Antibodies against PVRIG or TIGIT, bispecific antibodies constructed therefrom, methods of making and uses thereof

Abstract

The present invention relates to anti-PVRIG antibodies, anti-TIGIT antibodies, and multispecific / bispecific antibodies against PVRIG and TIGIT. The present invention also relates to nucleic acid molecules encoding the antibodies of the invention, expression vectors and host cells for expressing the antibodies of the invention. The present disclosure also provides for the use of the antibodies of the invention in the treatment of a proliferative disorder (e.g., a cancer or tumor), an infection, or sepsis in a subject.

Description

[0001] Priority Statement

[0002] This application claims the benefits of PCT / CN2023 / 120035, filed on September 20, 2023, and PCT / CN2023 / 125925, filed on October 23, 2023, the entire contents of which are incorporated herein by reference.

[0003] Statement regarding electronic archiving of sequence lists

[0004] The XML text format sequence list, named IEC240426PCT_sequencelisting_240920.xml and generated on September 20, 2024, and submitted with this application, is 765kb in size, and its disclosure is incorporated herein by reference. Technical Field

[0005] This application generally relates to antibodies. More specifically, this application relates to antibodies against PVRIG or TIGIT, multispecific antibodies constructed therefrom, their preparation methods, and their uses. Background Technology

[0006] The immune system's response to pathogens is controlled by various regulatory mechanisms to maintain tolerance to self-antigens and protect tissue integrity. Several inhibitory receptor-mediated signaling pathways have been described as contributing to immune homeostasis, while also defending against infection and transformation of cells.

[0007] In many solid tumors, effector T cells exhibit a depleted phenotype within the tumor microenvironment (TME) and are unable to mediate effective antitumor responses. These depleted T cells can be identified by increased surface expression of co-inhibitory receptors such as PD-1 and CTLA-4. Antibodies that inhibit the interaction of these co-inhibitory receptors with their homologous ligands have shown clinical efficacy in patients with advanced cancer.

[0008] Recently, a poliovirus receptor (PVR)-like protein co-signaling network involving several immune checkpoint receptors (i.e., DNAM-1 (CD226), TIGIT, CD96 (TACLILE), and CD112R (PVRIG)) has been discovered, which interact with their ligands CD155 (PVR / Necl-5), CD112 (PVRL2 / neconin-2), CD111 (PVRL1 / neconin-1), CD113 (PVRL3 / neconin-3), and connexin 4.

[0009] Of these receptors, TIGIT is the best characterized. It is a transmembrane protein containing the known ITIM domain in the intracellular portion of the receptor, a transmembrane domain, and an immunoglobulin variable domain in the extracellular portion. It has a high affinity for PVR and a weaker affinity for PVRL2 and PVRL3. TIGIT is an inhibitory receptor, primarily found on natural killer (NK) cells and CD8+ cells. + T cells, CD4 + TIGIT is expressed on T cells and regulatory T cells (Tregs). DNAM / CD226, a known co-stimulatory receptor, is also expressed on NK and T cells, competing with TIGIT for CD155 and CD112 binding, but with lower affinity. This suggests tight control over the activation of these effector cells to avoid uncontrolled cytotoxicity to normal cells expressing the CD155 ligand. TIGIT expression is increased in tumor-infiltrating lymphocytes (TILs) and in disease environments such as HIV infection. TIGIT expression marks exhausted T cells, which have lower effector function compared to their TIGIT-negative counterparts. Several preclinical studies support the use of TIGIT blockade as a monotherapy or in combination with other immune checkpoint inhibitors for the treatment of advanced solid malignancies.

[0010] CD112R, formerly known as the poliovirus receptor-associated immunoglobulin domain-containing protein (PVRIG), is a novel member of the PVR-like co-signaling network. It is a 36-kD transmembrane monomer composed of a single extracellular IgV domain, a transmembrane domain, and a long intracellular domain. The human CD112R intracellular domain contains two tyrosine residues, one of which is part of an ITIM-like motif and a potential site for phosphatases. CD112R is an inhibitory receptor primarily expressed by T cells and NK cells, competing with the activating receptor CD226 for CD112 binding. The affinity of CD112 for CD112R interaction is higher than that for CD226 interaction, thereby effectively regulating CD226-mediated cell activation. Anti-CD112R antibodies that block CD112 interaction restrict inhibitory signaling downstream of CD112R while simultaneously promoting greater immune cell activation by enhancing the CD226-CD112 interaction. Both in vivo and in vitro data indicate that anti-CD112R antibodies can increase the proliferation, activation, and cytotoxicity of immune effector cells. Administration of anti-CD112R immunotherapy provides an opportunity to increase, enhance, and maintain the immune response.

[0011] Based on predictive analysis using the TCGA (Cancer Genome Atlas) dataset, PVRIG mRNA expression was detected in numerous cancer tissues. Its expression was increased in tumors rich in T cells and NK cells and showed correlation with TIGIT and PD-1 expression. Both PVRIG ligand CD112 and TIGIT ligand CD155 were expressed on tumor-infiltrating myeloid cells and upregulated on cancer cells, contributing to local suppression of immune surveillance. Preclinical in vivo data indicated that blocking PVRIG had limited inhibitory effects on tumor growth, while the inhibitory effect was enhanced when used in combination with TIGIT or PD-1 blockade, suggesting that the PVRIG-PVRL2 and TIGIT-PVR pathways are non-redundant inhibitory signaling pathways.

[0012] Mounting evidence suggests that this novel co-signaling network axis, upon binding to ligands, co-stimulates and co-inhibits NK and T cell activation to eliminate cancer cells. Therefore, PVRIG and / or TIGIT-specific antagonistic antibodies capable of inhibiting PVRIG and / or TIGIT receptor activity represent an opportunity to mitigate immunosuppressive effects associated with the tumor microenvironment, thereby enhancing antitumor immune responses against tumor cells. Summary of the Invention

[0013] This invention relates to novel anti-PVRIG antibodies and their antigen-binding fragments. The anti-PVRIG antibody or its antigen-binding fragment of this invention can block the binding of PVRIG to its homologous ligand CD112.

[0014] This invention also relates to novel anti-TIGIT antibodies and their antigen-binding fragments. The anti-TIGIT antibody or its antigen-binding fragment of this invention can block the binding of TIGIT to its homologous ligand PVR.

[0015] This invention also relates to multispecific antibodies against PVRIG and TIGIT, and antigen-binding fragments thereof. The multispecific antibodies of this invention can block the binding of PVRIG to its homologous ligand CD112 and the binding of TIGIT to its homologous ligand CD155.

[0016] Therefore, in a first aspect, the present invention provides an anti-PVRIG antibody or an antigen-binding fragment thereof comprising three CDRs of one of the antibodies listed in Table 1 or three CDRs contained in one of the antibodies listed in Table 2.

[0017] In a second aspect, the present invention provides an anti-PVRIG antibody or an antigen-binding fragment thereof comprising the amino acid sequence of the VH of one of the antibodies listed in Table 2. Preferably, the anti-PVRIG antibody of the present invention comprises the amino acid sequence of the heavy chain of one of the antibodies listed in Table 3.

[0018] In a third aspect, the present invention provides a humanized form of an anti-PVRIG antibody as defined above, or an antigen-binding fragment thereof. Preferably, the humanized anti-PVRIG antibody or its antigen-binding fragment comprises three CDRs of one of the antibodies listed in Table 1 or Table 4a, or three CDRs of one of the antibodies listed in Table 2. More preferably, the humanized anti-PVRIG antibody or its antigen-binding fragment comprises the amino acid sequence of the VH of one of the antibodies listed in Table 4b or Table 4c. Most preferably, the humanized anti-PVRIG antibody or its antigen-binding fragment comprises the amino acid sequence of the heavy chain of one of the antibodies listed in Table 4d or Table 4e.

[0019] In a fourth aspect, the present invention relates to affinity maturation of the anti-PVRIG antibody of the present invention. Therefore, the present invention also provides an affinity-matured form of one of the anti-PVRIG antibodies as defined above, preferably a humanized antibody, or an antigen-binding fragment thereof. Preferably, the affinity-matured anti-PVRIG antibody or its antigen-binding fragment comprises three CDRs of one of the antibodies listed in Table 5a or Table 5b. More preferably, the affinity-matured anti-PVRIG antibody or its antigen-binding fragment comprises the amino acid sequence of the VH of one of the antibodies listed in Table 6a. Most preferably, the affinity-matured anti-PVRIG antibody or its antigen-binding fragment comprises the amino acid sequence of the heavy chain of one of the antibodies listed in Table 6b.

[0020] In a fifth aspect, the present invention provides an anti-TIGIT antibody or an antigen-binding fragment thereof comprising six CDRs of one of the antibodies listed in Table 7 or six CDRs contained in one of the antibodies listed in Table 8a.

[0021] In a sixth aspect, the present invention provides an anti-TIGIT antibody or an antigen-binding fragment thereof comprising VH and VL, said VH and VL comprising HCDR and LCDR of one of the antibodies listed in Table 7 or six CDRs contained in one of the antibodies listed in Table 8a. Preferably, the anti-TIGIT antibody of the present invention comprises the amino acid sequences of VL and VH of one of the antibodies listed in Table 8a. More preferably, the anti-TIGIT antibody of the present invention comprises the amino acid sequences of the heavy chain and light chain of one of the antibodies listed in Table 8b.

[0022] In a seventh aspect, the present invention provides a humanized form of one of the anti-TIGIT antibodies as defined above, or an antigen-binding fragment thereof. In one embodiment, the humanized anti-TIGIT antibody comprises six CDRs of one of the antibodies listed in Table 7 or six CDRs contained in one of the antibodies listed in Table 8a. In another embodiment, the humanized anti-TIGIT antibody comprises six CDRs of one of the antibodies listed in Table 9a or Table 9b or six CDRs contained in one of the antibodies listed in Table 10a or Table 10b. In yet another embodiment, the humanized anti-TIGIT antibody comprises the amino acid sequences of VL and VH of one of the antibodies listed in Table 10a or Table 10b. Preferably, the humanized anti-TIGIT antibody comprises the amino acid sequences of the light and heavy chains of one of the antibodies listed in Table 10c or Table 10d.

[0023] In an eighth aspect, the present invention provides a multispecific antibody or antigen-binding fragment thereof comprising a first portion binding to PVRIG and a second portion binding to TIGIT. In one embodiment, the first portion may be the anti-PVRIG antibody or antigen-binding fragment of the present invention. In another embodiment, the second portion may be the anti-TIGIT antibody or antigen-binding fragment of the present invention. In this invention, the term "multispecific" includes bispecific, trispecific, etc.

[0024] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the heavy chain and / or light chain of an antibody of the present invention, or an antigen-binding fragment thereof or a portion thereof. The antibody may be the anti-PVRIG antibody of the present invention, the anti-TIGIT antibody of the present invention, or the multispecific antibody of the present invention, preferably the bispecific antibody of the present invention. In one embodiment, the present invention provides a nucleic acid molecule encoding the heavy chain of an anti-PVRIG antibody of the present invention as defined above, or an antigen-binding fragment thereof or a portion thereof. In another embodiment, the present invention provides a nucleic acid molecule encoding the heavy chain and / or light chain of an anti-TIGIT antibody of the present invention as defined above, or an antigen-binding fragment thereof or a portion thereof. In yet another embodiment, the present invention provides a nucleic acid molecule encoding the heavy chain and / or light chain of a multispecific antibody of the present invention as defined above.

[0025] In a tenth aspect, the present invention provides a vector comprising a nucleic acid of the present invention operatively linked to a promoter. In one embodiment, the nucleic acid encodes the heavy chain of an anti-PVRIG antibody of the present invention, or an antigen-binding fragment thereof, or a portion thereof. In another embodiment, the nucleic acid encodes the heavy chain and / or light chain of an anti-TIGIT antibody of the present invention, or an antigen-binding fragment thereof, or a portion thereof. In yet another embodiment, the nucleic acid encodes the heavy chain and / or light chain of a multispecific antibody of the present invention, or a portion thereof.

[0026] In an eleventh aspect, the present invention also provides host cells transformed using the vector of the present invention. The host cells may be prokaryotic cells, fungal cells, or mammalian cells, such as CHO cells.

[0027] In a twelfth aspect, the present invention provides an immunoconjugate comprising an antibody or antigen-binding fragment of the present invention non-covalently or covalently conjugated to another portion (preferably a chemical portion that improves the properties of the antibody or antigen-binding fragment of the present invention). The chemical portion may be another therapeutic agent, BSA, an Fc fragment, a PEG molecule, etc. The antibody may be the anti-PVRIG antibody of the present invention, the anti-TIGIT antibody of the present invention, or the multispecific antibody of the present invention, preferably the bispecific antibody of the present invention. In one embodiment, the immunoconjugate comprises the anti-PVRIG antibody of the present invention or its antigen-binding fragment. In another embodiment, the immunoconjugate comprises the multispecific antibody of the present invention against PVRIG and TIGIT, or its antigen-binding fragment, preferably the bispecific antibody of the present invention against PVRIG and TIGIT, or its antigen-binding fragment.

[0028] In a thirteenth aspect, the present invention provides pharmaceutical compositions comprising the antibodies, immunoconjugates, nucleic acids, or host cells of the present invention, and optionally pharmaceutically acceptable excipients. As described herein, the antibody may be an anti-PVRIG antibody, an anti-TIGIT antibody, and / or a multispecific antibody against both PVRIG and TIGIT, preferably a bispecific antibody. Optionally, the pharmaceutical compositions of the present invention may comprise one or more additional therapeutic agents. One or more such additional therapeutic agents include, but are not limited to, chemotherapeutic agents, immunotherapeutic agents, radiotherapeutic agents, cytotoxic agents, etc. Preferably, the immunotherapeutic agent may be a PD-L1 inhibitor, an ADCC-enabled antibody, or a combination thereof. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is LAE005. In some embodiments, the ADCC-enabled antibody is an anti-EGFR antibody and / or an EGFR-MET bispecific antibody. In some embodiments, the anti-EGFR antibody is cetuximab. In some embodiments, the anti-EGFR / C-Met bispecific antibody is ervaltimab.

[0029] In a fourteenth aspect, the present invention provides combination therapies in which the antibody of the present invention or its antigen-binding fragment is used in combination with one or more additional therapeutic agents. One or more such additional therapeutic agents include, but are not limited to, chemotherapeutic agents, immunotherapeutic agents, radiotherapeutic agents, cytotoxic agents, etc. Preferably, the immunotherapeutic agent may be a PD-L1 inhibitor, an ADCC-enabled antibody, or a combination thereof. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is LAE005. In some embodiments, the ADCC-enabled antibody is an anti-EGFR antibody and / or an EGFR-MET bispecific antibody. In some embodiments, the anti-EGFR antibody is cetuximab. In some embodiments, the anti-EGFR / C-Met bispecific antibody is ervaltimab.

[0030] In a fifteenth aspect, the present invention provides methods for preparing antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, immunoconjugates, pharmaceutical compositions and kits of the present invention, and their use in the treatment, diagnosis and / or monitoring of proliferative diseases (e.g., cancer or tumors), infections or sepsis.

[0031] In a sixteenth aspect, the present invention provides a method for preventing the suppression of immune cells or the activation of immune cell-mediated responses, comprising contacting immune cells with an antibody or antigen-binding fragment thereof of the present invention, an immune conjugate, a nucleic acid, a carrier, or a pharmaceutical composition. In some embodiments, the immune cells are NK cells or T cells. In some embodiments, the T cells are CD8+ T cells or Treg cells. In some embodiments, the immune cells express PVRIG and / or TIGIT. In some embodiments, the immune cell-mediated response is an anti-tumor response. In some embodiments, the tumor cells express CD112 and / or CD155.

[0032] Other aspects and advantages of the invention will become apparent from the following detailed description of the invention. Attached Figure Description

[0033] Figures 1-11 Involving anti-PVRIG antibodies, including:

[0034] Figure 1 The results of an ELISA assay testing the binding activity of some anti-PVRIG sdAbs to human PVRIG protein are shown.

[0035] Figure 2This study demonstrates the ability of some anti-PVRIG sdAbs to inhibit the binding of human PVRIG to PVRL2 compared to IgG4 isotype control antibodies. The anti-PVRIG antibody dilution was mixed with huPVRIG-hFC-biotin at 4°C for 1 h. HEK293 cells overexpressing human PVRL2 were diluted in FACS buffer and incubated with the antibody mixture for 1 h. The ability of the anti-PVRIG antibodies to block the binding of PVRIG to PVRL2 was assessed by the mean fluorescence intensity (MFI) of PE-labeled streptavidin.

[0036] Figure 3 The dose-dependent binding of some anti-PVRIG sdAbs to cynomolgus monkey PVRIG, as assessed by ELISA, was demonstrated.

[0037] Figure 4 This study describes how anti-PVRIGsdAb enhances NFAT promoter-driven luciferase expression in Jurkat-PVRIG T cells upon stimulation with CHOK1 cells expressing anti-CD3 scFV and CD112 on the cell surface.

[0038] Figures 5A-5B Epitope binning results for selective anti-PVRIG sdAbs are shown. Specifically, 5A shows a competitive ELISA of P-AIL-15 against other reference antibodies for the hu-PVRIG-his protein. Incubation with 10 mg / ml P-AIL-15 was observed not to block the binding of the reference Ab to the hu-PVRIG-his protein. 5B shows a competitive ELISA of P-AIL-32 against other reference antibodies for the hu-PVRIG-his protein, revealing that incubation with 10 mg / ml P-AIL-32 partially blocked the binding of the reference Ab to the hu-PVRIG-his protein.

[0039] Figures 6A-6B Characterization of the humanized variant of P-AIL-A15 is shown. 6A. The humanized variant of P-AIL-A15 enhances NFAT promoter-driven luciferase expression in Jurkat-PVRIG T cells upon stimulation with CHOK1 cells expressing anti-CD3 scFV and CD112 on the cell surface. 6B. The role of the humanized variant of P-AIL-A15 in enhancing NK cell-mediated cytotoxicity against MOLM-13 cells is shown.

[0040] Figure 7 The results showed that when CHOK1 cells expressing anti-CD3 scFV and CD112 on the cell surface were stimulated, the humanized variant of P-AIL-A32 enhanced NFAT promoter-driven luciferase expression in Jurkat-PVRIG T cells.

[0041] Figures 8A-8B The characteristics of the P-AIL-A32 PTM variant are shown. Figure 8A The exemplary P-AIL-A32hu17 PTM variant demonstrates blocking activity against the interaction between human PVRIG and PVRL2 compared to the IgG4 isotype control antibody. Figure 8B This demonstrates that when CHOK1 cells expressing anti-CD3 scFV and CD112 on the cell surface are stimulated, the exemplary P-AIL-A32hu17PTM enhances NFAT promoter-driven luciferase expression in Jurkat-PVRIG T cells.

[0042] Figure 9 This demonstrates that when CHOK1 cells expressing anti-CD3 scFV and CD112 on the cell surface are stimulated, the P-AIL-A32 affinity maturation variant enhances NFAT promoter-driven luciferase expression in Jurkat-PVRIG T cells.

[0043] Figure 10 The role of the P-AIL-A32 affinity maturation variant in enhancing NK cell-mediated cytotoxicity against MOLM-13 cells was demonstrated.

[0044] Figure 11 The binding activity of the P-AIL-A32 affinity maturation variant against cyno-PVRIG was described. ELISA was performed by coating 96-well ELISA plates with 2 µg / ml cyno-PVRIG-Fc protein.

[0045] Figure 12-Figure 1 8 involves anti-TIGIT antibodies, among which:

[0046] Figure 12 The binding activity of several exemplary anti-TIGIT antibodies against engineered 293F cells overexpressing human TIGIT is described. Data show that the absolute MFI (MFI) measurement as a function of increasing antibody concentration indicates the strength of the antibody's binding affinity to TIGIT.

[0047] Figure 13 This study demonstrates the ability of an exemplary anti-TIGIT antibody to block the interaction between human TIGIT and PVR. Anti-TIGIT antibody dilution was mixed with HEK293 cells overexpressing huTIGIT at room temperature for 1 h. Then, huCD155-ECD-mFC was added to the cells and incubated at 4 °C for 1 h. The ability of the anti-TIGIT antibody to block TIGIT binding to PVR was assessed by the mean fluorescence intensity (MFI) of PE-labeled streptavidin.

[0048] Figure 14 This paper describes how some exemplary anti-TIGIT mAb enhances NFAT promoter-driven luciferase expression in Jurkat / TIGIT / CD226 T cells upon stimulation with CHOK1 cells expressing anti-CD3 scFV and PVR on the cell surface.

[0049] Figure 15A The exemplary anti-TIGIT mAb was shown to bind in a dose-dependent manner to 293F cells engineered to overexpress cynomolgus monkey TIGIT. Figure 15B This demonstrates the ability of an exemplary anti-TIGIT mAb to block the interaction between cyno-TIGIT and cyno-PVR.

[0050] Figure 16 This study demonstrated the effect of anti-TIGIT monoclonal antibody in enhancing primary NK cell-mediated cytotoxicity against HSCs. Primary NK cells were co-cultured with calcein-AM-labeled HSCs at a ratio of 10:1 for 4 hours in the presence of anti-TIGIT antibody. After co-culture, cells were lysed for fluorescence detection.

[0051] Figure 17 The exemplary anti-TIGIT antibody enhances IL-2 release in Jurkat / TIGIT cells in response to CHOK1 cells that stably express anti-CD3 scFV and PVR on their cell surface.

[0052] Figures 18A-18C Characterization of humanized anti-TIGIT monoclonal antibodies T-MIL-A60 and T-MIL-A169 is shown. Figure 18A Blocking data for the humanized T-MIL-A60 variant are shown. Figure 18B This study demonstrates that the humanized variant of T-MIL-A60 enhances NFAT promoter-driven luciferase expression in Jurkat-TIGIT cells in response to CHO cells that stably express anti-CD3 scFV and CD155 on their cell surface. Figure 18C The NFAT-luciferase data for the humanized T-MIL-A169 antibody are shown.

[0053] Figures 19-25 This involves bispecific antibodies against PVRIG and TIGIT, among which:

[0054] Figure 19 The structures of some exemplary forms of the bispecific antibodies of the present invention are shown.

[0055] Figures 20A-20B The exemplary bispecific antibody was shown to bind in a dose-dependent manner to TIGIT or PVRIG in cynomolgus monkeys. Figure 20AThe binding ability of the bispecific antibody to 293F cells overexpressing cyno-TIGIT was demonstrated. Figure 20B The binding ability of an exemplary bispecific antibody to cynomolgus monkey PVRIG, as evaluated by ELISA, is shown.

[0056] Figures 21A-21B The effect of an exemplary bispecific antibody on enhancing NFAT promoter-driven luciferase expression is shown. Figure 21A This demonstrates an exemplary bispecific antibody that enhances NFAT promoter-driven luciferase expression in Jurkat / TIGIT / CD226 T cells that stably express anti-CD3 scFV and PVR on the cell surface of CHO cells. Figure 21B This demonstrates an exemplary bispecific antibody that enhances NFAT promoter-driven luciferase expression in Jurkat / PVRIG T cells that stably express CHO cells with anti-CD3 scFV and PVRL2 on their cell surface.

[0057] Figure 22 This demonstrates an exemplary bispecific antibody-enhanced response in primary CD8 cells of CHOK1 cells stably expressing anti-CD3 scFV, PVR, and PVRL2 on their cell surface. + IFN-γ release in T cells.

[0058] Figures 23A-23B The exemplary bispecific antibody was demonstrated to enhance NK-92MI cell-mediated cytotoxicity against MOLM-13 or A375 cells. Figure 23A The protein expression of PVR and PVRL2 on MOLM-13 cells was shown by FACS. Figure 23B The study demonstrates a cytotoxicity assay in which NK-92MI cells were co-cultured with calcein-AM-labeled MOLM-13 cells or A375 cells for 4 hours in the presence of an exemplary bispecific antibody, and the cells were lysed after co-culture for fluorescence detection.

[0059] Figure 24This study demonstrated the activity of PVRIG, TIGIT, and PD-L1 blockade as monotherapy and in combination therapy in human CD8+ T cells. In a co-culture system consisting of primary CD8+ T cells and CHOK1 / PD-L1 / CD155 / CD112 / OKT3 cells, the combination of the bispecific antibody BsAb05 and P-AIL-A32 sdAb with T-MIL-A60 mAb enhanced LAE005-induced IFNγ release from primary human CD8+ T cells. Compared to the combination of P-AIL-A32 sdAb and T-MIL-A60 mAb, BsAb05 exhibited a stronger effect in increasing cytokine production.

[0060] Figure 25 The PVRIG / TIGIT bispecific antibodies BsAb05 or BsAb17 were shown to enhance NK cell-mediated antibody-dependent cytotoxicity (ADCC) against human malignant melanoma A375 cell lines serially diluted with cetuximab or ervantuximab.

[0061] Figure 26 shows the plasma concentration-time curves for BsAb05 and BsAb17. The antibodies were injected into the tail vein of mice at a dose of 10 mg / kg, and blood was collected continuously. Serum concentrations were determined by ELISA using coatings of 2 mg / ml huPVRIG-his (26A) or 2 mg / ml huTIGIT-his (26B) protein.

[0062] Figures 27A-27C This study demonstrated the effects of the anti-PVRIG / TIGIT bispecific antibody on tumor volume, tumor weight, and TIGIT expression in CD8+ TILs in an A375 xenograft model with human PBMCs. Human PBMCs were co-cultured with MMC-treated A375 cells for 5 days. Activated huPBMCs were then seeded into NSG mice along with fresh A375 cells. When the tumor volume reached 50-80 mmHg... 3 At that time, the mice were randomly divided into 4 groups (n=8). Figure 27A and Figure 27B The effects of the experimental antibody on tumor volume and tumor weight were shown. Figure 27C The surface expression of TIGIT on CD8+ TILs was shown after treatment with experimental antibodies or isotype controls. BsAb05 and BsAb17 exhibited significant antitumor activity in the HubBMC A375 xenograft model. P<0.05, P<0.01, P<0.0001. Detailed Implementation

[0063] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any materials and methods similar to or equivalent to those described herein may be used to practice this invention. For definitions and terminology in the art, as well as other methods known to those skilled in the art, practitioners may refer to Sambrook et al. (1989) *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Press, Plainsview, NY; Ausubel et al. (1999) *Current Protocols in Molecular Biology* (Supplement 47), John Wiley & Sons, New York; Murphy et al. (1995) *Virus Taxonomy*, Springer Verlag: 79-87; and Harlow and Lane. Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998).

