μL / well of 1M HCl was added to stop the reaction. The OD450nm was then read using a microplate reader (Biotek, Powerwave HT). The experimental results demonstrated that both the purified mouse monoclonal antibody and the control antibody RG6058 effectively blocked the binding of human TIGIT protein to CHO-K1-CD155 cells.

[0142] Table 1 below shows that all 13 antibodies exhibited good TIGIT binding ability and blocking effect on the interaction between TIGIT and CD155, and their binding ability was comparable to or superior to that of the anti-TIGIT control antibody Roche RG6058.

[0143] [Table 1]

[0144] Example 4 Identification of anti-human TIGIT chimeric antibodies The above mouse antibodies were identified by constructing human-mouse chimeric antibodies, and four chimeric antibodies, TIGIT-CHI-002, TIGIT-CHI-005, TIGIT-CHI-006, and TIGIT-CHI-070, were identified. Table 2 shows the VH / VL sequences of the chimeric antibodies, Table 3 shows the results of KABAT analysis of the chimeric antibodies, and Table 4 shows the results of IMGT analysis of the chimeric antibodies.

[0145] [Table 2] [Table 3] [Table 4]

[0146] (a) Detection of binding activity of anti-TIGIT chimeric antibodies to human TIGIT ECD-mFc and cynomolgus monkey TIGIT ECD-mFc Sheep anti-mouse IgG antibody (Jackson, CAT:115-006-071) was diluted to 4 μg / mL in pH 7.4 PBS (Hyclone, CAT#SH30256) and added at 50 μL / well to an ELISA plate (Corning, CAT#9018). The plate was left at 4°C overnight, and the coating solution was shaken off. 250 μL / well of 5% nonfat dry milk (Seiko Seiko, CAT#A600669-0250)-PBS was added. The plate was then incubated at 37°C for 2-4 hours. The plate was then washed three times with 0.05% Tween 20-PBS (Seiko Seiko, CAT#A100777-0500, B548117-0500) in a plate washer (Biotek, CAT#405TUS). 50 μL / well of human TIGIT antibody was added. ECD-mFc (internally constructed, working concentration 30 ng / mL) and cynomolgus monkey TIGIT ECD-mFc (internally constructed, working concentration 30 ng / mL) were added and incubated overnight at 4°C. The plates were washed three times with 0.05% Tween 20-PBS in a plate washer. 50 μL / well of the antibody to be tested (diluted to 13 nM with 1% BSA and diluted to 12 concentration points in a 3-fold serial gradient) was added and incubated for 1.5 to 2 hours at 37°C. The plates were washed three times with 0.05% Tween 20-PBS in a plate washer. 1:5000 dilution of the antibody was diluted with 1% BSA (Seiko Seiko Bio, CAT# A500023-01) in a 3-fold serial gradient. HRP enzyme-conjugated antibody (Merck, Cat# AP113P) was diluted in 0.00% Tween 20-PBS and added to the ELISA plate at 50 μL / well. The plate was then washed three times with 0.05% Tween 20-PBS in a plate washer. 50 μL / well of TMB color development solution (KPL, Cat# 52-00-03) was added and incubated at room temperature for 7–10 minutes. 50 μL / well of 1 M HCl was added to stop the reaction. The OD at 450 nm was measured using a microplate reader (Biotek, Powerwave HT). The experimental results were consistent with those of the control antibody Roche RG6058, demonstrating that the anti-TIGIT chimeric antibody could bind to human TIGIT ECD-mFc (Figure 1) and cynomolgus monkey TIGIT ECD-mFc (Figure 2).

[0147] (b) FACS detection of binding activity of anti-TIGIT chimeric antibodies to CHO-K1 cells expressing high, intermediate, or low levels of human TIGIT and CHO-K1 cynomolgus monkey TIGIT. Cells were collected, washed once with PBS (Hyclone, CAT#SH30256), and diluted to 2 × 10 in 1% BSA-PBS. 5 The cells were resuspended in 50 μL / well, and the antibody was diluted to 80 nM in 1% BSA-PBS and diluted to 12 concentration points in a 3-fold serial gradient. 50 μL of the cells and 50 μL of the antibody dilution to be tested were mixed and incubated at 4°C for 60 minutes. After washing twice with PBS, 100 μL / well of Alexa Fluor® 647 fluorescein-labeled secondary antibody (1:800) (Jackson, CAT# 109-605-088) was added. The cells were resuspended, incubated at 4°C for 40 minutes, washed twice with PBS, and resuspended in 1% BSA-PBS, 100 μL / well. The cell samples were analyzed using a flow cytometer (BD, Canto II). The experimental results were consistent with those of the control antibody Roche RG6058, and showed that the anti-TIGIT chimeric antibody could effectively bind to the CHO-K1-TIGIT high-expressing cell line (Figure 3), the CHO-K1-TIGIT medium-expressing cell line (Figure 4), the CHO-K1-TIGIT low-expressing cell line (Figure 5), and the CHO-K1-cynomolgus TIGIT cell line (Figure 6).

[0148] (c) Blocking of the interaction between Bio-CD155-His and the CHO-K1 human TIGIT overexpressing cell line by anti-TIGIT chimeric antibodies was detected by FACS. Cells were collected, washed once with PBS (Hyclone, CAT#SH30256), and diluted to 2 × 10 in 1% BSA-PBS. 5The cells were resuspended in 40 μL / well. The antibody was diluted to 210 nM in 1% BSA-PBS and diluted threefold to 12 concentration points. Bio-CD155-His (Yiqiao Shenzhou, 10109-H08H) was diluted to 3 μg / mL in 1% BSA-PBS. 40 μL of the antibody dilution and 40 μL of the Bio-CD155-His dilution were then mixed with 40 μL of the diluted antibody and 40 μL of the diluted Bio-CD155-His solution and incubated at 4°C for 60 minutes. The cells were washed twice with PBS, and 100 μL / well of APC-labeled streptavidin (working dilution 1:1700, Biolegend, 405243) was added. The cells were resuspended and incubated at 4°C for 40 minutes. The cells were washed twice with PBS and resuspended in 1% BSA-PBS at 100 μL / well. The cell samples were then analyzed using a flow cytometer (BD, Canto II). As shown in Figure 7, both the anti-TIGIT chimeric antibody and the control antibody RG6058 can effectively block the binding of Bio-CD155-His protein to CHO-K1-human TIGIT cells.

[0149] (d) Blocking of the interaction between TIGIT ECD-mFc and CHO-K1 CD155 by anti-TIGIT chimeric antibodies detected by FACS. CHO-K1 CD155 cells were harvested, washed once with PBS (Hyclone, CAT#SH30256), and diluted to 2 × 10 in 1% BSA-PBS. 5The cells were resuspended in 40 μL / well. The antibody to be tested was diluted to 600 nM in 1% BSA-PBS and diluted 2.5-fold to 12 concentration points. TIGIT ECD-mFc was diluted to 6 μg / mL in 1% BSA-PBS. 40 μL of the diluted antibody solution and 40 μL of the diluted TIGIT ECD-mFc solution were then mixed with 40 μL of the cells and incubated for 60 minutes at 4°C. The cells were washed twice with PBS, and 100 μL / well of Alexa Fluor® 647 fluorescein-labeled secondary antibody (working dilution 1:800, Jackson, CAT#115-605-003) was added. The cells were resuspended and incubated for 40 minutes at 4°C. The cells were washed twice with PBS and resuspended in 1% BSA-PBS, 100 μL / well. The cell samples were analyzed using a flow cytometer (BD, Canto II). As shown in Figure 8, both the anti-TIGIT chimeric antibody and the control antibody RG6058 can effectively block the binding of human TIGIT protein to CHO-K1-CD155 cells.

[0150] Table 5 shows a summary of the TIGIT chimeric antibody identification.

[0151] [Table 5]

[0152] (e) Detection of PVRIG and TIGIT expression on NK cells and PVR and PVRL2 expression on WIDR cells. Expression of PVRIG and TIGIT was detected in NK cells (natural killer cells) by FACS. First, NK cells were counted using a cell counter (Beckman Coulter, Vi-CELL). Three flow tubes were prepared. 1E+5 NK cells were added to each flow tube. The cells were washed twice with PBS and the supernatant was removed. One tube was prepared as an unstained tube and 300 μL of staining buffer (PBS + 2% FBS) was added to each tube. 100 μL of staining solution (PBS + 1% Zombie Violet (Biolegend, 423114)) was added to each of the other two tubes. The cells were then washed twice with staining buffer. The supernatant was removed. 50 μL of Fc inhibitor (staining buffer + Fcx blocker (Biolegend, 422302)) was added to each tube and mixed thoroughly. The tubes were then incubated at 4°C for 15 minutes. Next, add staining solution to each tube. 50 μL of 2* staining solution (Staining buffer + PE-Cy7 Mouse anti-hCD3 detection antibody + PE Mouse anti-hCD56 detection antibody + APC Mouse anti-hTIGIT detection antibody + AF488 Rabbit anti-hPVRIG detection antibody, CD3 detection antibody Biolegend 300316, CD56 detection antibody Biolegend 318306, TIGIT detection antibody Biolegend 372706, PVRIG detection antibody RD FAB93651G) was added to the first tube, and 50 μL of 2* isotype control staining solution (Staining buffer + PE-Cy7 Mouse anti-hCD3 detection antibody + PE Mouse anti-hCD56 detection antibody + APC Mouse IgG2a κ isotype control antibody + AF488 Rabbit IgG κ isotype control antibody, APC mIgG2a κ isotype control antibody Biolegend) was added to the second tube. 400222, AF488 Rabbit IgG κ isotype control antibody RD IC1051G) was added and mixed uniformly, and then incubated at 4°C for 30 minutes.After the incubation period, the cells were washed twice with staining buffer, centrifuged, and then 300 μL of staining buffer was added and mixed thoroughly. Detection was then performed on-machine (Thermo Attune NxT). Finally, the percentage of CD56+CD3-negative cells in the Zombie Violet-negative cell population and the APC and AF488 signals of the CD56+CD3-negative cells in the Zombie Violet-negative cell population were measured. Figure 9A shows that PVRIG and TIGIT were expressed on the surface of NK cells from both donors, donor-010 and donor-050.

[0153] Expression of PVR and PVRL2 in WIDR cells was detected using FACS. WIDR cells were first digested with trypsin to prepare a cell suspension, and then counted using a cell counter (Beckman Coulter, Vi-CELL). Three flow tubes were prepared, and 1E+5 cells were added to each tube. The cells were then washed twice with PBS. After centrifugation, the supernatant was removed. One tube was filled with 300 μL of staining buffer (PBS + 2% FBS) to prepare it as an unstained tube. The other two tubes were filled with 100 μL of staining solution (PBS + 1% Zombie Violet (Biolegend, 423114)) and mixed thoroughly. The tubes were then incubated at room temperature for 15 minutes. Next, the cells were washed twice with staining buffer, the supernatant was removed, and the staining solution was added to each tube. 100 μL of the staining solution (Staining buffer + PerCP-Cy5.5 Mouse anti-hPVR detection antibody + APC Mouse anti-hPVRL2 detection antibody, PVR detection antibody Biolegend 337612, PVRL2 detection antibody Biolegend 337412) was added to the first tube, and 100 μL of the isotype control staining solution (Staining buffer + PerCP-Cy5.5 Mouse IgG1 κ isotype control antibody + APC κ Mouse IgG1 isotype control antibody, PerCP-Cy5.5 mIgG1 κ isotype control antibody Biolegend 400150, APC mIgG1 κ isotype control antibody Biolegend 400122) was added to the second tube. After uniform mixing, the cells were incubated at 4 ° C for 30 minutes. After the time had elapsed, the cells were washed twice with staining buffer, centrifuged, and then 300 μL of staining buffer was added and mixed thoroughly. Then, on-machine detection was performed (Thermo Attune NxT). Finally, PerCP-Cy5.5 and APC signals were read in the Zombie Violet-negative cell population. Figure 9B shows that PVR and PVRL2 were highly expressed on the surface of WIDR cells.

[0154] (f) Enhancement of NK cell function by anti-TIGIT chimeric antibodies as detected by NK cell degranulation assay The effect of the test antibody on the NK cell (natural killer cell) degranulation process was demonstrated by detecting CD107a signaling by FACS. PBMCs were resuscitated the day before, and NK cells (Stemcell, 17955) were sorted and stimulated overnight with 200 IU / mL human interleukin-2 (RD, 202-IL) and 10 ng / mL human interleukin-12 (Peprotech, 200-12-50UG). The seeding experiment was performed on the second day. The antibody was first diluted to a maximum concentration of 275 nM (4x concentration) in assay buffer (RPMI 1640-Glutamax + 10% FBS + 1x P / S), followed by a 10-fold gradient dilution in assay buffer. 50 μL of the diluted antibody was added per well to an ultra-low-binding 96-well U-bottom plate (Costar, 7007) for use. Next, NK cells were counted using a cell counter (Beckman Coulter, Vi-CELL). A fixed number of NK cells were collected, centrifuged at 350 g for 5 minutes, and the supernatant discarded. The cells were then resuspended in assay buffer to a density of 0.5E+6 cells / mL. Protein transport inhibitor (Invitrogen, 00498093) and APC mouse anti-human detection antibody (Biolegend, 328620) were added to the cell suspension. 50 μL / well of the treated NK cell suspension was added to the drug-coated 96-well U-bottom plate, mixed evenly, and incubated at room temperature for 15 minutes. During incubation, target cells (WIDR) were digested with trypsin to form a cell suspension. The target cells were counted using a cell counter (Beckman Coulter, Vi-CELL). An appropriate amount of cells was removed, centrifuged at 200 g for 5 minutes, the supernatant discarded, and the cells were resuspended in assay buffer to a density of 0.25E+6 cells / mL. After incubation, 100 μL of the target cell suspension was added to each well of the plate. Each well contained 25,000 NK cells, 25,000 target cells, and various concentrations of the test antibody. Wells containing only NK cells served as resting controls, and wells containing both NK cells and target cells served as no-drug controls. Each well was mixed evenly and then placed in a 37°C incubator for 16 hours.Detection of CD107a status by FACS staining: Cells in a well plate were transferred to a 96-well V-bottom plate in parallel, washed twice with PBS, and the supernatant discarded. Each well was added with staining solution (PBS + 2% FBS + 1% concentration of zombie violet (Biolegend, 423114) + PE mouse anti-CD56 detection antibody (Biolegend, 318306)). After uniform mixing, the cells were incubated at 4°C for 30 minutes. After the incubation, the cells were washed twice with staining buffer, the supernatant discarded, and each well was resuspended in 150 μL of staining buffer. Detection was performed on-machine (Thermo Attune NxT). The percentage of strongly CD107a-positive cells among CD56-positive cells was finally determined. A higher percentage of strongly CD107a-positive cells indicated stronger NK cell shedding and activation. Figure 10 shows that the negative control anti-HEL-hIgG1 had no effect on CD107a on the surface of NK cells, while the three test antibodies all increased the expression of CD107a on NK cells to different degrees, indicating that the three test antibodies could effectively promote NK activation.

[0155] (g) Detection of the NK cell killing-promoting effect of anti-TIGIT chimeric antibodies against target cells by NK cell killing experiments The effect of test antibodies on natural killer cell (NK) cell killing function was estimated by detecting the level of target cell (WIDR) degradation by FACS. PBMCs were resuscitated the day before, and NK cells (Stemcell, 17955) were selected and stimulated overnight with 200 IU / mL human interleukin-2 (RD, 202-IL) and 10 ng / mL human interleukin-12 (Peprotech, 200-12-50UG). The seeding experiment was performed on the second day. The antibodies were first diluted to a maximum concentration of 275 nM (4x concentration) in assay buffer (RPMI 1640-Glutamax + 10% FBS + 1x P / S), followed by a 10-fold gradient dilution in assay buffer. 50 μL of the diluted antibodies were added per well to an ultra-low binding 96-well U-bottom plate (Costar, 7007) for use. WIDR target cells were digested with trypsin to prepare a cell suspension. WIDR cells were counted using a cell counter (Beckman Coulter, Vi-CELL). An appropriate amount of cells was removed and centrifuged at 200 g for 5 minutes. The supernatant was discarded and resuspended in an appropriate amount of PBS. CellTrace Violet (Invitrogen, C34557A) staining solution was added to the WIDR suspension to a final CellTrace Violet concentration of 5 μM. The WIDR suspension with the staining solution was mixed thoroughly and placed in a 37°C incubator for 10 minutes, with shaking during incubation. At the same time, NK cells were counted using a cell counter. A certain number of NK cells were removed and centrifuged at 350 g for 5 minutes. The supernatant was discarded and resuspended in assay buffer to a density of 0.5E+6 cells / mL. 50 μL of the treated NK cell suspension was added per well to a drug-coated 96-well U-bottom plate, mixed thoroughly, and incubated at room temperature for 15 minutes. After WIDR cell staining was completed, the reaction was stopped by adding 5 times the volume of complete medium (MEM + 10% FBS + 1*P / S + 1*non-essential amino acid + 1*monosodium glutamate) to the cell suspension, and the mixture was centrifuged at 200g for 5 minutes. The supernatant was discarded, and the cells were resuspended in assay buffer to a density of 0.25E+6 cells / mL.After incubation with NK cells and drugs, 100 μL of WIDR cell suspension was added to each well plate. Each well contained 25,000 NK cells, 25,000 WIDR cells, and various concentrations of the test antibody. Wells containing only WIDR cells served as resting controls, while wells containing both NK and WIDR served as drug-free controls. After mixing, each well was placed in a 37°C incubator and incubated for 4 hours. FACS staining was used to detect WIDR cell lysis. Staining solution (PBS + PI (Propidium Iodide, Invitrogen, P3566)) was added to each well, mixed thoroughly, and incubated at room temperature for 20 minutes. After the incubation time, the cells were detected on-machine (Thermo Attune NxT) and the percentage of PI-positive cells among CTV-positive cells was finally determined. A higher percentage of PI-positive cells indicated a stronger killing effect of NK cells. Figure 11 shows that the negative control anti-HEL-hIgG1 had no significant effect on NK cell killing, that all four chimeric antibodies tested were able to effectively promote NK cell killing of WIDR target cells, and that the killing-promoting effects of the four chimeric antibodies on NK cells were greater than or equal to that of the positive control RG6058-hIgG1.

[0156] (h) Functional improvement of anti-TIGIT chimeric antibodies on antigen-specific CD8 T cells as detected by CMV antigen-recall assay In an experimental system using PBMCs from anti-CMV IgG-positive donors, CMV pp65-specific CD8 T cells induced by CMV pp65 (495-503) polypeptide as effector cells and pp65-pulsed colo205 tumor cell line as target cells, we detected the functional improvement effect of TIGIT antibody on antigen-specific CD8 T cells.

[0157] After PBMC resuscitation, 2 × 10 cells were cultured in complete medium (RPMI1640-Glutamax + 5% AB serum + 1% P / S + (1×) 2-β-mercaptoethanol) containing 1 mg / mL CMV pp65(495-503) polypeptide (Anaspec, Cat. No. AS-28328), 2 ng / mL human IL-2 (R&D, Cat. No. IL-202), and 10 ng / mL human IL-7 (Peprotech, Cat. No. 200-07). 6 The cells were resuspended in 100 μg / mL of PP65 / mL and seeded into 6-well plates at 5 mL / well and cultured at 37°C, 5% CO for 6 days. On day 6, all cells were harvested, pp65 and IL-7 in the medium were removed, the cells were split into two, and resuspended in complete medium containing 100 IU / mL of human IL-2. Culture was continued for 2 days. On day 8, all cells were harvested, resuspended in complete medium containing 100 IU / mL of human IL-2, and the cell density was increased to 2 × 10 6 The total cell volume was adjusted to 1 mL / mL and culture was continued. On day 11, all cells were harvested and flow cytometry was performed to detect the percentage of CD8 T cells and CMV pp65(495-503)-specific CD8 T cells in PBMCs (Figure 12A), as well as the expression of PVRIG, TIGIT, and PD-1 in the cells (Figure 12B). Flow cytometry detection antibodies were Livedead Near IR (Invitrogen, Catalog No. L34976), CD8-PerCp Cy5.5 (BD, Catalog No. 565310), CD3-PE-Cy7 (Biolegend, Catalog No. 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Catalog No. TS-0010-1C), PVRIG-AF488 (R&D, Catalog No. FAB93651G-100UG), TIGIT-APC (Biolegend, Catalog No. 372706), and PD-1-BV421 (BD, Catalog No. 562516).

[0158] After the induction, PBMCs were isolated using a CD8 T cell selection reagent kit (Stemcell, Cat. No. 17953). CD8 T cells were isolated as effector cells and resuspended in AIM-V at a cell density of 0.4 × 10 6 The concentration was adjusted to 1 / mL. Colo205 was used as the target cell line, and TrypLE TM The cells were digested with Express Enzyme (Gibco, Cat. No. 12605010) and resuspended in AIM-V (Gibco, Cat. No. 31035-025) containing 50 ng / mL pp65 at a cell density of 1 × 10 6 The cells were then diluted to 0.2 × 10 cells / mL and incubated at 37°C and 5% CO for 1–2 hours, then centrifuged at 250 g for 5 minutes, and the supernatant was discarded. 6 The cells were resuspended at 10000 / mL, and flow cytometry detection of high expression of PVRL2 and PVR in Colo205 cells is shown in Figure 12C. TIGIT antibody or positive control was diluted to 280 nM in AIM-V. 50 μL of antibody, 50 μL of CD8 T cells, and 100 μL of pp65-treated Colo205 cells were added sequentially to a low-binding 96-well U-bottom plate (Corning, catalog no. 7007). The plate was gently mixed with a pipette and incubated at 37°C and 5% CO2 for 18 hours. The final drug concentrations in this system were 70 nM, 20,000 / well for CD8 T cells, and 20,000 / well for Colo205 cells. After incubation, the plate was centrifuged at 400 g to collect the supernatant, and human IFN-γ levels in the supernatant were detected using an ELISA kit (Dacowei, catalog no. 1110003). In this system, the positive control was RG6058-hIgG1, and the negative control was no treatment. As shown in Figure 12D, after the treatment of the three TIGIT test antibodies, there was no statistically significant difference in IFN-γ secretion in the cell supernatant compared to RG6058-hIgG1, but all were significantly higher than the no-treatment group. The percentages on each bar graph indicate the percentage of improvement in IFN-γ secretion compared to the no-treatment group.

[0159] Flow cytometry detection antibodies for PVRL2 and PVR expression in Colo205 were livedead-BV421 (Invitrogen, Cat. No. L34964), PVRL2-APC (Biolegend, Cat. No. 337412), PVR-PerCp Cy5.5 (Biolegend, Cat. No. 337612), and PD-L1-PE-Cy7 (BD, Cat. No. 558017).

[0160] Example 5 Humanization of anti-human TIGIT monoclonal antibody By aligning the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database, heavy and light chain variable region germline genes with high homology to mouse antibodies were screened as templates. The CDRs of the mouse antibody were then grafted onto the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Where necessary, key amino acids in the framework sequences were mutated back to their corresponding amino acids in the mouse antibody to maintain the original affinity, resulting in humanized anti-TIGIT monoclonal antibodies. The CDR amino acid residues of the antibody were generally identified and annotated using the Kabat numbering system.

[0161] 1. Humanization of TIGIT-002 The humanized light chain templates for the murine antibody TIGIT-002 were IGKV2-29*02 / IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV4-38~2*01 and IGHJ6*01. The CDRs of the murine antibody TIGIT-002 were grafted onto the human templates to obtain the corresponding humanized versions. If necessary, key amino acids in the FR region sequence of the humanized TIGIT-002 antibody were mutated to the corresponding amino acids in the murine antibody to ensure the original affinity. The specific mutated sequences are shown in Table 6.

[0162] [Table 6]

[0163] The specific sequences of the variable regions of the TIGIT-002 humanized antibody are as follows:

[0164] The TIGIT-002.VL1 amino acid sequence is as shown in SEQ ID NO:66. DIVMTQTPLSLSVTPGQPASISCKASQNVRTAVAWYLQKPGQSPQLMIYSASYRYTGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCQQYYTTPWTFGGGTKVEIK

[0165] The TIGIT-002.VL2 amino acid sequence is as shown in SEQ ID NO:67. DIVMTQTPLSLSVTPGQPASISCKASQNVRTAVAWYQQKPGQSPQLMIYSASYRYTGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCQQYYTTPWTFGGGTKVEIK

[0166] The TIGIT-002.VL3 amino acid sequence is as shown in SEQ ID NO:68. DIVMTQSPDSLAVSLGERATINCKASQNVRTAVAWYQQKPGQSPKLMIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPWTFGGGTKVEIK

[0167] The TIGIT-002.VH1 amino acid sequence is as shown in SEQ ID NO:69. EVQLQESGPGLVKPSETLSLTCAVSGYSITSDSWNWIRQPPGKGLEWIGYISYSGNTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDFSNYGGAVDYWGQGTTVTVSS

[0168] The TIGIT-002.VH2 amino acid sequence is as shown in SEQ ID NO:70. EVQLQESGPGLVKPSETLSLTCAVSGYSITSDSWNWIRQPPGKKLEWIGYISYSGNTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDFSNYGGAVDYWGQGTTVTVSS

[0169] The TIGIT-002.VH3 amino acid sequence is as shown in SEQ ID NO:71. EVQLQESGPGLVKPSETLSLTCAVSGYSITSDSWNWIRQPPGKKLEWMGYISYSGNTYYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARLDFSNYGGAVDYWGQGTTVTVSS

[0170] The TIGIT-002.VH4 amino acid sequence is as shown in SEQ ID NO:72. EVQLQESGPGLVKPSETLSLTCAVSGYSITSDSWNWIRQPPGKKLEYIGYISYSGNTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDFSNYGGAVDYWGQGTTVTVSS

[0171] The humanized light chain template IGKV2-29*02 amino acid sequence is as shown in SEQ ID NO:73. DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQSPQLLIYEVSSRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGIHLP

[0172] The humanized light chain template IGKV4-1*01 amino acid sequence is as shown in SEQ ID NO:74. DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP

[0173] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:75. FGGGTKVEIK

[0174] The amino acid sequence of the humanized heavy chain template IGHV4-38~2*01 is as shown in SEQ ID NO:76. QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR

[0175] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is as shown in SEQ ID NO:77. WGQGTTVTVSS

[0176] The present invention designs restorative mutations in the light and heavy chain variable regions of the above-mentioned 002 humanized antibodies, and then selects and cross-combines different light and heavy chain sequences to ultimately obtain multiple 002 humanized antibodies, the amino acid sequences of which in the variable regions are as follows:

[0177] [Table 7]

[0178] According to the Kabat numbering system, the analysis results of the above 12 humanized antibody VH and VL sequences are shown in Table 8.

[0179] [Table 8]

[0180] 2. Humanization of TIGIT-006 The humanized light chain templates for the murine antibody TIGIT-006 were IGKV2-29*02 / IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV4-38~2*01 and IGHJ6*01. The CDRs of the murine antibody TIGIT-006 were grafted onto the human templates to obtain the corresponding humanized versions. If necessary, key amino acids in the FR region sequence of the humanized TIGIT-006 antibody were mutated to the corresponding amino acids in the murine antibody to ensure the original affinity. The specific mutated sequences are shown in Table 9.

[0181] [Table 9]

[0182] The specific sequences of the variable regions of the TIGIT-006 humanized antibody are as follows:

[0183] The TIGIT-006.VL1 amino acid sequence is as shown in SEQ ID NO:78. DIVMTQTPLSLSVTPGQPASISCRASQGVSTTIAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYSSPFTFGGGGTKVEIK

[0184] The TIGIT-006.VL2 amino acid sequence is as shown in SEQ ID NO:79. DIVMTQTPLSLSVTPGQPASISCRASQGVSTTIAWYQQKPGQSPKLLIYSASYRYTGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCQQYYSSPFTFGGGGTKVEIK

[0185] The TIGIT-006.VL3 amino acid sequence is as shown in SEQ ID NO:80. DIVMTQSPDSLAVSLGERATINCRASQGVSTTIAWYQQKPGQSPKLLIYSASYRYTGVPRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPFTFGGGGTKVEIK

[0186] The TIGIT-006.VH1 amino acid sequence is as shown in SEQ ID NO:81. EVQLQESGPGLVKPSETLSLTCAVSGYSITSDYWNWIRQPPGKGLEWIGYISYSGRTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARGDYSNYGGAMYDWGQGTTVTVSS

[0187] The TIGIT-006.VH2 amino acid sequence is as shown in SEQ ID NO:82. EVQLQESGPGLVKPSETLSLTCAVSGYSMTSDYWNWIRQPPGKGLEWIGYISYSGRTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARGDYSNYGGAMYDWGQGTTVTVSS

[0188] The TIGIT-006.VH3 amino acid sequence is as shown in SEQ ID NO:83. EVQLQESGPGLVKPSETLSLTCAVSGYSMTSDYWNWIRQPPGKKLEWIGYISYSGRTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARGDYSNYGGAMYDWGQGTTVTVSS

[0189] The TIGIT-006.VH4 amino acid sequence is as shown in SEQ ID NO:84. EVQLQESGPGLVKPSETLSLTCAVSGYSMTSDYWNWIRQPPGKKLEWMGYISYSGRTYYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARGDYSNYGGAMYDWGQGTTVTVSS

[0190] The TIGIT-006.VH5 amino acid sequence is as shown in SEQ ID NO:85. EVQLQESGPGLVKPSETLSLTCAVSGYSMTSDYWNWIRQPPGKKLEYIGYISYSGRTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARGDYSNYGGAMYDWGQGTTVTVSS

[0191] The humanized light chain template IGKV2-29*02 amino acid sequence is as shown in SEQ ID NO:73. DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQSPQLLIYEVSSRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGIHLP

[0192] The humanized light chain template IGKV4-1*01 amino acid sequence is as shown in SEQ ID NO:74. DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP

[0193] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:75. FGGGTKVEIK

[0194] The amino acid sequence of the humanized heavy chain template IGHV4-38~2*01 is as shown in SEQ ID NO:76. QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR

[0195] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is as shown in SEQ ID NO:77. WGQGTTVTVSS

[0196] The present invention selects and cross-combines different light and heavy chain sequences from the above-mentioned TIGIT-006 humanized antibody light and heavy chain variable region restoration mutations to ultimately obtain multiple TIGIT-006 humanized antibodies, each with the following variable region amino acid sequences:

[0197] [Table 10]

[0198] According to the Kabat numbering system, the analysis results of the 15 humanized antibody VH and VL sequences are shown in Table 11.

[0199] [Table 11]

[0200] 3. Humanization of TIGIT-005 The humanized light chain templates for the mouse antibody TIGIT-005 were IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV1-3*01 and IGHJ6*01. The CDRs of the mouse antibody TIGIT-005 were grafted onto the human templates to obtain the corresponding humanized versions. If necessary, to ensure the original affinity, key amino acids in the FR region sequence of the humanized antibody TIGIT-005 were mutated back to the corresponding amino acids in the mouse antibody. The specific mutated sequences are shown in Table 12.

[0201] [Table 12]

[0202] The specific sequences of the variable regions of the TIGIT-005 humanized antibody are as follows:

[0203] The TIGIT-005.VL1 amino acid sequence is as shown in SEQ ID NO:86. DIVMTQSPDSLAVSLGERATINCKASQHVSNGVAWYQHKPGQSPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYNTPHTFGGGTKVEIK

[0204] The TIGIT-005.VH2 amino acid sequence is as shown in SEQ ID NO:87. EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPGQRLEWMGVINPGSGGTNYKEKFKGRVTITADKSSSTAYMELSSLRSEDTAVYYCARGEYFFFDYWGQGTTVTVSS

[0205] The humanized light chain template IGKV4-1*01 amino acid sequence is as shown in SEQ ID NO:74. DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP

[0206] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:75. FGGGTKVEIK

[0207] The amino acid sequence of the humanized heavy chain template IGHV1-3*01 is as shown in SEQ ID NO:106. QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR

[0208] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is as shown in SEQ ID NO:77. WGQGTTVTVSS

[0209] The TIGIT-005 antibody has a site NG that is susceptible to chemical modification, and the inventors mutated NG to eliminate the risk of modification. In five examples, the inventors mutated NG of 005.VL1, respectively. The mutation sites are indicated by bold lines, and the sequences after mutation are as follows:

[0210] TIFF0007727086000022.tif25170

[0211] TIFF0007727086000023.tif26170

[0212] TIFF0007727086000024.tif26170

[0213] TIFF0007727086000025.tif26170

[0214] TIFF0007727086000026.tif25170

[0215] The present invention designed the above-mentioned TIGIT-005 humanized antibody light chain and heavy chain variable region restoration mutations, and then selected and cross-combined different light chain and heavy chain sequences to finally obtain six TIGIT-005 humanized antibodies, the amino acid sequences of the variable regions of each antibody being as follows:

[0216] [Table 13]

[0217] According to the Kabat numbering system, the analysis results of the six humanized antibody VH and VL sequences are shown in Table 14.

[0218] [Table 14]

[0219] 4. Humanization of TIGIT-070 The humanized light chain templates for the mouse antibody TIGIT-070 were IGKV1-39*01 / IGKV4-1*01 and IGKJ3*01, and the humanized heavy chain templates were IGHV1-3*01 and IGHJ6*01. The CDRs of the mouse antibody TIGIT-070 were grafted onto the human templates to obtain the corresponding humanized versions. If necessary, to ensure the original affinity, key amino acids in the FR region sequence of the humanized antibody TIGIT-070 were mutated back to the corresponding amino acids in the mouse antibody. The specific mutated sequences are shown in Table 15.

[0220] [Table 15]

[0221] The specific sequences of the variable regions of the TIGIT-070 humanized antibody are as follows:

[0222] The TIGIT-070.VL1 amino acid sequence is as shown in SEQ ID NO:98. DIQMTQSPSSLSASVGDRVTITCRVSENIYSYLAWYQQKPGKSPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGNPLTFGPGTKVDIK

[0223] The TIGIT-070.VL2 amino acid sequence is as set forth in SEQ ID NO:99. DIQMTQSPSSLSASVGDRVTITCRVSENIYSYLAWYQQKPGKSPKLLVYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGNPLTFGPGTKVDIK

[0224] The TIGIT-070.VL3 amino acid sequence is as shown in SEQ ID NO:100. DIVMTQSPDSLAVSLGERATINCRVSENIYSYLAWYQQKPGQSPKLLVYNAKTLAEGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHHYGNPLTFGPGTKVDIK

[0225] The TIGIT-070.VH1 amino acid sequence is as set forth in SEQ ID NO:101. EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQRLEWMGRIDPDSGGSKYNEKFKSRVTITVDKSASTAYMELSSLRSEDTAVYYCAREGHYGFYSDYWGQGTTVTVSS

[0226] The TIGIT-070.VH2 amino acid sequence is as shown in SEQ ID NO:102. EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQRLEWMGRIDPDSGGSKYNEKFKSRVTITVDKLSSTAYMELSSLRSEDTAVYYCAREGHYGFYSDYWGQGTTVTVSS

[0227] The TIGIT-070.VH3 amino acid sequence is as shown in SEQ ID NO:103. EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGRIDPDSGGSKYNEKFKSRVTITVDKLSSTAYMELSSLRSEDTAVYYCAREGHYGFYSDYWGQGTTVTVSS

[0228] The humanized light chain template IGKV1-39*01 amino acid sequence is as shown in SEQ ID NO:104. DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYST

[0229] The humanized light chain template IGKV4-1*01 amino acid sequence is as shown in SEQ ID NO:74. DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP

[0230] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:105. FGPGTKVDIK

[0231] The amino acid sequence of the humanized heavy chain template IGHV1-3*01 is as shown in SEQ ID NO:106. QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR

[0232] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is as shown in SEQ ID NO:77. WGQGTTVTVSS

[0233] The present invention selects and cross-combines different light and heavy chain sequences from the above-mentioned TIGIT-070 humanized antibody light and heavy chain variable region restoration mutations to finally obtain multiple TIGIT-070 humanized antibodies, each with the following variable region amino acid sequences:

[0234] [Table 16]

[0235] The analysis of the nine humanized antibody VH and VL sequences according to the Kabat numbering system is shown in Table 17.

[0236] [Table 17]

[0237] 5. Identification of humanized variants of anti-human TIGIT monoclonal antibodies The affinity of the above humanized antibodies for human TIGIT ECD-His was detected by BIAcore. Furthermore, the binding activity of the above humanized antibodies for human TIGIT ECD-mFc, CHO-K1 human TIGIT, CHO-K1 cynomolgus monkey TIGIT, cynomolgus monkey TIGIT ECD-mFc, and the blocking effect of the above humanized antibodies on the binding of human TIGIT ECD-mFc to CHO-K1 CD155 were detected according to the methods described in Example 4(a), (b), and (d). The results are shown in Table 18.

[0238] The four humanized antibodies, TIGIT-002-H4L3, TIGIT-005-H2L1d, TIGIT-006-H5L3, and TIGIT-070-H1L1, maintained the affinity, binding activity, and blocking effect equivalent to those of the chimeric antibody, were not significantly affected by humanization, and were generally superior to RG6058.

[0239] [Table 18]

[0240] Example 6 Identification of anti-TIGIT humanized antibodies (a) Detection of binding activity of anti-TIGIT humanized antibodies to human TIGIT ECD-mFc and cynomolgus monkey TIGIT ECD-mFc The experimental method was described in Example 4(a). The experimental results were consistent with those of the control antibody Roche RG6058, and showed that the anti-TIGIT humanized antibody could effectively bind to human TIGIT ECD-mFc (Figure 13) and cynomolgus monkey TIGIT ECD-mFc (Figure 14).

[0241] (b) FACS detection of binding activity of anti-TIGIT humanized antibodies to CHO-K1 cells with high, intermediate, or low levels of human TIGIT expression and CHO-K1 cynomolgus monkey TIGIT. The experimental method was as described in Example 4(b). The experimental results were consistent with those of the control antibody Roche RG6058, and showed that the anti-TIGIT humanized antibody could effectively bind to the CHO-K1-TIGIT high-expressing cell line ( FIG. 15 ), the CHO-K1-TIGIT medium-expressing cell line ( FIG. 16 ), the CHO-K1-TIGIT low-expressing cell line ( FIG. 17 ), and the CHO-K1-cynomolgus TIGIT cell line ( FIG. 18 ).