[0064] 1. definition

[0065] The definitions and explanations of the relevant terms or expressions are as follows.

[0066] As used herein, the term “about” refers to a quantity, level, value, size, or amount that varies by as much as 10% (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) relative to a reference quantity, level, value, size, or amount.

[0067] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations such as “comprises” or “comprising” shall be understood to imply inclusion of the stated element or whole or group of elements or wholes, but not to exclude any other element or whole or group of elements or wholes.

[0068] "Composed of..." means including and limited to anything following the phrase "composed of...". Therefore, the phrase "composed of..." indicates that the listed element is necessary or mandatory, and no other elements exist. "Substantially composed of..." means including any element listed after this phrase, and is limited to other elements that do not interfere with or contribute to the activities or actions specified in the disclosure of the listed element. Therefore, the phrase "substantially composed of..." indicates that the listed element is necessary or mandatory, but other elements are optional and may be present or absent depending on whether they affect the activities or actions of the listed element.

[0069] As used herein, the singular forms “a,” “an,” and “the” include the plural aspect unless the context clearly indicates otherwise. For example, “host cell” includes a single host cell, as well as two or more host cells; and so on.

[0070] The terms “antibody,” “immunoglobulin,” and “Ig” are used interchangeably herein and in the broadest sense to include, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having multiple or single epitope specificity, recombinant antibodies, single-domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human forms of antibodies having full-length heavy and / or light chains. As used herein, VHH refers to a domain antibody derived from the variable region of a pure heavy chain antibody. Exemplary single-domain antibodies include, but are not limited to, antibodies naturally lacking a light chain, such as antibodies from camel species (e.g., llamas and alpacas), single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, alpacas, goats, rabbits, and cattle. VHH can also be derived from species other than camelids that produce naturally occurring heavy-chain antibodies lacking the light chain. Antibodies also include antibody fragments (and / or peptides containing antibody fragments) that retain PVRIG and / or TIGIT binding characteristics. Non-limiting examples of antibody fragments include antigen-binding and / or effector regions of antibodies, such as Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, bivariate domain IgG (DVD-Ig), single variable domain, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, biantibodies, bi-biantibodies, disulfide-linked Fvs (dsFv), single-domain antibodies (e.g., nanobodies), or other fragments (e.g., fragments composed of non-covalently coupled heavy and light chain variable regions). Generally, the variable (V) region domain can be any suitable arrangement of immunoglobulin heavy (VH) and / or light (VL) variable domains. For example, antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the V region domain can be a dimer containing VHH-VHH, VH-VH, VH-VL, or VL-VL dimers that bind PVRIG and / or TIGIT. If desired, VH and VL can be covalently coupled directly or via a linker to form a single-chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as being included in the category of “antibody fragments.” Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of the antibody. CDRs (also known as “minimum recognition units” or “hypervariates”) can be obtained by constructing polynucleotides encoding one or more target CDRs.For example, such polynucleotides can be prepared by using polymerase chain reaction to synthesize variable regions using the mRNA of antibody-producing cells as templates (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al., (ed.), p. 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (ed.), p. 137, Wiley-Liss, Inc. (1995)). For example, antibody fragments can also be incorporated into single-domain antibodies, macrobodies, microbodies, intracellular antibodies, biantibodies, triantibodies, tetraantibodies, variable domains (v-NARs) of neoantigen receptors, and double single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, antibodies containing VH and / or VL contain light chain and / or heavy chain constant regions, such as one or more constant regions, including one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, antibodies may include any of the epitope-binding fragments described above. The antibodies described herein can be any class of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0071] In this invention, "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein, which comprises two heavy (H) polypeptide chains and two light (L) polypeptide chains linked together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified as κ and λ light chains. The heavy chains can be classified as μ, δ, γ, α, and ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H 1. C H 2 and C H 3) Composition. Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L Composed of ) V H and V L The regions can be further divided into highly variable regions (called complementary determinant regions (CDRs)), which are separated from each other by relatively conservative regions (called framing regions (FRs)). Each V H and V L It consists of 3 CDRs and 4 FRs in the following order: from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V) of each heavy / light chain pair... H and V L These amino acids form antigen-binding sites. The distribution of amino acids in different regions or domains follows the definition in the Kabat sequence of the immunogenic protein (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-883. Antibodies can be different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0072] As used herein, the term "antibody" or "Ab" also includes single-domain antibodies (abbreviated as "sdAb"), which can be produced, for example, in camels and alpacas, or recombinantly. These are commonly referred to as VHH antibodies and contain only the heavy chain, with the same heavy chain structure as antibodies produced in mice or humans. VHH antibodies are also known in the art as nanobodies.

[0073] Variable regions and CDRs in antibody sequences can be identified according to general rules developed in the art (as described above, such as the Kabat numbering system) or by comparing the sequence with a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001, and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described on the “Abysis” website at www.bioinf.org.uk / abs (maintained by AC Martin of the Department of Biochemistry and Molecular Biology, University College London, London, England) and the VBASE2 website at www.vbase2.org, and are accessible through these websites, as described in Retter et al., Nucl. Acids Res., 33 (Database Issues): D671-D674 (2005). Preferably, the Abysis database is used for sequence analysis, which integrates sequence data from Kabat, IMGT, and the Protein Database (PDB), as well as structural data from the PDB. See Dr. Andrew C. Martin's book chapter. Protein Sequence and Structure Analysis of Antibody Variable Domains In: Antibody Engineering Lab Manual (Edited by: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available at bioinforg.uk / abs. The Abysis database website also includes general rules developed for identifying CDRs, which can be used in accordance with the teachings herein. Unless otherwise stated, all CDRs listed herein are derived according to the Kabat numbering system. However, CDRs of the antibodies of this invention defined by other numbering systems, such as the Chothia numbering system, the AbM numbering system, etc., are also included in this invention.)

[0074] The terms “residue numbering according to Kabat” or “amino acid position numbering as in Kabat” and their variations refer to the numbering system for heavy chain or light chain variable regions used in antibody editing, as described by Kabat et al. (see above). Using this numbering system, the actual linear amino acid sequence can contain fewer or more amino acids, corresponding to a shortening or insertion into the FR or CDR of the variable domain. For example, the heavy chain variable domain can contain a single amino acid insert after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). For a given antibody, the Kabat number of the residues can be determined by comparing the homologous regions of the antibody sequence with sequences numbered according to “standard” Kabat. When dealing with residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system (e.g., Kabat et al., ibid.) is typically used. When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is commonly used (e.g., the EU index reported by Kabat et al., ibid.). “EU index as in Kabat” refers to the residue numbering of human IgG1 EU antibodies. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0075] As used herein, the term "heavy chain," when used to refer to antibodies, refers to a polypeptide chain of approximately 50–70 kDa, wherein the N-terminal portion comprises a variable region of approximately 120–130 or more amino acids, and the C-terminal portion comprises a constant region. Based on the amino acid sequence of the heavy chain's constant region, the constant region can be one of five different types (e.g., isotypes) known as alfa (α), delta (δ), epsilon (ε), gamma (γ), and muon (μ). The different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while µ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains respectively produce five well-known antibody classes (e.g., isotypes): IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4.

[0076] As used herein, the term "light chain" when referring to antibodies refers to a polypeptide chain of approximately 25 kDa, wherein the N-terminal portion comprises a variable region of approximately 100 to approximately 110 or more amino acids, and the C-terminal portion comprises a constant region. Light chains are approximately 211 to 217 amino acids in length. Based on the amino acid sequence of the constant domain, there are two distinct types, referred to as kappa (κ) or lamuda (λ).

[0077] The CDR region is well known to those skilled in the art and has been defined by well-known numbering systems. For example, the Kabat Complementarity Determination Region (CDR) is based on sequence variability and is the most commonly used (see, for example, Kabat et al., ibid.; Nick Deschacht et al., J Immunol 2010; 184:5696-5704). Chothia, on the other hand, refers to the position of the structural loop (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia HCDR1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; if both 35A and 35B exist, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop, and was modeled using Oxford Molecular's AbM antibody modeling software (see, for example, [link to software]). Antibody Engineering Volume 2 (edited by Kontermann and Dübel, second edition 2010) uses the "contact" hypervariable region based on the analysis of existing complex crystal structures. Another widely adopted and developed general-purpose numbering system is the ImMunoGeneTics (IMGT) Information System. ®(Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is a comprehensive information system focusing on immunoglobulins (IG), T cell receptors (TCR), and major histocompatibility complex (MHC) in humans and other vertebrates. In this paper, CDRs are referred to in terms of both amino acid sequence and position within the light or heavy chain amino acids. Since the “position” of CDRs in the variable domain structure of immunoglobulins is conserved across species and exists in structures called loops, CDRs and framework residues can be easily identified by using a numbering system that aligns variable domain sequences based on structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into receptor frameworks typically derived from human antibodies. Honegger and Plückthun, J. Mol. Biol. 309: 657-70 (2001) have developed another numbering system (AHon). The correspondences between numbering systems, including, for example, the Kabat numbering and the IMGT unique numbering system, are well known to those skilled in the art (see, for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.). The following table illustrates, exemplified, residues from each of these hypervariable regions or CDRs according to various numbering systems.

[0078] Exemplary CDRs based on various numbering systems

[0079]

[0080] The boundaries of a given CDR can vary depending on the protocol used for identification. Therefore, unless otherwise stated, the terms "CDR" and "complementation-determining region" for a given antibody or its regions, such as variable regions, and for a single CDR of an antibody or its regions (e.g., HCDR1, HCDR2), should be understood to include the complementation-determining region as defined by any of the known protocols described above. In some cases, a protocol for identifying a particular CDR or CDR, such as a CDR defined by the IMGT, Kabat, Chothia, or Contact method, is specified. In other cases, a specific amino acid sequence of the CDR is given. It should be noted that CDR regions can also be defined by combinations of various numbering systems (e.g., ...). ， The CDR can be defined using a combination of the Kabat and Chothia numbering systems, or a combination of the Kabat and IMGT numbering systems. Therefore, terms such as “CDR1 as shown in a particular VH” include, but are not limited to, any CDR1 as defined by the exemplary CDR numbering systems described above. Once a variable zone (e.g., VH or VL) is given, those skilled in the art will understand that CDRs within that zone can be defined by different numbering systems or combinations thereof.

[0081] The high-variable region may include the following "extended high-variable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH.

[0082] The term "constant region" or "constant domain" refers to the carboxyl-terminal portion of the light and heavy chains, which does not directly participate in antibody-antigen binding but exhibits various effector functions, such as interaction with Fc receptors. The term also refers to the portion of an immunoglobulin molecule with a more conserved amino acid sequence compared to other parts of the immunoglobulin (i.e., the variable regions containing antigen-binding sites). Constant regions may include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0083] The term "frame" or "FR" refers to the variable domain residues flanking the CDR. FR residues are found in, for example, chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, biantibodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region residues or CDR residues.

[0084] The antibodies in this invention include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, monovalent antibodies, bivalent or multivalent antibodies, single-domain antibodies, monospecific antibodies, bispecific or multispecific antibodies, single-chain antibodies, nanobodies, etc.

[0085] The terms “antigen-binding portion” or “antigen-binding fragment” are used interchangeably in the specification of this application and refer to a polypeptide containing a full-length antibody fragment that retains the ability to specifically bind to the antigen specifically bound by the full-length antibody and / or competes with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Under certain conditions, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and such polypeptides that contain at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., the antibody against PVRIG or TIGIT provided in this application) by conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) and their specificity can be screened in the same manner as intact antibodies.

[0086] As used herein, the term "monoclonal antibody" or "mAb" refers to a formulation of an antibody molecule consisting of a single molecule. Monoclonal antibodies exhibit single binding specificity and affinity for a specific epitope.

[0087] The term "humanized antibody" refers to an antibody in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human frame sequence. Further frame region modifications can be performed within the human frame sequence.

[0088] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0089] As used herein, the term “recombinant antibody” refers to an antibody prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from animals that are transgenic with respect to immunoglobulin genes of another species, antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from a library of recombinant combined antibodies, or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of immunoglobulin gene sequences with other DNA sequences.

[0090] As used herein, the term “monospecific” for antibodies refers to an antibody having one or more binding sites, each of which binds the same epitope of the same antigen.

[0091] As used herein, the term "multispecific antibody" refers to an antibody capable of binding to two or more antigenic determinants (e.g., epitopes) through two or more distinct antigen-binding sites, each antigen-binding site being formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), or each antigen-binding site being formed by a pair of VHH domains binding to different antigens or different epitopes on the same antigen. In some embodiments, a multispecific antibody is capable of binding two or more antigenic determinants (e.g., epitopes) simultaneously. In some embodiments, a multispecific antibody binds one or more antigens (or epitopes) on one of its binding arms (a pair of heavy / light chains) and binds one or more different antigens (or epitopes) on its second binding arm (a different pair of heavy / light chains). In some embodiments, a multispecific antibody may have two or more distinct antigen-binding arms (in terms of both specificity and CDR sequence) and is monovalent for each antigen it binds. Multispecific antibodies include, for example, antibodies produced by quadromatechnology (Milstein & Cuello (1983) Nature 305(5934):537-40), by chemical conjugation of two or more different monoclonal antibodies (Staerz et al. (1985) Nature 314(6012):628-31), or by introducing a mutant into the Fc region via a knob-into-hole or similar method (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA90(14): 6444–6448). Recently, various recombinant multispecific antibody forms have been developed, for example, by fusing IgG antibodies with two or more single-chain domains (see, Kontermann RE, mAbs 4:2, (2012) 1-16). WO2009080251 and WO2009080252 describe multispecific antibodies in which the variable domains VL and VH or the constant domains CL and CH1 are replaced with each other. The term "multispecific antibody" in this invention encompasses bispecific antibodies, trispecific antibodies, etc.

[0092] As used herein, the term "bispecific antibody" refers to an antibody capable of binding to two antigenic determinants (e.g., epitopes) through two distinct antigen-binding sites (e.g., two binding sites formed by an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), each binding a different antigen). In some embodiments, a bispecific antibody is capable of binding to two antigenic determinants (e.g., epitopes) simultaneously. In some embodiments, a bispecific antibody binds to one antigen (or epitope) on one of its binding arms (a pair of heavy / light chains) and to a different antigen (or epitope) on its second binding arm (a different pair of heavy / light chains). Such a bispecific antibody may have a 1+1 form (containing a binding site for a first antigen or epitope and a binding site for a second antigen or epitope). Other forms of bispecific antibodies may be 2+1 or 1+2 (containing two binding sites for a first antigen or epitope and a binding site for a second antigen or epitope) or 2+2 (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific antibody contains two antigen-binding sites, each site can bind a different epitope. Such a bispecific antibody can bind two different epitopes on the same antigen (e.g., epitopes on PVRIG or TIGIT). In some embodiments, a "bispecific" antibody can specifically bind two different antigenic determinants through, for example, two binding sites formed by an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), each binding a different antigen. In some embodiments, a bispecific antibody may have two different antigen-binding arms (in terms of both specificity and CDR sequence) and is monovalent for each antigen it binds to. Bispecific antibodies include, for example, antibodies produced by tetravalent somatic tumor technology (Milstein & Cuello (1983) Nature 305(5934):537-40), by chemical conjugation of two different monoclonal antibodies (Staerz et al. (1985) Nature 314(6012):628-31), or by introducing a mutated knob-in-hole or similar method into the Fc region (Holliger et al. (1993) Proc.Natl.Acad.Sci.USA90(14): 6444–6448). Recently, various recombinant bispecific antibody forms have been developed, for example, by fusing two or more single-chain domains with IgG antibody forms (see, Kontermann RE, mAbs 4:2, (2012) 1-16). Bispecific antibodies in which the variable domains VL and VH or the constant domains CL and CH1 are replaced by each other are described in WO2009080251 and WO2009080252.

[0093] As used herein, the terms "anti-PVRIG antibody," "aPVRIG antibody," or "an antibody against PVRIG" refer to an antibody capable of binding PVRIG (e.g., human or mouse PVRIG) as defined herein. These terms are used interchangeably in this invention. Preferably, the anti-PVRIG antibody is a single-domain antibody (sdAb) or a VHH-type antibody.

[0094] As used herein, the terms "anti-TIGIT antibody," "TIGIT antibody," or "antibody against TIGIT" refer to an antibody capable of binding TIGIT (e.g., human or mouse TIGIT) as defined herein. These terms are used interchangeably in this invention.

[0095] As used in this article, the term "high affinity" for IgG antibodies refers to the affinity for the K+ of target antigens such as PVRIG or TIGIT. D 1 x 10 -7 M or lower, preferably 5 x 10 -8 M or lower, or even better 1x10 -8 M or lower, or even better, 5 x 10 -9 M or lower, or even better, 1 x 10 -9 M or lower antibodies.

[0096] The “on-rate”, “rate of association”, “association rate”, or “kon” of the specific antibody-antigen interaction according to this application can be determined using surface plasmon resonance technology, such as BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ).

[0097] The "off-rate" or "rate of dissociation" or "koff" of the specific antibody-antigen interaction according to this application can also be determined using surface plasmon resonance technology.

[0098] As used in this article, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.

[0099] As used in this article, the term "EC" 50 Also known as the "half-maximum effective concentration," it refers to the concentration of a drug, antibody, or agent that induces half the response between baseline and maximum after a specific exposure time. In the specification of this application, EC... 50 It is expressed in units of “nM”.

[0100] As used herein, the term "competitive binding" refers to the interaction between two antibodies in their binding to their target. If the binding of the first antibody to its homologous epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody to its homologous epitope in the absence of the second antibody, then the first and second antibodies compete for binding. Alternatively, the binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody; this is possible but not guaranteed. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody may not inhibit the binding of the first antibody to its corresponding epitope. However, when each antibody detectably inhibits the binding of another antibody to its homologous epitope, regardless of the degree of inhibition—whether equal, greater, or less—the antibodies are said to "cross-competitively" bind to one or more of their respective epitopes.

[0101] As used herein, the ability to "inhibit binding" refers to the ability of an antibody or its antigen-binding fragment to inhibit the binding of two molecules (e.g., human PVRIG and anti-PVRIG antibody, or human TIGIT and anti-TIGIT antibody) to any detectable level. In some embodiments, the binding of the two molecules can be inhibited by the antibody or its antigen-binding fragment by at least 50%. In some embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0102] As used herein, the term "separated" refers to a state obtained artificially from its natural state. If a "separated" substance or component exists in nature, it may be due to changes in its natural environment, the separation of the substance from its natural environment, or both. For example, an unseparated polynucleotide or polypeptide naturally exists in a living animal; a high-purity copy of the same polynucleotide or polypeptide separated from this natural state is called a separated polynucleotide or polypeptide. The term "separated" does not exclude the presence of artificial or synthetic mixtures, nor does it exclude other impurities that do not affect the activity of the separated substance.

[0103] As used herein, the term "vector" refers to a nucleic acid vector in which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. A vector may have elements of carried genetic material that are expressed in a host cell by transformation, transduction, or transfection into a host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (such as SV40 virus). A vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may contain an origin of replication.

[0104] As used herein, the term "host cell" refers to a cellular system that can be engineered to produce a target protein, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term includes not only the specific test cell but also its progeny. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may differ from the parent cells but are still included within the scope of the term "host cell."

[0105] As used herein, the terms “homology” or “identity” refer to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence alignment and comparison. “Identity percentage” refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably resolved using a specific mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, ed.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., ed.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAMJ. Applied Math. 48:1073.

[0106] As used in this article, the term "humanization" refers to altering certain residues in the constant region of an animal-derived antibody to better conform its structure to that of a human antibody. "Humanization" produces antibodies with lower immunogenicity that fully retain the antigen-binding properties of the original molecule. To preserve all the antigen-binding properties of the original antibody, the structure of its binding site must be faithfully replicated in the "humanized" form. This could potentially be achieved by transplanting the binding site of a nonhuman antibody onto the human framework in the following ways: (a) by transplanting the entire nonhuman variable region onto the human constant region to generate a chimeric antibody (Morrison et al., Proc. Natl. Acad. Sci., USA 81:6801 (1984); Morrison and O1, Adv. Immunol. 44:65 (1988) (which preserves ligand-binding properties but also the immunogenicity of the nonhuman variable domain); (b) by transplanting only the nonhuman CDR into the human framework and constant region, with or without preserving key framework residues (Jones et al., Nature, 321:522 (1986); Verhoeyen et al., Science 239:1539 (1988)); or (c) by transplanting the entire nonhuman variable domain (to preserve ligand-binding properties) and by intelligently replacing exposed residues with a human-like surface (to reduce antigenicity) (Padlan, Molec. Immunol. 28:489 (1991)).

[0107] Humanization via CDR transplantation typically involves transplanting only the animal fragment's CDR onto the human framework and constant regions. Theoretically, this should essentially eliminate immunogenicity (unless allotype or idiotype differences are present). However, some framework residues of the original antibody have been reported to need to be retained (Riechmann et al., Nature 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10,029 (1989)). The framework residues that need to be retained can be identified through computer modeling. Alternatively, key framework residues can potentially be identified by comparing them to known antibody-binding site structures (Padlan, Molec. Immun. 31(3):169-217 (1994)). The present invention also includes partially humanized antibodies, wherein six CDRs and a limited number of structural amino acids of the heavy and light chains of a mouse monoclonal antibody are transplanted into a human IgG scaffold lacking CDRs by recombinant technology (Jones et al., Nature 321:522-525 (1986)).

[0108] The term "affinity maturation" or "affinity-matured antibody" refers to an antibody that has one or more alterations in one or more hypervariable regions, resulting in increased affinity for the antigen compared to a parent antibody without such alterations. Preferred affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced by methods known in the art. Marks et al., Bio / Technology 10:779-783 (1992), describe affinity maturation via VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described below: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al. J. Immunol. 154(7):3310-9 (1995); and Hawkins et al. J. Mol. Biol 226: 889-896 (1992).

[0109] As used herein, the term "transfection" refers to a method by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. In a specific embodiment, the human ActRIIB gene is transfected into 293F cells.

[0110] As used herein, the terms “hybridoma” and “hybridoma cell line” are used interchangeably. When referring to the terms “hybridoma” and “hybridoma cell line,” they also include subclones and progeny cells of the hybridoma.

[0111] As used herein, the term “SPR” or “surface plasmon resonance” refers to and includes optical phenomena that allow for the analysis of real-time, biospecific interactions by detecting changes in protein concentration in a biosensor matrix, for example, using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see Example 5 and Jönsson, U. et al. (1993). Ann. Biol. Clin. 51:19-26; Jönsson, U. et al. (1991) Biotechniques 11:620-627; Johnsson, B. et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B. et al. (1991) Anal. Biochem. 198:268-277.

[0112] As used herein, the term “subject” refers to any vertebrate, including mammals and birds, especially humans, that requires treatment or diagnosis according to the present invention.

[0113] As used in this article, the term “cancer” refers to any solid tumor or non-solid tumor mediated by the growth, proliferation, or metastasis of malignant cells, such as leukemia, and causes medical symptoms.

[0114] As used in the context of treating a disease, the terms "treatment," "treating," or "treated" generally refer to the treatment and therapy of a person or animal in which some desired therapeutic effect is achieved, such as inhibiting the progression of the disease, including a reduction in the rate of progression, cessation of the rate of progression, regression of the disease, improvement of the disease, and cure of the disease. It also includes treatment as a preventative measure (i.e., prevention, avoidance). In the context of cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cells, or some combination thereof. For tumors, "treatment" includes the removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0115] As used herein, the term "effective amount" refers to an amount of an active compound or a material, composition, or dosage form containing the active compound that, when administered according to the desired treatment regimen, is effective in producing a desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, "effective amount," when used to treat muscle metabolic disorders or ailments, refers to an antibody or its antigen-binding portion present at an amount or concentration that is effective in treating said disorder or ailment.

[0116] As used in this article, the terms “prevent,” “prevention,” or “preventing” refer to preventing or delaying the onset of a disease, or preventing the manifestation of its clinical or subclinical symptoms.

[0117] As used herein, the term "pharmaceutically acceptable" means that the medium, diluent, excipient and / or its salt are chemically and / or physically compatible with other components in the formulation and physiologically compatible with the recipient.

[0118] As used herein, the term “pharmaceutically acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, as is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro Ar, 19th edition, Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0119] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with or before an antigen, can enhance the immune response to the antigen or alter the type of immune response in the organism. There are various adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0120] Those skilled in the art will understand that this disclosure is open to variations and modifications, except those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any and all combinations of said steps or features, or any two or more said steps or features.

[0121] 2. Anti-PVRIG antibody

[0122] This invention relates to anti-PVRIG antibodies and their antigen-binding fragments. Specifically, this application relates to alpaca or camel antibodies against PVRIG, obtained using phage display technology and recombinant antibody technology. Such antibodies are all covered by this invention.

[0123] A. VHH antibody

[0124] VHH antibodies, also known as single-domain antibodies or nanobodies, are antigen-binding fragments derived from pure heavy-chain antibodies. These antibodies lack light chains and have been naturally found in various species, including camels and cartilaginous fish. This invention provides anti-PVRIG antibodies or antigen-binding fragments thereof comprising VHH fragments.