[0242] (c) Blocking of the interaction between Bio-CD155-His and CHO-K1 human TIGIT by anti-TIGIT humanized antibodies detected by FACS. The experimental method was as described in Example 4(c). As shown in Figure 19, both the anti-TIGIT humanized antibody and the control antibody RG6058 can effectively block the binding of Bio-CD155-His protein to CHO-K1-human TIGIT cells.

[0243] (d) Blocking of the interaction between TIGIT ECD-mFc and CHO-K1 CD155 by anti-TIGIT humanized antibodies detected by FACS. The experimental method was as described in Example 4(d). As shown in Figure 20, both the anti-TIGIT humanized antibody and the control antibody RG6058 can effectively block the binding of human TIGIT protein to CHO-K1-CD155 cells.

[0244] (e) Blocking effect of anti-TIGIT humanized antibody on the interaction between TIGIT ECD-mFc and CHO-K1 CD112 detected by FACS. CHO-K1 CD112 cells were harvested, washed once with PBS (Hyclone, CAT#SH30256), and diluted to 2 × 10 in 1% BSA-PBS. 5The humanized antibody was diluted to 600 nM in 1% BSA-PBS and diluted to 12 concentration points in a 2.5-fold serial gradient. TIGIT ECD-mFc was diluted to 6 μg / mL in 1% BSA-PBS. 40 μL of the cells were then mixed with 40 μL of the antibody dilution and 40 μL of the TIGIT ECD-mFc dilution, incubated at 4°C for 60 minutes, washed twice with PBS, and Alexa Fluor® 647 fluorescein-labeled secondary antibody (working dilution 1:800, Jackson, CAT#115-605-003) was added. The cells were resuspended in 100 μL / well, incubated at 4°C for 40 minutes, washed twice with PBS, and resuspended in 1% BSA-PBS, 100 μL / well. The cell samples were analyzed using a flow cytometer (BD, Canto II). As shown in Figure 21, both the anti-TIGIT humanized antibody and the control antibody RG6058 can effectively block the binding of human TIGIT protein to CHO-K1-CD112 cells.

[0245] (f) Detection of binding activity of anti-TIGIT humanized antibodies to human PBMCs by FACS Fresh human PBMCs (AllCells, PB004-C) were taken and the cells were diluted to 5 × 10 5 The cells were adjusted to a concentration of 100 ng / mL, and SEA (Toxin Technology, Inc., CAT: AT101) was added at the same time to 100 ng / mL. The cells were cultured at 37°C, 5% CO for 3 days. After 3 days, the cells were harvested, washed once with PBS (Hyclone, CAT# SH30256), added with Fc Block (BD, 564220), incubated at 4°C for 10 minutes, washed twice with PBS, and diluted to 2 × 10 with 1% BSA-PBS. 5The humanized antibody was diluted to 80 nM in 1% BSA-PBS and diluted to 12 concentration points in a 3-fold serial gradient. 50 μL of cells were mixed with 50 μL of the antibody diluent and incubated at 4°C for 60 minutes. After washing twice with PBS, Alexa Fluor® 647 fluorescein-labeled secondary antibody (working dilution 1:800, Jackson, CAT#109-605-088) was added, resuspended at 100 μL / well, incubated at 4°C for 60 minutes, washed twice with PBS, and resuspended at 100 μL / well in 1% BSA-PBS. The cell samples were analyzed using a flow cytometer (BD, Canto II). As shown in Figure 22, the anti-TIGIT humanized antibody, consistent with the control antibody Roche RG6058, can effectively bind to human PBMCs after SEA stimulation.

[0246] Table 19 summarizes the properties of the four humanized antibodies. The four humanized antibodies, TIGIT-002-H4L3, TIGIT-005-H2L1d, TIGIT-006-H5L3, and TIGIT-070-H1L1, had similar binding tendencies to PBMCs and stable transfected cell lines expressing high, medium, or low levels of human TIGIT. TIGIT-002-H4L3 and TIGIT-005-H2L1d were more potent than TIGIT-006-H5L3 and TIGIT-070-H1L1, both of which were more potent than RG6058. The monovalent affinities for ECD-His were 0.0994 nM, 0.0852 nM, 0.1145 nM, and 0.2505 nM, respectively, and for RG6058 was 0.1560 nM. All four antibodies showed strong cross-reactivity with cynomolgus monkey TIGIT. In vitro blocking characterization revealed that all four humanized antibodies exhibited significant blocking ability against the interactions of TIGIT-CD155 and TIGIT-CD112.

[0247] [Table 19]

[0248] (g) Detection of the promoting effect of anti-TIGIT humanized antibodies on NK cell killing target cells by NK cell killing experiments The experimental method was described in Example 4(g). Figure 23 shows that the negative control anti-HA HcAb-hIgG1 had no significant effect on NK cell killing, and the four humanized antibodies TIGIT-002-H4L3, TIGIT-005-H2L1d, TIGIT-006-H5L3, and TIGIT-070-H1L1 were able to promote NK cell killing to different degrees, of which TIGIT-002-H4L3 and TIGIT-005-H2L1d had comparable NK cell killing-promoting abilities to the unhumanized chimeric antibodies TIGIT-CHI-002 and TIGIT-CHI-005.

[0249] (h) Functional improvement of anti-TIGIT humanized antibodies against antigen-specific CD8 T cells detected by CMV antigen-recall assay After PBMC resuscitation, 2 × 10 PBMCs were cultured in complete medium (RPMI1640-Glutamax + 5% AB serum + 1% P / S + 1 × 2-β-mercaptoethanol) containing 1 mg / mL CMV pp65(495-503) polypeptide (Anaspec, Cat. No. AS-28328), 2 ng / mL human IL-2 (R&D, Cat. No. IL-202), and 10 ng / mL human IL-7 (Peprotech, Cat. No. 200-07). 6 The cells were resuspended in 100 μg / mL of PP65 / mL and seeded into 6-well plates at 5 mL / well and cultured at 37°C, 5% CO for 6 days. On day 6, all cells were harvested, pp65 and IL-7 in the medium were removed, the cells were split into two, and resuspended in complete medium containing 100 IU / mL of human IL-2. Culture was continued for 2 days. On day 8, all cells were harvested, resuspended in complete medium containing 100 IU / mL of human IL-2, and the cell density was increased to 2 × 10 6The total cell volume was adjusted to 1 mL / mL and culture was continued. On day 11, all cells were harvested and flow cytometry was performed to detect the percentage of CD8+ T cells in PBMCs, the percentage of CMV pp65(495-503)-specific CD8+ T cells (Figure 24A), and the expression of PVRIG, TIGIT, PD-1, and CD226 in the cells (Figure 24B). Flow cytometry detection antibodies were Livedead Near IR (Invitrogen, Catalog No. L34976), CD8-PerCp Cy5.5 (BD, Catalog No. 565310), CD3-PE-Cy7 (Biolegend, Catalog No. 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Catalog No. TS-0010-1C), PVRIG-AF488 (R&D, Catalog No. FAB93651G-100UG), TIGIT-APC (Biolegend, Catalog No. 372706), and PD-1-BV421 (BD, Catalog No. 562516).

[0250] After the induction, PBMCs were isolated using a CD8 T cell selection reagent kit (Stemcell, Cat. No. 17953). CD8 T cells were isolated as effector cells and resuspended in AIM-V at a cell density of 0.4 × 10 6 The concentration was adjusted to / mL. After selection, the purity and CD226 expression of CD8 cells were detected. Colo205 cells were used as target cells, and TrypLE TM The cells were digested with Express Enzyme (Gibco, Cat. No. 12605010) and resuspended in AIM-V (Gibco, Cat. No. 31035-025) containing 20 ng / mL pp65 at a cell density of 1 × 10 6 The cells were then diluted to 0.5 × 10 cells / mL and incubated at 37°C and 5% CO for 3 hours, then centrifuged at 250 g for 5 minutes, and the supernatant was discarded. 6The anti-TIGIT humanized antibody or positive control was diluted to 280 nM in AIM-V. 50 μL of antibody, 50 μL of CD8 T cells, and 100 μL of pp65-treated colo205 cells were added sequentially to a low-binding 96-well U-bottom plate (Corning, catalog no. 7007). The plate was gently mixed with a pipette and incubated at 37°C and 5% CO2 for 18 hours. The final drug concentrations in this system were 70 nM, 20,000 / well for CD8 T cells, and 50,000 / well for colo205 cells. After incubation, the plate was centrifuged at 400 g to collect the supernatant, and the human IFN-γ levels in the supernatant were detected using an ELISA kit (Daco-Wei, catalog no. 1110003). In this system, the positive controls were RG6058-hIgG1 and the unhumanized TIGIT antibody (TIGIT-CHI-002), and the negative control was no treatment. As shown in Figure 24C, the secretion of IFN-γ in the cell supernatant after the treatment with the four TIGIT humanized antibodies (TIGIT-002-H4L3, TIGIT-005-H2L1d, TIGIT-006-H5L3, and TIGIT-070-H1L1) was not statistically significantly different from that of RG6058-hIgG1 (One-way ANOVA Analysis). The secretion of IFN-γ in the cell supernatant after the treatment with the four anti-TIGIT humanized antibodies was not statistically significantly different from that of the chimeric antibody (TIGIT-CHI-002) before TIGIT humanization (One-way ANOVA Analysis). However, all of the four anti-TIGIT humanized antibodies were significantly higher than that of the no-treatment group. The percentages on each bar graph indicate the percentage increase in IFN-γ secretion compared to the no-treatment group.

[0251] After sorting, flow cytometry detection antibodies for the purity of CD8 T cells and CD226 expression therein were livedead-BV421 (Invitrogen, Cat. No. L34964), CD8-FITC (BD, Cat. No. 555366), CD226-PE-Cy7 (Biolegend, Cat. No. 338316). Flow cytometry detection antibodies for PVRL2, PVR, PD-L1, and HLA-A2 expression in Colo205 were livedead-BV421 (Invitrogen, Cat. No. L34964), PVRL2-APC (Biolegend, Cat. No. 337412), PVR-PerCp Cy5.5 (Biolegend, Cat. No. 337612), PD-L1-PE-Cy7 (BD, Cat. No. 558017), and HLA-A2-PE (Biolegend, Cat. No. 343306).

[0252] Example 7 Production of PVRIG alpaca VHH antibody Two healthy adult alpacas (purchased through Chengdu Apac Co., Ltd.) were selected. The first immunization was administered with complete Freund's adjuvant (CFA, purchased from SIGMA, catalog number: F5881) mixed with human PVRIG recombinant protein (Acro Biosystems, catalog number: PVG H5257). The subsequent three immunizations were administered with incomplete Freund's adjuvant (IFA, purchased from SIGMA, catalog number: F5506) mixed with the same human PVRIG recombinant protein as the first immunization. All four immunizations were administered subcutaneously. Ten mL of blood was collected before immunization to serve as a negative serum control. Another 10 mL of blood was collected after the second immunization to detect serum antibody titers. After the third and fourth immunizations, 50 mL of peripheral blood was collected and lymphocytes were isolated. Five mL of RNA iso Plus (Takara, catalog number 9109) was added depending on the lymphocyte amount, and the resulting solution was dispensed into 1.5 mL EP tubes and stored at -80°C. Total RNA was extracted from the cryopreserved lymphocytes and reverse-transcribed into cDNA. The cDNA was then used as a template for two rounds of VHH PCR amplification. Phage libraries were constructed by enzymatic digestion of the VHH products amplified in the second PCR. The established bacterial libraries were collected and analyzed for insertion rate and diversity by sequencing.

[0253] Two rounds of affinity panning were performed on the phage to identify phage clones that specifically bound to the target protein, human PVRIG-his (AcroBiosystems, catalog number PVG-H52H4). The best clones that showed strong binding to human PVRIG-His protein during panning were sequenced and then cloned into an expression vector by homologous recombination. The CH2 and CH3 constant regions were both derived from human IgG1, and the complete expression sequence was signal peptide-VHH-hinge region-CH2-CH3. After a series of physical, chemical, and functional screenings, a total of 13 positive candidate antibody molecules were obtained. The CDRs of these sequences were analyzed using IMGT and KABAT software, respectively. The corresponding sequence information is shown in Tables 20-22 below. Table 20 lists the VHH sequences of the candidate antibody molecules, Table 21 lists the IMGT analysis results, and Table 22 lists the KABAT analysis results.

[0254] [Table 20] [Table 21] [Table 22]

[0255] Example 8 ELISA detection of specific binding of anti-PVRIG antibodies to human and cynomolgus monkey PVRIG proteins An ELISA plate was pre-coated with 0.5 μg / mL of human PVRIG-his (AcroBiosystems, Catalog No. PVG-H52H4) or cynomolgus monkey PVRIG protein (Novoprotein, Catalog No. C09B) at 100 μL / well, and a test anti-PVRIG antibody (composed of human IgG1-Fc bound to the VHH described in Example 7) was gradient diluted (starting concentration 20 nM, 3.33-fold gradient dilution or 3 nM, 3-fold gradient dilution). 100 μL / well of the sample was added and incubated with shaking at room temperature for 1.5 hours. After washing the plate, mouse anti-human IgG Fc-HRP (Jackson ImmunoResearch, catalog no. 209-035-098) working solution (1:10,000 dilution) was added, and 100 μL / well of sample was added. The plate was then incubated with shaking at room temperature for 1.0 hour. The plate was then washed, and the HRP substrate TMB (Thermo, catalog no. 34029) was added to develop the color. The reaction was terminated with stop solution, and the absorbance was read using a microplate reader (MD i3x). The antibody binding curve was plotted with the antibody concentration as the abscissa and the corresponding OD value as the ordinate, and a four-parameter fitting (GraphPad Prism 9) was performed to calculate the EC50 value. The smaller the EC50 value, the stronger the antibody's ability to bind to human / cynomolgus PVRIG. The positive control antibodies were COM701-hIgG1 (Patent No.: US20180244774A1), COM701-hIgG4 (Patent No.: US20180244774A1), and SRF813-hIgG1 (Patent No.: US20200040081A1), and the negative control antibodies were anti-HA HcAb-hIgG1 (Chengdu Apac, Catalog No. NBR022), anti-CD38 HcAb-hIgG1 (in-house), and anti-Fluorescein-hIgG1 (in-house).

[0256] The amino acid sequence corresponding to COM701 is shown below.

[0257] COM701VH sequence number 236. QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNINWVRQAPGQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREDKTARNAMDYWGQGTLVTVSS

[0258] COM701 VL sequence number 237. DIQMTQSPSSLSASVGDRVTITCRVSENIYSNLAWYQQKPGKAPKLLIYEATNLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWGTPYTFGQGTKLEIK

[0259] SRF813 VH SEQ ID NO:238. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSAAISWVRQAPGQGLEWMGNIIPIVGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDTGRGYTRHFWFDPWGQGTLVTVSS

[0260] SRF813 VL sequence number 239. DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDILYTFGGGTKVEIK

[0261] The binding results of the 13 anti-PVRIG antibodies to human PVRIG protein are shown in Figure 26A and Table 23, and the binding results to cynomolgus monkey PVRIG are shown in Figure 26B and Table 23. The data showed that all tested antibodies were able to specifically bind to human or cynomolgus monkey PVRIG protein.

[0262] [Table 23]

[0263] Example 9 FACS detection of binding of anti-PVRIG antibodies to FlpinCHO-PVRIG cells and FlpinCHO-cyno PVRIG cells CHO-K1 stable cells transfected with human or cynomolgus monkey PVRIG high-expression plasmids (designated FlpinCHO-PVRIG and FlpinCHO-cyno PVRIG, respectively) were used. The full-length human PVRIG plasmid (NCBI Ref Seq: NP_076975) and full-length cynomolgus monkey PVRIG plasmid (NCBI Ref Seq: XP_014989941) were synthesized from common organisms. Experiments were performed when the cell density was less than 80%. The cell culture medium was discarded, washed with PBS, and digested with 1 mL of Versene (Gibico, 15040-066) for 8–10 minutes. The digestion was terminated with Ham's F12 (Gibico, 21127-022) complete medium containing 10% FBS, and the cell suspension was then obtained. After counting with a cell counter (Beckman Coulter, Vi-CELL), an appropriate amount of cell suspension was taken, centrifuged at 350 × g to remove the supernatant, washed twice with PBS, and stained with the live / dead cell dye Zombie Violet (Biolegend, 423114) and incubated at room temperature for 20 minutes. After incubation, staining was stopped with staining buffer (2% FBS + PBS), centrifuged at 350 × g to remove the supernatant, washed twice, and then stained with 2 × 10 cells in staining buffer. 6The cells were resuspended at a density of 1000 cells / mL and seeded into a 96-well plate. 50 μL of cell suspension was added to each well and prepared for use. The antibody was diluted 3.3-fold in staining buffer, starting from a maximum concentration of 46 nM (2x concentration). The diluted antibody was added to wells already containing 50 μL of cell suspension and shaken at 400 rpm for 1 minute on a microwell plate shaker to thoroughly mix the antibody and cells. The wells were then incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with 200 μL of staining buffer, centrifuged at 350 × g for 5 minutes, and the supernatant was discarded. PE goat anti-human IgG Fc antibody (ebioscience, 12-4998-82) was diluted 250-fold in staining buffer and added to the washed cell wells at a volume of 100 μL / well. The antibody was mixed evenly and stained at 4°C for 30 minutes. After staining, the cells were washed twice with staining buffer. Finally, the cells were resuspended in 200 μL of staining buffer, and signals were detected on-line using a flow cytometer (BD Canto II). A stronger fluorescent signal indicated stronger antibody binding to PVRIG. Antibody binding curves were plotted with antibody concentration as the abscissa and the corresponding multiple of the mean fluorescence intensity (MFI) as the ordinate. AUC values were calculated using four-parameter fitting (GraphPad Prism 9). A higher AUC value indicated stronger binding of the test antibody to FlpinCHO-PVRIG and FlpinCHO-cyno PVRIG cells. As shown in Figures 27A and 27B, all test antibodies were able to bind to overexpressed human / cynomolgus PVRIG on the cell surface. Antibody binding activity was normalized as a percentage relative to the positive molecules COM701-hIgG1 and SRF813-hIgG1; a higher percentage indicated stronger antibody binding activity. The results in Table 24 showed that the binding activity of the test antibodies PVRIG-A11, A35, A43, A105, A117, and A118 to human PVRIG was stronger than that of COM701-hIgG1 and SRF813-hIgG1. Of these, the binding activity of PVRIG-A105 and A117 to cynomolgus monkey PVRIG was also stronger than that of COM701-hIgG1 and SRF813-hIgG1.

[0264] [Table 24]

[0265] Example 10 BIAcore detection of the affinity of anti-PVRIG antibodies to human PVRIG protein Specific binding between the test anti-PVRIG antibody and human PVRIG protein was detected using Biacore. This experiment used a Protein A chip, and the time required for the chip to capture diluted antibody was measured manually to achieve a saturated antigen binding Rmax of 50 RU. Human PVRIG protein (Human PVRIG-His, Acro C227P1-9ARF1-T4) was gradient-diluted to 20, 10, 5, 2.5, and 1.25 nM. The affinity of the antibody for the antigen was measured using multi-cycle kinetics. In each cycle, the antibody was injected, followed by injections of PVRIG protein at gradient concentrations, allowing the antigen-antibody binding and dissociation processes to occur. After each cycle, the Protein A chip was regenerated (removing proteins from the chip) using glycine pH 1.5. The antibody-antigen affinity KD was fitted using BIAcore T200 analysis software. The results in Table 25 demonstrated that all of the tested anti-PVRIG antibodies bound specifically to the human PVRIG protein with relatively high affinity levels.

[0266] [Table 25]

[0267] Example 11 ELISA detection of anti-PVRIG antibodies blocking PVRIG binding to PVRL2 An ELISA plate was pre-coated with 0.5 μg / mL human PVRIG-his protein (AcroBiosystems, Catalog No. PVG-H52H4) at 100 μL / well. All test anti-PVRIG antibodies were diluted 2-fold starting from a maximum concentration of 16 nM. Equal volumes of the diluted antibodies and 18 ng / mL human PVRL2-mFc (AcroBiosystems, Catalog No. CD2-H5257) were mixed and added to the ELISA plate at 100 μL / well. The mixture was incubated at room temperature with shaking for 2.0 hours. After washing the plate, the mixture was incubated with sheep anti-mouse IgG Fc-HRP (Jackson ImmunoResearch, Catalog No. 115-035-071) working solution (1:10,000 dilution) was added, and 100 μL / well of the solution was incubated at room temperature with shaking for 1.0 hour. The plate was then washed, and the HRP substrate TMB (Thermo, Catalog No. 34029) was added to develop color. The reaction was terminated by adding stop solution, and the absorbance values were read using a microplate reader (MD i3X). Antibody inhibition curves were plotted with antibody concentration as the abscissa and the corresponding OD value as the ordinate, and four-parameter fitting (GraphPad Prism 9) was performed to calculate IC50 values. The smaller the IC50 value, the stronger the antibody's ability to inhibit human PVRIG binding to human PVRL2. The positive and negative control antibodies were the same as those used in Example 8. Blocking curves for the 13 test antibodies are shown in Figure 28, and the inhibitory activities are listed in Table 26. Figure 28 and Table 26 show that all of the tested antibodies were able to significantly inhibit the binding of human PVRIG to human PVRL2 protein.

[0268] [Table 26]

[0269] Example 12: Detection by FACS of anti-PVRIG antibodies blocking binding of CHO-K1-CD112 cells to human PVRIG-mFc protein CHO-K1 stable cells (designated CHO-K1-CD112) transfected with a human CD112 high-expression plasmid (NP_001036189.1 / NCBI Ref Seq:Q92692) were used for experiments at cell density less than 80%. The cell culture medium was discarded, washed with PBS, and digested with 1 mL of trypsin (Gibico, 25200-72) for 2 minutes. The digestion was stopped with Ham's F12 (Gibico, 21127-022) complete medium containing 10% FBS, and the cell suspension was then obtained. After counting with a cell counter (Beckman Coulter, Vi-CELL), an appropriate amount of cell suspension was taken, centrifuged at 350 × g, and the supernatant was removed. The cells were washed twice with PBS and then stained with the live / dead cell dye Zombie Violet (Biolegend, 423114) and incubated at room temperature for 20 minutes. After incubation, staining was stopped with staining buffer (2% FBS + PBS), centrifuged at 350 × g, and the supernatant was removed. The cells were washed twice and then stained at 1 × 10 cells with staining buffer. 6The cells were resuspended to a density of 1000 cells / mL and ready for use. A 96-well plate was seeded with 50 μL of cell suspension per well and ready for use. A working solution of human PVRIG-mFc protein (Acro, PVG-H5253) was prepared using staining buffer to a concentration of 1 μg / mL (4x concentration). 50 μL of the PVRIG-mFc working solution was added to the 96-well plate at a concentration of 1 μg / mL. The antibody was diluted 3-fold using staining buffer, starting from a maximum concentration of 275 nM (4x concentration). The diluted antibody was added to wells already containing 50 μL of PVRIG-mFc. The wells were shaken at 400 rpm on a microwell plate shaker for 1 minute to thoroughly mix the antibody and PVRIG-mFc protein, and then incubated at 4°C for 30 minutes. After incubation, 100 μL of the prepared cell suspension was added directly to each well. The mixture was gently mixed evenly with a pipette head and then incubated at 4°C for 30 minutes. After incubation, cells were washed twice with 200 μL / well of staining buffer and centrifuged at 350 × g for 5 minutes. The supernatant was discarded. A 250-fold dilution of PE goat anti-mouse IgG Fc antibody (Biolegend, 405337) was added to the washed cell wells at a volume of 100 μL / well, mixed evenly, and stained at 4°C for 30 minutes. After staining, cells were washed twice with staining buffer and finally resuspended in 200 μL of staining buffer. Signals were detected on-machine using a flow cytometer (BD Canto II). A weaker fluorescent signal indicated a stronger antibody's ability to compete for binding of CHO-K1-CD112 cells to PVRIG-mFc protein. Antibody binding curves were plotted with antibody concentration as the abscissa and the corresponding mean fluorescence intensity (MFI) as the ordinate. A four-parameter fitting (GraphPad Prism 9) was performed to calculate the antibody IC50 and binding curve AUC values. The smaller the IC50 and AUC values, the stronger the antibody's competitive ability to block the binding of CHO-K1-CD112 cells to PVRIG-mFc protein, i.e., the stronger the blocking effect of the antibody. As shown in Figure 29, all of the tested antibodies were able to block the binding of CHO-K1-CD112 cells to human PVRIG-mFc protein.The competitive blocking activity of the test antibodies was normalized as a percentage relative to the control molecules COM701-hIgG1 and SRF813-hIgG1 antibodies, with smaller percentage values indicating stronger blocking activity of the antibody. The results in Table 27 show that the blocking activity of the test antibodies PVRIG-A11, A15, A30, and A50 was stronger than that of COM701-hIgG1 and SRF813-hIgG1.

[0270] [Table 27]

[0271] Example 13 Detection of expression of PVRIG and TIGIT on the surface of NK cells, and PVR and PVRL2 on the surface of tumor cell lines Reh and WIDR Expression of PVRIG and TIGTI on NK (natural killer) cells was detected by FACS. The experimental method was as described in Example 4(e). The results are shown in Figure 9A. PVRIG and TIGTI were expressed on the surface of NK cells from both donors, donor-010 and donor-050.

[0272] The expression of PVR and PVRL2 in Reh / WIDR cells was detected by FACS. The experimental method was as described in Example 4(e), except that Reh was directly sprayed onto the cells to form a cell suspension. The expression of PVR and PVRL2 on the surface of WIDR cells is shown in Figure 9B. Figure 30 shows that PVR expression on the surface of Reh cells was negative, and PVRL2 expression was positive.

[0273] Example 14: Detection of the functional promoting effect of anti-PVRIG antibody on NK cells by NK cell degranulation experiment The effect of the test antibodies on the NK cell (natural killer cell) CD107a signal was detected by FACS to demonstrate the effect of the test antibodies on the NK cell degranulation process. The experimental method was as described in Example 4(f), except that Reh cells were directly mixed uniformly to form a cell suspension. Figure 31 shows that the negative control anti-HA HcAb-hIgG1 had no effect on NK cell CD107a expression, while the 12 PVRIG test antibodies and the control antibodies COM701-hIgG1 and SRF813-hIgG1 all increased NK cell CD107a expression to varying degrees, indicating that both the test and control antibodies could effectively promote NK cell activation.

[0274] Example 15: Detection of PVRIG antibody-mediated NK cell killing effect on tumor cell lines by NK cell killing experiments The effect of the test antibodies on NK cell killing function was reflected by detecting the level of target cell (WIDR) destruction by FACS. The experimental method was as described in Example 4(g). Figure 32 shows that the negative control anti-HA HcAb-hIgG1 had no effect on NK cell killing, and all 13 test antibodies could effectively promote NK cell killing of WIDR target cells. In addition to PVRIG-A35 and A43, the killing-promoting functions of the remaining 11 test antibodies against WIDR cells were all higher than or equivalent to the control antibody COM701-hIgG1.

[0275] Example 16: Detection of the functional improvement effect of anti-PVRIG antibodies on antigen-specific CD8 T cells by CMV antigen-recall assay In an experimental system in which PBMCs from anti-CMV IgG-positive donors were used with CMV pp65(495-503) polypeptide-induced CMV pp65-specific CD8 T cells as effector cells and pp65-pulsed colo205 tumor cell lines as target cells, we detected the functional improvement effect of anti-PVRIG antibodies on antigen-specific CD8 T cells.

[0276] CMV IgG+ PBMCs were resuscitated and cultured at 2 × 10 in complete medium (RPMI1640-Glutamax + 5% AB serum + 1% P / S + (1×) 2-β-mercaptoethanol) containing 1 mg / mL CMV pp65(495-503) polypeptide (Anaspec, Cat. No. AS-28328), 2 ng / mL human IL-2 (R&D, Cat. No. IL-202), and 10 ng / mL human IL-7 (Peprotech, Cat. No. 200-07). 6 The cells were resuspended in 100 μg / mL of PP65 polypeptide and IL-7 in a 6-well plate at 5 mL / well and cultured at 37°C and 5% CO for 6 days. On day 6, all the cells were harvested, the pp65 polypeptide and IL-7 in the medium were removed, the cells were split into two, and resuspended in complete medium containing 100 IU / mL of human IL-2. Culture was continued for 2 days. On day 8, all the cells were harvested, resuspended in complete medium containing 100 IU / mL of human IL-2, and the cell density was increased to 2 × 10 6 The total volume was adjusted to 1 mL / mL and culture was continued. On day 11, all cells were harvested, and the percentage of CD8 T cells in PBMCs, the percentage of CMV pp65(495-503)-specific CD8 T cells, and the expression of PVRIG, TIGIT, and PD-1 in these cells were detected by flow cytometry. As shown in Figure 33A and B, after pp65 induction, the percentage of CMV pp65(495-503)-specific CD8 T cells exceeded 80%, and as shown in Figure 33B, pp65+CD8+ T cells (donor 021) expressed different levels of PVRIG, TIGIT, PD-1, and CD226. Flow cytometry detection antibodies were Live / dead Near IR (Invitrogen, Cat. No. L34976), CD8-PerCp Cy5.5 (BD, Cat. No. 565310), CD3-PE-Cy7 (Biolegend, Cat. No. 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Cat. No. TS-0010-1C), PVRIG-AF488 (R&D, Cat. No. FAB93651G-100UG), TIGIT-APC (Biolegend, Cat. No. 372706), and PD-1-BV421 (BD, Cat. No. 562516).

[0277] After the induction, PBMCs were isolated using a CD8 T cell selection reagent kit (Stemcell, Cat. No. 17953). CD8 T cells were isolated as effector cells and resuspended in AIM-V at a cell density of 0.4 × 10 6 The concentration was adjusted to / mL. The purity of CD8 T cells after sorting and the expression of CD226 were detected. Colo205 cells were used as target cells, and TrypLE TM The cells were digested with Express Enzyme (Gibco, Cat. No. 12605010) and resuspended in AIM-V (Gibco, Cat. No. 31035-025) containing 20 ng / mL pp65 at a cell density of 1 × 10 6 The cells were diluted to 0.5 × 10 cells / mL and incubated at 37°C and 5% CO for 3 hours, then centrifuged at 250 g for 5 minutes and the supernatant was discarded. 6 The cells were resuspended at 10000 / mL and analyzed by flow cytometry to detect high expression of PVRL2, PVR, and HLA-A2 in Colo205 cells (Figure 33C). Anti-PVRIG antibody or a negative control was diluted to 280 nM in AIM-V. 50 μL of antibody, 50 μL of CD8 T cells, and 100 μL of pp65-treated Colo205 cells were added sequentially to a low-binding 96-well U-bottom plate (Corning, Cat. No. 7007). The plate was gently mixed with a pipette and incubated at 37°C and 5% CO2 for 18 hours. The final drug concentrations in this system were 70 nM, with 20,000 CD8 T cells and 50,000 Colo205 cells per well. After incubation, the plate was centrifuged at 400 g to collect the supernatant, and the human IFN-γ levels in the supernatant were detected using an ELISA kit (Dacowei, Cat. No. 1110003). In this system, the positive controls were COM701-hIgG4 and SRF813-hIgG1, and the negative control was no treatment. As shown in Figure 33D, compared with the no treatment group, IFN-γ secretion in the cell supernatant was significantly increased after the action of most of the test PVRIG antibodies.

[0278] After sorting, flow cytometry detection antibodies were used to check the purity of CD8 T cells and their CD226 expression: livedead-BV421 (Invitrogen, catalog no. L34964), CD8-FITC (BD, catalog no. 555366), and CD226-PE-Cy7 (Biolegend, catalog no. 338316). Flow cytometry detection antibodies for Colo205 cell PVRL2, PVR, PD-L1, and HLA-A2 expression were livedead-BV421 (Invitrogen, Cat. No. L34964), PVRL2-APC (Biolegend, Cat. No. 337412), PVR-PerCp Cy5.5 (Biolegend, Cat. No. 337612), PD-L1-PE-Cy7 (BD, Cat. No. 558017), and HLA-A2-PE (Biolegend, Cat. No. 343306).

[0279] Example 17 Humanization of alpaca anti-human PVRIG antibody By aligning the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database, heavy chain variable region germline genes highly homologous to the alpaca antibody were screened as templates, and the CDRs of the alpaca antibody were grafted onto the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. If necessary, key amino acids in the framework sequences were mutated back to the corresponding amino acids in the alpaca antibody to maintain the original affinity, thereby obtaining a humanized anti-PVRIG monoclonal antibody.

[0280] 1. Humanization of PVRIG-A50 The humanized heavy chain templates for the alpaca antibody PVRIG-A50 were IGHV3-23*04 and IGHJ3*01. The CDRs of the alpaca antibody PVRIG-A50 were grafted onto the human templates to obtain the corresponding humanized versions, in which the antibody CDR amino acids were determined and annotated according to the Kabat numbering system. Where necessary, key amino acids in the FR region sequence of the humanized PVRIG-A50 antibody were mutated back to the corresponding amino acids in the alpaca antibody to ensure the original affinity. The PVRIG-A50 antibody contains the NG site, which is susceptible to chemical modification, and the NLS glycosylation site, which is susceptible to glycosylation. The inventors mutated the NG / NLS site to eliminate the risk of modification. The specific design is shown in Table 28.

[0281] [Table 28]

[0282] The specific sequences of the variable regions of the PVRIG-A50 humanized antibody are as follows:

[0283] The A50.VH1 (PVRIG-A50-H1) amino acid sequence is as set forth in SEQ ID NO:198. EVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDMSWVRQAPGKGLEWVSTINSNGGRTSYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVEGDPHNFGLENLSLRDFGSWGQGTMVTVSS

[0284] The A50.VH1a (PVRIG-A50-H1a) amino acid sequence is as set forth in SEQ ID NO:199. EVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDMSWVRQAPGKGLEWVSTINSDGGRTSYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVEGDPHNFGLENLSLRDFGSWGQGTMVTVSS

[0285] The A50.VH1b (PVRIG-A50-H1b) amino acid sequence is as set forth in SEQ ID NO:200. EVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDMSWVRQAPGKGLEWVSTINSDGGRTSYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVEGDPHNFGLESLSLRDFGSWGQGTMVTVSS

[0286] The A50.VH1c (PVRIG-A50-H1c) amino acid sequence is as set forth in SEQ ID NO:201. EVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDMSWVRQAPGKGLEWVSTINSDGGRTSYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVEGDPHNFGLENLALRDFGSWGQGTMVTVSS

[0287] The A50.VH1d (PVRIG-A50-H1d) amino acid sequence is as set forth in SEQ ID NO:202. EVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDMSWVRQAPGKGLEWVSTINSNGGRTSYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVEGDPHNFGLESLSLRDFGSWGQGTMVTVSS

[0288] The A50.VH1e (PVRIG-A50-H1e) amino acid sequence is as set forth in SEQ ID NO:203. EVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDMSWVRQAPGKGLEWVSTINSNAGRTSYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVEGDPHNFGLESLSLRDFGSWGQGTMVTVSS

[0289] The A50.VH2a (PVRIG-A50-H2a) amino acid sequence is as set forth in SEQ ID NO:204. EVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDMSWVRQAPGKGLEWVSTINSNAGRTSYVDSVKGRFTISRDNTKNTLYLQMNSLRAEDTAVYYCVEGDPHNFGLESLSLRDFGSWGQGTMVTVSS

[0290] The humanized heavy chain template IGHV3-23*04 amino acid sequence is as shown in SEQ ID NO:205. EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

[0291] The humanized heavy chain template IGHJ3*01 amino acid sequence is as shown in SEQ ID NO:206. WGQGTMVTVSS

[0292] According to the Kabat numbering system, the analysis results of the seven humanized antibody VH sequences are shown in Table 29.

[0293] [Table 29]

[0294] 2. Humanization of PVRIG-A105 The humanized heavy chain templates for the alpaca antibody PVRIG-A105 were IGHV3-7*01 and IGHJ3*01. The CDRs of the alpaca antibody PVRIG-A105 were grafted onto the human templates to obtain the corresponding humanized versions, in which the antibody CDR amino acids were determined and annotated according to the IMGT numbering system. Where necessary, key amino acids in the FR region sequence of the humanized PVRIG-A105 antibody were mutated back to the corresponding amino acids in the alpaca antibody to ensure the original affinity. The PVRIG-A105 antibody contains two free cysteines, and the inventors mutated the Cys to improve antibody stability. The specific design is shown in Table 30.

[0295] [Table 30]

[0296] The specific sequences of the variable regions of the PVRIG-A105 humanized antibody are as follows: The A105.VH1 (PVRIG-A105-H1) amino acid sequence is as set forth in SEQ ID NO:211.

[0297] EVQLVESGGGLVQPGGSLRLSCAASGRTFDRHTMSWFRQAPGKEREFVATASRIPGDTYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAATSAYCSEVDCYEKGSWYDNWGQGTMVTVSS

[0298] The A105.VH2 (PVRIG-A105-H2) amino acid sequence is as set forth in SEQ ID NO:212. EVQLVESGGGLVQPGGSLRLSCAASGRTFDRHTMTWFRQAPGKEREFVATASRIPGDTYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAATSAYCSEVDCYEKGSWYDNWGQGTMVTVSS

[0299] The A105.VH3 (PVRIG-A105-H3) amino acid sequence is as set forth in SEQ ID NO:213. EVQLVESGGGLVQPGGSLRLSCAASGRTFDRHTMTWFRQAPGKEREFVATASRIPGDTYYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAATSAYCSEVDCYEKGSWYDNWGQGTMVTVSS

[0300] The A105.VH4 (PVRIG-A105-H4) amino acid sequence is set forth in SEQ ID NO:214. EVQLVESGGGLVQPGGSLRLSCAASGRTFDRHTMTWFRQAPGKEREFVATASRIPGDTYYSHSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAATSAYCSEVDCYEKGSWYDNWGQGTMVTVSS

[0301] The A105.VH5 (PVRIG-A105-H5) amino acid sequence is as set forth in SEQ ID NO:215. EVQLVESGGGLVQPGGSLRLSCAASGRTFDRHTMTWFRQAPGKEREFVATASRIPGDTYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAATSAYCSEVDCYEKGSWYDNWGQGIQVTVSS

[0302] The A105.VH3a (PVRIG-A105-H3a) amino acid sequence is as set forth in SEQ ID NO:216. EVQLVESGGGLVQPGGSLRLSCAASGRTFDRHTMTWFRQAPGKEREFVATASRIPGDTYYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAATSAYSSEVDSYEKGSWYDNWGQGTMVTVSS

[0303] The humanized heavy chain template IGHV3-7*01 amino acid sequence is as shown in SEQ ID NO:217. EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR

[0304] The humanized heavy chain template IGHJ3*01 amino acid sequence is as shown in SEQ ID NO:206. WGQGTMVTVSS

[0305] According to the IMGT numbering system, the analysis results of the six humanized antibody VH sequences are shown in Table 31.