[0125] In some embodiments, the isolated anti-PVRIG antibody of the present invention comprises a heavy chain variable region (VH), which includes:

[0126] (1) Heavy chain CDR1 (HCDR1) of SEQ ID NO: 1, heavy chain CDR2 (HCDR2) of SEQ ID NO: 2 and heavy chain CDR3 (HCDR3) of SEQ ID NO: 3;

[0127] (2) HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7 and HCDR3 of SEQ ID NO: 8;

[0128] (3) HCDR1 of SEQ ID NO: 11, HCDR2 of SEQ ID NO: 12 and HCDR3 of SEQ ID NO: 13;

[0129] (4) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 17 and HCDR3 of SEQ ID NO: 18;

[0130] (5) HCDR1 of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22 and HCDR3 of SEQ ID NO: 23;

[0131] (6) HCDR1 of SEQ ID NO: 26, HCDR2 of SEQ ID NO: 27 and HCDR3 of SEQ ID NO: 28;

[0132] (7) HCDR1 of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32 and HCDR3 of SEQ ID NO: 33;

[0133] (8) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 36 and HCDR3 of SEQ ID NO: 18;

[0134] (9) HCDR1, HCDR2 and HCDR3 of the same antibodies as those listed in Tables 2, 4b and 4c.

[0135] (10) HCDR1, HCDR2 and HCDR3 are identical in all respects to HCDR1, HCDR2 and HCDR3 listed in one or more of the above (1) and (9) except that there are up to 5 (e.g. up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more HCDRs.

[0136] (11) HCDR of an anti-PVRIG antibody that binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(9) above; or

[0137] (12) HCDR of an anti-PVRIG antibody that competes with one of the antibodies defined in (1)-(9) above for binding to PVRIG.

[0138] The antibodies listed in Table 2 include: P-AIL-A8, P-AIL-A11, P-AIL-A15, P-AIL-A32, P-AIL-A82, P-AIL-B352, and P-AIL-C242.

[0139] The antibodies listed in Tables 4b and 4c include: P-AIL-A15-Hu01, P-AIL-A15-Hu02, P-AIL-A15-Hu03, P-AIL-A15-Hu04, P-AIL-A15-Hu06, P-AIL-A15-Hu07, P-AIL-A15-Hu08, P-AIL-A15-Hu09, P-AIL-A15-Hu10, P-AIL-A15-Hu11, and P-AIL-A3. 2-Hu09, P-AIL-A32-Hu10, P-AIL-A32-Hu11, P-AIL-A32-Hu12, P-AIL-A32-Hu13, P-AIL-A32-Hu14, P-AI L-A32-Hu15, P-AIL-A32-Hu16, P-AIL-A32-Hu17, P-AIL-A32-Hu18, P-AIL-A32-Hu19 and P-AIL-A32-Hu20.

[0140] In some embodiments, the anti-PVRIG antibody of the present invention comprises a heavy chain variable (VH) region, which includes, is substantially composed of, or is composed of the following amino acid sequence:

[0141] (1) The amino acid sequences selected from the following groups: SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29 or SEQ ID NO: 34;

[0142] (2) An amino acid sequence identical to the amino acid sequence of the VH of the antibody selected from the antibodies listed in Table 2;

[0143] (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of (1) or (2) above;

[0144] (4) Compared with the amino acid sequence of (1) or (2) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids added, deleted and / or substituted amino acid sequences.

[0145] (5) An amino acid sequence identical to the VH of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(2) above; or

[0146] (6) An amino acid sequence identical to the amino acid sequence of the VH of the anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1)-(2) above for binding to PVRIG.

[0147] In some embodiments, the anti-PVRIG antibody of the present invention comprises a heavy chain, which contains, is substantially composed of, or is composed of the following amino acid sequence:

[0148] (1) The amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35;

[0149] (2) An amino acid sequence identical to the amino acid sequence of the heavy chain of an antibody selected from the antibodies listed in Table 3;

[0150] (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of (1) or (2) above;

[0151] (4) Compared with the amino acid sequence of (1) or (2) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids added, deleted and / or substituted amino acid sequences.

[0152] (5) An amino acid sequence identical to the amino acid sequence of the anti-PVRIG antibody heavy chain, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(2) above; or

[0153] (6) An amino acid sequence identical to the amino acid sequence of the anti-PVRIG antibody heavy chain, wherein the antibody competes with one of the antibodies defined in (1)-(2) above for binding to PVRIG.

[0154] Preferably, the anti-PVRIG antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region, said heavy chain variable region comprising:

[0155] (1) HCDR1 of SEQ ID NO: 11, HCDR2 of SEQ ID NO: 12 and HCDR3 of SEQ ID NO: 13;

[0156] (2) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 17 and HCDR3 of SEQ ID NO: 18;

[0157] (3) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 36 and HCDR3 of SEQ ID NO: 18;

[0158] (4) HCDR1, HCDR2 and HCDR3 of antibodies containing VH, wherein the VH contains, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 14, 19 or 48;

[0159] (5) HCDR1, HCDR2 and HCDR3 of antibodies P-AIL-A15, P-AIL-A32 and P-AIL-A32-Hu10 as shown in Table 2 or Table 4b.

[0160] (6) HCDR1, HCDR2 and HCDR3 are identical in all respects to HCDR1, HCDR2 and HCDR3 listed in one of (1)-(4) above, except that there are up to 5 (e.g. up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more HCDRs.

[0161] (7) A CDR of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(5) above; or

[0162] (8) A CDR of an anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1)-(5) above for binding to PVRIG.

[0163] More preferably, the anti-PVRIG antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region, which contains, is substantially composed of, or is composed of the following amino acid sequence:

[0164] (1) The amino acid sequence of SEQ ID NO: 14 or 19;

[0165] (2) The amino acid sequence of VH of antibody P-AIL-A15 or P-AIL-A32 as shown in Table 2;

[0166] (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence defined in (1) or (2) above;

[0167] (4) Compared with the amino acid sequence defined in (1) or (2) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acid additions, deletions and / or substitutions.

[0168] (5) An amino acid sequence identical to the amino acid sequence of the anti-PVRIG antibody heavy chain, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(2) above; or

[0169] (6) An amino acid sequence identical to the amino acid sequence of the anti-PVRIG antibody heavy chain, wherein the antibody competes with one of the antibodies defined in (1)-(2) above for binding to PVRIG.

[0170] In this invention, the percentage homology or identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4, which has been incorporated into the ALIGN program (version 2.0). Alternatively, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), using a Blossum 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com).

[0171] Alternatively or additionally, the protein sequences disclosed herein can also be used as “query sequences” to search public databases for, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) as described by Altschul et al. (1990) J. MoI. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. For gap alignments to be obtained for comparative purposes, gap BLAST can be used as described by Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0172] Preferably, the anti-PVRIG antibody or its antigen-binding fragment contains a heavy chain variable (VH) region comprising, substantially comprising, or consisting of the amino acid sequence of SEQ ID NO: 14 or 19.

[0173] In other embodiments, the amino acid sequence of the heavy chain variable region of the anti-PVRIG antibody may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequence shown above.

[0174] In some other embodiments, the isolated antibody or its antigen-binding moiety may contain amino acid modifications (e.g., conserved substitutions) in the variable regions of the heavy and / or light chains. It will be understood in the art that certain conserved sequence modifications that do not alter antigen binding can be made. See, for example, Brummell et al. (1993) Biochem 32:1180-8; deWildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O' Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0175] As described above, the term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain (e.g., a physically or functionally similar residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.)) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residues are preferably substituted with another amino acid residue from the same side chain family. The methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997), which are incorporated herein by reference).

[0176] B. Humanized anti-PVRIG antibody

[0177] This invention also relates to the humanized form of the aforementioned anti-PVRIG antibody (referred to as "humanized antibody") and its antigen-binding fragment. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "input" residues, which are typically derived from an "input" variable domain.

[0178] In some cases, humanized antibodies are constructed via CDR grafting, where the amino acid sequences of six CDRs of the VH and VL of a parental non-human antibody (e.g., rodent) or three CDRs of the VH of a parental VHH-type antibody (e.g., camel or alpaca antibody) are grafted onto the human antibody framework. For example, Padlan et al. ( FASEB J(9:133-139, 1995) determined that only about one-third of the residues in the CDR actually contact the antigen, and referred to these residues as "specificity-determining residues," or SDRs. In SDR transplantation techniques, only SDR residues are transplanted onto the human antibody framework (see, for example, Kashmiri et al., 9:133-139, 1995). Methods 36: 25-34, 2005). To maintain antibody binding and functional activity, additional frame region modifications can be performed within the human frame sequence.

[0179] The frame regions described in this paper are defined based on the boundaries of the CDR numbering system. In other words, if the CDR is determined by, for example, Kabat, IMGT, or Chothia, then the frame region is a variable region consisting of amino acid residues surrounding the CDR in the form of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. For example, FR1 is defined as the N-terminal amino acid residue of the CDR1 amino acid residue as defined by, for example, the Kabat, IMGT, or Chothia numbering system; FR2 is defined as the amino acid residue between the CDR1 and CDR2 amino acid residues as defined by, for example, the Kabat, IMGT, or Chothia numbering system; FR3 is defined as the amino acid residue between the CDR2 and CDR3 amino acid residues as defined by, for example, the Kabat, IMGT, or Chothia numbering system; and FR4 is defined as the C-terminal amino acid residue of the CDR3 amino acid residue as defined by, for example, the Kabat, IMGT, or Chothia numbering system.

[0180] The humanized antibodies of the present invention, combined with PVRIG, can be produced using techniques known to those skilled in the art (Zhang et al.). Molecular Immunology , 42(12): 1445-1451, 2005; Hwang et al., Methods , 36(1):35-42, 2005; Dall'Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today , 21(8): 397-402, 2000, and U.S. Patent Nos. 6,180,370, 6,054,927, 5,869,619, 5,861,155, 5,712,120 and 4,816,567).

[0181] Therefore, this invention provides a humanized form of the anti-PVRIG antibody as defined above and its antigen-binding fragment.

[0182] In some implementations, the humanized anti-PVRIG antibody includes a heavy chain variable region, said heavy chain variable region comprising:

[0183] (1) Contains the same heavy chain CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2, HCDR3) as the heavy chain CDR1, CDR2 and CDR3 regions of the antibodies selected from the antibodies listed in Table 1 and Table 4a.

[0184] (2) Contains the same heavy chain CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2, HCDR3) as the heavy chain CDR1, CDR2 and CDR3 regions of the antibodies selected from the antibodies listed in Table 2, Table 4b and Table 4c.

[0185] (3) The amino acid sequence of HCDR1, HCDR2 and HCDR3 is identical in all respects to the HCDR1, HCDR2 and HCDR3 listed in (1) or (2) above, except that there are up to 5 (e.g. up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more HCDRs.

[0186] (4) An amino acid sequence identical to the amino acid sequence of the heavy chain variable region of the antibody selected from the antibodies listed in Tables 2, 4b and 4c;

[0187] (5) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences listed in (1), (2) and (4) above, or

[0188] (6) Compared with one of the amino acid sequences listed in (1), (2) and (4) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acid sequences with addition, deletion and / or substitution.

[0189] (7) The amino acid sequence of the VH of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1), (2) and (4) above; or

[0190] (8) The amino acid sequence of VH of the anti-PVRIG antibody, which competes with one of the antibodies defined in (1), (2) and (4) above for binding to PVRIG.

[0191] Preferably, the humanized anti-PVRIG antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 of antibody P-AIL-A15 or P-AIL-A32 as listed in Table 1.

[0192] More preferably, the humanized anti-PVRIG antibody or its antigen-binding fragment comprises a VH, the VH comprising:

[0193] (1) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 36 and HCDR3 of SEQ ID NO: 18;

[0194] (2) Contains the same HCDR1, HCDR2 and HCDR3 amino acid sequences as the HCDR1, HCDR2 and HCDR3 regions of antibodies selected from those listed in Tables 4b and 4c;

[0195] (3) The amino acid sequence of HCDR1, HCDR2 and HCDR3 is identical in all respects to the HCDR1, HCDR2 and HCDR3 listed in (1) or (2) above, except that there are up to 5 (e.g. up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more HCDRs.

[0196] (4) An amino acid sequence identical to the amino acid sequence of the heavy chain variable region of the antibody selected from the antibodies listed in Tables 4b and 4c;

[0197] (5) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences listed in (1), (2) and (4) above.

[0198] (6) Compared with one of the amino acid sequences listed in (1), (2) and (4) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acid sequences with addition, deletion and / or substitution.

[0199] (7) An amino acid sequence identical to the amino acid sequence of the VH of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1), (2) and (4) above; or

[0200] (8) An amino acid sequence identical to the amino acid sequence of the VH of the anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1), (2) and (4) above for binding to PVRIG.

[0201] More preferably, the humanized anti-PVRIG antibody includes a heavy chain variable region containing the same amino acid sequence as the heavy chain variable region of the antibody selected from those listed in Tables 4b and 4c.

[0202] Most preferably, the humanized anti-PVRIG antibody comprises a heavy chain variable region comprising, substantially comprising, or comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 37-58.

[0203] In some embodiments, the humanized anti-PVRIG antibody comprises a heavy chain, which contains, is substantially composed of, or is composed of the following amino acid sequence:

[0204] (1) Select the amino acid sequence of the group consisting of SEQ ID NO: 59-80;

[0205] (2) An amino acid sequence identical to the amino acid sequence of the heavy chain of an antibody selected from the antibodies listed in Tables 4d and 4e;

[0206] (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences listed in (1) or (2) above.

[0207] (4) Compared with one of the amino acid sequences listed in (1) or (2) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acid sequences with addition, deletion and / or substitution.

[0208] (5) An amino acid sequence identical to the amino acid sequence of the anti-PVRIG antibody heavy chain, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1) or (2) above; or

[0209] (6) An amino acid sequence identical to the amino acid sequence of the anti-PVRIG antibody heavy chain, wherein the antibody competes with one of the antibodies defined in (1) or (2) above for binding to PVRIG.

[0210] C. Affinity-matured anti-PVRIG antibodies

[0211] In some implementations, antibodies may be affinity-matured to improve their properties, such as affinity, stability, or expression levels, compared to their respective parent antibodies. As with the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. Antibody libraries are displayed on the surface of an organism (e.g., bacteriophage, bacteria, yeast, or mammalian cells) or bound to their encoding mRNA or DNA (e.g., covalently or non-covalently). Affinity selection of displayed antibodies allows the isolation of organisms or complexes carrying the genetic information encoding the antibody. Two or three rounds of mutation and selection using display methods such as phage display typically produce antibody fragments with affinity in the low nanomolar range. Affinity-matured antibodies can have nanomolar or even picomolar affinity for the target antigen.

[0212] Phage display is a widely used method for displaying and selecting antibodies. Antibodies are displayed on the surface of Fd or M13 phages as fusions of phage coat proteins. Selection involves exposing the phage to an antigen to allow the displayed antibody to bind to its target; this process is called “panning.” Phages bound to the antigen are recovered and used to infect bacteria to generate phages for further selection. For reviews, see, for example, Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002) and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0213] As described above, the terms “affinity maturation” and “affinity-matured” used in the context of antibodies refer to an antibody having one or more alterations in one or more hypervariable regions, and the one or more alterations resulting in increased affinity of the antibody for the antigen compared to a parent antibody that does not have said one or more alterations.

[0214] Therefore, the present invention provides an affinity-matured form of the anti-PVRIG antibody and its antigen-binding fragment as described above, wherein one or more amino acid residues in one or more hypervariable regions of the antibody are altered to enhance their binding affinity to the homologous antigen PVRIG.

[0215] In some embodiments, the affinity-matured anti-PVRIG antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 of the antibodies listed in Table 5a or Table 5b. Preferably, the affinity-matured anti-PVRIG antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region, said heavy chain variable region comprising:

[0216] (1) HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 82 and HCDR3 of SEQ ID NO: 18;

[0217] (2) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 83 and HCDR3 of SEQ ID NO: 18;

[0218] (3) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 84 and HCDR3 of SEQ ID NO: 18;

[0219] (4) HCDR1 of SEQ ID NO: 85, HCDR2 of SEQ ID NO: 86 and HCDR3 of SEQ ID NO: 18;

[0220] (5) HCDR1 of SEQ ID NO: 87, HCDR2 of SEQ ID NO: 88 and HCDR3 of SEQ ID NO: 18;

[0221] (6) HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 89 and HCDR3 of SEQ ID NO: 18;

[0222] (7) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 90 and HCDR3 of SEQ ID NO: 18;

[0223] (8) HCDR1 of SEQ ID NO: 91, HCDR2 of SEQ ID NO: 92 and HCDR3 of SEQ ID NO: 18;

[0224] (9) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 93 and HCDR3 of SEQ ID NO: 18;

[0225] (10) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 82 and HCDR3 of SEQ ID NO: 18;

[0226] (11) HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 84 and HCDR3 of SEQ ID NO: 18;

[0227] (12) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 96 and HCDR3 of SEQ ID NO: 18;

[0228] (13) HCDR1 of SEQ ID NO: 97, HCDR2 of SEQ ID NO: 98 and HCDR3 of SEQ ID NO: 18;

[0229] (14) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 99 and HCDR3 of SEQ ID NO: 18;

[0230] (15) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 100 and HCDR3 of SEQ ID NO: 18;

[0231] (16) HCDR1 of SEQ ID NO: 101, HCDR2 of SEQ ID NO: 99 and HCDR3 of SEQ ID NO: 18;

[0232] (17) HCDR1 of SEQ ID NO: 102, HCDR2 of SEQ ID NO: 103 and HCDR3 of SEQ ID NO: 18;

[0233] (18) HCDR1 of SEQ ID NO: 104, HCDR2 of SEQ ID NO: 105 and HCDR3 of SEQ ID NO: 18;

[0234] (19) HCDR1 of SEQ ID NO: 106, HCDR2 of SEQ ID NO: 105 and HCDR3 of SEQ ID NO: 18;

[0235] (20) HCDR1 of SEQ ID NO: 97, HCDR2 of SEQ ID NO: 107 and HCDR3 of SEQ ID NO: 18;

[0236] (21) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 108 and HCDR3 of SEQ ID NO: 18;

[0237] (22) HCDR1 of SEQ ID NO: 94, HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 18;

[0238] (23) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 110 and HCDR3 of SEQ ID NO: 18;

[0239] (24) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 98 and HCDR3 of SEQ ID NO: 18;

[0240] (25) HCDR1 of SEQ ID NO: 97, HCDR2 of SEQ ID NO: 100 and HCDR3 of SEQ ID NO: 18;

[0241] (26) An amino acid sequence comprising heavy chain CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2, HCDR3), wherein the heavy chain CDR1, CDR2 and CDR3 regions are identical to the heavy chain CDR1, CDR2 and CDR3 regions of antibodies selected from the antibodies listed in Tables 6a and 6b.

[0242] (27) HCDR1, HCDR2 and HCDR3 are identical in all respects to HCDR1, HCDR2 and HCDR3 listed in one of (1)-(26) above, except that there are at most 5 (e.g., at most 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more HCDRs.

[0243] (28) HCDR1, HCDR2, and HCDR3 of anti-PVRIG antibodies, said antibodies binding to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(26) above; or

[0244] (29) HCDR1, HCDR2 and HCDR3 of anti-PVRIG antibodies, said antibodies competing with one of the antibodies defined in (1)-(26) above for binding to PVRIG.

[0245] Preferably, the affinity-matured anti-PVRIG antibody or its antigen-binding fragment comprises a heavy chain variable region, which contains, is substantially composed of, or is composed of the following amino acid sequence:

[0246] (1) The amino acid sequences of the antibodies listed in Table 6a are selected;

[0247] (2) Select the amino acid sequence of the group consisting of SEQ ID NO: 111-123;

[0248] (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to (1) or (2) above.

[0249] (4) An amino acid sequence having one or more added, deleted and / or substituted amino acids compared to the amino acid sequence of (1) or (2) above;

[0250] (5) The amino acid sequence of the VH of the anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(2) above; or

[0251] (6) The amino acid sequence of the VH of the anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1)-(2) above for binding to PVRIG.

[0252] More preferably, the affinity-matured anti-PVRIG antibody or its antigen-binding fragment comprises a heavy chain, which contains, is substantially composed of, or is composed of the following amino acid sequence:

[0253] (1) The amino acid sequences of the antibodies listed in Table 6b are selected;

[0254] (2) Select the amino acid sequence of the group consisting of SEQ ID NO: 124-136;

[0255] (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to (1) or (2) above.

[0256] (4) An amino acid sequence having one or more added, deleted and / or substituted amino acids compared to the amino acid sequence of (1) or (2) above;

[0257] (5) The amino acid sequence of the heavy chain of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(2) above; or

[0258] (6) The amino acid sequence of the heavy chain of the anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1)-(2) above for binding to PVRIG.

[0259] In some embodiments, the present invention further provides a humanized antibody against PVRIG and its antigen-binding fragment, comprising a VH, the VH comprising, substantially comprising, or comprising the amino acid sequence of a reference antibody selected from the antibodies listed in Table 4c or Table 4e, except that the three CDRs in the reference antibody are replaced by a different set of three CDRs contained in the antibodies listed in Table 5a or Table 5b or Table 6a.

[0260] D. Variants of anti-PVRIG antibodies

[0261] The present invention also provides a variant of the anti-PVRIG antibody disclosed above or an antigen-binding fragment thereof, wherein one or more amino acid residues of one, two or all three HCDRs of the antibody are modified.

[0262] In some implementations, variants of the anti-PVRIG antibody disclosed above include:

[0263] (1) Contains the amino acid sequence X1FAVG (SEQ ID NO: 540), substantially composed of or composed of the amino acid sequence X1FAVG (SEQ ID NO: 540), and HCDR1, wherein X1 is S or A or L or G or P.

[0264] (2) Contains the amino acid sequence CSX3X4X5X6X7X8X9X 10YX 12 DSVKG (SEQ ID NO: 541), HCDR2 which is essentially composed of or consists of, wherein:

[0265] X3 is either S or T;

[0266] X4 is S, Y, or W;

[0267] X5 is T, A, G, M, R, or L;

[0268] X6 is either D or R;

[0269] X7 X8 X9 X 10 Is it NSTD, ESTD, NSPF, APTD, or NVLA; and

[0270] X 12 It is either D or A;

[0271] and

[0272] (3) Contains the amino acid sequence GWDCSGAVVYGVGH (SEQ ID NO: 18), and is substantially composed of or composed of HCDR3;

[0273] The amino acids are numbered according to the Kabat numbering system.

[0274] Preferably, X3 in HCDR2 is S or T; X4 or X5 in HCDR2 is YA or YG or YM or YR or ST or WG or WT or YL; X6 is D or R; and X 12 It is D or A. More specifically, X3, X4, X5, and X6 in HCDR2 are SYAD, SYGD, SYMD, SYRD, SSTD, SWGD, SYGR, TWTD, or SYLD.

[0275] More preferably, the variants of the anti-PVRIG antibody disclosed above include:

[0276] (1) Contains the amino acid sequence SFAVG (SEQ ID NO: 16), substantially composed of or composed of HCDR1;

[0277] (2) Containing the amino acid sequences CSSSTDNVLAYADSVKG (SEQ ID NO: 559), CSSSTDESTDYADSVKG (SEQ ID NO: 560), or CSSSTDAPTDYADSVKG (SEQ ID NO: 561), substantially composed of or composed of these sequences, HCDR2; and

[0278] (3) Contains the amino acid sequence GWDCSGAVVYGVGH (SEQ ID NO: 18), which is substantially composed of or composed of HCDR3.

[0279] In some implementations, variants of the anti-PVRIG antibody disclosed above include:

[0280] (1) Contains the amino acid sequence GFTLX5X6FAVG (SEQ ID NO: 542), and is substantially composed of or composed of HCDR1, wherein

[0281] X5 is E, V, D, H, or G; and

[0282] X6 is S, A, L, G, or P;

[0283] (2) Contains the amino acid sequence CSX3X4X5X6X7X8X9X 10 (SEQ ID NO: 543), HCDR2 which is essentially composed of or consists of, wherein:

[0284] X3 is either S or T;

[0285] X4 is S, Y, or W;

[0286] X5 is T, A, G, M, R, or L;

[0287] X6 is either D or R; and

[0288] X7 X8 X9 X 10 Is it NSTD, ESTD, NSPF, APTD, or NVLA; and

[0289] (3) Contains the amino acid sequence GWDCSGAVVYGVGH (SEQ ID NO: 18), and is substantially composed of or composed of HCDR3;

[0290] The amino acids are numbered according to the AbM numbering system.

[0291] Preferably, X3 in HCDR2 is S or T; X4 and X5 in HCDR2 are YA, YG, YM, YR, ST, WG, WT, or YL; and X6 is D or R. More specifically, X3, X4, X5, and X6 in HCDR2 are SYAD, SYGD, SYMD, SYRD, SSTD, SWGD, SYGR, TWTD, or SYLD.

[0292] This invention also provides humanized forms of the anti-PVRIG antibody variants or their antigen-binding fragments as disclosed above. In some embodiments, the humanized form of the anti-PVRIG antibody variant comprises a VH, which contains, or is substantially composed of, the amino acid sequence of the VH of a reference antibody selected from, or composed of, the antibodies listed in Table 4c or Table 4e, except that the three CDRs in the reference antibody are replaced by the three HCDRs as described above. Those skilled in the art will understand how such humanized forms of antibodies are generated. Such antibodies are all covered by this invention.

[0293] E. Removal of post-translational modifications (PTM) of anti-PVRIG antibodies

[0294] In some embodiments, the anti-PVRIG antibody of the present invention may be modified to remove post-translational modifications (PTMs), thereby improving its properties, such as stability, binding affinity, pharmacokinetics, and immunogenicity. Examples of PTMs include oxidation, deamidation, isomerization, and glycosylation. Specifically, mutations may be introduced into the CDR to remove potential glycosylation sites. Such modified antibodies are also included in the present invention.

[0295] 3. Anti-TIGIT antibody

[0296] This invention relates to anti-TIGIT antibodies and antigen-binding fragments thereof. Antibodies can be prepared using recombinant DNA methods in bacterial, eukaryotic, or plant cells (see, for example, U.S. Patent No. 4,816,567). Alternatively, for example, the anti-TIGIT antibodies of this invention can be produced using hybridoma techniques first described by Kohler et al., Nature 256:495 (1975), or can be isolated from phage antibody libraries using techniques described by Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies expressed therefrom are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al., eds., 5th edition 2002). Antibodies from various sources are included in this invention.