[0306] [Table 31]

[0307] 3. Humanization of PVRIG-A118 The humanized heavy chain templates for the alpaca antibody PVRIG-A118 were IGHV3-7*01 and IGHJ3*01, and the CDRs of the alpaca antibody PVRIG-A118 were grafted onto the human templates to obtain the corresponding humanized versions, in which the antibody CDR amino acids were determined and annotated according to the IMGT numbering system. Where necessary, key amino acids in the FR region sequence of the humanized antibody PVRIG-A118 were mutated back to the corresponding amino acids in the alpaca antibody to ensure the original affinity. The specific design is shown in Table 32.

[0308] [Table 32]

[0309] The specific sequences of the variable regions of the PVRIG-A118 humanized antibody are as follows:

[0310] The A118.VH1 (PVRIG-A118-H1) amino acid sequence is as set forth in SEQ ID NO:219. EVQLVESGGGLVQPGGSLRLSCAASETYFDLYVMGWYRQAPGKDRELVATITYTGSIYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCNADPSGLGRKVYWGQGTMVTVSS

[0311] The A118.VH2 (PVRIG-A118-H2) amino acid sequence is as set forth in SEQ ID NO:220. EVQLVESGGGLVQPGGSLRLSCAASETYFDLYVMGWYRQAPGKDRELVATITYTGSIKYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCNADPSGLGRKVYWGQGTMVTVSS

[0312] The A118.VH3 (PVRIG-A118-H3) amino acid sequence is as set forth in SEQ ID NO:221. EVQLVESGGGLVQPGGSLRLSCAASETYFDLYVMGWYRQAPGKDRELVATITYTGSIKYVDSVKGRFTISRGDAKNSLYLQMNSLRAEDTAVYYCNADPSGLGRKVYWGQGTMVTVSS

[0313] The A118.VH4 (PVRIG-A118-H4) amino acid sequence is set forth in SEQ ID NO:222. EVQLVESGGGLVQPGGSLRLSCAASETYFDLYVMGWYRQAPGKDRELVATITYTGSIKYVDSVKGRFTISRGDAKNSLSLQMNSLRAEDTAVYYCNADPSGLGRKVYWGQGTMVTVSS

[0314] The A118.VH5 (PVRIG-A118-H5) amino acid sequence is set forth in SEQ ID NO:223. EVQLVESGGGLVQPGGSLRLSCAASETYFDLYVMGWYRQAPGKDRELVATITYTGSIKYVDSVKGRFTISRGDAKNSVYLQMNSLRAEDTAVYYCNADPSGLGRKVYWGQGTMVTVSS

[0315] The A118.VH6 (PVRIG-A118-H6) amino acid sequence is set forth in SEQ ID NO:224. EVQLVESGGGLVQPGGSLRLSCAASETYFDLYVMGWYRQAPGKDRELVATITYTGSIKIVDSVKGRFTISRGDAKNSLYLQMNSLRAEDTAVYYCNADPSGLGRKVYWGQGTMVTVSS

[0316] The A118.VH7 (PVRIG-A118-H7) amino acid sequence is as set forth in SEQ ID NO:225. EVQLVESGGGLVQPGGSLRLSCAASETYFDLYVMGWYRQAPGKDRELVATITYTGSIKYVDSVKGRFTISRGDAKNSLYLQMNSLRAEDTAVYFCNADPSGLGRKVYWGQGTMVTVSS

[0317] The humanized heavy chain template IGHV3-7*01 amino acid sequence is as shown in SEQ ID NO:217. EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR

[0318] The humanized heavy chain template IGHJ3*01 amino acid sequence is as shown in SEQ ID NO:206. WGQGTMVTVSS

[0319] According to the IMGT numbering system, the analysis results of the seven humanized antibody VH sequences are shown in Table 33.

[0320] [Table 33]

[0321] Example 18 ELISA detection of specific binding of PVRIG humanized antibodies to human and cynomolgus monkey PVRIG proteins An ELISA plate was pre-coated with 0.5 μg / mL of human PVRIG-his (AcroBiosystems, Cat. No. PVG-H52H4) or cynomolgus monkey PVRIG protein (Novoprotein, Cat. No. C09B) at 100 μL / well. A test anti-PVRIG humanized antibody was gradient diluted (starting concentration 3 nM, 3-fold gradient dilution). 100 μL / well of the sample was added and incubated at room temperature with shaking for 1.5 hours. After washing the plate, a working solution of mouse anti-human IgG Fc-HRP (Jackson ImmunoResearch, Cat. No. 209-035-098) (1:10,000 dilution) was added. 100 μL / well of the sample was added and incubated at room temperature with shaking for 1.0 hour. The plate was further washed. HRP substrate TMB (Thermo, Cat. No. 34029) was added to develop color. The reaction was stopped with stop solution and then read using a microplate reader (MD Absorbance values were read using a 3x chromatograph. Antibody binding curves were plotted with antibody concentration as the abscissa and the corresponding OD value as the ordinate, and EC50 values were calculated using four-parameter fitting (GraphPad Prism 9). The smaller the EC50 value, the stronger the antibody's ability to bind to human / cynomolgus PVRIG. The binding efficiencies of all humanized antibodies were normalized to their corresponding unhumanized parental antibodies. A percentage value higher than 100% indicated that the binding efficiencies of the humanized antibodies were superior to those of the parental antibodies. As shown in Figures 34A and 34B and Table 34, most humanized antibodies, PVRIG-A50, PVRIG-A105, and PVRIG-A118, all specifically bound to human / cynomolgus PVRIG protein.

[0322] [Table 34]

[0323] Example 19 FACS detection of binding of PVRIG humanized antibodies to FlpinCHO-PVRIG cell-surface human PVRIG and FlpinCHO-cyno PVRIG cell-surface cynomolgus monkey PVRIG The experimental method was as described in Example 9. A larger AUC value indicated a stronger binding ability of the humanized antibody to FlpinCHO-human / cyno PVRIG cells. As shown in Figure 35A, the binding of most humanized test antibodies PVRIG-A50, A105, and A118 to human PVRIG on the FlpinCHO-PVRIG cell surface was comparable to the binding ability of their parental antibodies. Figure 35B showed that the binding of most humanized test antibodies PVRIG-A50 and A105 to cynomolgus monkey PVRIG was relatively good and comparable to the binding ability of their parental antibodies, except that the binding of the humanized test antibody PVRIG-A118 to cynomolgus monkey PVRIG was significantly lower than that of the corresponding parental antibody. The binding activity of the humanized antibodies was normalized to a percentage relative to the control molecules COM701-hIgG1 and SRF813-hIgG1 antibodies, and as shown in Table 35, a higher percentage value indicated stronger antibody binding activity.

[0324] [Table 35]

[0325] Example 20 BIAcore detection of the affinity of PVRIG humanized antibodies to human PVRIG protein For the experimental method, see Example 10. The results in Table 36 showed that specific binding existed between all the tested PVRIG humanized antibodies and human PVRIG protein, and the affinity level was relatively high.

[0326] [Table 36]

[0327] Example 21 ELISA detection of anti-PVRIG humanized antibodies blocking binding of PVRIG to PVRL2 The experimental methods were as described in Example 11. The inhibitory effects of the humanized antibodies were all normalized to the corresponding unhumanized parental antibodies, and percentage values higher than 100% indicated that the inhibitory effect of the humanized antibodies was superior to that of the parental antibodies. The blocking curves of the humanized antibodies are shown in Figure 36, and the inhibitory activities are shown in Table 37. As shown in Figure 36 and Table 37, most of the humanized antibodies, PVRIG-A50, PVRIG-A105, and PVRIG-A118, all significantly inhibited the binding of human PVRIG to human PVRL2.

[0328] [Table 37]

[0329] Example 22 FACS detection of PVRIG humanized antibody blocking binding of CHO-K1-CD112 cells to human PVRIG-mFc protein The experimental method was described in Example 12. The experimental results are shown in Figure 37, which shows that most of the humanized antibodies can block the binding of CHO-K1-CD112 cells to the human PVRIG-mFc protein. The blocking activity of the humanized antibodies was normalized as a percentage relative to the control molecules COM701-hIgG1 and SRF813-hIgG1 antibodies, with a smaller percentage indicating a stronger blocking effect of the antibody.

[0330] [Table 38]

[0331] Example 23: Detection of PVRIG humanized antibody-mediated NK cell killing effect against tumor cell lines by NK cell killing experiments The experimental method was as described in Example 4(g). The experimental results showed that all of the humanized antibodies in Figure 38 were able to promote NK cell killing of target cells to different degrees. Figure 38A shows that the seven humanized antibodies PVRIG-A50-H1b and PVRIG-A50-H2a were comparable in promoting NK cell killing of target cells to the parental antibody PVRIG-A50 before humanization. Figure 38B shows that the seven humanized antibodies PVRIG-A118-H3, H4, H5, and H6 were comparable in promoting NK cell killing of target cells to the parental antibody PVRIG-A118 before humanization. Figure 38C shows that the five humanized antibodies PVRIG-A105-H1, H2, and H3 were comparable in promoting NK cell killing of target cells to the parental antibody PVRIG-A105 before humanization.

[0332] Example 24: CMV antigen-recall assay detection of the functional improvement effect of anti-PVRIG humanized antibodies on antigen-specific CD8 T cells PBMCs were resuscitated and diluted to 2 × 10 in complete medium (RPMI 1640-Glutamax + 5% AB serum + 1% P / S + 1 × 2-β-mercaptoethanol) containing 1 mg / mL CMV pp65(495-503) polypeptide (Anaspec, Cat. No. AS-28328), 2 ng / mL human IL-2 (R&D, Cat. No. IL-202), and 10 ng / mL human IL-7 (Peprotech, Cat. No. 200-07). 6 The cells were resuspended in 100 μg / mL of PP65 / mL and seeded into 6-well plates at 5 mL / well and cultured at 37°C, 5% CO for 6 days. On day 6, all cells were harvested, pp65 and IL-7 in the medium were removed, the cells were split into two, and resuspended in complete medium containing 100 IU / mL of human IL-2. Culture was continued for 2 days. On day 8, all cells were harvested, resuspended in complete medium containing 100 IU / mL of human IL-2, and the cell density was increased to 2 × 10 6On day 11, all cells were harvested, and the percentage of CD8 T cells in PBMCs, the percentage of CMV pp65(495-503)-specific CD8 T cells (Figure 24A), and the expression levels of PVRIG, TIGIT, and PD-1 in the cells (Figure 24B) were detected by flow cytometry. Flow cytometry detection antibodies were Livedead Near IR (Invitrogen, Catalog No. L34976), CD8-PerCp Cy5.5 (BD, Catalog No. 565310), CD3-PE-Cy7 (Biolegend, Catalog No. 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Catalog No. TS-0010-1C), PVRIG-AF488 (R&D, Catalog No. FAB93651G-100UG), TIGIT-APC (Biolegend, Catalog No. 372706), and PD-1-BV421 (BD, Catalog No. 562516).

[0333] After the induction, PBMCs were isolated using a CD8 T cell selection reagent kit (Stemcell, Catalog No. 17953). The CD8 T cells were used as effector cells and resuspended in AIM-V at a cell density of 0.4 × 10 6 The concentration was adjusted to / mL. The purity of CD8 T cells after selection and the expression of CD226 were detected. Colo205 was used as the target cell, and TrypLE TM The cells were digested with Express Enzyme (Gibco, Cat. No. 12605010) and resuspended in AIM-V medium (Gibco, Cat. No. 31035-025) containing 20 ng / mL of pp65 to a cell density of 1 × 10 6 The cells were cultured in AIM-V medium at a concentration of 0.5 × 10 6The PVRIG humanized antibody or negative control antibody was diluted to 280 nM in AIM-V medium. 50 μL of antibody, 50 μL of CD8 T cells, and 100 μL of pp65-treated colo205 cells were added sequentially to a low-binding 96-well U-bottom plate (Corning, catalog no. 7007). After uniform mixing, the plate was incubated for 18 hours at 37°C and 5% CO2. The final drug concentrations were 70 nM, with CD8 T cells at 20,000 / well and colo205 cells at 50,000 / well. After incubation, the plate was centrifuged at 400 g to collect the supernatant. Human IFN-γ levels in the supernatant were measured using an ELISA kit (Daco, catalog no. 1110003). The positive control was the parental PVRIG antibody (untreated), and the negative control was no treatment. As shown in Figure 39, the humanized PVRIG antibody significantly increased IFN-γ levels in cell supernatants compared with the untreated group. Among the tested antibodies, the effect of PVRIG-A105-H2 was significantly lower than that of PVRIG-A105 (*p<0.05, one-way ANOVA analysis). The remaining humanized antibodies were not statistically significantly different from their respective parental antibodies (one-way ANOVA analysis). Flow cytometry detection of CD8 T cell purity and CD226 expression after selection was performed with live / dead-BV421 (Invitrogen, Catalog No. L34964), CD8-FITC (BD, Catalog No. 555366), and CD226-PE-Cy7 (Biolegend, Catalog No. 338316). Flow cytometry detection of PVRL2, PVR, PD-L1, and HLA-A2 expression in Colo205. Antibody information was live / dead-BV421 (Invitrogen, Cat. No. L34964), PVRL2-APC (Biolegend, Cat. No. 337412), PVR-PerCp Cy5.5 (Biolegend, Cat. No. 337612), PD-L1-PE-Cy7 (BD, Cat. No. 558017), and HLA-A2-PE (Biolegend, Cat. No. 343306).

[0334] Example 25 Design of anti-PVRIG x TIGIT humanized bispecific antibody constructs For two anti-PVRIG humanized VHH antibodies (PVRIG-A50-H1b, PVRIG-A105-H1) and two anti-TIGIT humanized monoclonal antibodies (TIGIT-002-H4L3, TIGIT-005-H2L1d), the anti-PVRIG humanized VHH antibodies were linked to the N-terminus of the anti-TIGIT humanized antibody heavy chains via a G4S linker peptide to generate anti-PVRIG x TIGIT humanized bispecific antibodies designated LC-BsAb-002, LC-BsAb-006, LC-BsAb-009, and LC-BsAb-010, respectively ( Figure 40 ). Table 39 shows the sequences of the four bispecific antibody heavy chain fusion polypeptides (HC) and light chain polypeptides (LC).

[0335] [Table 39]

[0336] Positive control antibodies were also constructed at the same time as constructing the double antibodies. The anti-TIGIT positive control antibody was Roche's RG6058-hIgG1, and the anti-PVRIG positive control antibody was Compugen's COM701-hIgG4. The corresponding amino acid sequences were as described above.

[0337] Example 26 ELISA detection of specific binding of anti-PVRIG x TIGIT humanized bispecific antibodies to human and cynomolgus monkey PVRIG proteins An ELISA plate was pre-coated with 1.0 μg / mL human PVRIG (AcroBiosystems, Catalog No. PVG-H52H4) or 0.5 μg / mL cynomolgus PVRIG (Novoprotein, Catalog No. C09B) at 50 μL / well, and the test antibody was gradient diluted (starting concentration 13 nM, 3-fold gradient dilution, 12 concentration points). 50 μL / well of the sample was added and incubated at 37°C for 2.0 hours. After washing the plate, goat anti-human IgG (Goat anti-human IgG) was added. Fc-HRP (Merck, Catalog No. AP113P) working solution (1:5000 dilution) was added, and 50 μL / well of sample was added. The plate was then incubated at 37°C for 1.0 hour. The plate was then washed, and the HRP substrate TMB (KPL, Catalog No. 5120-0077) was added to develop color at 37°C for 10 minutes. The reaction was terminated with stop solution, and the absorbance was read using a microplate reader (PE, Ensight-HH3400). The antibody binding curve was plotted with the antibody molar concentration as the abscissa and the corresponding OD value as the ordinate. A four-parameter fitting (GraphPad Prism 9) was performed to calculate the EC50 value. A smaller EC50 value indicated a stronger antibody binding ability to human or cynomolgus monkey PVRIG. The positive control antibodies were COM701-hIgG1, PVRIG-A50-H1b, and PVRIG-A105-H1, and the negative control antibody was anti-Fluorescein-hIgG1 (in-house). The binding results of the four humanized biantibodies to human PVRIG protein are shown in Figure 41 and Tables 40 and 41, and the binding results to cynomolgus monkey PVRIG are shown in Figure 42 and Tables 40 and 41. The data showed that all four humanized biantibodies could specifically bind to human or cynomolgus monkey PVRIG protein.The binding of LC-BsAb-002 and LC-BsAb-006 to human PVRIG protein was slightly weaker than that of their corresponding monoclonal PVRIG-A50-H1b and the positive control COM701-hIgG1, while the binding of LC-BsAb-009 and LC-BsAb-010 to human or cynomolgus PVRIG protein was essentially equivalent to that of their monoclonal counterpart PVRIG-A105-H1 and superior to that of the positive control COM701-hIgG1.

[0338] [Table 40] [Table 41]

[0339] Example 27 ELISA detection of specific binding of anti-PVRIG x TIGIT humanized bispecific antibodies to human and cynomolgus monkey TIGIT proteins An ELISA plate was pre-coated with 4.0 μg / mL sheep anti-mouse IgG Fc (Jackson, Cat. No. 115-006-071) at 50 μL / well. After sealing and washing, 30 ng / mL human TIGIT ECD-mFc (in-house) or cynomolgus monkey TIGIT ECD-mFc (in-house) was added at 50 μL / well and incubated at 37°C for 2 hours. After washing the plate, 50 μL / well of gradient-diluted test antibodies (starting concentration 13 nM, 3-fold gradient dilution, 12 concentration points) were added and incubated at 37°C for 2 hours. After washing the plate, sheep anti-human IgG Fc-HRP (Merck, Catalog No. AP113P) working solution (1:5000 dilution) was added, and 50 μL / well of sample was added. The plate was then incubated at 37°C for 1.0 hour. The plate was then washed, and the HRP substrate TMB (KPL, Catalog No. 5120-0077) was added to develop color. The reaction was terminated with stop solution, and the absorbance was read using a microplate reader (PE, Ensight-HH3400). The antibody binding curve was plotted with the antibody molar concentration as the abscissa and the corresponding OD value as the ordinate. A four-parameter fitting (GraphPad Prism 9) was performed to calculate the EC50 value. A smaller EC50 value indicated a stronger antibody binding ability to human or cynomolgus monkey TIGIT. The positive control antibodies were RG6058-hIgG1, TIGIT-002-H4L3, and TIGIT-005-H2L1d, and the negative control antibody was anti-Fluorescein-hIgG1 (in-house). The binding results of the four humanized biantibodies to human TIGIT protein are shown in Figure 43 and Tables 42 and 43, and the binding results to cynomolgus TIGIT are shown in Figure 44 and Tables 42 and 43. The data showed that all four humanized biantibodies could specifically bind to human or cynomolgus TIGIT protein.The binding of LC-BsAb-002 and 009 to human TIGIT protein was superior to that of the positive control RG6058-hIgG1 and equivalent to that of their corresponding monoclonal antibody TIGIT-002-H4L3. The binding of LC-BsAb-009 to cynomolgus monkey TIGIT protein was essentially equivalent to that of its corresponding monoclonal antibody TIGIT-002-H4L3 and the positive control RG6058-hIgG1, while LC-BsAb-002 was slightly weaker than the corresponding monoclonal antibody and the positive control. The binding of LC-BsAb-006 and 010 to human / cynomolgus monkey TIGIT protein was superior to that of their corresponding monoclonal antibody TIGIT-005-H2L1d and the positive control RG6058-hIgG1.

[0340] [Table 42] [Table 43]

[0341] Example 28 FACS detection of binding activity of anti-PVRIG x TIGIT humanized bispecific antibodies to FlpinCHO human PVRIG and FlpinCHO cynomolgus monkey PVRIG CHO-K1 stable cells (ATCC® CCL-61) transfected with a human or cynomolgus monkey PVRIG high-expression plasmid TMThese were designated FlpinCHO-hPVRIG, FlpinCHO-cyno PVRIG, human PVRIG full-length plasmid (NCBI Ref Seq: NP_076975), and cynomolgus monkey PVRIG full-length plasmid (NCBI Ref Seq: XP_014989941), respectively. All were synthesized from common organisms. Experiments were performed when the cell density was less than 80%. The cell culture medium was discarded, washed with PBS, and digested with 1 mL of Versene (Gibico, 15040-066) for 8–10 minutes. Digestion was terminated with Ham's F12 (Gibico, 21127-022) complete medium containing 10% FBS, and the cell suspension was obtained. After counting, an appropriate amount of cell suspension was taken, centrifuged at 350 × g to remove the supernatant, washed twice with PBS, and then stained with the viable cell dye Zombie Violet (Biolegend, 423114) and incubated at room temperature for 20 minutes. After incubation, staining was stopped with staining buffer (2% FBS + PBS), centrifuged at 350 × g to remove the supernatant, washed twice, and then stained with 2 × 10 cells in staining buffer. 6The cells were resuspended at a density of 1000 cells / mL and seeded into a 96-well plate. 50 μL of cell suspension was added to each well and prepared for use. The antibody was diluted 3.3-fold in staining buffer, starting from a maximum concentration of 46 nM (2x concentration). The diluted antibody was added to wells already containing 50 μL of cell suspension and shaken at 400 rpm for 1 minute on a microwell plate shaker to thoroughly mix the antibody and cells. The wells were then incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with 200 μL of staining buffer, centrifuged at 350 × g for 5 minutes, and the supernatant was discarded. PE goat anti-human IgG Fc antibody (ebioscience, 12-4998-82) was diluted 250-fold in staining buffer and added to the washed cell wells at a volume of 100 μL / well. The antibody was mixed evenly and stained at 4°C for 30 minutes. After staining, the cells were washed twice with staining buffer in the same manner, and finally resuspended in 200 μL of staining buffer. Signals were detected on-machine using a flow cytometer (BD Canto II). A stronger signal indicated a stronger antibody binding ability to PVRIG. Figure 45 shows that all four humanized biantibodies had excellent human PVRIG-binding activity and could also bind well to cynomolgus monkey PVRIG (Figure 46), and all were superior to their corresponding anti-PVRIG humanized monoclonal antibodies.

[0342] Example 29 FACS detection of binding activity of anti-PVRIG x TIGIT humanized bispecific antibodies to CHO-K1 human TIGIT (high / medium / low expressing strains) and CHO-K1 cynomolgus monkey TIGIT cells Cells were collected, washed once with PBS (Hyclone, SH30256), and then diluted to 2 × 10 in 1% BSA-PBS. 5The cells were resuspended in 100 μL / well, the antibody was diluted to 80 nM in 1% BSA-PBS, and serially diluted 3-fold to 12 concentration points. 50 μL of the diluted antibody solution was mixed with 50 μL of the cells and incubated at 4°C for 60 minutes, washed twice with PBS, and Alexa Fluor® 647 fluorescein-labeled secondary antibody (1:800) (Jackson, 109-605-088) was added. The cells were resuspended in 100 μL / well, incubated at 4°C for 40 minutes, washed twice with PBS, and resuspended in 1% BSA-PBS, 100 μL / well. The cell samples were analyzed using a flow cytometer (BD, Canto II). All four humanized biantibodies had good binding activity to CHO-K1 human TIGIT (high / medium / low expressing cells, Figures 47, 48, 49) and CHO-K1 cynomolgus monkey TIGIT cells (Figure 50).

[0343] Example 30: Detection by HTRF of blocking of interaction between PVRIG protein and PVRL2 protein by anti-PVRIG x TIGIT humanized bispecific antibody PVRIG-mFc (ACRO Biosystems, catalog number PVG-H5253) and Bio-CD112-His (Sino Biological, catalog number 10005-H08H) were diluted to 0.5 μg / mL, respectively, and Streptavidin-Tb cryptate (Cisbio, catalog number) and PAb anti-mouse IgG-XL665 (Cisbio, catalog number) were diluted to 20 μg / mL and 0.8 μg / mL, respectively. The starting concentration of the antibodies to be tested was 120 nM, and a 3-fold gradient dilution was performed, resulting in a total of 12 concentration points. The diluted PVRIG-mFc, Bio-CD112-His, Streptavidin-Tb Crytate, and PAb anti-mouse IgG-XL665 were mixed at a ratio of 1:1:1:1 and added to a 384-well plate (PE, Cat. No. 6007299) at 10 μL / well. Then, gradient-diluted antibodies to be tested were added at 10 μL / well. The plate was centrifuged at 1500 rpm for 30 seconds, incubated at 37°C for 1 hour, and read at wavelengths of 665 nm and 620 nm using a microplate reader (PE, Envision2105). The formula: Ratio = Signal 665 nm / Signal 620 nm x 10 4Data were converted according to the formula below. Using four-parameter fitting with the molar antibody concentration as the abscissa and the ratio as the ordinate, the IC50 was calculated. A smaller IC50 indicated a stronger blocking effect of the antibody on PVRIG binding to PVRL2. The positive controls in this experiment were COM701-hIgG1, PVRIG-A50-H1b, and PVRIG-A105-H1, and the negative control antibody was anti-Fluorescein-hIgG1 (in-house). The blocking effects of the four humanized biantibodies on PVRIG-PVRL2 binding are shown in Figure 51 and Tables 44 and 45. The data demonstrated that all four humanized biantibodies could block human PVRIG binding to the PVRL2 protein. Among them, the blocking effect of LC-BsAb-002 was superior to that of its corresponding monoclonal PVRIG-A50-H1b and the positive control COM701-hIgG1; the blocking effect of LC-BsAb-006 was superior to that of its corresponding monoclonal PVRIG-A50-H1b but slightly lower than that of the positive control COM701-hIgG1; and the blocking effects of LC-BsAb-009 and LC-BsAb-010 were lower than that of their corresponding monoclonal PVRIG-A105-H1 and the positive control COM701-hIgG1.

[0344] [Table 44] [Table 45]

[0345] Example 31 FACS detection of anti-PVRIG x TIGIT humanized bispecific antibody blocking binding of CHO-K1 human CD112 cells to human PVRIG-mFc protein CHO-K1 stable cells (designated CHO-K1-CD112) transfected with a human CD112 high-expression plasmid (NP_001036189.1 / NCBI Ref Seq:Q92692) were used for experiments at cell density less than 80%. The cell culture medium was discarded, washed with PBS, and digested with 1 mL of trypsin (Gibico, 25200-72) for 2 minutes. The digestion was stopped with Ham's F12 (Gibico, 21127-022) complete medium containing 10% FBS, and the cell suspension was then obtained. After counting with a cell counter (Beckman Coulter, Vi-CELL), an appropriate amount of cell suspension was taken, centrifuged at 350 × g, and the supernatant was removed. The cells were washed twice with PBS and then stained with the live / dead cell dye Zombie Violet (Biolegend, 423114) and incubated at room temperature for 20 minutes. After incubation, staining was stopped with staining buffer (2% FBS + PBS), centrifuged at 350 × g, and the supernatant was removed. The cells were washed twice and then stained at 1 × 10 cells with staining buffer. 6The cells were resuspended to a density of 1000 cells / mL and ready for use. A 96-well plate was seeded with 50 μL of cell suspension per well and ready for use. A working solution of human PVRIG-mFc protein (Acro, PVG-H5253) was prepared using staining buffer to a concentration of 1 μg / mL (4x concentration). 50 μL of the PVRIG-mFc working solution was added to the 96-well plate at a concentration of 1 μg / mL. The antibody was diluted 3-fold using staining buffer, starting from a maximum concentration of 275 nM (4x concentration). The diluted antibody was added to wells already containing 50 μL of PVRIG-mFc. The wells were shaken at 400 rpm on a microwell plate shaker for 1 minute to thoroughly mix the antibody and PVRIG-mFc protein, and then incubated at 4°C for 30 minutes. After incubation, 100 μL of the prepared cell suspension was added directly to each well. The mixture was gently mixed evenly with a pipette head and then incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with 200 μL / well of staining buffer, centrifuged at 350 × g for 5 minutes, and the supernatant was discarded. A 250-fold dilution of PE goat anti-mouse IgG Fc antibody (Biolegend, 405337) was added to the washed cell wells at a volume of 100 μL / well, mixed evenly, and stained at 4°C for 30 minutes. After staining, the cells were washed twice with staining buffer and finally resuspended in 200 μL of staining buffer. Signals were detected on-machine using a flow cytometer (BD Canto II). A weaker fluorescent signal indicated a stronger ability of the antibody to block CHO-K1 human CD112 cell binding to PVRIG-mFc protein. Figure 52 shows that all four humanized biantibodies were able to block CHO-K1 human CD112 cell binding to human PVRIG-mFc protein.

[0346] Example 32: Detection by ELISA of the blocking of the binding activity of human TIGIT to CHO-K1 CD155 by anti-PVRIG x TIGIT humanized bispecific antibodies CHO-K1 CD155 cells constructed in Example 2 were collected and cultured at a concentration of 5 × 10 in 10% FBS-DMEM / F12 medium (Excell, FSP500; Gibco, 11330).5 The culture supernatant was shaken off, and a cell fixative (Biyuntian, P0098) was added. 50 μL / well of the culture was fixed at room temperature for 1 hour. The plate was washed once with 0.05% Tween 20-PBS in a plate washer. 5% nonfat dry milk-PBS was added, and the plate was incubated at 37°C for 2-4 hours at 37°C for 250 μL / well. The plate was washed three times with 0.05% Tween 20-PBS in a plate washer. ECD-mFc (working concentration 100 ng / mL) and the sample were mixed and incubated for 0.5 hours. The antigen-antibody mixture was then added to the cell plate at 50 μL / well and incubated at 37°C for 1.5 to 2 hours. The plate was then washed three times with 0.05% Tween 20-PBS in a plate washer. HRP enzyme-labeled antibody (Jackson, 11) was then incubated in 1% BSA (Seiko Seiko, A500023-0100)-PBS at a dilution ratio of 1:5000. The plate was diluted with 50 μL of 5-035-003 and added to the cell plate. The plate was then incubated at 37°C for 1 hour at 50 μL / well with 0.05% Tween 20-PBS. TMB color development solution (KPL, 52-00-03) was added at 50 μL / well and incubated at 37°C for 10 minutes. The reaction was stopped by adding 50 μL / well of 1 M HCl. The OD450nm was then read using a microplate reader (Biotek, Powerwave HT). Figure 53 shows that all four humanized biantibodies can block the binding of human TIGITN to CHO-K1 CD155.

[0347] Example 33: Detection by FACS of the blocking of the binding activity of Bio-CD155-His to CHO-K1 human TIGIT by anti-PVRIG x TIGIT humanized bispecific antibodies Cells were collected, washed once with PBS (Hyclone, SH30256), and then diluted to 2 × 10 in 1% BSA-PBS. 5The cells were resuspended in 40 μL / well. The antibody was diluted to 210 nM in 1% BSA-PBS and serially diluted three-fold to 12 concentration points. Bio-CD155-His (Yiqiao Shenzhou, 10109-H08H) was diluted to 3 μg / mL in 1% BSA-PBS. 40 μL of the antibody dilution and 40 μL of the Bio-CD155-His dilution were then mixed with 40 μL of the diluted antibody and 40 μL of the diluted Bio-CD155-His solution and incubated at 4°C for 60 minutes. After washing twice with PBS, APC-labeled streptavidin (working dilution 1:1700, Biolegend, 405243) was added, resuspended in 100 μL / well, incubated at 4°C for 40 minutes, washed twice with PBS, resuspended in 100 μL / well of 1% BSA-PBS, and the cell samples were analyzed using a flow cytometer (BD, Canto II). Figure 54 shows that all four humanized biantibodies can block the binding of Bio-CD155-His to CHO-K1 human TIGIT.

[0348] Example 34 Detection of binding activity of anti-PVRIG x TIGIT humanized bispecific antibodies to human PBMCs by FACS Fresh human PBMCs (AllCells, PB004-C) were taken and the cells were diluted to 5 × 10 5 The cells were adjusted to 100 ng / mL and simultaneously added with 100 ng / mL SEA (Toxin Technology, Inc., AT101) and cultured at 37°C, 5% CO for 3 days. After 3 days, the cells were harvested, washed once with PBS (Hyclone, SH30256), added with Fc Block (BD, 564220), incubated at 4°C for 10 minutes, washed twice with PBS, and diluted to 2 × 10 with 1% BSA-PBS. 5The humanized antibodies were diluted to 80 nM in 1% BSA-PBS and serially diluted threefold to 12 concentration points. 50 μL of the cells were mixed with 50 μL of the antibody diluent and incubated at 4°C for 60 minutes. After washing twice with PBS, Alexa Fluor® 647 fluorescein-labeled secondary antibody (working dilution 1:800, Jackson, 109-605-088) was added, the cells were resuspended at 100 μL / well, incubated at 4°C for 60 minutes, washed twice with PBS, and resuspended in 100 μL / well of 1% BSA-PBS. The cell samples were analyzed using a flow cytometer (BD, Canto II). Figure 55 shows that all four humanized biantibodies have good binding activity to human PBMCs.

[0349] Example 35 BIAcore detection of the affinity of anti-PVRIG x TIGIT humanized bispecific antibodies to human, cynomolgus monkey, and mouse TIGIT and PVRIG proteins This experiment used a Protein A chip and manually measured the time required for the chip to capture diluted antibodies so that the saturation binding antigen Rmax was 50 RU. Human, cynomolgus monkey, and mouse TIGIT and PVRIG proteins were gradient diluted to 20, 10, 5, 2.5, and 1.25 nM. The affinity of the antibodies for the antigens was measured using multi-cycle kinetics. In each cycle, the antibody was injected, followed by gradient injections of human, cynomolgus monkey, and mouse TIGIT and PVRIG proteins to allow the binding and dissociation processes between the antigen and antibody to occur. After each cycle, the Protein A chip was regenerated (removing proteins from the chip) using glycine pH 1.5. The antibody-antigen affinity KD was fitted using BIAcore T200 analysis software. The results in Table 46 showed that there was specific binding between the two humanized double antibodies and human and cynomolgus monkey TIGIT and PVRIG proteins, with relatively high affinity levels, but they did not bind to mouse TIGIT and PVRIG proteins.

[0350] [Table 46]

[0351] Example 36 BIAcore detection of co-binding of anti-PVRIG x TIGIT humanized bispecific antibodies to human TIGIT and PVRIG proteins The simultaneous binding properties of bispecific antibodies to two antigens were characterized using BIAcore. Antibodies LC-BsAb-002 and LC-BsAb-006 were captured on a Protein A chip. Then, his-tagged proteins of TIGIT and PVRIG were injected, respectively. Then, TIGIT and PVIRIG, and PVRIG and TIGIT were injected sequentially. The antibody and antigen binding signals were recorded. Finally, the chip was regenerated with glycine pH 1.5. The mobile phase was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) at a flow rate of 30 μL / min. The binding time for each antigen was 300 s, the regeneration time was 30 s, the detection temperature was 25°C, and the analytical concentrations of hTIGIT and hPVRIG were 20 nM and 50 nM, respectively. Data were analyzed using BIAcore 8K analysis software (version 2.0), recording the antibody capture level and binding responses (RU) for different antigens. The stoichiometric ratio of antigen-antibody molecules was calculated based on the antigen-antibody molecular weight, and the number of antigens that could bind to an antibody molecule was estimated. To confirm the interaction between antibody LC-BsAb-002 and the antigens TIGIT and PVRIG, four steps of detection were performed, each reaching saturation with each antigen: binding to the hTIGIT antigen alone, binding to the hPVRIG antigen alone, binding to hTIGIT first and then to hPVRIG, and binding to hPVRIG first and then to hTIGIT. Antibody-antigen binding curves were collected, and the capture levels of the two bispecific antibodies and the binding signals of TIGIT and PVIRG in each test were recorded. The stoichiometric ratio of antigen-antibody molecules was calculated based on this, as shown in Table 47. Figure 56, A and B, show the antibody-antigen binding curves for LC-BsAb-002 and LC-BsAb-006 bound to TIGIT and PVIRG, respectively, and for TIGIT and PVIRG, respectively, when injected sequentially.As shown in Table 47 and Figures 56A and 56B, the binding signals generated by sequential injection of TIGIT and PVIRG were almost identical to those generated by single injection of TIGIT and PVIRG, and the binding signals generated by sequential forward and reverse injection of TIGIT and PVIRG were also almost identical. This indicates that LC-BsAb-002 and LC-BsAb-006 can simultaneously bind to hTIGIT and hPVRIG, and there is no mutual influence between the two antigens. Taking into consideration the molecular weights of the antibody and antigen, as well as the antibody capture level and antigen binding level, LC-BsAb The stoichiometric ratios of TIGIT to LC-BsAb-002 were preliminarily estimated to be 1.76, 1.86, 2.14, and 2.18, respectively. The stoichiometric ratios of the two antigen-antibody antibodies were all close to 2, and taking into account the errors caused by the detection method, the inventors estimated that one LC-BsAb-002 or one LC-BsAb-006 biantibody molecule could simultaneously bind to two TIGIT molecules and two PVRIG molecules.

[0352] [Table 47]

[0353] Example 37: Detection of the functional enhancing effect of anti-PVRIG x TIGIT humanized bispecific antibodies on NK cells by NK cell degranulation experiments The expression level of CD107a on NK cells was detected by FACS to show the effect of the test antibody on NK cell activation (FIG. 57A shows the experimental protocol).

[0354] A. Expression of PVRIG, TIGIT, and WIDR cell surface PVR and PVRL2 in NK cells (natural killer cells) was detected by FACS.