[0297] A. Monoclonal antibodies

[0298] In some embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:

[0299] (1) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 137-139, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 140-142, substantially composed of or composed of therein;

[0300] (2) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 147-149, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 150-152, substantially composed of or composed of therein;

[0301] (3) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 157-159, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 160-162, substantially composed of or composed of therein;

[0302] (4) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 167-169, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 170-172, substantially composed of or composed of therein;

[0303] (5) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 177-179, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 180-182, substantially composed of or composed of therein;

[0304] (6) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 187-189, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 190-192, substantially composed of or composed of therein;

[0305] (7) The VH region comprises HCDR1, HCDR2 and HCDR3, each of which contains the amino acid sequence of SEQ ID NO: 197-199, and is substantially composed of or composed of; and the VL region comprises LCDR1, LCDR2 and LCDR3, each of which contains the amino acid sequence of SEQ ID NO: 200-202, and is substantially composed of or composed of;

[0306] (8) The VH region comprises HCDR1, HCDR2 and HCDR3, each of which contains the amino acid sequence of SEQ ID NO: 207-209, is substantially composed of or is composed of; and the VL region comprises LCDR1, LCDR2 and LCDR3, each of which contains the amino acid sequence of SEQ ID NO: 210-212, is substantially composed of or is composed of;

[0307] (9) The VH region comprises HCDR1, HCDR2 and HCDR3, each of which contains the amino acid sequence of SEQ ID NO: 217-219, is substantially composed of or is composed of; and the VL region comprises LCDR1, LCDR2 and LCDR3, each of which contains the amino acid sequence of SEQ ID NO: 220-222, is substantially composed of or is composed of;

[0308] (10) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 227-229; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 230-232;

[0309] (11) The VH region comprises HCDR1, HCDR2 and HCDR3, each of which contains the amino acid sequence of SEQ ID NO: 237-239, is substantially composed of or is composed of; and the VL region comprises LCDR1, LCDR2 and LCDR3, each of which contains the amino acid sequence of SEQ ID NO: 240-242, is substantially composed of or is composed of;

[0310] (12) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 247-249; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 250-252;

[0311] (13) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 257-259; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 260-262;

[0312] (14) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 267-269; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 270-272;

[0313] (15) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 277-279; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 280-282;

[0314] (16) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 287-289; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 290-292;

[0315] (17) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 297-299; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 300-302;

[0316] (18) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 307-309; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 310-312;

[0317] (19) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 317-319; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 320-322;

[0318] (20) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 327-329; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 330-332;

[0319] (21) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 337-339; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 340-342;

[0320] (22) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 347-349; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 350-352;

[0321] (23) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 357-359; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 360-362;

[0322] (24) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 367-369; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 370-372;

[0323] (25) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 377-379; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 380-382;

[0324] (26) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 387-389; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 390-392;

[0325] (27) The VH region comprises HCDR1, HCDR2, and HCDR3, each comprising, substantially comprising, or comprising the amino acid sequence of SEQ ID NO: 397-399; and the VL region comprises LCDR1, LCDR2, and LCDR3, each comprising, substantially comprising, or comprising the amino acid sequence of SEQ ID NO: 400-402; or

[0326] (28) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 407-409; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 410-412;

[0327] (29) The VH region contains the same HCDR1, HCDR2 and HCDR3 as the reference antibody selected from Table 7 or Table 8a; and the VL region contains the same LCDR1, LCDR2 and LCDR3 as the reference antibody;

[0328] (30) Except for the presence of up to 5 (e.g., up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the VH region contains the same HCDR1, HCDR2 and HCDR3 as the reference antibody selected from Table 7 or Table 8a and the VL region contains the same LCDR1, LCDR2 and LCDR3 as the reference antibody;

[0329] (31) The VH region and the VL region contain the same HCDR and LCDR of the anti-TIGIT antibody, which binds to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(29) above; or

[0330] (32) The heavy chain variable region and the light chain variable region contain the same HCDR and LCDR of the anti-TIGIT antibody, which competes with one of the antibodies defined in (1)-(29) above for binding to TIGIT.

[0331] The antibodies listed in Tables 7 and 8a include: T-MIL-A8, T-MIL-A9, T-MIL-A18, T-MIL-A20, T-MIL-A23, T-MIL-A28, T-MIL-A47, T-MIL-A53, T-MIL-A60, T-MIL-A69, T-MIL-A71, T-MIL-A86, T-MIL-A89, and T-MIL-A90. T-MIL-A93, T-MIL-A101, T-MIL-A105, T-MIL-A109, T-MIL-A116, T-MIL-A125, T-MIL-A165, T-MIL-A169, T-MIL-A170, T-MIL-A172, T-MIL-A174, T-MIL-A180, T-MIL-A181 and T-MIL-A187.

[0332] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:

[0333] (1) The VH contains the amino acid sequence of SEQ ID NO: 143, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 144, is substantially composed of or is composed of therein;

[0334] (2) The VH contains the amino acid sequence of SEQ ID NO: 153, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 154, is substantially composed of or is composed of therein;

[0335] (3) The VH contains the amino acid sequence of SEQ ID NO: 163, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 164, is substantially composed of or is composed of therein;

[0336] (4) The VH contains the amino acid sequence of SEQ ID NO: 173, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 174, is substantially composed of or is composed of therein;

[0337] (5) The VH contains the amino acid sequence of SEQ ID NO: 183, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 184, is substantially composed of or is composed of therein;

[0338] (6) The VH contains the amino acid sequence of SEQ ID NO: 193, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 194, is substantially composed of or is composed of therein;

[0339] (7) The VH contains the amino acid sequence of SEQ ID NO: 203, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 204, is substantially composed of or is composed of therein;

[0340] (8) The VH contains the amino acid sequence of SEQ ID NO: 213, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 214, is substantially composed of or is composed of therein;

[0341] (9) The VH contains the amino acid sequence of SEQ ID NO: 223, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 224, is substantially composed of or is composed of therein;

[0342] (10) The VH contains the amino acid sequence of SEQ ID NO: 233, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 234, is substantially composed of or is composed of therein;

[0343] (11) The VH contains the amino acid sequence of SEQ ID NO: 243, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 244, is substantially composed of or is composed of therein;

[0344] (12) The VH contains the amino acid sequence of SEQ ID NO: 253, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 254, is substantially composed of or is composed of therein;

[0345] (13) The VH contains the amino acid sequence of SEQ ID NO: 263, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 264, is substantially composed of or is composed of therein;

[0346] (14) The VH contains the amino acid sequence of SEQ ID NO: 273, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 274, is substantially composed of or is composed of therein;

[0347] (15) The VH contains the amino acid sequence of SEQ ID NO: 283, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 284, is substantially composed of or is composed of therein;

[0348] (16) The VH contains the amino acid sequence of SEQ ID NO: 293, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 294, is substantially composed of or is composed of therein;

[0349] (17) The VH contains the amino acid sequence of SEQ ID NO: 303, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 304, is substantially composed of or is composed of therein;

[0350] (18) The VH contains the amino acid sequence of SEQ ID NO: 313, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 314, is substantially composed of or is composed of therein;

[0351] (19) The VH contains the amino acid sequence of SEQ ID NO: 323, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 324, is substantially composed of or is composed of therein;

[0352] (20) The VH contains the amino acid sequence of SEQ ID NO: 333, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 334, is substantially composed of or is composed of therein;

[0353] (21) The VH contains the amino acid sequence of SEQ ID NO: 343, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 344, is substantially composed of or is composed of therein;

[0354] (22) The VH contains the amino acid sequence of SEQ ID NO: 353, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 354, is substantially composed of or is composed of therein;

[0355] (23) The VH contains the amino acid sequence of SEQ ID NO: 363, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 364, is substantially composed of or is composed of therein;

[0356] (24) The VH contains the amino acid sequence of SEQ ID NO: 373, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 374, is substantially composed of or is composed of therein;

[0357] (25) The VH contains the amino acid sequence of SEQ ID NO: 383, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 384, is substantially composed of or is composed of therein;

[0358] (26) The VH contains the amino acid sequence of SEQ ID NO: 393, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 394, is substantially composed of or is composed of therein;

[0359] (27) The VH contains the amino acid sequence of SEQ ID NO: 403, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 404, is substantially composed of or is composed of therein;

[0360] (28) The VH contains the amino acid sequence of SEQ ID NO: 413, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 414, is substantially composed of or is composed of therein;

[0361] (29) The VH and the VL contain the same amino acid sequence as the antibodies selected from those listed in Table 8a;

[0362] (30) The VH and the VL contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the same amino acid sequence as the VH and VL listed in any one of (1)-(29) above.

[0363] (31) Compared with the amino acid sequences of VH and VL listed in any one of (1)-(29) above, the VH and VL contain one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids added, deleted and / or substituted.

[0364] (32) The VH and VL contain the same amino acid sequences as the VH and VL of the anti-TIGIT antibody, and the antibody binds to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(29) above; or

[0365] (33) The VH and the VL contain the same amino acid sequences as the VH and VL of the anti-TIGIT antibody, which competes with one of the antibodies defined in (1)-(29) above for binding to TIGIT.

[0366] In some embodiments, the anti-TIGIT antibody of the present invention comprises a heavy chain and a light chain, wherein:

[0367] (1) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 145, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 146;

[0368] (2) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 155, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 156.

[0369] (3) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 165, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 166.

[0370] (4) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 175, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 176.

[0371] (5) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 185, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 186.

[0372] (6) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 195, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 196;

[0373] (7) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 205, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 206;

[0374] (8) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 215, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 216;

[0375] (9) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 225, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 226;

[0376] (10) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 235, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 236;

[0377] (11) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 245, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 246;

[0378] (12) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 255, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 256;

[0379] (13) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 265, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 266;

[0380] (14) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 275, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 276;

[0381] (15) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 285, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 286;

[0382] (16) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 295, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 296;

[0383] (17) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 305, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 306;

[0384] (18) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 315, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 316;

[0385] (19) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 325, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 326;

[0386] (20) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 335, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 336;

[0387] (21) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 345, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 346;

[0388] (22) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 355, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 356;

[0389] (23) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 365, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 366;

[0390] (24) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 375, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 376;

[0391] (25) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 385, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 386;

[0392] (26) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 395, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 396;

[0393] (27) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 405, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 406;

[0394] (28) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 415, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 416;

[0395] (29) The heavy chain and the light chain contain the same amino acid sequence as the antibody selected from the antibodies listed in Table 8b;

[0396] (30) The heavy chain and the light chain contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the same amino acid sequence as the corresponding heavy chain and light chain listed in any one of (1)-(29) above.

[0397] (31) The heavy chain and the light chain comprise an amino acid sequence having one or more amino acids compared to the amino acid sequence of the corresponding heavy chain and light chain listed in any one of (1)-(29) above, preferably an amino acid sequence with the addition, deletion and / or substitution of 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids.

[0398] (32) The heavy chain and the light chain contain the same amino acid sequences as the corresponding heavy chain and light chain of the anti-TIGIT antibody, and the antibody binds to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(29) above; or

[0399] (33) The heavy chain and the light chain contain the same amino acid sequences as the corresponding heavy chain and light chain of the anti-TIGIT antibody, and the antibody competes with one of the antibodies defined in (1)-(29) above for binding to TIGIT.

[0400] Preferably, the anti-TIGIT antibody or its antigen-binding fragment comprises a VH region and a VL region, wherein:

[0401] (1) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 217-219, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 220-222, substantially composed of or composed of therein;

[0402] (2) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 347-349, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 350-352, substantially composed of or composed of therein;

[0403] (3) The VH region and the VL region contain HCDR1, HCDR2 and HCDR3 of the antibody and LCDR1, LCDR2 and LCDR3, the antibody contains VH and VL, the VH contains the amino acid sequence of SEQ ID NO: 223, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 224, is substantially composed of or is composed of therein;

[0404] (4) The VH region and the VL region contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the antibody, the antibody contains VH and VL, the VH contains the amino acid sequence of SEQ ID NO: 353, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 354, is substantially composed of or is composed of therein;

[0405] (5) The VH region and the VL region contain the same HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as those of the antibodies T-AIL-A60 or T-AIL-A169 shown in Table 8a or Table 8b.

[0406] (6) The VH region and the VL region contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 that are otherwise identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 listed in one of (1)-(5) above, except that there are at most 5 (e.g., at most 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more CDRs;

[0407] (7) The VH region and the VL region respectively contain the same corresponding HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as the anti-TIGIT antibody, and the anti-TIGIT antibody binds to the same or overlapping epitopes on the PVRIG with one of the antibodies defined in (1)-(5) above; or

[0408] (8) The VH region and the VL region contain the same corresponding HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as the anti-TIGIT antibody, which competes with one of the antibodies defined in (1)-(5) above for binding to TIGIT.

[0409] More preferably, the anti-TIGIT antibody or its antigen-binding fragment comprises a VH region and a VL region, wherein:

[0410] (1) The VH region contains, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 223, and the VL region contains, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 224;

[0411] (2) The VH region contains, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 353, and the VL region contains, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 354;

[0412] (3) The VH region and the VL region contain the same amino acid sequences as the VH and VL of the antibodies T-AIL-A60 or T-AIL-A169 shown in Table 8a or Table 8b;

[0413] (4) The VH region and the VL region contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the corresponding amino acid sequence of the antibody as defined in (1)-(3) above;

[0414] (5) The VH region and the VL region contain an amino acid sequence identical to the corresponding amino acid sequence of the antibody as defined in (1)-(3) above, except for the presence of one or more amino acids in one or both of the VH region and the VL region, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, through addition, deletion and / or substitution.

[0415] (6) The VH region and the VL region contain amino acid sequences identical to the corresponding VH and VL amino acid sequences of the anti-TIGIT antibody, and the antibody binds to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(3) above; or

[0416] (7) The VH region and the VL region contain the same amino acid sequences as the corresponding VH and VL amino acid sequences of the anti-TIGIT antibody, which competes with one of the antibodies defined in (1)-(3) above for binding to TIGIT.

[0417] Most preferably, the anti-TIGIT antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences of SEQ ID NO: 223 and 224, respectively substantially composed of therein or respectively composed of therein, or respectively comprise the amino acid sequences of SEQ ID NO: 353 and 354, respectively substantially composed of therein or respectively composed of therein.

[0418] In some other embodiments, the isolated antibody or its antigen-binding moiety may contain amino acid modifications (e.g., conserved substitutions) in the variable regions of the heavy and / or light chains. It will be understood in the art that certain conserved sequence modifications that do not remove antigen binding can be made. See, for example, Brummell et al. (1993) Biochem 32:1180-8; deWildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O' Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43. The term "conservative substitution" has the same meaning as described above for anti-PVRIG antibodies.

[0419] Preferably, the anti-TIGIT antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0420] (1) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 225, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 226;

[0421] (2) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 355, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 356.

[0422] (3) The heavy chain and the light chain contain the same amino acid sequences as the heavy chain and light chain of the antibody T-AIL-A60 or T-AIL-A169 shown in Table 8b;

[0423] (4) The heavy chain and the light chain contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the same amino acid sequence as the corresponding amino acid sequence of the antibody as defined in (1)-(3) above;

[0424] (5) The heavy chain and the light chain comprise an amino acid sequence identical to the corresponding amino acid sequence of the antibody as defined in (1)-(3) above, except for the presence of one or more amino acids in one or both of the heavy chain and the light chain, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, through addition, deletion and / or substitution.

[0425] (6) The heavy chain and the light chain contain amino acid sequences identical to the corresponding heavy chain and light chain amino acid sequences of the anti-TIGIT antibody, and the antibody binds to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(3) above; or

[0426] (7) The heavy chain and the light chain contain the same amino acid sequences as the corresponding heavy chain and light chain amino acid sequences of the anti-TIGIT antibody, and the antibody competes with one of the antibodies defined in (1)-(3) above for binding to TIGIT.

[0427] Most preferably, the anti-TIGIT antibody or its antigen-binding fragment of the present invention comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise the amino acid sequences of SEQ ID NO: 225 and 226, respectively, and are substantially composed of therein or respectively, or respectively comprise the amino acid sequences of SEQ ID NO: 355 and 356, respectively, and are substantially composed of therein or respectively.

[0428] B. Humanized anti-TIGIT antibody

[0429] This invention also relates to a humanized form of the aforementioned anti-TIGIT antibody, referred to as a "humanized antibody," and its antigen-binding fragment. The term "humanized antibody" has the same meaning as described above for the anti-PVRIG antibody. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "input" residues, which are typically derived from an "input" variable domain.

[0430] In some cases, humanized antibodies are constructed via CDR grafting, where the amino acid sequences of the six CDRs of the parental non-human antibody (e.g., rodent) are grafted onto the human antibody framework. For example, Padlan et al. ( FASEB J(9:133-139, 1995) determined that only about one-third of the residues in the CDR actually contact the antigen, and referred to these residues as "specificity-determining residues," or SDRs. In SDR transplantation techniques, only SDR residues are transplanted onto the human antibody framework (see, for example, Kashmiri et al., 9:133-139, 1995). Methods 36: 25-34, 2005). To maintain antibody binding and functional activity, additional frame region modifications can be performed within the human frame sequence.

[0431] The frame regions described in this paper are defined based on the boundaries of the CDR numbering system. In other words, if the CDR is determined by, for example, Kabat, IMGT, or Chothia, then the frame region is a variable region consisting of amino acid residues surrounding the CDR in the form of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. For example, FR1 is defined as the N-terminal amino acid residue of the CDR1 amino acid residue as defined by, for example, the Kabat, IMGT, or Chothia numbering system; FR2 is defined as the amino acid residue between the CDR1 and CDR2 amino acid residues as defined by, for example, the Kabat, IMGT, or Chothia numbering system; FR3 is defined as the amino acid residue between the CDR2 and CDR3 amino acid residues as defined by, for example, the Kabat, IMGT, or Chothia numbering system; and FR4 is defined as the C-terminal amino acid residue of the CDR3 amino acid residue as defined by, for example, the Kabat, IMGT, or Chothia numbering system.

[0432] The humanized antibodies of the present invention, in combination with TIGIT, can be produced using techniques known to those skilled in the art (Zhang et al.). Molecular Immunology , 42(12): 1445-1451, 2005; Hwang et al., Methods , 36(1):35-42, 2005; Dall'Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today , 21(8): 397-402, 2000, and U.S. Patent Nos. 6,180,370, 6,054,927, 5,869,619, 5,861,155, 5,712,120 and 4,816,567).

[0433] Therefore, this disclosure also provides humanized forms of anti-TIGIT antibodies as defined above and their antigen-binding fragments.

[0434] In some embodiments, the humanized anti-TIGIT antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise:

[0435] (1) The heavy chain CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2, HCDR3) and light chain CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2, LCDR3) of the antibodies selected from those listed in Table 7.

[0436] (2) The heavy chain CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, HCDR3) and light chain CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, LCDR3) of antibodies selected from those listed in Table 8a, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3; or

[0437] (3) The heavy chain CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2, HCDR3) and the light chain CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2, LCDR3) are otherwise identical to (1) or (2) above, except that there are up to 5 (e.g., up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more CDRs.

[0438] In some embodiments, the humanized anti-TIGIT antibody of the present invention or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of antibody T-MIL-A60 or T-MIL-A169 as listed in Table 7 or Table 8a.

[0439] In some implementations, the humanized anti-TIGIT antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise:

[0440] (1) The heavy chain CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, HCDR3) and light chain CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, LCDR3) of the antibodies selected from those listed in Table 9a or Table 9b are identical to those of the antibodies listed in Table 9a or Table 9b.

[0441] (2) The heavy chain CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, HCDR3) and light chain CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, LCDR3) of antibodies selected from those listed in Table 10a or Table 10b are identical to those of the antibodies listed in Table 10a or Table 10b; or

[0442] (3) The heavy chain CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2, HCDR3) and the light chain CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2, LCDR3) are otherwise identical to (1) or (2) above, except that there are up to 5 (e.g., up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more CDRs.

[0443] In some embodiments, the humanized anti-TIGIT antibody or its antigen-binding fragment of the present invention comprises VH and VL, wherein:

[0444] (1) The VH includes HCDR1 of SEQ ID NO: 217, HCDR2 of SEQ ID NO: 218 and HCDR3 of SEQ ID NO: 219, and the VL includes LCDR1 of SEQ ID NO: 220, LCDR2 of SEQ ID NO: 221 and LCDR3 of SEQ ID NO: 222;

[0445] (2) The VH includes HCDR1 of SEQ ID NO: 347, HCDR2 of SEQ ID NO: 348 and HCDR3 of SEQ ID NO: 349, and the VL includes LCDR1 of SEQ ID NO: 350, LCDR2 of SEQ ID NO: 351 and LCDR3 of SEQ ID NO: 352;

[0446] (3) The VH includes HCDR1 of SEQ ID NO: 217, HCDR2 of SEQ ID NO: 417 and HCDR3 of SEQ ID NO: 219, and the VL includes LCDR1 of SEQ ID NO: 220, LCDR2 of SEQ ID NO: 221 and LCDR3 of SEQ ID NO: 222;

[0447] (4) The VH includes HCDR1 of SEQ ID NO: 217, HCDR2 of SEQ ID NO: 218 and HCDR3 of SEQ ID NO: 219, and the VL includes LCDR1 of SEQ ID NO: 418, LCDR2 of SEQ ID NO: 419 and LCDR3 of SEQ ID NO: 222;

[0448] (5) The VH includes HCDR1 of SEQ ID NO: 217, HCDR2 of SEQ ID NO: 417 and HCDR3 of SEQ ID NO: 219, and the VL includes LCDR1 of SEQ ID NO: 418, LCDR2 of SEQ ID NO: 419 and LCDR3 of SEQ ID NO: 222;

[0449] (6) The VH includes HCDR1 of SEQ ID NO: 347, HCDR2 of SEQ ID NO: 348 and HCDR3 of SEQ ID NO: 349, and the VL includes LCDR1 of SEQ ID NO: 350, LCDR2 of SEQ ID NO: 420 and LCDR3 of SEQ ID NO: 352;

[0450] (7) The VH includes HCDR1 of SEQ ID NO: 347, HCDR2 of SEQ ID NO: 421 and HCDR3 of SEQ ID NO: 349, and the VL includes LCDR1 of SEQ ID NO: 350, LCDR2 of SEQ ID NO: 351 and LCDR3 of SEQ ID NO: 352;

[0451] (8) The VH includes HCDR1 of SEQ ID NO: 347, HCDR2 of SEQ ID NO: 421 and HCDR3 of SEQ ID NO: 349, and the VL includes LCDR1 of SEQ ID NO: 350, LCDR2 of SEQ ID NO: 420 and LCDR3 of SEQ ID NO: 352;

[0452] (9) The VH and the VL comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of antibodies selected from those listed in Table 10a or Table 10b; or

[0453] (10) The VH and the VL comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of antibodies selected from the antibodies defined in (1)-(9) above, except that they contain at most 5 (e.g., at most 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more CDRs.

[0454] In some embodiments, the humanized anti-TIGIT antibody or its antigen-binding fragment of the present invention comprises VH and VL, wherein:

[0455] (1) The VH contains the amino acid sequence of SEQ ID NO: 422, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 423, is substantially composed of or is composed of therein;

[0456] (2) The VH contains the amino acid sequence of SEQ ID NO: 424, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 425, is substantially composed of or is composed of therein;

[0457] (3) The VH contains the amino acid sequence of SEQ ID NO: 426, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 427, is substantially composed of or is composed of therein;

[0458] (4) The VH contains the amino acid sequence of SEQ ID NO: 428, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 429, is substantially composed of or is composed of therein;

[0459] (5) The VH contains the amino acid sequence of SEQ ID NO: 430, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 431, is substantially composed of or is composed of therein;

[0460] (6) The VH contains the amino acid sequence of SEQ ID NO: 432, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 433, is substantially composed of or is composed of therein;

[0461] (7) The VH contains the amino acid sequence of SEQ ID NO: 434, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 435, is substantially composed of or is composed of therein;

[0462] (8) The VH contains the amino acid sequence of SEQ ID NO: 436, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 437, is substantially composed of or is composed of therein;

[0463] (9) The VH contains the amino acid sequence of SEQ ID NO: 438, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 439, is substantially composed of or is composed of therein;

[0464] (10) The VH contains the amino acid sequence of SEQ ID NO: 440, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 441, is substantially composed of or is composed of therein;

[0465] (11) The VH contains the amino acid sequence of SEQ ID NO: 442, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 443, is substantially composed of or is composed of therein;

[0466] (12) The VH contains the amino acid sequence of SEQ ID NO: 444, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 445, is substantially composed of or is composed of therein;

[0467] (13) The VH contains the amino acid sequence of SEQ ID NO: 446, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 447, is substantially composed of or is composed of therein;

[0468] (14) The VH contains the amino acid sequence of SEQ ID NO: 448, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 449, is substantially composed of or is composed of therein;

[0469] (15) The VH comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 450, and the VL comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 451;

[0470] (16) The VH contains the amino acid sequence of SEQ ID NO: 452, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 453, is substantially composed of or is composed of therein;

[0471] (17) The VH contains the amino acid sequence of SEQ ID NO: 454, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 455, is substantially composed of or is composed of therein;

[0472] (18) The VH contains the amino acid sequence of SEQ ID NO: 456, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 457, is substantially composed of or is composed of therein;

[0473] (19) The VH contains the amino acid sequence of SEQ ID NO: 458, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 459, is substantially composed of or is composed of therein;

[0474] (20) The VH and VL comprise the corresponding amino acid sequences of the VH and VL of antibodies selected from those listed in Table 10a or Table 10b.

[0475] (21) The VH and the VL contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the corresponding amino acid sequences of the VH and VL of the antibodies selected from those defined in (1)-(20) above.

[0476] (22) The VH and the VL contain amino acid sequences identical to the corresponding amino acid sequences of the antibodies selected from the antibodies defined in (1)-(20) above, except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, through addition, deletion and / or substitution.

[0477] (23) The VH and VL comprise the amino acid sequences of the VH and VL of the anti-TIGIT antibody, wherein the antibody binds to the same or overlapping epitopes on TIGIT with an antibody selected from those defined in (1)-(20) above; or

[0478] (24) The VH and VL comprise the amino acid sequences of the VH and VL of the anti-TIGIT antibody, which competes with an antibody selected from the antibodies defined in (1)-(20) above for binding to TIGIT.