[0355] First, NK cells were counted using a cell counter (Beckman Coulter, Vi-CELL). Three flow tubes were taken, and 1e+5 NK cells were added to each flow tube. The cells were washed twice with PBS. The supernatant was removed. One tube was prepared as an unstained tube by adding 300 μL of staining buffer (PBS + 2% FBS). 100 μL of staining solution (PBS + 1% Zombie Violet (Biolegend, 423114)) was added to each of the other two tubes. The cells were then washed twice with staining buffer. The supernatant was removed. 50 μL of Fc inhibitor (staining buffer + Fcx blocker (Biolegend, 422302)) was added to each tube. The cells were mixed thoroughly and incubated at 4°C for 15 minutes. Next, add staining solution to each tube. 50 μL of 2* staining solution (Staining buffer + PE-Cy7 Mouse anti-hCD3 detection antibody + PE Mouse anti-hCD56 detection antibody + APC Mouse anti-hTIGIT detection antibody + AF488 Rabbit anti-hPVRIG detection antibody, CD3 detection antibody Biolegend 300316, CD56 detection antibody Biolegend 318306, TIGIT detection antibody Biolegend 372706, PVRIG detection antibody RD FAB93651G) was added to the first tube, and 50 μL of 2* isotype control staining solution (Staining buffer + PE-Cy7 Mouse anti-hCD3 detection antibody + PE Mouse anti-hCD56 detection antibody + APC Mouse IgG2a κ isotype control antibody + AF488 Rabbit IgG κ isotype control antibody, APC mIgG2a κ isotype control antibody Biolegend) was added to the second tube. 400222, AF488 Rabbit IgG κ isotype control antibody RD IC1051G) was added, mixed uniformly, and then incubated at 4°C for 30 minutes. After the time had passed, the cells were washed twice with staining buffer, centrifuged, and then 300 μL of staining buffer was added and mixed uniformly.Next, on-machine detection (Thermo Attune NxT) was performed, and finally, the proportion of CD56-positive CD3-negative cells in the Zombie Violet-negative cell group and the APC and AF488 signals of the CD56-positive CD3-negative cell group in the Zombie Violet-negative cell group were read.

[0356] WIDR cells were digested with trypsin to prepare a cell suspension, and counted using a cell counter (Beckman Coulter, Vi-CELL). Three flow tubes were prepared, and 1e+5 cells were added to each. PBS was added and the cells were washed twice. After centrifugation, the supernatant was removed, and 300 μL of staining buffer (PBS + 2% FBS) was added to one tube to prepare it as an unstained tube. 100 μL of staining solution (PBS + 1% Zombie Violet (Biolegend, 423114)) was added to each of the other two tubes. After uniform mixing, the tubes were incubated at room temperature for 15 minutes. Next, the cells were washed twice with staining buffer, the supernatant was removed, and the staining solution was added to each tube. 100 μL of the staining solution (Staining buffer + PerCP-Cy5.5 Mouse anti-hPVR detection antibody + APC Mouse anti-hPVRL2 detection antibody, PVR detection antibody Biolegend 337612, PVRL2 detection antibody Biolegend 337412) was added to the first tube, and 100 μL of the isotype control staining solution (Staining buffer + PerCP-Cy5.5 Mouse IgG1 κ isotype control antibody + APC κ Mouse IgG1 isotype control antibody, PerCP-Cy5.5 mIgG1 κ isotype control antibody Biolegend 400150, APC mIgG1 κ isotype control antibody Biolegend 400122) was added to the second tube. After uniform mixing, the cells were incubated at 4 ° C for 30 minutes. After the time interval, the cells were washed twice with staining buffer, centrifuged, and then 300 μL of staining buffer was added and mixed thoroughly. Detection was then performed on-machine (Thermo Attune NxT). Finally, the PerCP-Cy5.5 and APC signals of the Zombie Violet-negative cells were read. Figure 57B shows that the NK cells used in the experiment expressed certain levels of PVRIG and TIGIT, while the target cells, WIDR, highly expressed the ligands PVR and PVRL2.

[0357] B. NK cell degranulation experiment (WIDR as target cells).

[0358] Human PBMCs were resuscitated the day before the experiment, and human NK cells were selected using a selection reagent kit (Stemcell, 17955). They were stimulated overnight with 200 IU / mL h-IL2 (R&D, 202-IL) and 10 ng / mL h-IL12 (Peprotech, 200-12-50UG). The following day, the cells were seeded. Antibodies were diluted to a maximum concentration of 275 nM (4x concentration) in assay buffer (RPMI 1640-Glutamax + 10% FBS + 1x P / S). Subsequently, a 10-fold gradient dilution was performed in assay buffer. 50 μL of the diluted antibodies were added per well to an ultra-low binding 96-well U-bottom plate (Costar, 7007) for use. Next, NK cells were counted using a cell counter (Beckman Coulter, Vi-CELL). A fixed number of NK cells were collected and centrifuged at 350 g for 5 minutes. The supernatant was discarded and resuspended in assay buffer to a density of 0.5E+6 cells / mL. A protein transport inhibitor (Invitrogen, 00498093) and an APC mouse anti-human detection antibody (Biolegend, 328620) were added to the cell suspension. 50 μL of the treated NK cell suspension was added to each well of a 96-well U-bottom plate coated with the drug, mixed evenly, and incubated at room temperature for 15 minutes. During incubation, WIDR target cells were digested with trypsin to prepare a cell suspension (which was then uniformly mixed directly with the Reh cells). The target cells were counted using a cell counter (Beckman Coulter, Vi-CELL). An appropriate amount of cells was removed and centrifuged at 200 x g for 5 minutes. The supernatant was discarded and the cells were resuspended in assay buffer to a density of 0.25e+6 cells / mL. After incubation, 100 µL of the target cell suspension was added to well plates containing 25,000 NK cells, 25,000 target cells, and various concentrations of test antibody. Wells containing only NK cells served as resting controls, and wells containing only NK and WIDR cells served as drug-free controls. Each well was mixed uniformly and then placed in a 37°C incubator for 16 hours.Finally, FACS staining was performed. The cells in the well plate were transferred in parallel to a 96-well V-bottom plate, washed twice with PBS, and the supernatant discarded. Each well was added with staining solution (PBS + 2% FBS + 1* concentration of zombie violet (Biolegend, 423114) + PE mouse anti-CD56 detection antibody (Biolegend, 318306)). After uniform mixing, the wells were incubated at 4°C for 30 minutes. After the time had elapsed, the cells were washed twice with staining buffer, the supernatant discarded, and each well was resuspended in 150 μL of staining buffer. Detection was performed on-machine (Thermo Attune NxT). Finally, the percentage of strongly CD107a-positive cells among CD56-positive cells was measured. A higher percentage of strongly CD107a-positive cells indicated stronger NK cell shedding and activation. Figure 57C shows that the negative control anti-Fluorescein-hIgG1 had no effect on CD107a expression on NK cells, while the candidate humanized biantibodies were all able to enhance CD107a expression on NK cells to different degrees, indicating that the test antibodies could effectively promote NK cell activation.

[0359] C. NK cell degranulation experiment (TF-1 as target cells).

[0360] The experimental method is described in Example 37B, and the experimental results are shown in Figure 57D. The effect of the humanized biantibody on NK cell degranulation was superior to that of the PVRIG positive control antibody COM701-hIgG4 and the TIGIT positive control antibody RG6058-hIgG1, and equivalent to that of the combination group of COM701-hIgG4 and RG6058-hIgG1. At the same time, the promoting effect of the humanized biantibody on NK cell degranulation was also superior to that of its PVRIG arm antibody PVRIG-A50-H1b and TIGIT arm antibody TIGIT-002-H4L3, and equivalent to that of the combination group of PVRIG-A50-H1b and TIGIT-002-H4L3.

[0361] Example 38 Detection of anti-PVRIG x TIGIT humanized bispecific antibody-mediated NK cell killing against tumor cell lines by NK cell killing experiments The effect of the test antibody on the killing function of NK cells against target cells was demonstrated by detecting the level of destruction of target cells (WIDR) by FACS.

[0362] PBMCs were resuscitated the day before the experiment, and NK cells were selected using a selection reagent kit (Stemcell, 17955). NK cells were stimulated overnight with 200 IU / mL h-IL2 (RD, 202-IL) and 10 ng / mL h-IL12 (Peprotech, 200-12-50UG). The next day, the seeding experiment was performed. The expression levels of PVRIG and TIGIT were detected in three NK donors (Donor-050, Donor-831, and Donor-715) using the method described in Example 13. The antibodies to be tested were diluted to a maximum concentration of 275 nM (4x concentration) in assay buffer (RPMI1640-Glutamax + 10% FBS + 1x P / S), then diluted 10-fold with assay buffer. 50 μL of the diluted antibodies were added to an ultra-low binding 96-well U-bottom plate (Costar, 7007) for use. Next, the target WIDR cells were digested with trypsin to prepare a cell suspension. WIDR cells were counted using a cell counter (Beckman Coulter, Vi-CELL). An appropriate amount of cells was taken and centrifuged at 200 g for 5 minutes. The supernatant was discarded and the cells were resuspended in an appropriate amount of PBS. CellTrace Violet (Invitrogen, C34557A) staining solution was added to the cells to a final CellTrace Violet concentration of 5 μM. The WIDR suspension with the staining solution was mixed uniformly and placed in a 37°C incubator for 10 minutes, with shaking during incubation. A portion of the WIDR cells was removed and the expression levels of PVR and PVRL2 in WIDR cells were detected using the method described in Example 13. At the same time, NK cells were counted using a cell counter. A certain number of NK cells were taken and centrifuged at 350 g for 5 minutes. The supernatant was discarded and the cells were resuspended in assay buffer to a density of 0.5e+6 cells / mL. The treated NK cell suspension was added to the drug-coated 96-well U-bottom plate at 50 μL / well, mixed evenly, and then incubated at room temperature for 15 minutes.After staining of WIDR cells, the reaction was stopped by adding 5x the volume of complete medium (MEM + 10% FBS + 1*P / S + 1*non-essential amino acid + 1*monosodium glutamate) to the cell suspension. The mixture was centrifuged at 200xg for 5 minutes, the supernatant discarded, and the cells were resuspended in assay buffer to a density of 0.25e+6 cells / mL. After incubation with NK and drugs, 100µL of the WIDR cell suspension was added to well plates. Each well contained 25,000 NK cells, 25,000 WIDR cells, and various concentrations of the test antibody. Wells containing only WIDR cells served as resting controls, and wells containing NK cells and WIDR served as drug-free controls. Each well was mixed thoroughly and placed in a 37°C incubator for 4 hours. Finally, FACS staining was performed by adding staining solution (PBS + PI (Propidium Iodide, Invitrogen, P3566)) to each well, mixing thoroughly, and incubating at room temperature for 20 minutes. After the time course, on-machine detection (Thermo Attune NxT) was performed to determine the percentage of PI-positive cells among CTV-positive cells. The higher the percentage of PI-positive cells, the stronger the NK cell killing activity. Figure 58A shows that all three NK donors (Donor-050, Donor-831, and Donor-715) express certain levels of PVRIG and TIGIT. Figure 58B shows that the target cells WIDR express high levels of PVR and PVRL2. Figure 58C shows the experimental protocol for the NK cell killing experiment against WIDR cells. Figure 58D shows that the negative control anti-Fluorescein-hIgG1 had no significant effect on NK cell killing, and that all two humanized biantibodies tested effectively promoted NK cell killing (derived from three donors) against WIDR cells. The table shows the EC50 and area under the curve (AUC) for the killing of WIDR cells by the two humanized biantibodies using different NK donors. Using TF-1 as the target cell, the anti-PVRIG x TIGIT humanized bispecific antibody-mediated NK cell killing against TF-1 was measured. The results are shown in Figure 58E, which show that the humanized bispecific antibody can promote NK cell killing of tumor cells, and its activity is superior to that of the PVRIG positive control antibody COM701-hIgG4, the TIGIT positive control antibody RG6058-hIgG1, and the combination of COM701-hIgG4 and RG6058-hIgG1. At the same time, the activity of the humanized bispecific antibody is also superior to that of the PVRIG arm antibody PVRIG-A50-H1b, the TIGIT arm antibody TIGIT-002-H4L3, and the combination of PVRIG-A50-H1b and TIGIT-002-H4L3.

[0363] Example 39 Detection of the direct killing effect of anti-PVRIG x TIGIT humanized bispecific antibody-mediated human Treg cells by NK cell ADCC experiments The level of target cell (Treg) destruction was detected by FACS, demonstrating the direct ADCC killing effect of the test antibody on NK cell target cells (Fig. 59A).

[0364] PBMCs were resuscitated the day before the experiment, and NK cells were selected as effector cells using a selection reagent kit (Stemcell, 17955). NK cells were stimulated overnight with 200 IU / mL h-IL2 (RD, 202-IL) and 10 ng / mL h-IL12 (Peprotech, 200-12-50UG) and seeded the following day. Regulatory T cells isolated from PBMCs were expanded in vitro for 12 days using Dynabeads (Gibco, 11129D) as target cells (Stemcell, 18063). The expanded Treg cells were then used in the experiment. Expression of TIGIT and PVRIG in Treg cells was detected using the methods and reagents described in Example 13 before the experiment. Effector cells and target cells were co-incubated at a ratio of 5:1, and then a series of gradient dilutions of the test humanized bispecific antibody or isotype control anti-fluorescein-hIgG1 or anti-fluorescein-hIgG4 antibody were added. The cells were incubated in an incubator at 37°C for 4 hours, and then stained with PI. Finally, the percentage of PI-positive Treg cells was read, thereby evaluating the ADCC (antibody-dependent cell-mediated cytotoxicity) killing effect of the test bispecific antibody on the target Treg cells. The results showed that the isolated and expanded human Treg cells highly expressed TIGIT and PVRIG (Figure 59B), and that the test humanized biantibody showed concentration-dependent ADCC killing of Treg cells only in the hIgG1 Fc form, while the corresponding hIgG4 Fc form showed no obvious ADCC effect on Treg cells, comparable to the two negative control antibodies anti-Fluorescein-hIgG1 and anti-Fluorescein-hIgG4 (Figure 59C). The table shows the EC50 and AUC of the ADCC killing effect of the two test humanized antibodies in the hIgG1 Fc form on Treg cells.

[0365] Example 40 ADCP activity of anti-PVRIG x TIGIT humanized bispecific antibodies Monocytes were isolated from donor PBMCs and induced to differentiate into macrophages with 75 ng / mL GM-CSF for 7 days. They were labeled with CellTrace Violet and used as effector cells. Human Treg cells selected from human PBMCs using a regulatory T cell isolation reagent kit were expanded in vitro using Dynabeads Human Treg Expander and activated for 13 days to prepare target cells, which were then labeled with CFSE dye. Effector and target cells were co-incubated at a 4:1 ratio. Serially diluted samples of the antibody-in-test, the negative control Hel hIgG1 antibody, its single-arm antibodies (PVRIG-A50-H1b and TIGIT-002-H4L3), or a combination of the two monoclonal antibodies were added and incubated for 4 hours at 37°C. After incubation, the cell dye PI was added, and the proportion of CellTrace Violet-positive cells among CFSE-positive Treg cells was detected by flow cytometry to evaluate the ADCP activity of the antibody-in-test.

[0366] As shown in Figure 60A, the antibodies to be tested activated the ADCP activity of Treg cells in a dose-dependent manner. PVRIG-A50-H1b or COM701-hIgG4 had almost no ADCP activity. TIGIT-002-H4L3 or RG6058-hIgG1 showed dose-dependent ADCP activity. According to the area under the curve (AUC) of the ADCP curve, the ADCP activity of the antibodies to be tested was slightly weaker than that of TIGIT-002-H4L3 and the combination of two single-arm antibodies (PVRIG-A50-H1b + TIGIT-002-H4L3). The ADCP effect of the antibodies to be tested was comparable to that of RG6058-hIgG1 and the combination of two positive antibodies (COM701-hIgG4 + RG6058-hIgG1). E of the ADCP curve max According to the results, the ADCP activity of the antibody under test is comparable to that of two single-arm antibody combinations (PVRIG-A50-H1b + TIGIT-002-H4L3) and two positive antibody combinations (COM701-hIgG4 + RG6058-hIgG1) (Figure 60B).

[0367] Example 41 Effect of anti-PVRIG x TIGIT humanized bispecific antibodies on cytokine release in human PBMCs from healthy donors In this experiment, we investigated the effects of the test antibodies on cytokine secretion in unstimulated PBMCs from healthy volunteers. PBMCs from three healthy volunteers were incubated with the test antibodies in either liquid or solid phase for 24 hours, and then the secretion levels of five cytokines, IFN-γ, IL-2, IL-6, IL-10, and TNF-α, in the PBMC supernatants were detected by flow cytometry. Lipopolysaccharide and CD3 monoclonal antibodies served as positive controls, while Anti-Hel hIgG1 antibody served as a negative control. RG6058-hlgG1 and COM701-hlgG4 served as monoclonal controls with TIGIT and PVRIG terminals, respectively.

[0368] The results showed that after 24 hours of incubation with unstimulated PBMCs from three healthy volunteers, the positive control CD3 monoclonal antibody, either in liquid or solid phase, or with LPS in liquid phase, increased the secretion levels of five cytokines (IFN-γ, IL-2, IL-6, IL-10, and TNF-α) in PBMCs to different degrees. After 24 hours of incubation with unstimulated PBMCs in liquid phase with various concentrations of test antibodies, the secretion levels of IFN-γ, IL-2, IL-6, IL-10, and TNF-α in PBMCs were comparable to those of the negative control or below the detection limit. Under solid-phase conditions, after incubation of various concentrations of the test antibody with unstimulated PBMCs for 24 hours, the secretion levels of IFN-γ, IL-2, and IL-10 in PBMCs were equivalent to or below the detection limit of the negative control. After the action of the test antibody at a high concentration (2850 nM) under solid-phase conditions, the secretion levels of TNF-α and IL-6 were significantly higher than or equivalent to the negative control. However, compared with RG6058-hlgG1 and COM701-hlgG4 under the same conditions, the test antibody did not further increase the secretion of five cytokines, IFN-γ, IL-2, IL-6, IL-10, and TNF-α, in unstimulated PBMCs from healthy individuals in vitro.

[0369] From the above, compared to the TIGIT-terminated monoclonal RG6058-hIgG1 and the PVRIG-terminated monoclonal COM701-hIgG4, the antibody under test under the same conditions does not further increase the secretion of five cytokines, IFN-γ, IL-2, IL-6, IL-10, and TNF-α, in unstimulated PBMCs from healthy individuals in vitro.

[0370] Example 42: CMV antigen-recall assay detection of the functional enhancing effect of anti-PVRIG x TIGIT humanized bispecific antibodies on antigen-specific CD8 T cells Principle of this experiment: In an experimental system using PBMCs from CMV IgG-positive donors, CMV pp65-specific CD8 T cells induced by CMV pp65 (495-503) polypeptide were used as effector cells, and colo205 cells pulsed with pp65 were used as target cells. The functional enhancing effect of anti-PVRIG & TIGIT dual antibodies on pp65-specific CD8 T cells was examined (Figure 61A).

[0371] After PBMC resuscitation, 2 × 10 cells were cultured in complete medium (RPMI1640-Glutamax + 5% AB serum + 1% P / S + (1×) 2-β-mercaptoethanol) containing 1 μg / mL CMV pp65(495-503) polypeptide (Anaspec, Cat. No. AS-28328), 2 ng / mL human IL-2 (R&D, Cat. No. IL-202), and 10 ng / mL human IL-7 (Peprotech, Cat. No. 200-07). 6 The cells were resuspended in 100 μg / mL of PP65 / mL and seeded into 6-well plates at 5 mL / well and cultured at 37°C, 5% CO for 6 days. On day 6, all cells were harvested, pp65 and IL-7 in the medium were removed, the cells were split into two, and resuspended in complete medium containing 100 IU / mL of human IL-2. Culture was continued for 2 days. On day 8, all cells were harvested, resuspended in complete medium containing 100 IU / mL of human IL-2, and the cell density was increased to 2 × 10 6The total cell volume was adjusted to 1 mL / mL and culture was continued. On day 11, all cells were collected and the percentage of CMV pp65(495-503)-specific CD8 T cells and the expression of PVRIG, TIGIT, and PD-1 in the cells were detected by flow cytometry (Figure 61B). Flow cytometry detection antibodies were Livedead Near IR (Invitrogen, Catalog No. L34976), CD8-PerCp Cy5.5 (BD, Catalog No. 565310), CD3-PE-Cy7 (Biolegend, Catalog No. 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Catalog No. TS-0010-1C), PVRIG-AF488 (R&D, Catalog No. FAB93651G-100UG), TIGIT-APC (Biolegend, Catalog No. 372706), and PD-1-BV421 (BD, Catalog No. 562516).

[0372] After the induction, PBMCs were isolated using a CD8 selection reagent kit (Stemcell, Cat. No. 17953). CD8 cells were used as effector cells and resuspended in AIM-V medium at a cell density of 0.4 × 10 6 / mL (if cryopreserved after CD8 induction, the cell number was adjusted to 0.7 × 10 6 The purity and CD226 expression of the selected CD8 cells were examined. Colo205 cells were used as target cells, and TrypLE TM The cells were digested with Express Enzyme (Gibco, Cat. No. 12605010) and resuspended in AIM-V (Gibco, Cat. No. 31035-025) containing 20 ng / mL pp65 at a cell density of 1 × 10 6 The cells were then diluted to 0.5 × 10 cells / mL and incubated at 37°C and 5% CO for 3 hours, then centrifuged at 250 g for 5 minutes, and the supernatant was discarded. 6The cells were resuspended at 10000 / mL and their PVRL2, PVR, and PD-L1 expression was detected by flow cytometry (Figure 61B). The antibodies to be tested (humanized biantibody and Tecentriq) were diluted to 280 nM in AIM-V medium. 50 μL of antibody, 50 μL of CD8, and 100 μL of pp65-treated colo205 were added sequentially to a low-binding 96-well U-bottom plate (Corning, catalog no. 7007). The plates were gently mixed with a pipette and incubated at 37°C and 5% CO2 for 18 hours. The final drug concentrations in this system were 70 nM, CD8 20,000 / well, and colo205 50,000 / well. After incubation, the plates were centrifuged at 400 g to collect the supernatant, and the human IFN-γ levels in the supernatant were detected using an ELISA kit (Dacowei, catalog no. 1110003). In this system, the positive controls were COM701-hIgG4 and RG6058-hIgG1, and the negative control was no treatment. The flow cytometry detection antibodies for CD8+ T cell purity after selection were livedead-BV421 (Invitrogen, Cat. No. L34964) and CD8-FITC (BD, Cat. No. 555366).

[0373] As shown in Figure 61C and Table 48, there was no statistically significant difference in the IFN-γ release levels at each concentration of the two bibody molecules LC-BsAb-002 and LC-BsAb-006. The factor release at high concentrations of the bibody molecules (LC-BsAb-002 at 70 nM, LC-BsAb-006 at 70 and 7 nM) was significantly higher than that of the combined combinations under the same conditions. There was no significant difference at each concentration between the combined combinations corresponding to the two candidate molecules and the combined combination of the positive control antibodies RG605-hIgG1 and COM701-hIgG4. The overall trend indicated that the two bibody molecules had a better effect on promoting IFN-γ release from CD8 T cells than the combined positive control antibodies RG605-hIgG1 and COM701-hIgG4.

[0374] As shown in Figure 61D, after the combination of two dual antibody molecules, LC-BsAb-002 and LC-BsAb-006, with Tencentriq, human IFN-γ secretion was significantly increased compared to the dual antibody alone (t-test, **P<0.01). The combination of three drugs, PVRIG monoclonal, TIGIT monoclonal, and PD-L1 monoclonal, significantly increased human IFN-γ secretion compared to the combination of two drugs, PVRIG monoclonal and TIGIT monoclonal (t-test, *P<0.05). The percentages on the bars in the figure represent the percentage increase compared to the anti-TIGIT positive control antibody RG6058-hIgG1 IFN-γ.

[0375] [Table 48]

[0376] Example 43: CMV antigen-recall assay detection of the functional enhancement of antigen-specific CD8 T cells after combination use of anti-PVRIG x TIGIT humanized bispecific antibody and Tecentriq Experimental principle: Same as in Example 40 (A in Figure 61).

[0377] Induction of antigen-specific CD8 T cells: The same as in Example 40. On the day of inoculation, the expression of PVRIG, TIGIT, and PD-1 was detected by flow cytometry (Figure 62A).

[0378] After induction, PBMCs were isolated using a CD8 T cell selection reagent kit (Stemcell, Cat. No. 17953). CD8 T cells were used as effector cells and resuspended in AIM-V medium at a cell density of 0.8 × 10 6 The number of CD8 T cells in the microwell plate was adjusted depending on the percentage of antigen-specific CD8 T cells. Colo205 cells were pretreated overnight with IFN-γ at a final concentration of 100 ng / mL in complete medium and added as target cells. TrypLE TMAfter digestion with Express Enzyme (Gibco, Cat. No. 12605010), and washing twice, the cells were resuspended in AIM-V (Gibco, Cat. No. 31035-025) containing 20 ng / mL pp65 to a cell density of 1 × 10 6 The cells were then diluted to 0.5 × 10 cells / mL and incubated at 37°C and 5% CO for 3 hours, then centrifuged at 250 g for 5 minutes, and the supernatant was discarded. 6 The cells were resuspended at 0.05 µg / mL, and their PVRL2, PVR, and PD-L1 expression was detected by flow cytometry (Figure 62A). The antibodies to be tested (humanized biantibody, Tecentriq, combination of biantibody and Tecentriq, combination of two positive control monoclonals (COM701-hIgG4 and RG6058-hIgG1), and combination of three monoclonals (COM701-hIgG4, RG6058-hIgG1, and Tecentriq)) were diluted to 280 nM (4x) in AIM-V medium as a starting concentration, followed by a 10-fold gradient dilution for a total of six concentration points. 50 μL of antibody, 50 μL of CD8, and 100 μL of pp65-treated colo205 were added sequentially to a low-binding 96-well U-bottom plate (Corning, catalog no. 7007). The plates were gently mixed with a pipette and incubated at 37°C, 5% CO for 18 hours. The final drug concentrations in this system were 70 nM, 7 nM, 0.7 nM, 0.07 nM, 0.007 nM, and 0.0007 nM, respectively. CD8 was 40,000 / well, and colo205 was 50,000 / well. After co-incubation, the plates were centrifuged at 400 g to collect the supernatant. Human IFN-γ levels in the supernatant were detected using an ELISA kit (Daco-Wei, catalog no. 1110003).

[0379] As shown in Figure 62B and Table 49, the IFN-γ fitting curves were sorted by AUC, as follows: LC-BsAb-002 + Tecentriq > RG6058-hIgG1 + COM701-hIgG4 + Tecentriq > LC-BsAb-002 > RG6058-hIgG1 + COM701-hIgG4 > Tecentriq. A larger AUC indicates stronger efficacy. The combination of LC-BsAb-002 and Tencentiq significantly increased human IFN-γ secretion compared with the dual antibody LC-BsAb-002 alone, and the triple combination of COM701-hIgG4, RG6058-hIgG1, and Tecentriq significantly increased human IFN-γ secretion compared with the dual combination of COM701-hIgG4 and RG6058-hIgG1.

[0380] [Table 49]

[0381] Example 44 Evaluation of the efficacy of anti-PVRIG x TIGIT humanized bispecific antibodies in vivo in mice 5 × 10 A375 cells 6 5 × 10 PBMC cells were inoculated subcutaneously into the right lateral skin of 5-6 week-old female NPG mice (strain: NPG, Beijing Weitongda Biotechnology Co., Ltd.) at a concentration of 0.1 mL / cell. The day after A375 cell inoculation, 5 × 10 PBMC cells were added to the right lateral skin of the mice. 6 The tumors were injected into the tail vein of mice at a concentration of 0.2 mL / cell. 3Once the tumors reached the tumor size, 56 mice were screened according to tumor volume and randomly assigned to seven groups (8 mice per group): vehicle (PBS), RG6058-hIgG1 (10 mg / kg), COM701-hIgG4 (10 mg / kg), RG6058-hIgG1 + COM701-hIgG4 (10 mg / kg + 10 mg / kg), Tecentriq (5 mg / kg, lot number HK65567, Roche), LC-BsAb-002 (11.7 mg / kg), and LC-BsAb-006 (11.7 mg / kg). All groups were administered intraperitoneally, twice weekly for four consecutive doses. The experiment was terminated three days after the final dose. During the administration and observation period, mouse body weights and tumor volumes were measured three times weekly and recorded. Tumor volume (longest diameter × shortest diameter) was then calculated. 2 / 2) and growth inhibition rate (TGI TV (%) = (1-(Tn-T0) / (Vn-V0)) × 100% was calculated.

[0382] Efficacy results: As shown in Figure 63, after administration of the test candidate molecules LC-BsAb-002 and LC-BsAb-006, they had a clear inhibitory effect on A375 tumor growth, and the inhibition level was similar to that of the combination of positive molecules RG6058-hIgG1 + COM701-hIgG4 and Tecentriq. On day 13 of group administration, the tumor growth inhibitor (TGI) and differences between each test drug group and the negative control PBS group were analyzed (Table 11). The TGI of LC-BsAb-002 and LC-BsAb-006 were 82.16% and 78.59%, respectively, and were significantly higher than PBS (P<0.005). The TGI levels were higher than those of RG6058-hIgG1 (TGI=42.55) and COM701-hIgG4 (TGI=0.23%) alone, and comparable to the combination of RG6058-hIgG1 and COM701-hIgG4 (TGI=83.32%). The growth curves of single mouse tumors are shown in Figure 64 and show the same trends as Figure 63.

[0383] [Table 50]

[0384] Body weight results: As shown in Figure 65 and Table 51, except for the control molecule Tecentriq, which showed significant body weight loss and toxicity and side effects, the body weight change trends of the remaining control and candidate molecules LC-BsAb-002 and LC-BsAb-006 were basically consistent with those of PBS, and the subsequent weight loss was due to GVHD caused by PBMC reconstitution.

[0385] [Table 51]

[0386] The final results showed that the anti-PVRIG x TIGIT humanized bispecific antibody molecules LC-BsAb-002 and LC-BsAb-006 had a significant inhibitory effect on the growth of subcutaneously xenografted A375 tumors, with tumor inhibition superior to that of the positive control antibodies RG6058-hIgG1 and COM701-hIgG4 alone and equivalent to that of the positive control antibody Tecentriq in combination with RG6058-hIgG1 + COM701-hIgG4. At the same time, no toxicity or side effects were observed during administration or observation, demonstrating the safety and tolerability of the candidate molecules in this model.

[0387] Example 45 In vivo efficacy evaluation of anti-PVRIG x TIGIT humanized bispecific antibodies in combination with Tecentriq in mice 5 × 10 A375 cells 6 5.5 × 10 Hu PBMC cells were inoculated subcutaneously into the right lateral skin of 5-6 week-old female NPG mice (strain: NPG, Beijing Weitongda Biotechnology Co., Ltd.) at a concentration of 0.1 mL / cell. The day after A375 cell inoculation, 5.5 × 10 Hu PBMC cells were inoculated. 6 The tumor was injected into the tail vein of mice in vivo at a concentration of 0.2 mL / 1000 cells / 1000 cells / 0.2 mL. 3Once tumors reached the tumor size, 45 mice were screened according to tumor volume and randomly assigned to five groups: vehicle (PBS, 9 mice), LC-BsAb-002 (11.7 mg / kg, 9 mice), LC-BsAb-002 (5.9 mg / kg, 9 mice), Tecentriq (3 mg / kg, 10 mice, lot No. HK65567, Roche), and LC-BsAb-002 + Tecentriq (5.9 mg / kg + 3 mg / kg, 8 mice). All groups were administered intraperitoneally, twice weekly for five consecutive doses. The experiment was terminated three days after the final dose. During the administration and observation period, mouse body weights and tumor volumes were measured three times weekly and recorded. Tumor volume (longest diameter × shortest diameter) was then calculated. 2 / 2) and growth inhibition rate (TGI TV (%) = (1-(Tn-T0) / (Vn-V0)) × 100% was calculated.

[0388] Drug efficacy results: As shown in Figure 66, administration of the test candidate molecule LC-BsAb-002 had a significant inhibitory effect on A375 tumor growth, and the higher the dose, the stronger the inhibitory effect on A375 tumor growth. Meanwhile, the combined use of LC-BsAb-002 and Tecentriq showed a significantly greater inhibitory effect on A375 tumor growth than LC-BsAb-002 or Tecentriq alone. On Day 17 of group administration, the tumor growth inhibitor (TGI) and differences between each test drug group and the negative control PBS group were analyzed (Table 52). The TGI of LC-BsAb-002 (11.7 mg / kg), LC-BsAb-002 (5.9 mg / kg), and Tecentriq (3 mpk) were 66.56%, 60.51%, and 41.53%, respectively, showing significant differences compared to the PBS group (P<0.0001, P=0.0003, and P=0.0015). The TGI of the LC-BsAb-002 and Tecentriq combination group was 80.44%, showing a significant difference compared to the PBS group (P<0.0001) and superior to the TGI of LC-BsAb-002 (5.9 mg / kg) and Tecentriq (3 mpk) alone. The growth curves of single mouse tumors are shown in FIG. 67, and the growth trends of tumors in each group were the same as in FIG.

[0389] [Table 52]

[0390] Body weight results: As shown in Figure 68 and Table 53, the LC-BsAb-002 + Tecentriq combination group showed a certain decrease in body weight, but no obvious toxicity or side effects were observed. The weight change trends of mice in the Tecentriq group, LC-BsAb-002 (11.7 mpk), and LC-BsAb-002 (5.9 mpk) groups were basically consistent with those of the PBS group.

[0391] [Table 53]

[0392] The final results showed that the anti-PVRIG x TIGIT humanized bispecific antibody molecule LC-BsAb-002 significantly inhibited the growth of subcutaneously xenografted A375 tumors, with the inhibitory effect demonstrating a dose-dependent relationship with increasing dose. Meanwhile, the tumor inhibitory effect of LC-BsAb-002 in combination with Tecentriq was significantly superior to that of either agent alone, demonstrating significant combination efficacy. At the same time, no toxicity or side effects of the candidate molecule were observed during administration and observation, demonstrating the safety and tolerability of the candidate molecule in this model.

Claims

1. 1. An anti-PVRIG / anti-TIGIT bispecific antibody, (a) a first antigen-binding portion of an anti-TIGIT antibody, the first antigen-binding portion comprising two heavy chains and two light chains; In its heavy chain variable region (VH), HCDR1 is the sequence set forth in SEQ ID NO: 21; HCDR2 is the sequence set forth in SEQ ID NO: 22; HCDR3 is the sequence set forth in SEQ ID NO: 23; In its light chain variable region (VL), LCDR1 is the sequence set forth in SEQ ID NO: 18; LCDR2 is the sequence set forth in SEQ ID NO: 19; a first antigen-binding portion, wherein LCDR3 is the sequence set forth in SEQ ID NO: 20; (b) a second antigen-binding moiety comprising a VHH that specifically binds to PVRIG, CDR1 of said VHH is the sequence set forth in SEQ ID NO: 168; CDR2 is the sequence set forth in SEQ ID NO: 207; and a second antigen-binding portion, wherein CDR3 is the sequence set forth in SEQ ID NO: 208; Bispecific antibodies.

2. the VH of the first antigen-binding portion comprises the sequence set forth in SEQ ID NO: 72; the VL of the first antigen-binding portion comprises the sequence set forth in SEQ ID NO: 68; The second antigen-binding portion comprises the sequence set forth in SEQ ID NO:

200. The bispecific antibody of claim 1.

3. the first antigen-binding portion is a full-length antibody; the C-terminus of the second antigen-binding portion is fused to the N-terminus of at least one heavy chain of the first antigen-binding portion; The bispecific antibody of claim 1.

4. the heavy chain fusion polypeptide comprises, from N-terminus to C-terminus, PVRIG VHH-(G4S)4 Linker-TIGIT VH-CH1-hinge-CH2-CH3; the light chain polypeptide comprises, from N-terminus to C-terminus, TIGIT VL-CL; The bispecific antibody of claim 3.

5. the amino acid sequence of the heavy chain fusion polypeptide is set forth in SEQ ID NO:227; The amino acid sequence of the light chain polypeptide is set forth in SEQ ID NO:

226. The bispecific antibody of claim 4.

6. is a humanized antibody, The bispecific antibody according to any one of claims 1 to 5.

7. specifically binds to human or monkey PRVIG or TIGIT protein, has a KD of more than 1.00E-7M for binding to human or monkey PRVIG, has a KD of more than 1.00E-8M for binding to human or monkey PRVIG, and can simultaneously bind to TIGIT and PRVIG; The bispecific antibody according to any one of claims 1 to 5.

8. further coupled to a therapeutic agent or tracer, the therapeutic agent is selected from a drug, a toxin, a radioisotope, a chemotherapeutic agent, or an immunomodulator; The tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, and a photosensitizer; The anti-PVRIG / anti-TIGIT bispecific antibody according to any one of claims 1 to 5.

9. A gene encoding the bispecific antibody of any one of claims 1 to 5. Isolated nucleic acid fragments.

10. 1. A pharmaceutical composition comprising: A method for producing a recombinant human ovarian tumor suppressor comprising administering to a subject the bispecific antibody of any one of claims 1 to 5 and a pharmaceutically acceptable vector. Pharmaceutical compositions.

11. 1. A pharmaceutical composition comprising:

10. A method for producing a nucleic acid fragment comprising the steps of: Pharmaceutical compositions.

12. further comprising an additional therapeutic agent, wherein said additional therapeutic agent is a PD-1 binding antagonist; the PD-1 binding antagonist is selected from a PD-1 binding antagonist, a PD-L1 binding antagonist, or a PD-L2 binding antagonist; the PD-1 binding antagonist is selected from MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108; the PD-L1 binding antagonist is selected from MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), Tecentriq, or MSB0010718C (avelumab); The PD-L2 binding antagonist is selected from an anti-PD-L2 antibody or an immunoadhesin. The pharmaceutical composition of claim 10.

13. further comprising an additional therapeutic agent, wherein said additional therapeutic agent is a PD-1 binding antagonist; the PD-1 binding antagonist is selected from a PD-1 binding antagonist, a PD-L1 binding antagonist, or a PD-L2 binding antagonist; the PD-1 binding antagonist is selected from MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108; the PD-L1 binding antagonist is selected from MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), Tecentriq, or MSB0010718C (avelumab); The PD-L2 binding antagonist is selected from an anti-PD-L2 antibody or an immunoadhesin. The pharmaceutical composition of claim 11.

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