[0479] In some embodiments, the humanized anti-TIGIT antibody or its antigen-binding fragment of the present invention comprises a heavy chain and a light chain, wherein:

[0480] (1) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 460, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 461;

[0481] (2) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 462, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 463;

[0482] (3) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 464, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 465;

[0483] (4) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 466, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 467;

[0484] (5) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 468, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 469.

[0485] (6) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 470, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 471;

[0486] (7) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 472, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 473;

[0487] (8) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 474, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 475;

[0488] (9) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 476, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 477;

[0489] (10) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 478, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 479;

[0490] (11) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 480, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 481;

[0491] (12) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 482, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 483;

[0492] (13) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 484, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 485;

[0493] (14) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 486, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 487;

[0494] (15) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 488, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 489;

[0495] (16) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 490, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 491;

[0496] (17) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 492, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 493;

[0497] (18) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 494, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 495;

[0498] (19) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 496, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 497;

[0499] (20) The heavy chain and the light chain comprise the corresponding amino acid sequences of the heavy chain and light chain of the antibody selected from the antibodies listed in Table 10c or Table 10d.

[0500] (21) The heavy chain and the light chain contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the corresponding amino acid sequences of the heavy chain and light chain of the antibody selected from the antibodies defined in (1)-(20) above.

[0501] (22) The heavy chain and the light chain comprise an amino acid sequence identical to the corresponding amino acid sequence of the heavy chain and light chain of the antibody selected from the antibodies defined in (1)-(20) above, except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, through addition, deletion and / or substitution.

[0502] (23) The heavy chain and the light chain comprise the amino acid sequences of the heavy chain and light chain of the anti-TIGIT antibody, wherein the antibody binds to the same or overlapping epitopes on TIGIT with an antibody selected from those defined in (1)-(20) above; or

[0503] (24) The heavy chain and the light chain comprise the amino acid sequences of the heavy chain and light chain of the anti-TIGIT antibody, the antibody competing with an antibody selected from the antibodies defined in (1)-(20) above for binding to TIGIT.

[0504] In some embodiments, the present invention also provides humanized anti-TIGIT antibodies and their antigen-binding fragments, comprising VH and VL, wherein the VH and VL comprise, are substantially composed of, or are composed of the amino acid sequences of the VH and VL of a reference antibody selected from the antibodies listed in Table 10a, except that the six CDRs of the reference antibody are replaced by a different set of six CDRs contained in the antibodies listed in Table 9a or Table 10a. Such humanized antibodies or their antigen-binding fragments can be constructed using conventional techniques known in the art, such as chemical synthesis, genetic recombination, etc., which are also included in this invention.

[0505] In some embodiments, the present invention also provides humanized anti-TIGIT antibodies and their antigen-binding fragments, comprising VH and VL, wherein the VH and VL comprise, are substantially composed of, or are composed of the amino acid sequences of the VH and VL of a reference antibody selected from the antibodies listed in Table 10b, except that the six CDRs in the reference antibody are those listed in Table 9b, or a different set of six CDRs contained in the antibodies listed in Table 10b. Such humanized antibodies or their antigen-binding fragments can be constructed using conventional techniques known in the art, such as chemical synthesis, genetic recombination, etc., which are also included in this invention.

[0506] C. Affinity-matured anti-TIGIT antibodies

[0507] Optionally, the anti-TIGIT antibodies of the present invention may also be affinity-matured to enhance their binding affinity to the homoantigen—TIGIT. As described above, the terms "affinity maturation" and "affinity-matured" as used in the context of antibodies refer to an antibody having one or more alterations in one or more hypervariable regions, and such alterations result in increased antibody affinity for the antigen compared to a parent antibody without such alterations. Those skilled in the art can substitute one or more CDRs in the humanized anti-TIGIT antibodies to enhance their binding affinity to the homoantigen TIGIT. Therefore, affinity-matured anti-TIGIT antibodies or their antigen-binding fragments are also included in the present invention.

[0508] D. Variants of anti-TIGIT antibodies

[0509] The present invention also provides variants of the anti-TIGIT antibody disclosed above or antigen-binding fragments thereof, wherein one or more amino acid residues of one, two, three, four, five or all six CDRs of the antibody are modified.

[0510] In some implementations, variants of the anti-TIGIT antibody disclosed above include VH and VL, wherein:

[0511] The VH includes:

[0512] (1) Contains the amino acid sequence NYIMS (SEQ ID NO: 217), and is substantially composed of or composed of HCDR1;

[0513] (2) Contains the amino acid sequence TITSGGGNAYYX 12 DSVX 16 G (SEQ ID NO: 544), HCDR2 which is essentially composed of or consists of, wherein:

[0514] X12 It is P or A; and

[0515] X 16 It is R or K;

[0516] and

[0517] (3) Contains the amino acid sequence WLISFYAMDY (SEQ ID NO: 219), substantially composed of or composed of HCDR3; and

[0518] The VL includes:

[0519] (1) Contains the amino acid sequence X1ASQNINVWLS (SEQ ID NO: 545), is substantially composed of or is composed of the amino acid sequence X1ASQNINVWLS (SEQ ID NO: 545), and is LCDR1, wherein X1 is H or R;

[0520] (2) Contains the amino acid sequence KASNLX6X7 (SEQ ID NO: 546), is substantially composed of or is composed of LCDR2, wherein X6X7 is HT or ES;

[0521] and

[0522] (3) Contains the amino acid sequence QQGQSYPLT (SEQ ID NO: 222), and is substantially composed of or composed of LCDR3;

[0523] The amino acids are numbered according to the Kabat numbering system.

[0524] This invention also provides humanized forms of the anti-TIGIT antibody variants or their antigen-binding fragments as disclosed above. In some embodiments, the humanized forms of the anti-TIGIT antibody variants comprise VH and VL, said VH and VL comprising, or substantially comprising, the amino acid sequences of the VH and VL of a reference antibody selected from the antibodies listed in Table 10a, except that the six CDRs in the reference antibody are replaced by the six CDRs as described above. Those skilled in the art will understand how such humanized forms of antibodies are generated. Such antibodies are all covered by this invention.

[0525] In some implementations, variants of the anti-TIGIT antibody disclosed above include VH and VL, wherein:

[0526] The VH includes:

[0527] (1) Contains the amino acid sequence NTYMH (SEQ ID NO: 347), which is substantially composed of or composed of HCDR1;

[0528] (2) Contains the amino acid sequence RIDPANGNTKY X 12 X13 KFQG (SEQ ID NO: 547), HCDR2 which is essentially composed of or consists of, wherein X 12 X 13 Is it AP or SQ; and

[0529] (3) Contains the amino acid sequence EGDDGYYEDY (SEQ ID NO: 349), substantially composed of or composed of HCDR3; and

[0530] The VL includes:

[0531] (1) Contains the amino acid sequence RASENIYSYLA (SEQ ID NO: 350), and LCDR1 which is substantially composed of or composed of the amino acid sequence RASENIYSYLA;

[0532] (2) Containing the amino acid sequence NAKTLX6X7 (SEQ ID NO: 548), substantially composed of or composed of the amino acid sequence LCDR2, wherein X6X7 is AE or QS; and

[0533] (3) Contains the amino acid sequence QHHYGNPFT (SEQ ID NO: 352), and is substantially composed of or composed of LCDR3;

[0534] The amino acids are numbered according to the Kabat numbering system.

[0535] This invention also provides humanized forms of the anti-TIGIT antibody variants or their antigen-binding fragments as disclosed above. In some embodiments, the humanized forms of the anti-TIGIT antibody variants comprise VH and VL, said VH and VL comprising, or substantially comprising, the amino acid sequences of the VH and VL of a reference antibody selected from the antibodies listed in Table 10b, except that the six CDRs in the reference antibody are replaced by the corresponding six CDRs as described above. Those skilled in the art will understand how such humanized forms of antibodies are generated. Such antibodies are all covered by this invention.

[0536] E. Removal of post-translational modifications (PTM) of anti-TIGIT antibodies

[0537] In some embodiments, the anti-TIGIT antibody of the present invention may be modified to remove post-translational modifications (PTMs), thereby improving its properties, such as stability, binding affinity, pharmacokinetics, and immunogenicity. Examples of PTMs include oxidation, deamidation, isomerization, and glycosylation. Such modified antibodies are also included in the present invention.

[0538] 4. Multispecific antibodies against PVRIG and TIGIT

[0539] The present invention also relates to multispecific antibodies capable of binding both PVRIG and TIGIT. Therefore, the present invention provides multispecific antibodies comprising a first antibody portion binding human PVRIG and a second antibody portion binding human TIGIT, and optionally one or more additional antibody portions.

[0540] A. Primary Antibody Section

[0541] The first antibody portion may be the anti-PVRIG antibody of the present invention as described above, or its antigen-binding fragment thereof, and / or the second antibody portion may be the anti-TIGIT antibody of the present invention as described above, or its antigen-binding fragment thereof. Furthermore, anti-PVRIG antibodies and their antigen-binding fragments known in the art, as well as anti-TIGIT antibodies and their antigen-binding fragments known in the art, are also applicable to the present invention.

[0542] In some embodiments, the first antibody portion binding PVRIG is the anti-PVRIG antibody of the present invention as described above, or its antigen-binding fragment. The first antibody portion comprises at least one immunoglobulin monovariable domain (such as VHH), which includes:

[0543] (1) HCDR1, HCDR2 and HCDR3 of the first reference antibodies selected from the antibodies defined in Tables 1, 4a, 5a and 5b

[0544] (2) HCDR1, HCDR2 and HCDR3 that are identical to the antibodies selected from those listed in Tables 2, 4b, 4c and 6a.

[0545] (3) HCDR1, HCDR2 and HCDR3 are identical in all respects to HCDR1, HCDR2 and HCDR3 listed in one or more of the above (1) or (2) except that there are up to 5 (e.g. up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more HCDRs.

[0546] (4) HCDR1, HCDR2, and HCDR3 of anti-PVRIG antibodies, said antibodies binding to the same or overlapping epitopes on PVRIG as one of the antibodies defined in (1) or (2) above; or

[0547] (5) HCDR1, HCDR2 and HCDR3 of anti-PVRIG antibodies, said antibodies competing with one of the antibodies defined in (1) or (2) above for binding to PVRIG.

[0548] Table 1 provides the amino acid sequences of the HCDRs of the anti-PVRIG antibodies of the present invention, as determined by the Kabat coding system. Those skilled in the art will understand that the boundaries of antibody CDRs can be determined using other antibody numbering systems known in the art (e.g., the Chothia numbering system, the AbM numbering system, the IMGT numbering system, or the Contact numbering system). Embodiments relating to CDRs determined by such antibody numbering systems are also covered in this invention.

[0549] In some embodiments, the first antibody portion binding PVRIG is an anti-PVRIG antibody or its antigen-binding fragment, comprising:

[0550] (1) Contains the amino acid sequence X1FAVG (SEQ ID NO: 540), substantially composed of or composed of the amino acid sequence X1FAVG (SEQ ID NO: 540), and HCDR1, wherein X1 is S or A or L or G or P.

[0551] (2) Contains the amino acid sequence CSX3X4X5X6X7X8X9X 10 YX 12 DSVKG (SEQ ID NO: 541), HCDR2 which is essentially composed of or consists of, wherein:

[0552] X3 is either S or T;

[0553] X4 is S, Y, or W;

[0554] X5 is T, A, G, M, R, or L;

[0555] X6 is either D or R;

[0556] X7 X8 X9 X 10 Is it NSTD, ESTD, NSPF, APTD, or NVLA; and

[0557] X 12 It is either D or A;

[0558] and

[0559] (3) Contains the amino acid sequence GWDCSGAVVYGVGH (SEQ ID NO: 18), and is substantially composed of or composed of HCDR3;

[0560] The amino acids are numbered according to the Kabat numbering system.

[0561] Preferably, X3 in HCDR2 is S or T; X4 or X5 in HCDR2 is YA or YG or YM or YR or ST or WG or WT or YL; X6 is D or R; and X 12It is D or A. More specifically, X3, X4, X5, and X6 in HCDR2 are SYAD, SYGD, SYMD, SYRD, SSTD, SWGD, SYGR, TWTD, or SYLD.

[0562] More preferably, the first antibody portion binding PVRIG is an anti-PVRIG antibody or its antigen-binding fragment, comprising:

[0563] (1) Contains the amino acid sequence SFAVG (SEQ ID NO: 16), substantially composed of or composed of HCDR1;

[0564] (2) Containing the amino acid sequences CSSSTDNVLAYADSVKG (SEQ ID NO: 559), CSSSTDESTDYADSVKG (SEQ ID NO: 560), or CSSSTDAPTDYADSVKG (SEQ ID NO: 561), substantially composed of or composed of these sequences, HCDR2; and

[0565] (3) Contains the amino acid sequence GWDCSGAVVYGVGH (SEQ ID NO: 18), which is substantially composed of or composed of HCDR3.

[0566] In some embodiments, the first antibody portion is an anti-PVRIG antibody or its antigen-binding fragment, which contains a VH containing, or substantially consists of, the amino acid sequence of the VH of a reference antibody selected from, or consisting of, the reference antibody, except that the three CDRs in the reference antibody are replaced by the three HCDRs mentioned above.

[0567] In some embodiments, the first antibody portion binding PVRIG is an anti-PVRIG antibody or its antigen-binding fragment, comprising:

[0568] (1) Contains the amino acid sequence GFTLX5X6FAVG (SEQ ID NO: 542), and is substantially composed of or composed of HCDR1, wherein

[0569] X5 is E, V, D, H, or G; and

[0570] X6 是 S or A or L or G or P;

[0571] (2) Contains the amino acid sequence CSX3X4X5X6X7X8X9X 10 (SEQ ID NO: 543), HCDR2 which is essentially composed of or consists of, wherein:

[0572] X3 is either S or T;

[0573] X4 is S, Y, or W;

[0574] X5 is T, A, G, M, R, or L;

[0575] X6 is either D or R; and

[0576] X7 X8 X9 X 10 Is it NSTD, ESTD, NSPF, APTD, or NVLA; and

[0577] (3) Contains the amino acid sequence GWDCSGAVVYGVGH (SEQ ID NO: 18), and is substantially composed of or composed of HCDR3;

[0578] The amino acids are numbered according to the AbM numbering system.

[0579] Preferably, X3 in HCDR2 is S or T; X4 and X5 in HCDR2 are YA, YG, YM, YR, ST, WG, WT, or YL; and X6 is D or R; more specifically, X3, X4, X5, and X6 in HCDR2 are SYAD, SYGD, SYMD, SYRD, SSTD, SWGD, SYGR, TWTD, or SYLD.

[0580] In some embodiments, the first antibody portion is an anti-PVRIG antibody or its antigen-binding fragment, which contains a VH containing, or substantially consists of, the amino acid sequence of the VH of a reference antibody selected from, or consisting of, the reference antibody, except that the three CDRs in the reference antibody are replaced by the three HCDRs mentioned above.

[0581] In some embodiments, the first antibody portion is a VHH-type anti-PVRIG antibody or its antigen-binding fragment, comprising VH, wherein the VH comprises:

[0582] (1) The amino acid sequences of the antibodies selected from those listed in Tables 2, 4b, 4c and 6a;

[0583] (2) An amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with (1) above;

[0584] (3) Except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, the amino acid sequence is the same as the amino acid sequence in (1) above.

[0585] (4) An amino acid sequence identical to the VH amino acid sequence of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with an antibody selected from those defined in (1) above; or

[0586] (5) An amino acid sequence identical to the amino acid sequence of the VH of the anti-PVRIG antibody, wherein the antibody competes with an antibody selected from the antibodies defined in (1) above for binding to PVRIG.

[0587] In some embodiments, the first antibody portion is a VHH-type anti-PVRIG antibody that comprises a heavy chain, said heavy chain comprising:

[0588] (1) The amino acid sequences of the antibodies selected from those listed in Tables 3, 4d, 4e and 6b;

[0589] (2) An amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with (1) above;

[0590] (3) Except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, the amino acid sequence is the same as the amino acid sequence in (1) above.

[0591] (4) An amino acid sequence identical to the amino acid sequence of the heavy chain of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with an antibody selected from those defined in (1) above; or

[0592] (5) An amino acid sequence identical to the amino acid sequence of the heavy chain of an anti-PVRIG antibody, wherein the antibody competes with an antibody selected from those defined in (1) above for binding to PVRIG.

[0593] The first reference anti-PVRIG antibody may be P-AIL-A8, P-AIL-A11, P-AIL-A15, P-AIL-A32, P-AIL-A82, P-AIL-B352, or P-AIL-C242. The amino acid sequences of their CDRs according to the Kabat numbering system are shown in Table 1. This invention also covers embodiments related to CDRs determined by other numbering systems. Preferably, the first reference anti-PVRIG antibody may be a humanized anti-PVRIG antibody listed in Table 4b or 4c, or an affinity-matured variant listed in Table 6a.

[0594] B. Second Antibody Section

[0595] In this invention, the second antibody portion of the multispecific antibody that binds to TIGIT is the anti-TIGIT antibody or its antigen fragment as described above, which may be single-chain or comprise two heavy chains and two light chains. Alternatively, anti-TIGIT antibodies or their antigen-binding fragments known in the art may also be used as the second antibody portion in this invention.

[0596] In some implementations, the second antibody portion comprises a VH region and a VL region, wherein:

[0597] (1) The VH region and the VL region respectively contain the corresponding HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 of the second reference antibody listed in Table 7, Table 9a and Table 9b;

[0598] (2) The VH region and the VL region contain the corresponding HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 of the second reference antibody selected from those listed in Tables 8a, 10a and 10b.

[0599] (3) The VH region and the VL region contain HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 of antibodies selected from antibodies defined in (1) or (2) above, except that there are at most 5 (e.g., at most 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more CDRs.

[0600] (4) The VH region and the VL region contain HCDR1, HCDR2, and HCDR3 of the anti-TIGIT antibody, as well as LCDR1, LCDR2, and LCDR3, wherein the antibody binds to the same or overlapping epitopes on TIGIT by an antibody selected from those defined in (1) or (2) above; or

[0601] (5) The VH region and the VL region contain HCDR1, HCDR2 and HCDR3 of anti-PVRIG antibodies and LCDR1, LCDR2 and LCDR3, the antibodies competing with one of the antibodies defined in (1) or (2) above for binding to PVRIG.

[0602] Tables 7, 9a, and 9b provide the amino acid sequences of the HCDR and LCDR of the anti-TIGIT antibodies of the present invention, as determined by the Kabat numbering system. Those skilled in the art will understand that the boundaries of antibody CDRs can be determined using other antibody numbering systems known in the art (e.g., the Chothia numbering system, the AbM numbering system, the IMGT numbering system, or the Contact numbering system). Embodiments associated with CDRs determined by such antibody numbering systems are also covered in this invention.

[0603] In some embodiments, the second antibody portion binding TIGIT is an anti-TIGIT antibody or its antigen-binding fragment, comprising VH and VL, wherein:

[0604] The VH includes:

[0605] (1) Contains the amino acid sequence NYIMS (SEQ ID NO: 217), and is substantially composed of or composed of HCDR1;

[0606] (2) Contains the amino acid sequence TITSGGGNAYYX 12 DSVX 16 G (SEQ ID NO: 544), HCDR2 which is essentially composed of or consists of, wherein:

[0607] X 12 It is P or A; and

[0608] X 16 It is R or K;

[0609] and

[0610] (3) Contains the amino acid sequence WLISFYAMDY (SEQ ID NO: 219), substantially composed of or composed of HCDR3; and

[0611] The VL includes:

[0612] (1) Contains the amino acid sequence X1ASQNINVWLS (SEQ ID NO: 545), is substantially composed of or is composed of the amino acid sequence X1ASQNINVWLS (SEQ ID NO: 545), and is LCDR1, wherein X1 is H or R;

[0613] (2) Contains the amino acid sequence KASNLX6X7 (SEQ ID NO: 546), is substantially composed of or is composed of LCDR2, wherein X6X7 is HT or ES;

[0614] and

[0615] (3) Contains the amino acid sequence QQGQSYPLT (SEQ ID NO: 222), and is substantially composed of or composed of LCDR3;

[0616] The amino acids are numbered according to the Kabat numbering system.

[0617] In some embodiments, the second antibody portion is an anti-TIGIT antibody or its antigen-binding fragment, comprising VH and VL, wherein the VH and VL comprise, or substantially comprise, the amino acid sequences of the reference antibody selected from those described in Table 10a, except that the six CDRs in the reference antibody are replaced by the six CDRs as described above.

[0618] In some embodiments, the second antibody portion binding TIGIT is an anti-TIGIT antibody or its antigen-binding fragment, comprising VH and VL, wherein:

[0619] The VH includes:

[0620] (1) Contains the amino acid sequence NTYMH (SEQ ID NO: 347), which is substantially composed of or composed of HCDR1;

[0621] (2) Contains the amino acid sequence RIDPANGNTKYX 12 X 13 KFQG (SEQ ID NO: 547), HCDR2 which is essentially composed of or consists of, wherein X 12 X 13 Is it AP or SQ; and

[0622] (3) Contains the amino acid sequence EGDDGYYEDY (SEQ ID NO: 349), substantially composed of or composed of HCDR3; and

[0623] The VL includes:

[0624] (1) Contains the amino acid sequence RASENIYSYLA (SEQ ID NO: 350), and LCDR1 which is substantially composed of or composed of the amino acid sequence RASENIYSYLA;

[0625] (2) Containing the amino acid sequence NAKTLX6X7 (SEQ ID NO: 548), substantially composed of or composed of the amino acid sequence LCDR2, wherein X6X7 is AE or QS; and

[0626] (3) Contains the amino acid sequence QHHYGNPFT (SEQ ID NO: 352), and is substantially composed of or composed of LCDR3;

[0627] The amino acids are numbered according to the Kabat numbering system.

[0628] In some embodiments, the second antibody portion is an anti-TIGIT antibody or its antigen-binding fragment, comprising VH and VL, wherein the VH and VL comprise, or substantially comprise, the amino acid sequences of the reference antibody selected from those described in Table 10a, except that the six CDRs in the reference antibody are replaced by the six CDRs as described above.

[0629] In some implementations, the second antibody portion comprises a VH region and a VL region, wherein:

[0630] (1) The VH region and the VL region contain the same amino acid sequences as the VH and VL of the second reference antibody selected from the antibodies listed in Tables 8a, 10a and 10b;

[0631] (2) The VH region and the VL region contain amino acid sequences that have at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the corresponding amino acid sequences of the VH and VL of the second reference antibody selected from the antibodies listed in Tables 8a, 10a and 10b.

[0632] (3) The VH region and the VL region contain amino acid sequences identical to the corresponding amino acid sequences of the VH and VL of the second reference antibody selected from the antibodies listed in Tables 8a, 10a and 10b, except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, through addition, deletion and / or substitution.

[0633] (4) The VH region and the VL region contain amino acid sequences identical to the corresponding amino acid sequences of the VH and VL regions of the second reference antibody, and the second reference antibody binds to the same or overlapping epitopes on TIGIT with the second reference antibody selected from the antibodies listed in Tables 8a, 10a and 10b; or

[0634] (5) The VH region and the VL region contain the same amino acid sequences as the corresponding amino acid sequences of the VH and VL of the second reference antibody, which competes with antibodies selected from those listed in Tables 8a, 10a and 10b for binding to TIGIT.

[0635] In some implementations, the second antibody portion comprises a heavy chain and a light chain, wherein:

[0636] (1) The heavy chain and the light chain contain the same amino acid sequence as the heavy chain and light chain of the second reference antibody selected from the antibodies listed in Tables 8b, 10c and 10d;

[0637] (2) The heavy chain and the light chain comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the corresponding amino acid sequences of the heavy chain and light chain of the second reference antibody selected from the antibodies listed in Tables 8b, 10c and 10d.

[0638] (3) The heavy chain and the light chain comprise an amino acid sequence identical to the corresponding amino acid sequence of the heavy chain and light chain of the second reference antibody selected from the antibodies listed in Tables 8b, 10c and 10d, except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, through addition, deletion and / or substitution.

[0639] (4) The heavy chain and the light chain contain the same amino acid sequences as the corresponding amino acid sequences of the heavy chain and light chain of the second reference antibody, and the second reference antibody binds to the same or overlapping epitopes on TIGIT with the second reference antibody selected from the antibodies listed in Tables 8b, 10c and 10d; or

[0640] (5) The heavy chain and the light chain contain the same amino acid sequences as the corresponding amino acid sequences of the heavy chain and the light chain of the second reference antibody, which competes with antibodies selected from those listed in Tables 8b, 10c and 10d for binding to TIGIT.

[0641] The second reference anti-TIGIT antibody can be T-MIL-A8, T-MIL-A9, T-MIL-A18, T-MIL-A20, T-MIL-A23, T-MIL-A28, T-MIL-A47, T-MIL-A53, T-MIL-A60, T-MIL-A69, T-MIL-A71, T-MIL-A86, T-MIL-A89, or T-MIL-A90. , T-MIL-A93, T-MIL-A101, T-MIL-A105, T-MIL-A109, T-MIL-A116, T-MIL-A125, T-MIL-A165, T-MIL-A169, T-MIL-A170, T-MIL-A172, T-MIL-A174, T-MIL-A180, T-MIL-A181, or T-MIL-A187. The second reference anti-TIGIT antibody can also be T-MIL-A60-Hu01, T-MIL-A60-Hu02, T-MIL-A60-Hu03, T-MIL-A60-Hu04, T-MIL-A60-Hu05, T-MIL-A60-Hu06, T-MIL-A60-Hu07, T-MIL-A169-Hu01, T-MIL-A169-Hu02, T-MIL-A169-Hu03, or T-MIL-A169-Hu04. T-MIL-A169-Hu05, T-MIL-A169-Hu06, T-MIL-A169-Hu07, T-MIL-A169-Hu08, T-MIL-A169-Hu09, T-MIL-A169-Hu10, T-MIL-A169-Hu11, or T-MIL-A169-Hu12.

[0642] Tables 7, 9a, and 9b provide the amino acid sequences of the HCDR and LCDR of some anti-TIGIT antibodies of the present invention, as determined by the Kabat numbering system. Those skilled in the art will understand that the boundaries of the antibody's CDR can be determined by other antibody numbering systems known in the art (e.g., the Chothia numbering system, the AbM numbering system, the IMGT numbering system, or the Contact numbering system). Embodiments associated with CDRs determined by such antibody numbering systems are also covered in this invention.

[0643] C. Forms of multispecific antibodies

[0644] The multispecific antibodies against PVRIG and TIGIT of the present invention can be in single-chain or double-chain form. In some embodiments, the multispecific antibody is in single-chain form, wherein the first antibody portion and the second antibody portion are located on a single polypeptide. They can be linked together by a linker. For example, both the first antibody portion and the second antibody portion can be scFv, and they can be linked in a direction from the N-terminus to the C-terminus. In other embodiments, the multispecific antibody is in double-chain form, wherein the VH region of the first antibody portion is linked to the N-terminus and / or C-terminus of the VH region and / or VL region of the second antibody portion.

[0645] The various variable regions are linked together via peptide linkers. Optionally, a single polypeptide may also include a C-terminal fragment (e.g., the Fc fragment of an immunoglobulin molecule such as IgG), which can be linked to the C-terminal variable region fragment via an additional peptide linker. This additional peptide linker, the C-terminal fragment, or both may contain one or more cysteine ​​residues to form a disulfide bond. Any peptide linker can be a G / S-rich peptide linker.

[0646] Alternatively, the multispecific antibodies against PVRIG and TIGIT may be in a double-chain form, comprising a first polypeptide and a second polypeptide. In some embodiments, each of the first and second polypeptides comprises a variable region of the anti-PVRIG first antibody moiety and a variable region of the anti-TIGIT second antibody moiety. The variable region of the anti-PVRIG first antibody moiety may be N-terminal, and the variable region of the anti-TIGIT second antibody moiety may be C-terminal. Alternatively, the variable region of the anti-PVRIG first antibody moiety may be C-terminal, and the variable region of the anti-TIGIT second antibody moiety may be N-terminal. In some cases, the first polypeptide also comprises a first C-terminal fragment. Additionally or optionally, the second polypeptide also comprises a second C-terminal fragment. The first C-terminal fragment and the second C-terminal fragment form a dimer.

[0647] In some instances, the first C-terminal fragment is the first Fc fragment of the first IgG molecule. The second C-terminal fragment is the second Fc fragment of the second IgG molecule. The first Fc fragment and the second Fc fragment form the IgG Fc region. In some instances, the first Fc fragment contains a first CH2 domain and a first CH3 domain, and the second Fc fragment contains a second CH2 domain and a second CH3 domain.

[0648] In some specific embodiments, the multispecific antibody comprises a first VH region of the anti-PVRIG antibody of the present invention, and / or a second VH region and a second VL region of the anti-TIGIT antibody of the present invention. The first VH region is linked to the N-terminus, C-terminus, N-terminus, or C-terminus of the second VH region, and in each case, they are linked by a peptide linker.

[0649] In some specific embodiments, the multispecific antibody of the present invention comprises a second antibody portion in the form of an antibody or an antigen-binding fragment thereof and two first antibody portions in the form of VHH or an antigen-binding fragment thereof; the second antibody portion comprises two heavy chains (HC) and two light chains (LC), wherein in the second antibody portion, the VH of one HC forms an antigen-binding site with the VL of one LC, and the VH of the other HC forms an antigen-binding site with the VL of the other LC.

[0650] In some specific embodiments, in the multispecific antibodies of this disclosure, one VHH first antibody portion is located at the N-terminus of the HC or LC of the second antibody portion, and another VHH first antibody portion is located at the C-terminus of the HC or LC of the second antibody portion.

[0651] In some specific embodiments, in the multispecific antibodies disclosed herein, the VHH type first antibody portion is located at the N-terminus of the two HCs or two LCs of the second antibody portion; or, the VHH type first antibody is located at the C-terminus of the two HCs or two LCs of the second antibody portion.

[0652] In some specific embodiments, in the multispecific antibodies disclosed herein, the VHH type first antibody portion is located at the N-terminus of the two HCs of the second antibody portion; or, the VHH type first antibody portion is located at the C-terminus of the two HCs of the second antibody portion.

[0653] In some specific embodiments, the second antibody portion of this disclosure can be linked to 1, 2, 3, 4, 5, 6, 7 or 8 VHH first antibody portions. The VHH first antibody portions can be the same or different. They can all be linked to the N-terminus of the HC of the second antibody portion, or all of them can be linked to the C-terminus of the HC of the second antibody portion, or all of them can be linked to the N-terminus of the LC of the second antibody portion, or all of them can be linked to the C-terminus of the LC of the second antibody portion, or any combination of the N-terminus, C-terminus, N-terminus and C-terminus of the HC of the second antibody portion.

[0654] D. Optional peptide linkers between antibody moieties

[0655] The first antibody portion of the multispecific antibody disclosed herein can be directly or via one or more peptide linkers linked to the N-terminus or C-terminus of each HC or LC of the second antibody portion. As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein.

[0656] Suitable non-immunogenic adaptor peptides can be, for example, (G) m S n )x (GGNGT) x Or (YGNGT) x , where m and n are independent integers from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), and x is an independent integer selected from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0657] In some embodiments, one or more peptide linkers are each independently selected from (G4S)n, (SG4)n, or G4(SG4)n peptide linkers, where "n" is typically a number between 1 and 10, typically a number between 1 and 4, and particularly 2. The peptide linkers can be selected from the group consisting of: GGGGS, GGGGSGGGGS, SGGGGSGGG, (G4S)3 or GGGGSGGGGSGGGGS, G4(SG4)2 or GGGGS GGGGS GGGG, (G4S)4 or GGGGS GGGGS GGGGS GGGGS, (G4S)5 or GGGGS GGGGSGGGGS GGGGS GGGGS or (G4S)6 or GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS. Peptide linkers can also be selected from the sequences GSPGSSSSGS, GSGSGSGS, GSGSGNGS, GGSGSGSG, GGSGSG, GGSG, GGSGNGSG, GGNGSGSG, and GGNGSG. Peptide linkers of particular interest include (G4S)1 or GGGGS, (G4S)2 or GGGGSGGGGS, and (G4S)3 or GGGGSGGGGSGGGGS.

[0658] E. Forms of the first and second antibody portions

[0659] In some embodiments, the HC portion of the second antibody moiety of the multispecific antibody disclosed herein comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the LC portion comprises a light chain variable region (VL) and a light chain constant region (CL). The second antibody moiety may be a full-length antibody or an antigen-binding fragment thereof.

[0660] In some embodiments, the HC of the second antibody portion of the multispecific antibody of this disclosure is an IgG isotype (e.g., IgG1, IgG2, IgG3, or IgG4), such as the IgG1 isotype; and / or the LC of the second antibody portion is a κ isotype.

[0661] In some embodiments, the two HCs of the second antibody portion of the multispecific antibody of the present invention contain the same CDR and / or the two LCs contain the same CDR. In some specific embodiments, the two HCs of the second antibody portion contain the same VH and / or the two LCs contain the same VL. In some specific embodiments, the two HCs of the second antibody portion have the same amino acid sequence, and / or the two LCs have the same amino acid sequence.

[0662] In some embodiments, the two VHH type first antibody moieties of the multispecific antibody of this disclosure have the same or different amino acid sequences. For example, the two VHH type first antibody moieties have the same amino acid sequence.

[0663] In some embodiments, the multispecific antibody of this disclosure comprises two first polypeptide chains and two second polypeptide chains, wherein for each polypeptide chain: a) each of the first polypeptide chains independently comprises the heavy chain (HC) of the first antibody and the second antibody; and b) each of the second polypeptide chains independently comprises the light chain (LC) of the second antibody; wherein the VHH is linked to the N-terminus and / or C-terminus of the HC of the first antibody via a linker.

[0664] Alternatively, i) each of the first polypeptide chains independently comprises a heavy chain (HC) of the second antibody; and ii) each of the second polypeptide chains independently comprises a light chain (LC) of a VHH-type first antibody portion and a second antibody portion; wherein the VHH is directly or via a linker attached to the N-terminus and / or C-terminus of the LC of the second antibody portion.

[0665] In some specific embodiments, the multispecific antibody disclosed herein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0666] In some implementations, the multispecific antibodies against PVRIG and TIGIT are bispecific antibodies.

[0667] In some implementations, the first antibody portion of the multispecific antibody is an anti-PVRIG antibody or its antigen-binding fragment as described above.

[0668] In some implementations, the second antibody portion of the multispecific antibody is an anti-TIGIT antibody or its antigen-binding fragment as described above.

[0669] Preferably, the multispecific antibody of the present invention comprises an amino acid sequence of an anti-PVRIG first antibody portion linked to an amino acid sequence of an anti-TIGIT second antibody portion, wherein the first antibody portion is a VHH antibody against PVRIG containing a CDR of one of the anti-PVRIG antibodies as defined above, and the second antibody portion contains a CDR of one of the anti-TIGIT antibodies as defined above.

[0670] In another embodiment, the anti-PVRIG first antibody portion comprises VH of P-AIL-A8, P-AIL-A11, P-AIL-A15, P-AIL-A32, P-AIL-A82, P-AIL-B352 and P-AIL-C242 provided in Table 2. In another embodiment, the first anti-PVRIG antibody portion comprises P-AIL-A15-Hu01, P-AIL-A15-Hu02, P-AIL-A15-Hu03, P-AIL-A15-Hu04, P-AIL-A15-Hu06, P-AIL-A15-Hu07, P-AIL-A15-Hu08, P-AIL-A15-Hu09, P-AIL-A15-Hu10, P-AIL-A15-Hu11, P-AIL-A32-Hu09, P-AIL-A32-Hu10, P-AIL-A32-Hu11, P-AIL-A32-Hu12, P-AIL-A32-Hu13, and P-AIL-A32-Hu09, as provided in Tables 4b, 4c, or 6a. 14. P-AIL-A32-Hu15, P-AIL-A32-Hu16, P-AIL-A32-Hu17, P-AIL-A32-Hu18, P-AIL- A32-Hu19, P-AIL-A32-Hu20, P-AIL-A32-012, P-AIL-A32-024, P-AIL-A32-029, P-AI VH of L-A32-031, P-AIL-A32-035, P-AIL-A32-061, P-AIL-A32-077, P-AIL-A32-081, P-AIL-A32-103, P-AIL-A32-114, P-AIL-A32-127, P-AIL-A32-131, or P-AIL-A32-132. In another embodiment, the first anti-PVRIG antibody comprises a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO: 37-58 and 111-123. Preferably, the first anti-PVRIG antibody portion comprises a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO: 111-123.

[0671] In another embodiment, the anti-TIGIT second antibody portion comprises T-MIL-A8, T-MIL-A9, T-MIL-A18, T-MIL-A20, T-MIL-A23, T-MIL-A28, T-MIL-A47, T-MIL-A53, T-MIL-A60, T-MIL-A69, T-MIL-A71, T-MIL-A86, T-MIL-A86, T-MIL-A9, T-MIL-A20, T-MIL-A23, T-MIL-A28, T-MIL-A47, T-MIL-A53, T-MIL-A60, T-MIL-A69, T-MIL-A71, T-MIL-A86 ...3, T-MIL-A28, T-MIL-A47, T-MIL-A53, T-MIL-A60, T-MIL- MIL-A89, T-MIL-A90, T-MIL-A93, T-MIL-A101, T-MIL-A105, T-MIL-A109, T-MIL-A116, T-MIL-A 125. T-MIL-A165, T-MIL-A169, T-MIL-A170, T-MIL-A172, T-MIL-A174, T-MIL-A180, T-MIL-A18 1. T-MIL-A187, T-MIL-A60-Hu01, T-MIL-A60-Hu02, T-MIL-A60-Hu03, T-MIL-A60-Hu04, T-MIL- A60-Hu05, T-MIL-A60-Hu06, T-MIL-A60-Hu07, T-MIL-A169-Hu01, T-MIL-A169-Hu02, T-MIL-A1 69-Hu03, T-MIL-A169-Hu04, T-MIL-A169-Hu05, T-MIL-A169-Hu06, T-MIL-A169-Hu07, T-MIL-A 6 CDRs of 169-Hu08, T-MIL-A169-Hu09, T-MIL-A169-Hu10, T-MIL-A169-Hu11 or T-MIL-A169-Hu12.

[0672] In another embodiment, the anti-TIGIT second antibody portion comprises the VH and VL of an antibody selected from the group consisting of: T-MIL-A8, T-MIL-A9, T-MIL-A18, T-MIL-A20, T-MIL-A23, T-MIL-A28, T-MIL-A47, T-MIL-A53, T-MIL-A60, T-MIL-A69, T-MIL-A71, T-MIL-A86, T-MIL-A86, etc., as provided in Table 8a. MIL-A89, T-MIL-A90, T-MIL-A93, T-MIL-A101, T-MIL-A105, T-MIL-A109, T-MIL-A116, T-MIL-A125, T- MIL-A165, T-MIL-A169, T-MIL-A170, T-MIL-A172, T-MIL-A174, T-MIL-A180, T-MIL-A181, or T-MIL-A187. In another embodiment, the anti-TIGIT first antibody comprises the VH and VL of an antibody selected from the group consisting of: T-MIL-A60-Hu01, T-MIL-A60-Hu02, T-MIL-A60-Hu03, T-MIL-A60-Hu04, T-MIL-A60-Hu05, T-MIL-A60-Hu06, T-MIL-A60-Hu07, and T-MIL-A169-Hu01 provided in Tables 10a and 10b. T-MIL-A169-Hu02, T-MIL-A169-Hu03, T-MIL-A169-Hu04, T-MIL-A169-Hu05, T-MIL-A169-Hu06, T-MIL-A1 69-Hu07, T-MIL-A169-Hu08, T-MIL-A169-Hu09, T-MIL-A169-Hu10, T-MIL-A169-Hu11 and T-MIL-A169-Hu12.

[0673] In another embodiment, the second anti-TIGIT antibody portion comprises:

[0674] (1) VH containing the amino acid sequence of SEQ ID NO: 422, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 423, substantially composed of or composed of therein;

[0675] (2) VH containing the amino acid sequence of SEQ ID NO: 424, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 425, substantially composed of or composed of therein;

[0676] (3) VH containing the amino acid sequence of SEQ ID NO: 426, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 427, substantially composed of or composed of therein;

[0677] (4) VH containing the amino acid sequence of SEQ ID NO: 428, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 429, substantially composed of or composed of therein;

[0678] (5) VH containing the amino acid sequence of SEQ ID NO: 430, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 431, substantially composed of or composed of therein;

[0679] (6) VH containing the amino acid sequence of SEQ ID NO: 432, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 433, substantially composed of or composed of therein;

[0680] (7) VH containing the amino acid sequence of SEQ ID NO: 434, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 435, substantially composed of or composed of therein;

[0681] (8) VH containing the amino acid sequence of SEQ ID NO: 436, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 437, substantially composed of or composed of therein;

[0682] (9) VH containing the amino acid sequence of SEQ ID NO: 438, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 439, substantially composed of or composed of therein;

[0683] (10) VH containing the amino acid sequence of SEQ ID NO: 440, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 441, substantially composed of or composed of therein;

[0684] (11) VH containing the amino acid sequence of SEQ ID NO: 442, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 443, substantially composed of or composed of therein;

[0685] (12) VH containing the amino acid sequence of SEQ ID NO: 444, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 445, substantially composed of or composed of therein;

[0686] (13) VH containing the amino acid sequence of SEQ ID NO: 446, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 447, substantially composed of or composed of therein;

[0687] (14) VH containing the amino acid sequence of SEQ ID NO: 448, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 449, substantially composed of or composed of therein;

[0688] (15) VH containing the amino acid sequence of SEQ ID NO: 450, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 451, substantially composed of or composed of therein;

[0689] (16) VH containing the amino acid sequence of SEQ ID NO: 452, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 453, substantially composed of or composed of therein;

[0690] (17) VH containing the amino acid sequence of SEQ ID NO: 454, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 455, substantially composed of or composed of therein;

[0691] (18) VH containing the amino acid sequence of SEQ ID NO: 456, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 457, substantially composed of or composed of therein;

[0692] (19) VH containing the amino acid sequence of SEQ ID NO: 458, substantially composed of or composed of therein, and VL containing the amino acid sequence of SEQ ID NO: 459, substantially composed of or composed of therein;

[0693] (20) VH and VL, which contain, are substantially composed of, or are composed of the corresponding amino acid sequences of the VH and VL of antibodies selected from those listed in Table 10a or Table 10b.

[0694] (21) VH and VL, which contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the corresponding amino acid sequences of the VH and VL of the antibodies selected from those defined in (1)-(20) above, are substantially composed of or constitute of the same amino acid sequences.

[0695] (22) VH and VL, which comprise, except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, the addition, deletion and / or substitution of which are identical to, substantially composed of or constituted by, the corresponding amino acid sequences of the antibodies selected from the antibodies defined in (1)-(20) above;

[0696] (23) VH and VL, comprising, substantially comprising, or consisting of the amino acid sequences of the anti-TIGIT antibody VH and VL, wherein the anti-TIGIT antibody binds to the same or overlapping epitopes on TIGIT with an antibody selected from those defined in (1)-(20) above; or

[0697] (24) VH and VL, which contain, are substantially composed of or consist of the amino acid sequences of VH and VL of the anti-TIGIT antibody, said anti-TIGIT antibody competitively binding to TIGIT with an antibody selected from the antibodies defined in (1)-(20) above.

[0698] The multispecific antibodies against PVRIG and TIGIT of the present invention can be bispecific. Preferably, the multispecific antibodies against PVRIG and TIGIT of the present invention comprise nine CDRs of antibodies selected from those listed in Table 12. More preferably, the multispecific antibodies against PVRIG and TIGIT of the present invention comprise VH and VL, which comprise the corresponding amino acid sequences of VH and VL of antibodies selected from those listed in Table 11. Most preferably, the multispecific antibodies against PVRIG and TIGIT of the present invention comprise a heavy chain and a light chain, which comprise the corresponding amino acid sequences of the heavy chain and light chain of antibodies selected from those listed in Table 11. The multispecific / bispecific antibodies of the present invention listed in Table 11 include: BsAb01, BsAb02, BsAb03, BsAb04, BsAb05, BsAb06, BsAb07, BsAb08, BsAb09, BsAb10, BsAb11, BsAb12, BsAb13, BsAb14, BsAb15, BsAb16, BsAb17, BsAb18, BsAb19, BsAb20, and BsAb21.

[0699] In some embodiments, the multispecific / bispecific antibody of the present invention comprises:

[0700] (1) The amino acid sequence comprising SEQ ID NO: 498, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 499, the light chain consisting of or consisting of therein;

[0701] (2) The amino acid sequence comprising SEQ ID NO: 500, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 501, the light chain consisting substantially of or consisting of therein;

[0702] (3) The amino acid sequence comprising SEQ ID NO: 502, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 503, the light chain consisting of or consisting of therein;

[0703] (4) The amino acid sequence comprising SEQ ID NO: 504, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 505, the light chain consisting substantially of or consisting of therein.

[0704] (5) The amino acid sequence comprising SEQ ID NO: 506, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 507, the light chain consisting of or consisting of therein.

[0705] (6) The amino acid sequence comprising SEQ ID NO: 508, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 509, the light chain consisting substantially of or consisting of therein.

[0706] (7) A heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 510 and substantially of it, and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 511.

[0707] (8) The amino acid sequence comprising SEQ ID NO: 512, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 513, the light chain consisting substantially of or consisting of therein;

[0708] (9) The amino acid sequence comprising SEQ ID NO: 514, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 515, the light chain consisting substantially of or consisting of therein.

[0709] (10) The amino acid sequence comprising SEQ ID NO: 516, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 517, the light chain consisting substantially of or consisting of therein.

[0710] (11) The amino acid sequence comprising SEQ ID NO: 518, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 519, the light chain consisting substantially of or consisting of therein.

[0711] (12) The amino acid sequence comprising SEQ ID NO: 520, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 521, the light chain consisting substantially of or consisting of therein;

[0712] (13) The amino acid sequence comprising SEQ ID NO: 522, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 523, the light chain consisting of or consisting of therein;

[0713] (14) The amino acid sequence comprising SEQ ID NO: 524, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 525, the light chain consisting of or consisting of therein;

[0714] (15) The amino acid sequence comprising SEQ ID NO: 526, the heavy chain consisting substantially of or composed of therein, and the amino acid sequence comprising SEQ ID NO: 527, the light chain consisting substantially of or composed of therein.

[0715] (16) The amino acid sequence comprising SEQ ID NO: 528, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 529, the light chain consisting substantially of or consisting of therein;

[0716] (17) The amino acid sequence comprising SEQ ID NO: 530, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 531, the light chain consisting of or consisting of therein.

[0717] (18) The amino acid sequence comprising SEQ ID NO: 532, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 533, the light chain consisting substantially of or consisting of therein;

[0718] (19) The amino acid sequence comprising SEQ ID NO: 534, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 535, the light chain consisting of or consisting of therein.

[0719] (20) The amino acid sequence comprising SEQ ID NO: 536, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 537, the light chain consisting substantially of or consisting of therein.

[0720] (21) The amino acid sequence comprising SEQ ID NO: 538, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 539, the light chain consisting substantially of or consisting of therein;

[0721] (22) Heavy chains and light chains, which contain, or are substantially composed of, the corresponding amino acid sequences of the heavy and light chains of antibodies selected from those listed in Table 11.

[0722] (23) Heavy and light chains comprising, substantially consisting of, or consisting of at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the corresponding amino acid sequences of the heavy and light chains of antibodies selected from those defined in (1)-(22) above; or

[0723] (24) Heavy and light chains comprising, substantially consisting of, or consisting of, the corresponding amino acid sequences of the heavy and light chains of antibodies selected from those defined in (1)-(22) above, except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, the addition, deletion and / or substitution of amino acids.

[0724] In the construction of multispecific antibodies containing two different antibodies (VH and / or VL), mismatch byproducts sometimes occur. One method to avoid the mismatch byproduct problem, the so-called 'button-in-pore', aims to alter the contact interface by introducing a mutation into the CH3 domain, thereby forcing the two different antibody heavy chains to pair. On one chain, a bulky amino acid is replaced with an amino acid with a short side chain to form a 'pore'. Conversely, an amino acid with a bulky side chain is introduced into the other CH3 domain to create a 'button'. By co-expressing these two heavy chains (and two identical light chains, which must be adapted to the two heavy chains), a high yield of heterodimer formation ('button-in-pore') relative to homodimer formation ('pore-in-pore' or 'button-in-button') has been observed (Ridgway JB, Presta LG, Carter P; and WO1996027011). The percentage of heterodimers can be further increased by reshaping the interaction surface of the two CH3 domains using phage display methods and introducing disulfide bridges to stabilize the heterodimer (Merchant AM et al., Nature Biotech 16 (1998) 677-681; Aτwell S, Ridgway JB, Wells JA, Carter P., J Mol Biol 270 (1997) 26-35). For example, a novel method using knob-in-hole technology is described in EP1870459A1. Xie, Z. et al., J Immunol Methods 286 (2005) 95-101 mentions the use of scFv combined with knob-in-hole technology for the FC portion of bispecific antibodies. Modular structures of antibodies have been developed to generate more than 60 different bispecific antibody forms. See Spiess et al. (2015) Molecular Immunology 67:95-106, which is incorporated herein by reference in its entirety.Therefore, in some respects, bispecific antibody forms are selected from crossMab, DAF (dual-action Fab) (two-in-one), DAF (quadruple-in-one), DutaMab, DT-IgG, Knobs-in-holes common LC, Knobs-in-holes assembly, charge pairs, Fab arm exchange, SEEDbody, Triomab, LUZ-Y (a bispecific antibody with a bright zipper that induces heterodimerization of two HCs), Fcab, orthogonal Fab, DVD-IgG (dual variable domain IgG), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG (Four-in-one), Nanobody, Nanobody-HSA, BiTE (Bispecific T cell Engager), Biantibody, DART (Dual-affinity-retargeting), TandAb (Tandab), scDiabody, scDiabody-CH3, Triple Body, Miniantibody, Minibody, TriBi Miniantibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-ScFv2, scFv-KIH, Fab-scFv-Fc, Quadrivalent HC Ab, scDiabody-Fc, Biantibody-Fc, Tandem scFv-Fc, Intracellular Antibody, Docking Locking Antibody Locck), ImmTAC, HSAbody, scDiabody-HSA, tandem scFv-toxin, IgG-IgG, Cov-X-Body and scFv1-PEG-scFV2.

[0725] In some implementations, the multispecific antibodies against PVRIG and TIGIT are selected from the antibodies listed in Table 11. Specifically, the multispecific antibodies against PVRIG and TIGIT are BsAb01, BsAb02, BsAb03, BsAb04, BsAb05, BsAb06, BsAb07, BsAb08, BsAb09, BsAb10, BsAb11, BsAb12, BsAb13, BsAb14, BsAb15, BsAb16, BsAb17, BsAb18, BsAb19, BsAb20, or BsAb21 as described in Table 11.

[0726] 5. Immunoconjugates

[0727] In some aspects, immunoconjugates of the antibodies of the present invention are also provided. "Immunocaloric conjugate" refers to the antibody provided herein conjugated to one or more heterologous molecules. In some embodiments, the anti-PVRIG antibody provided herein is an immunoconjugate. In some embodiments, the anti-TIGIT antibody provided herein is an immunoconjugate. In some embodiments, the multispecific antibody provided herein is an immunoconjugate.

[0728] In some embodiments, the antibody or antigen-binding fragment of the present invention is conjugated to one or more cytotoxic agents. Exemplary cytotoxic agents include chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant or animal-derived enzymatically active toxins or fragments thereof), and radioactive isotopes.

[0729] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody provided herein is conjugated to one or more drugs. In some instances, the anti-PVRIG antibody provided herein is an ADC. In some instances, the anti-TIGIT antibody provided herein is an ADC. In some other instances, the bispecific antibody provided herein is an ADC. Exemplary pharmaceutical products that can be conjugated with the antibodies of this invention include maytansinoids (see, for example, U.S. Patent Nos. 5,208,020 and 5,416,06); aurestatins, such as monomethylaurestatin pharmaceutical portions DE and DF (MMAE and MMAF) (see, for example, U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298); dollasstatin; galicarmycin or derivatives thereof (see, for example, U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., (1993) Cancer Research 53:3336-3342; and Lode et al., (1998) Cancer Res 58:2925-2928); anthracyclines, such as daunomycin or doxorubicin (see, for example, Kratz et al., (2006) Current Med. Chem 13:477-523; Jeffrey et al., (2006) Bioorganic & Med Chem Letters 16:358-362; Torgov et al., (2005) Bioconj Chem 16:717-721; Nagy et al., (2000) Proc Natl Acad Sci USA 97:829-834; Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., (2002) J Med Chem 45:4336-4343; and U.S. Patent No. 6,630,579), methotrexate, vindesine, and paclitaxel such as docetaxel, paclitaxel, larotaxel, teciltaxel, and ortataxel.

[0730] In some embodiments, the immunoconjugate comprises antibodies conjugated herein to the following substances: enzymatically active toxins or fragments thereof (such as diphtheria A chain, non-conjugated active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa)), ricin A chain, abrin A chain, sucralose root toxin A chain, α-arbusculin, tung oil protein (Aleurites fordii protein), caryophyllin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, jatropha toxin protein, croton toxin protein, sphagnum moss inhibitor, white tree toxin, mitogellin, localized aspergillin, phenolmycin, inomycin, and trichothecenes.

[0731] In some embodiments described herein, antibodies are conjugated with radioactive atoms to form radioconjugates. A variety of radioisotopes can be used to produce radioconjugates. Examples include radioisotopes of At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212, and Lu. When the radioconjugate is used for detection, it may contain radioactive atoms for scintillation imaging studies, such as tc99m or 1123, or spin-labeled atoms for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0732] The antibody and cytotoxic agent conjugates described herein can be prepared using a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyl dithio)propionate (SPDP), succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), iminotetrahydrothiophene (IT), bifunctional derivatives of imine esters (such as dimethyl diimide adipate HQ), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), bis-diazatonium derivatives (such as bis-(p-diazatonium benzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and biactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described by Vitetta et al., (1987) Science 238: 1098. Carbon-14 labeled l-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See, for example, WO94 / 11026. The adapter can be a “cleavable adapter” that facilitates the release of cytotoxic drugs into cells. For example, acid-instable adapters, peptidase-sensitive adapters, photostable adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., (1992) Cancer Res 52: 127-131; U.S. Patent No. 5,208,020).

[0733] Immunoconjugates or ADCs can be prepared using cross-linking agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC, and sulfon-SMPB, as well as SVSB (succinimide-(4-vinyl sulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0734] In one embodiment, an immunoconjugate comprising an anti-PVRIG antibody provided herein linked to a therapeutic agent or imaging agent is provided. In another embodiment, an immunoconjugate comprising an anti-TIGIT antibody provided herein linked to a therapeutic agent or imaging agent is provided. In yet another embodiment, an immunoconjugate comprising an antibody against PVRIG or TIGIT provided herein linked to a therapeutic agent or imaging agent is provided. In one embodiment, an immunoconjugate comprising a bispecific PVRIG / TIGIT antibody provided herein linked to a therapeutic agent or imaging agent is provided.

[0735] 6. Nucleic acid molecules

[0736] This invention also provides isolated nucleic acid molecules encoding the antibodies of this invention or their antigen-binding fragments. The antibodies of this invention may be anti-PVRIG antibodies, anti-TIGIT antibodies, or multispecific / bispecific antibodies as described above.

[0737] In some embodiments, the present invention also provides isolated nucleic acid molecules encoding the VH and / or VL regions of antibodies or antigen-binding fragments thereof as defined herein. In some instances, the nucleic acid molecule comprises a nucleic acid sequence encoding the VH region of an antibody listed in Tables 2, 4b, 4c, or 6a. In some other instances, the nucleic acid molecule comprises a nucleic acid sequence encoding the VH region of an antibody listed in Tables 8a, 10a, or 10b, and / or a nucleic acid sequence encoding the VL region of an antibody listed in Tables 8a, 10a, or 10b.

[0738] In some implementations, the nucleic acid molecule contains a nucleic acid sequence encoding the antibody heavy chain listed in Tables 3, 4d, 4e, or 6b.

[0739] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain of an antibody listed in Tables 8b, 10c, 10d, or 11 and / or a nucleic acid sequence encoding the light chain of the antibody.

[0740] The term "nucleic acid molecule" is used interchangeably with the term "polynucleotide" and includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide consists of a base, particularly 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. Typically, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms.

[0741] Furthermore, the nucleic acid molecules described herein may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having residues with derived sugar or phosphate backbones linked or chemically modified. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for the direct expression of the antibodies of the present invention in vitro and / or in vivo (e.g., in a host or patient). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA carrier and / or the expression of the encoding molecule, thereby allowing the mRNA to be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2101 823 B1).

[0742] 7. Carrier

[0743] The nucleic acids encoding the VH and / or VL of the antibody of the present invention can be cloned into an expression vector, with each nucleotide sequence effectively linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy chain and / or light chain is effectively linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy chain and light chain can be effectively linked to a single promoter, such that both the heavy chain and light chain are expressed by the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy chain and light chain coding sequences.

[0744] In some instances, the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cell expressing them, and the separated heavy and light chains can be mixed and incubated under suitable conditions that allow antibody formation.

[0745] Typically, a nucleic acid sequence encoding one or all strands of an antibody can be cloned into a suitable expression vector that is efficiently linked to a suitable promoter, using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to generate complementary ends on each molecule, which can then be paired with each other and ligated together using a ligase. Alternatively, synthetic nucleic acid adapters can be attached to the ends of the gene. These synthetic adapters contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell used to produce the antibody.

[0746] A variety of promoters can be used for the expression of the antibodies described herein, including but not limited to the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, Escherichia coli lac UV5 promoter and herpes simplex tk vims promoter.

[0747] Adjustable promoters can also be used. Such adjustable promoters include those that use E. coli lac repressors as transcription regulators to regulate transcription from mammalian cell promoters with lac operons [Brown, M. et al., Cell, 49:603-612 (1987)], and those that use tetracycline repressors (tetR) [Gossen, M. and Bujard, H., Proc.Natl.Acad.Sci.USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P. et al., Proc.Natl.Acad.Sci.USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16 or p65 using estradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0748] Adjustable promoters including repressors with operons can be used. In one embodiment, the *E. coli* lac repressor can act as a transcription regulator to regulate transcription from mammalian cell promoters with lac operons [M. Brown et al., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)], combining a tetracycline repressor (tetR) with a transcription activator (VP16) to produce the tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP16), which is then combined with a minimal promoter containing a tetO-derived major immediate early promoter from human cytomegalovirus (hCMV) to produce the tetR-tet operon system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operon is properly positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor alone (tetR), rather than a tetR-mammalian cell transcription factor fusion derivative, can act as a potent trans-regulator to control gene expression in mammalian cells (Yao et al., Human Gene Therapy, 10(16): 1392-1399(2003)). A particularly advantageous aspect of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivators or repressor fusion proteins (which may be cellularly toxic in some cases (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995))) to achieve its regulated function.

[0749] In addition, the vector may contain some or all of the following: selection marker genes, such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; enhancer / promoter sequences for the immediate early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 multitumor origin of replication and ColE1 for normal appendage replication; internal ribosome binding site (IRESe), multifunctional multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are known and available in the art.

[0750] Examples of polyadenylation signals that can be used to implement the methods described herein include, but are not limited to, human collagen I polyadenylation signals, human collagen II polyadenylation signals, and SV40 polyadenylation signals.

[0751] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any antibody can be introduced into suitable host cells to produce antibodies. Host cells can be cultured under suitable conditions to express the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered from cultured cells (e.g., from cells or culture supernatant) using conventional methods, such as affinity purification. If desired, the polypeptide chain of the antibody can be incubated under suitable conditions for a suitable period of time to produce antibodies.

[0752] In some embodiments, the method for preparing the antibodies of the present invention involves a recombinant expression vector encoding a heavy and light chain of an antibody against PVRIG or TIGIT, or two chains encoding a double-stranded bispecific antibody as described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions that allow for the expression of the two polypeptide chains that form the antibody, which can then be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow for antibody formation.

[0753] In one example, two recombinant expression vectors are provided: one encoding one of the two chains of a heavy chain or a double-chain bispecific antibody against PVRIG or TIGIT disclosed herein, and the other encoding the other chain of a light chain or a bispecific antibody against TIGIT. Both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) using conventional methods, such as calcium phosphate-mediated transfection. Alternatively, each expression vector can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow for antibody polypeptide chain expression. When both expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If desired, the polypeptide chain can be recovered from the host cell or culture medium and then incubated under suitable conditions that allow for antibody formation. When the two expression vectors are introduced into different host cells, each of them can be recovered from the respective host cell or the respective culture medium. The two polypeptide chains can then be incubated under conditions suitable for antibody formation.

[0754] Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover antibodies from culture media. For example, some antibodies can be separated by affinity chromatography using a matrix conjugated with protein A or protein G.

[0755] Any nucleic acid, heavy chain, light chain, or both of the heavy chain or light chain of the antibody against PVRIG or TIGIT or any bispecific antibody described herein, the vector containing the nucleic acid (e.g., an expression vector), and the host cell containing the vector are all within the scope of this disclosure.

[0756] The present invention also relates to vectors comprising the nucleic acid sequences disclosed above, wherein the nucleic acid sequences can be effectively linked to regulatory elements that drive the expression of the nucleic acid sequences in host cells.

[0757] 8. Host cell

[0758] The host cells disclosed in this disclosure can be any cells suitable for expressing the antibodies of this disclosure, such as mammalian cells. Mammalian host cells used for expressing the antibodies of this disclosure include Chinese hamster ovary (CHO) cells (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with the DHFR selection marker, for example, described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow the antibody to be expressed in the host cells or to secrete the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0759] 9. Pharmaceutical Compositions

[0760] In some aspects, the present invention relates to pharmaceutical compositions comprising at least one antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier.

[0761] The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions disclosed herein may also be administered in combination therapy with agents used to treat proliferative disorders (e.g., cancer or tumors), infections, or sepsis (e.g., anticancer agents). The antibodies of the present invention can improve symptoms of proliferative disorders, infections, or sepsis associated with overexpression of PVRIG and / or TIGIT.

[0762] In some implementations, cancer or tumor is selected from the group consisting of the following conditions or combinations thereof: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, stomach / gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial carcinoma, lung cancer (small cell lung cancer or non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cell carcinoma (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, and myelodysplastic syndrome (MDS). These conditions may be associated with aberrant expression of PVRIG and / or TIGIT. In some specific embodiments, the cancer or tumor is selected from the group consisting of the following cancers or combinations thereof: triple-negative breast cancer, gastric / stomach cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), Merkel cell carcinoma, MSI-high cancer, KRAS-mutant tumors, adult T-cell leukemia / lymphoma, and myelodysplastic syndromes (MDS). In some specific embodiments, the cancer or tumor is selected from the group consisting of the following cancers or combinations thereof: lung cancer (small cell lung cancer or non-small cell lung cancer), prostate cancer, breast cancer (e.g., triple-negative breast cancer), head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematologic malignancies, Merkel cell carcinoma, and MSI-high cancer, as well as any other disease or condition characterized by uncontrolled cell growth.

[0763] In some other implementations, the infection is a pathogen infection characterized by varying degrees of dysfunction in the virus-specific T-cell response, such as HIV, HCV, or HBV.

[0764] In some other implementations, sepsis includes severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, and sepsis syndrome.

[0765] Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic excipients, and other various components or combinations thereof known in the art.

[0766] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propylgalactose. As disclosed in this disclosure, in a solvent containing an antibody or antigen-binding fragment of this disclosure, the compositions of this disclosure contain one or more antioxidants, such as methionine, which can oxidize the reducing antibody or its antigen-binding fragment. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this disclosure provides compositions comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. This disclosure also provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, such that oxidation of the antibody or its antigen-binding fragment can be prevented, thereby extending their shelf life and / or increasing their activity.

[0767] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, or glucose injection and lactated Ringer's injection; non-aqueous media such as non-volatile oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents that inhibit or suppress fungal concentrations; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); sequestering agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents used as carriers can be incorporated into pharmaceutical compositions in multi-dose containers, such containers including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride. Suitable excipients may include, for example, water, saline, glucose, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubilizers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0768] The pharmaceutical compositions of the present invention can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal administration, or otherwise via implantation or inhalation. The subject compositions can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected according to the intended application and treatment regimen.

[0769] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups, or inhalers and their controlled-release forms.

[0770] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous liquids, isotonic liquids, pyrogen-free liquids, and sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise delivered (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of suitable isotonic carriers for such formulations include sodium chloride injection, Ringer's solution, or lactate Ringer's injection. Similarly, the specific dosing regimen, including dosage, timing, and repetition, will depend on the specific individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0771] The frequency of administration can be determined and adjusted during treatment, and is based on reducing the number of proliferative or tumorigenic cells, maintaining the reduction of such tumor-forming cells, reducing the proliferation of tumor-forming cells, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the subject therapeutic composition may be suitable.

[0772] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or adverse side effects. The chosen dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, route of administration, time of administration, rate of excretion of the compound, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the disease, and the patient's species, sex, age, weight, condition, general health status, and medical history. The amount of compound and route of administration will ultimately be determined by a physician, veterinarian, or clinician, although a dosage will generally be chosen to achieve local concentrations at the site of action to achieve the desired effect without causing substantial harmful or adverse side effects.

[0773] Generally, the antibodies or antigen-binding fragments thereof disclosed herein can be administered in a variety of ranges. These ranges include about 5 μg / kg body weight to about 100 mg / kg body weight per dose; about 50 μg / kg body weight to about 5 mg / kg body weight per dose; and about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose, and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, and at least about 10 mg / kg body weight.

[0774] In any case, the antibody or antigen-binding fragment thereof disclosed herein is preferably administered to the subject in need as required. The frequency of administration can be determined by those skilled in the art, such as attending physicians, based on considerations of the disease being treated, the age of the subject being treated, the severity of the disease being treated, and the general health condition of the subject being treated.

[0775] In some preferred embodiments, the treatment process involving the antibody or antigen-binding fragment thereof of this disclosure will include multiple doses of the selected drug over several weeks or months. More specifically, the antibody or antigen-binding fragment thereof of this disclosure may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it should be understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

[0776] For individuals who have received one or more administrations, the dosage and regimen of the disclosed therapeutic composition can also be determined empirically. For example, individuals can be given incremental doses of the therapeutic composition produced as described herein. In selected embodiments, the dosage can be gradually increased, decreased, or reduced based on empirically determined or observed side effects or toxicities. To assess the efficacy of the selected composition, biomarkers of a specific disease, condition, or ailment can be tracked as previously described. For cancer, these include direct measurement of tumor size by palpation or visual inspection, indirect measurement of tumor size by X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopic examination of tumor samples; measurement of reduction of indirect tumor biomarkers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein; improvement in speech, vision, breathing, or other tumor-related disabilities; enhanced appetite; or improvement in quality of life as measured by acceptable testing or prolonged survival. It will be apparent to those skilled in the art that the dosage will vary depending on the individual, the type of neoplastic ailment, the stage of the neoplastic ailment, whether the neoplastic ailment has begun to metastasize to other sites on the individual, and past and current concurrent treatments.

[0777] The compatible formulation, administered parenterally (e.g., intravenously), will contain the antibody or antigen-binding fragment thereof disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. In some selected embodiments, the concentration of the antibody or antigen-binding fragment thereof will include 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1 mg / ml. In other preferred embodiments, the concentration of the antibody or its antigen-binding fragment will include 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml.

[0778] 10. Applications / Indications

[0779] On the other hand, this document provides methods for using conjugates (e.g., anti-PVRIG antibodies, anti-TIGIT antibodies, multispecific / bispecific antibodies that bind both PVRIG and TIGIT) or compositions provided herein.

[0780] The antibodies disclosed herein (e.g., anti-PVRIG antibodies, anti-TIGIT antibodies, multispecific / bispecific antibodies binding both PVRIG and TIGIT) can be used in a variety of applications, including but not limited to therapeutic treatments such as the treatment of cancer, infection, or sepsis. In some embodiments, the therapeutic treatment includes immunotherapy for cancer. In some embodiments, the conjugates described herein can be used to activate, promote, increase, and / or enhance an immune response against cancer or cancer cells. In some embodiments, the conjugates described herein are used to activate, promote, increase, and / or enhance an immune response against tumors or tumor cells. The methods of use can be in vitro, ex vivo, or in vivo.

[0781] The cancer or tumor mentioned can be, but is not limited to, the following conditions or combinations thereof: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, stomach / gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial carcinoma, lung cancer (small cell lung cancer or non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cell carcinoma (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, and myelodysplastic syndrome (MDS). The above conditions may be associated with abnormal expression of PVRIG and / or TIGIT. In some specific embodiments, the cancer or tumor is selected from the group consisting of the following cancers or combinations thereof: triple-negative breast cancer, gastric / stomach cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), Merkel cell carcinoma, MSI-high cancer, KRAS-mutant tumors, adult T-cell leukemia / lymphoma, and myelodysplastic syndromes (MDS).

[0782] In some implementations, infection is a pathogen infection characterized by varying degrees of dysfunction in the virus-specific T-cell response, such as HIV, HCV, or HBV. In some implementations, sepsis includes severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, and sepsis syndrome.

[0783] Preferably, the disease is a proliferative disease; more preferably, the proliferative disease is cancer. More preferably, the cancer is selected from the group consisting of: lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematologic malignancies, and any other disease or condition characterized by uncontrolled cell growth.

[0784] In some embodiments, this document provides a method for inhibiting the interaction between a PVRIG (e.g., expressed on and / or in a first cell) and a ligand of the PVRIG (e.g., expressed on and / or in a second cell), comprising contacting the PVRIG (e.g., a first cell expressing the PVRIG) with a binding agent (e.g., an antibody or fragment thereof) provided herein. In some embodiments, this document provides the use of the binding agent provided herein for inhibiting the interaction between a PVRIG (e.g., expressed on and / or in a first cell) and a ligand of the PVRIG (e.g., expressed on and / or in a second cell), wherein said use includes binding the PVRIG (e.g., a first cell expressing the PVRIG) with a multispecific binding agent provided herein. ( For example, contact with an antibody or a fragment thereof. In some embodiments, the ligand is a CD112 molecule.

[0785] As used herein, the term "inhibition" means reduction or decrease. For example, in some embodiments, the binders provided herein inhibit the interaction between PVRIG and its ligand by 10%–99%. In other embodiments, the binders provided herein inhibit the interaction between PVRIG and its ligand by 100% (i.e., completely eliminate the interaction as measured by an assay). In some embodiments, PVRIG and its ligand are expressed on different cells. In some embodiments, PVRIG is expressed on a first cell, such as an immune cell. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a cytotoxic T cell, such as a CD8+ T cell.

[0786] In some embodiments, this document provides a method for inhibiting the interaction between TIGIT (e.g., expressed on and / or in a first cell) and a TIGIT ligand (e.g., expressed on and / or in a second cell), comprising contacting TIGIT (e.g., a first cell expressing TIGIT) with a conjugate (e.g., an antibody or fragment thereof) provided herein. In some embodiments, this document provides the use of the conjugate provided herein for inhibiting the interaction between TIGIT (e.g., expressed on and / or in a first cell) and a TIGIT ligand (e.g., expressed on and / or in a second cell), wherein said use includes contacting TIGIT (e.g., a first cell expressing TIGIT) with a multispecific conjugate provided herein. ( For example, contact with an antibody or a fragment thereof. In some embodiments, the ligand is a CD155 molecule.

[0787] As used herein, the term "inhibition" means reduction or decrease. For example, in some embodiments, the binders provided herein inhibit the interaction between TIGIT and its ligand by 10%–99%. In other embodiments, the binders provided herein inhibit the interaction between TIGIT and its ligand by 100% (i.e., the interaction is completely eliminated as measured by assay). In some embodiments, TIGIT and its ligand are expressed on different cells. In some embodiments, TIGIT is expressed on a first cell, such as an immune cell. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a cytotoxic T cell, such as a CD8+ T cell. In some embodiments, the T cell is a Treg cell.

[0788] Immune cells are cells in the immune system and can be cells of the lymphatic lineage. Non-restricted examples of lymphatic lineage cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer (NK) cells, and lymphocytes (B cells and T cells). T cells are a type of lymphocyte characterized by the expression of T cell receptors (TCRs). T cells play a central role in adaptive immune responses. T cell subtypes have multiple important functions in controlling and shaping immune responses. For example, cytotoxic T cells (also known as cytotoxic T lymphocytes and killer T cells) are T lymphocytes that kill certain cells, such as cancer cells, cells infected by intracellular pathogens (such as viruses or bacteria), or cells damaged in other ways. Most cytotoxic T cells express T cell receptors (TCRs) that recognize specific antigens. CD8+ T cells are a subset of MHC class I restricted T cells and are mediators of adaptive immunity, which are important for killing cancerous or virally infected cells. NK cells are a type of cytotoxic lymphocyte that is crucial to the innate immune system and belong to the innate lymphoid cell (ILC) family. In some implementations, NK cells can be identified by the presence of CD56 and the absence of CD3 (CD56+, CD3). In the absence of antibodies and MHC, NK cells have the ability to recognize and kill stress cells, thereby allowing for a faster immune response.

[0789] In some embodiments, CD112 is expressed on a second cell, such as a cancer cell or tumor cell. In some embodiments, CD155 is expressed on a second cell, such as a cancer cell or tumor cell.

[0790] In some embodiments, the present invention provides a method for activating, reducing or eliminating the number and / or activity of at least one type of regulatory T cell (Treg) in vitro and in vivo, comprising administering to a subject in need the anti-PVRIG antibody, anti-TIGIT antibody and / or multispecific / bispecific antibodies against PVRIG and TIGIT, immunoconjugates, nucleic acids, vectors, host cells and / or pharmaceutical compositions of the present invention as described above.

[0791] In other embodiments, this document provides methods for preventing the suppression of immune cells, such as suppression mediated by the interaction between PVRIG expressed on immune cells and its ligand (e.g., expressed on cancer or tumor cells), and / or suppression mediated by the interaction between TIGIT (e.g., expressed on immune cells) and its ligand (e.g., expressed on cancer or tumor cells). In other embodiments, this document provides methods for activating immune cell-mediated responses, such as antitumor responses. In some embodiments, the method includes binding immune cells to a binding agent provided herein. ( For example, contact with antibodies or fragments thereof. In some embodiments, the binding agents provided herein are used to prevent the inhibition or activation of immune cell-mediated responses. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are monocytes. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are cytotoxic T cells, such as CD8+ T cells. In some embodiments, the immune cells express PVRIG. Alternatively or optionally, the immune cells express TIGIT. In some embodiments, the immune cells mediate anti-cancer / tumor responses. In other embodiments, cancer or tumor cells express CD112. Alternatively or optionally, cancer or tumor cells express CD155.

[0792] This disclosure provides methods for activating an immune response in a subject using the binding agents described herein (e.g., anti-PVRIG antibodies, anti-TIGIT antibodies, or multispecific antibodies that bind to both PVRIG and TIGIT). In some embodiments, this disclosure provides methods for promoting an immune response in a subject using the binding agents described herein. In some embodiments, this disclosure provides methods for increasing an immune response in a subject using the binding agents described herein. In some embodiments, this disclosure provides methods for enhancing an immune response in a subject using the binding agents described herein. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes stimulation of monocytes. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes stimulation of macrophages. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes enhancement of macrophage phagocytosis. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes stimulation of dendritic cells. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes enhancement of FCR-mediated dendritic cell activity. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes enhancement of cell-mediated immunity. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes increasing effector T cell activity. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes increasing CD8+ T cell activity. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes increasing NK cell activity. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes enhancing NK cell activity. In some embodiments, the immune response is a result of antigen stimulation. In some embodiments, the antigenic stimulus is tumor cells. In some embodiments, the antigenic stimulus is cancer. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes enhancing cytokine release from immune cells. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes enhancing TNFα release from immune cells. In some embodiments, activation, promotion, increase, and / or enhancement of the immune response includes enhancing IFN-γ release from immune cells. In some embodiments, the antibodies described herein enhance antitumor immunity.

[0793] In some embodiments, the conjugates described herein (e.g., anti-PVRIG antibodies, anti-TIGIT antibodies, multispecific / bispecific antibodies, and / or their antigen-binding fragments) may be used in compositions and methods for treating diseases or conditions. Therefore, in some embodiments, this document provides a method for treating a subject's disease or condition, comprising administering to the subject the conjugate or pharmaceutical composition provided herein. In other embodiments, this document provides the use of the conjugate or pharmaceutical composition provided herein for treating a subject's disease or condition. In other embodiments, this document provides the conjugate or pharmaceutical composition provided herein for preparing a medicament for treating a disease or condition.

[0794] In some implementations, the treatments provided herein include relieving one or more symptoms associated with a disease or condition (e.g., cancer or tumor).

[0795] It also provides adjustments (e.g.) ， Methods for inhibiting, reducing, or preventing tumor growth in a subject. For example, the method includes administering to a subject a composition comprising a binder (e.g., an anti-PVRIG antibody, an anti-TIGIT antibody, a multispecific / bispecific antibody, and / or an antigen-binding fragment thereof) or another composition disclosed herein in an amount that effectively modulates tumor growth in the subject.

[0796] In some implementations, the disease or ailment is cancer or a tumor.

[0797] In some implementations, the cancer is blood cancer. In some implementations, the cancer is leukemia. In some implementations, the cancer is lymphoma.

[0798] In some implementations, cancer is a solid tumor.

[0799] As used in this article, “tumor” refers to any tumor-forming cell growth or proliferation, whether malignant or benign, and to all precancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals characterized by unregulated cell growth.

[0800] In some embodiments, the tumor or cancer is a solid tumor. In some embodiments, the tumor or cancer is not a solid tumor. In some embodiments, the tumor or cancer is a recurrent tumor or cancer. In some embodiments, the tumor or cancer is a metastatic tumor or cancer. In some embodiments, the tumor or cancer is a primary tumor or cancer. In some embodiments, the tumor or cancer has reached remission but may relapse. In some embodiments, the tumor or cancer is unresectable. Additionally or optionally, the tumor or cancer is resistant to chemotherapy or other anticancer therapies.

[0801] In some implementations, the cancer or tumor is selected from the group consisting of the following conditions or combinations thereof: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, stomach / gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial carcinoma, lung cancer (small cell lung cancer or non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cell carcinoma (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, and myelodysplastic syndrome (MDS). The above conditions may be associated with abnormal expression of PVRIG and / or TIGIT. In some specific embodiments, the cancer or tumor is selected from the group consisting of the following cancers or combinations thereof: triple-negative breast cancer, gastric / stomach cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), Merkel cell carcinoma, MSI-high cancer, KRAS-mutant tumors, adult T-cell leukemia / lymphoma, and myelodysplastic syndromes (MDS). In some specific embodiments, the cancer or tumor is selected from the group consisting of the following cancers or combinations thereof: lung cancer (small cell lung cancer or non-small cell lung cancer), prostate cancer, breast cancer (e.g., triple-negative breast cancer), head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematologic malignancies, Merkel cell carcinoma, and MSI-high cancer, as well as any other disease or condition characterized by uncontrolled cell growth.

[0802] The antibodies, pharmaceutical compositions, and methods disclosed herein can also be used to block the binding of TIGIT to its ligand PVR, or the binding of PVRIG to its ligand CD112, in vitro and in vivo.

[0803] The anti-PVRIG antibody, anti-TIGIT antibody, multispecific / bispecific antibodies against PVRIG and TIGIT, their antigen-binding fragments, immunoconjugates, expression vectors, host cells, and pharmaceutical compositions of the present invention can be used as part of a combination therapy, wherein one or more additional therapeutic agents may also be administered sequentially or simultaneously to the subject in need. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is a modulator of PD-L1 activity. In some embodiments, the additional therapeutic agent is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is LAE005.

[0804] Antibody-dependent cytotoxicity (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, is a cell-mediated immune defense mechanism in which effector cells of the immune system kill target cells whose membrane surface antigens have been bound by specific antibodies. Typical ADCC involves the activation of NK cells by antibodies in the multi-level progression of immune control. NK cells express Fcγ receptors. These receptors recognize and bind to the corresponding reciprocal portion of antibodies such as IgG, which bind to the surface of tumor cells or pathogen-infected target cells. The most common Fc receptors on the NK cell surface are CD16 or FcγRIII. Once an Fc receptor binds to the Fc region of an antibody, the NK cell releases cytotoxic factors that lead to the death of the target cells. In some embodiments, one or more therapeutic agents are antibodies that initiate ADCC. In some embodiments, one or more therapeutic agents are anti-EGFR antibodies or EGFR-MET bispecific antibodies. In some embodiments, the anti-EGFR antibody is cetuximab. In some embodiments, the anti-EGFR-Met bispecific antibody is ervaltimab. Suitable routes for administering the antibodies of the present invention (such as multispecific / bispecific antibodies, their antigen-binding fragments, immunoconjugates, expression vectors, host cells and pharmaceutical compositions, and / or one or more additional therapeutic agents) are well known in the art, such as intravenous injection (e.g., intravenous infusion), intratumoral injection, or injection adjacent to the tumor or cancer. Although more than one route can be used to administer the agent (e.g., antibody), a particular route may provide a more direct and effective response than another.

[0805] 11. Diagnostic uses and testing methods

[0806] Labeled binding molecules that specifically bind to antigens, such as labeled antibodies and their derivatives and analogs, can be used for diagnostic purposes, such as detecting, diagnosing, or monitoring diseases.

[0807] The antibodies described herein can be used to determine antigen levels in biological samples using classical immunohistochemical methods described herein or known to those skilled in the art (e.g., see Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay markers are known in the art, including enzyme markers such as glucose oxidase; radioisotopes such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent markers such as luminol; and fluorescent markers such as fluorescein and rhodamine, as well as biotin.

[0808] It should be understood in the art that the size of the subject and the imaging system used will determine the number of imaging fractions required to produce diagnostic images. In the case of radioisotope fractions, for human subjects, the amount of radioactivity injected is typically in the range of about 5 to 20 millicuries of 99Tc. The labeled antibody will then accumulate at the cellular site containing the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Tumor Imaging: The Radiochemical Detection of Cancer, Chapter 13, S.W. Burchiel and Barhodes, eds., Masson Publishing Inc. (1982)).

[0809] The antibodies, pharmaceutical compositions, and methods disclosed herein have a variety of in vitro and in vivo uses, including, for example, monitoring or detecting the expression levels of PVRIG and / or TIGIT, detecting the presence of human PVRIG and / or TIGIT in a sample, or measuring the amount of human PVRIG and / or TIGIT.

[0810] 12. Combined use with chemotherapy

[0811] Antibodies or their antigen-binding fragments can be used in combination with anticancer agents, cytotoxic agents, or chemotherapy agents.

[0812] The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat proliferative disorders such as cancer, including but not limited to cytotoxic agents, cell inhibitors, anti-angiogenic agents, debulking agents, chemotherapy agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and antimetastatic agents, and immunotherapy agents. It should be understood that, in selected embodiments as described above, such anticancer agents may comprise conjugates and may be bound to the disclosed site-specific antibody prior to administration. More specifically, in some embodiments, selected anticancer agents are linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such engineered conjugates are explicitly considered to be within the scope of this disclosure. In other embodiments, the disclosed anticancer agent is administered in combination with a site-specific conjugate comprising the various therapeutic agents described above.

[0813] As used herein, the term "cytotoxic agent" means a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell damage. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzyme-active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A), fungi (e.g., α-aspergillus, localized aspergillus), plants (e.g., arisaema toxin, ricin, saccharitoxin, quercetin, pokeweed antiviral protein, saponins, white tree toxin, mitoxin, trichosanthes pollen protein, barley toxin, tung oil protein, caryophyllin protein, pokeweed protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, sphagnum moschata inhibitor, white tree toxin, mitoxin, localized aspergillus, phenolmycin, neomycin and trichothecenes), or animals (e.g., cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including fragments and / or variants thereof).

[0814] For the purposes of this disclosure, "chemotherapy agents" include compounds (e.g., cytotoxic agents or cell inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemotherapeutic agents typically target intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancer cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Generally, chemotherapy agents may include any agent that inhibits or is designed to inhibit cancerous cells or cells that may become cancerous or produce tumorigenic progeny (e.g., TICs). Such agents are often administered in combination, for example, in regimens such as CHOP or FOLFIRI, and are often the most effective.

[0815] Examples of anticancer agents that can be used in combination with the site-specific constructs of this disclosure (either as components of a site-specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridine, ethyleneimine and methylmelamine, acetic acid, camptothecin, bryophyll, callystatin, CC-1065, cryptophycins, dosstatin, pyromycin, soft coral alcohol, pancratistatin, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, and dynemicin. n), bisphosphonates, esperamycin, chromophore antibiotics chromophores, aclacinomysins, actinomycins, autrarnycin, azaserine, bleomycins, actinomycin C, carabicin, carmimycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN ® Doxorubicin, epirubicin, isorubicin, idarubicin, mesorcinol, mitomycin, mycophenolic acid, nogaramycin, oligomycin, pepromycin, pofibromycin, puromycin, quelamycin, doxorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, fentostatin, zorubicin; antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, antiadrenergics, folic acid supplements such as florrin, acetoglucan lactone, aldehydes Phosphamide glycosides, aminolevulinic acid, emuramicin, acridine, bestrabucil, bisantrene, edatrazine, defofamine, demecoxine; diaciconone, efornithine, eletylamine, epomomycin, etogluconol, gallium nitrate, hydroxyurea, lentinan, lonidin, maytansine derivatives, mitoxantrone, mitoxantrone, mupirocin, diammonium nitrate, pentostatin, benzylmethionine, pirarubicin, loxoantrone, podophyllic acid, 2-ethylhydrazine, procarbazine, PSK ®Polysaccharide complexes (JHS Natural Products, Eugene, Or), razorcinol, cezofuran; spirogermanium; Alternaria alternifolia ketoacid; triaminoquinone; 2,2',2"-trichlorotriethylamine; trichothecene compounds (especially T-2 toxin, veraculin A, baculosporin A, and serpentin); urethan; vincristine; dacarbazine; mannomustine; dibromomannitol; dibromoeusol; piperbbromide; gasitocin; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes; cholecystokinin; gemzar ® Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs; Vincristine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Navelbine ® Vinorelbine; Noscholine; Teniposide; Idatraxa; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; Difluoromethylornithine; Retinoids; Capecitabine; Cobbutatin; Folic acid; Oxaliplatin; Inhibitors of PKC-α, Raf, H-Ras, EGFR, and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes antihormonal drugs used to modulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; and Traxatabine (a 1,3-dioxolane cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, PROLEUKIN ® rIL-2; LURTOTECAN ® Topoisomerase 1 inhibitor; ABARELIX ® rmRH; vinorelbine and espamycin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0816] 13. Combined use with radiotherapy

[0817] This disclosure also provides combinations of antibodies or antigen-binding fragments thereof with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as gamma radiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combined therapies using the targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed conjugates can be used in combination with targeted anticancer agents or other targeted agents. Typically, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered in single or multiple, continuous doses.

[0818] 14. Drug Packaging and Reagent Kits

[0819] Kits are also provided, which include, alone or in any combination thereof, anti-PVRIG antibodies, anti-TIGIT antibodies, bispecific PVRIG / TIGIT antibodies or antigen-binding fragments thereof as described herein, and optional instructions for use.

[0820] In some embodiments, a unit dose is provided, wherein the unit dose comprises a predetermined amount of a composition comprising, for example, an antibody or an antigen-binding fragment thereof, with or without one or more additional agents. In other embodiments, such a unit dose is provided in a disposable pre-filled syringe for injection. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, etc.; buffers such as phosphates, etc.; and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the conjugate composition may be provided as a lyophilized powder, which can be reconstituted by adding a suitable liquid (e.g., sterile water or a saline solution). In some preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with one or more containers indicates that the encapsulated conjugate composition is intended for the treatment of a selected oncological condition.

[0821] This disclosure also provides kits for producing single- or multi-dose administration units of site-specific conjugates and optionally one or more anticancer agents. The kits include containers and labels or packaging inserts on or associated with the containers. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be formed from a variety of materials such as glass or plastic and contain a pharmaceutically effective amount of the disclosed conjugates in conjugated or unconjugated form. In other preferred embodiments, one or more containers include sterile access ports (e.g., the container may be an intravenous infusion bag or a vial with a stopper that can be punctured by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally, one or more anticancer agents in the same or different containers. The kits may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, in addition to the antibodies or antigen-binding fragments thereof disclosed herein, such kits may contain any one or more of a range of anticancer agents such as chemotherapy or radiotherapy agents, anti-angiogenic agents; anti-metastatic agents, targeted anticancer agents, cytotoxic agents, and / or other anticancer agents.

[0822] More specifically, the kit may have a single container containing the disclosed antibody or its antigen-binding fragment, with or without additional components, or they may have different containers for each desired agent. When providing combination therapies for conjugation, the single solutions may be premixed in equimolar amounts or in a manner where one component is in greater quantity than another. Alternatively, the conjugates of the kit and any optional anticancer agents may be stored separately in different containers prior to administration to a patient. The kit may also include a second / third container for containing sterile, pharmaceutically acceptable buffers or other diluents, such as sterile water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0823] When the reagent kit components are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as one or more dry powders. When reagents or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in a separate container.

[0824] As briefly noted above, the kit may also include tools for administering antibodies or their antigen-binding fragments and any optional components to a patient, such as one or more needles, intravenous injection bags or syringes, or even eye droppers, pipettes or other similar devices, thereby injecting or introducing the preparation into an animal or applying it to a diseased area of ​​the body. The kits disclosed herein typically also include devices for containing vials, etc., and other sealed components for commercial sale, such as injection or blow-molded plastic containers in which the desired vials and other devices are placed and held.

[0825] The kit of the present invention can be used to treat or prevent proliferative diseases (e.g., cancer or tumors), infections or sepsis in subjects.

[0826] The antibodies of this invention can improve symptoms of proliferative disorders, infections, or sepsis associated with overexpression of PVRIG and / or TIGIT.

[0827] In some other implementations, the infection is a pathogen infection characterized by varying degrees of dysfunction in the virus-specific T-cell response, such as HIV, HCV, or HBV.

[0828] The foreword is an overview and therefore necessarily contains simplifications, generalizations, and omissions of details; thus, those skilled in the art will understand that this overview is merely illustrative and not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, and / or apparatuses and / or other subjects described herein will become apparent from the teachings set forth herein. This overview is provided to introduce, in a simplified form, a series of concepts that will be further described in the detailed description that follows. This overview is not intended to identify key or essential features of the claimed subject matter, nor is it intended to help determine the scope of the claimed subject matter.

[0829] 15. Tables and Sequence Lists

[0830] The amino acid and nucleotide sequences of the antibodies of this invention are provided below:

[0831]

[0832] Table 1. Amino acid sequences of the three CDRs of seven alpaca anti-PVRIG antibodies VH

[0833]

[0834] Table 2. Amino acid sequences of VH in 7 alpaca anti-PVRIG antibodies

[0835]

[0836] Table 3. Amino acid sequences of the H chain of seven alpaca anti-PVRIG antibodies

[0837]

[0838]

[0839] Table 4a. Amino acid sequences of the three CDRs of the VH of some humanized alpaca antibody P-AIL-A32

[0840]

[0841] Table 4b. Amino acid sequence of VH in humanized alpaca anti-PVRIG antibody P-AIL-A15

[0842]

[0843] Table 4c. Amino acid sequence of VH in humanized alpaca anti-PVRIG antibody P-AIL-A32

[0844]

[0845] Table 4d. Amino acid sequence of the H chain of humanized alpaca anti-PVRIG antibody P-AIL-A15

[0846]

[0847]

[0848] Table 4e. Amino acid sequence of the H chain of humanized alpaca anti-PVRIG antibody P-AIL-A32

[0849]

[0850]

[0851] Table 5a. Amino acid sequences of the three CDRs of affinity-mature alpaca anti-PVRIG antibody P-AIL-A32, numbered according to the Kabat numbering system.

[0852]

[0853] Table 5b. Amino acid sequences of the three CDRs of the affinity-mature alpaca anti-PVRIG antibody P-AIL-A32, numbered according to the AbM numbering system.

[0854]

[0855] Table 6a. Amino acid sequence of VH in humanized and affinity-matured alpaca anti-PVRIG antibody AIL-A32

[0856]

[0857] Table 6b. Amino acid sequence of the H chain of humanized and affinity-matured alpaca anti-PVRIG antibody AIL-A32.

[0858]

[0859]

[0860] Table 7. Amino acid sequences of the six CDRs of 28 mouse anti-TIGIT antibodies

[0861]

[0862]

[0863]

[0864]

[0865]

[0866]

[0867] Table 8a. Amino acid sequences of VH and VL of 28 mouse anti-TIGIT antibodies

[0868]

[0869]

[0870]

[0871]

[0872]

[0873] Table 8b. Amino acid sequences of the H and L chains of 28 mouse anti-TIGIT antibodies

[0874]

[0875]

[0876]

[0877]

[0878]

[0879]

[0880]

[0881]

[0882]

[0883]

[0884] Table 9a. Amino acid sequences of the six CDRs of some humanized anti-TIGIT antibody T-MIL-A60

[0885]

[0886] Table 9b. Amino acid sequences of the six CDRs of 12 humanized mouse anti-TIGIT antibodies T-MIL-A169

[0887]

[0888]

[0889] Table 10a. Amino acid sequences of VH and VL of seven humanized mouse anti-TIGIT antibodies T-MIL-A60

[0890]

[0891]

[0892] Table 10b. Amino acid sequences of VH and VL of 12 humanized mouse anti-TIGIT antibodies T-MIL-A169

[0893]

[0894]

[0895]

[0896] Table 10c. Amino acid sequences of the heavy and light chains of seven humanized mouse anti-TIGIT antibodies T-MIL-A60.

[0897]

[0898]

[0899]

[0900] Table 10d. Amino acid sequences of the heavy and light chains of 12 humanized mouse anti-TIGIT antibodies T-MIL-A169.

[0901]

[0902]

[0903]

[0904]

[0905]

[0906] Table 11. Amino acid sequences of the heavy and light chains of bispecific antibodies against PVRIG and TIGIT

[0907]

[0908]

[0909]

[0910]

[0911]

[0912]

[0913]

[0914]

[0915]

[0916]

[0917]

[0918] Table 12. Amino acid sequences of CDRs for 21 bispecific antibodies against PVRIG and TIGIT

[0919]

[0920]

[0921]

[0922]

[0923]

[0924]

[0925] Table 13. Amino acid sequence of the reference antibody used in the examples and figures:

[0926]

[0927] Example

[0928] The present disclosure, which is thus generally described, will be more readily understood by referring to the following embodiments, which are provided by way of example and are not intended to limit the present disclosure. The embodiments are not intended to represent all or only the experiments conducted.

[0929] Example 1

[0930] Example 1: Generation of human anti-PVRIG antibodies

[0931] The aim of this study was to isolate human antibodies that bind to PVRIG immuno-oncology targets with high affinity and specificity.

[0932] 1.1 PVRIG protein expression:

[0933] The human PVRIG ECD-Fc (NP_076975.2) or cynomolgus monkey PVRIG ECD-Fc (XP_005549281.1) fusion protein consists of the ECD of a human or cynomolgus monkey PVRIG fused with a human Fc. It was produced by transient transfection into CHO cells cultured for 6 days, followed by protein A purification of the cell harvest. The final product was prepared in PBS at pH 7.2. The expression vector used was the mammalian expression vector GSV0 (Sanyou Bio), in which the PVRIG gene was driven by the CMV promoter.

[0934] 1.2 PVRIG signal structure domain Ab:

[0935] Alpacas were immunized with 0.5 mg of human PVRIG-hFc protein, followed by three booster immunizations with human PVRIG or cyno-PVRIG protein. Total RNA was prepared from 100 ml of PBMCs from two different alpaca species. V HH gene was amplified from cDNA using PrimeSTAR Max DNA polymerase (TaKaRa) with primers specific to the V HH gene. HGene library. PCR products were purified by agarose gel electrophoresis and amplified again to add NcoI and NotI restriction sites. PCR products were digested with restriction endonucleases NcoI and NotI and purified by agarose gel electrophoresis. The resulting DNA fragments were ligated into plasmid PMID (Sanyou) digested with restriction endonucleases NcoI and NotI, and the ligated DNA was electroporated into *E. coli* strain SS320. A gene library containing 2.2 × 10⁻⁶ DNA fragments was obtained from the products of 23 ligation reactions and 15 electroporations. 9 Human phage display library of VHH gene of each member.

[0936] 1.3 Manual solid phage screening:

[0937] Thermo Scientific™ Immuno tubes (Thermo Fisher Scientific, catalog number 444202) are coated with huPVRIG-Fc or cyno-PVRIG-Fc (10 ug / ml) dissolved in PBS. After blocking with 5% skim milk powder in PBS, 5 x 10 μg / ml of the purified alpaca immunotherapy pool is added at room temperature. 12 One HuPVRIG-VHH phage was used for 1 hour. After several washing steps, the bound phage was eluted with 0.25% trypsin at room temperature for 20 minutes. As described above, three rounds of panning were performed, with phage amplification between each round.

[0938] 1.4 Liquid phage selection:

[0939] Phage antibody panning for huPVRIG or cyno-PVRIG was performed semi-automatically using the Kingfisher magnetic bead system. The beads were blocked with 5% BSA and then incubated with biotinylated PVRIG antigen. The phage library (1, 2) was mixed with biotinylated PVRIG-conjugated magnetic beads (300 nM) and gently rotated at room temperature for 1 hour to allow for PVRIG-specific phage binding. Non-specific bindings were removed with wash buffer (0.05% PBST). After washing, bound phages were eluted by incubation with 0.25% trypsin at room temperature for 20 minutes. Second and third rounds of panning were performed following the same procedure, except that the phage library was replaced with phage supernatant from the previous round of panning.

[0940] 1.5 Mixed solid / liquid phage selection:

[0941] Phage libraries were used for phage panning against biotinylated recombinant proteins of human PVRIG and cyno PVRIG. Three rounds of panning were performed using a mixture of solid phage panning (1.3) and liquid phage panning (1.4). After the final round of panning, binding of VHH antibodies to human PVRIG and / or Cyno PVRIG was screened in ELISA.

[0942] 1.6 HuPVRIG combined with VHH identification:

[0943] Clones obtained after three rounds of ELISA screening using solid phage panning (1.3), liquid phage panning (1.4), or a mixed solid phase (1.5) were directly bound to the huPVRIG antigen. ELISA was performed by coating 96-well ELISA plates (Costar) overnight at 4°C with 2 µg / ml huPVRIG protein (Sanyo), followed by blocking with 5% BSA for 1 h at room temperature, and then incubating with VHH supernatant diluted in PBS / 1% BSA / 0.05% Tween 20 for 1 h at room temperature. Positive VHH was detected using anti-Flag-HRP. The assay was performed using HRP chromogenic substrate (Sigma) according to the manufacturer's instructions. 223 positive hits were screened from an immune VHH library obtained from two alpacas. These clones were rearranged into new 96-well plates. They were grown overnight at 37°C, and the plasmid DNA was sequenced using heavy chain primers. The sequences were assembled and analyzed using SeqMan software. If more than one non-conserved difference exists in the heavy chain CDR3, the clone is considered sequence-unique. Forty-six uni...

Claims

1. An isolated anti-PVRIG antibody or its antigen-binding fragment, comprising a heavy chain variable (VH) region, said heavy chain variable region comprising: (1) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3; (2) HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7 and HCDR3 of SEQ ID NO: 8; (3) HCDR1 of SEQ ID NO: 11, HCDR2 of SEQ ID NO: 12 and HCDR3 of SEQ ID NO: 13; (4) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 17 and HCDR3 of SEQ ID NO: 18; (5) HCDR1 of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22 and HCDR3 of SEQ ID NO: 23; (6) HCDR1 of SEQ ID NO: 26, HCDR2 of SEQ ID NO: 27 and HCDR3 of SEQ ID NO: 28; (7) HCDR1 of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32 and HCDR3 of SEQ ID NO: 33; (8) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 36 and HCDR3 of SEQ ID NO: 18; (9) HCDR1, HCDR2 and HCDR3 that are identical to the HCDR1, HCDR2 and HCDR3 of the antibodies selected from those listed in Tables 2, 4b and 4c. (10) HCDR1, HCDR2 and HCDR3 that are otherwise identical to HCDR1, HCDR2 and HCDR3 listed in one of (1)-(9) above, except that they have at most 5 (e.g., at most 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more HCDRs. (11) HCDR of an anti-PVRIG antibody that binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(9) above; or (12) HCDR of an anti-PVRIG antibody that competes with one of the antibodies defined in (1)-(9) above for binding to PVRIG.

2. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable (VH) region, said heavy chain variable region comprising: (1) Contains the amino acid sequence X1FAVG (SEQ ID NO: 540), substantially composed of or composed of the amino acid sequence X1FAVG (SEQ ID NO: 540), and HCDR1, wherein X1 is S or A or L or G or P. (2) Contains the amino acid sequence CSX3X4X5X6X7X8X9X 10 YX 12 DSVKG (SEQ ID NO: 541), HCDR2 which is essentially composed of or consists of, wherein: X3 is either S or T; X4 is S, Y, or W; X5 is T, A, G, M, R, or L; X6 is either D or R; X7 X8 X9 X 10 Is it NSTD, ESTD, NSPF, APTD, or NVLA; and X 12 It is either D or A; and (3) Contains the amino acid sequence GWDCSGAVVYGVGH (SEQ ID NO: 18), and is substantially composed of or composed of HCDR3; The amino acids mentioned are numbered according to the Kabat numbering system; Preferably, X4 and X5 in HCDR2 are YA, YG, YM, YR, ST, WG, WT, or YL; more preferably, X3, X4, X5, and X6 in HCDR2 are SYAD, SYGD, SYMD, SYRD, SSTD, SWGD, SYGR, TWTD, or SYLD. More preferably, the isolated antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region, which includes: (1) Contains the amino acid sequence SFAVG (SEQ ID NO: 16), substantially composed of or composed of HCDR1; (2) Contains the amino acid sequences CSSSTDNVLAYADSVKG (SEQ ID NO: 559), CSSSTDESTDYADSVKG (SEQ ID NO: 560) or CSSSTDAPTDYADSVKG (SEQ ID NO: 561), or HCDR2 consisting essentially of or composed of these sequences; and (3) Contains the amino acid sequence GWDCSGAVVYGVGH (SEQ ID NO: 18), which is substantially composed of or composed of HCDR3.

3. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable (VH) region, said heavy chain variable region comprising: (1) Contains the amino acid sequence GFTLX5X6FAVG (SEQ ID NO: 542), and is substantially composed of or composed of HCDR1, wherein X5 is E, V, D, H, or G; and X6 is S, A, L, G, or P; (2) Contains the amino acid sequence CSX3X4X5X6X7X8X9X 10 (SEQ ID NO: 543), HCDR2 which is essentially composed of or consists of, wherein: X3 is either S or T; X4 is S, Y, or W; X5 is T, A, G, M, R, or L; X6 is either D or R; and X7 X8 X9 X 10 Is it NSTD, ESTD, NSPF, APTD, or NVLA? as well as (3) Contains the amino acid sequence GWDCSGAVVYGVGH (SEQ ID NO: 18), and is substantially composed of or composed of HCDR3; The amino acids mentioned are numbered according to the AbM numbering system; Preferably, X4 and X5 in HCDR2 are YA, YG, YM, YR, ST, WG, WT, or YL; more preferably, X3, X4, X5, and X6 in HCDR2 are SYAD, SYGD, SYMD, SYRD, SSTD, SWGD, SYGR, TWTD, or SYLD.

4. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable (VH) region, said heavy chain variable region comprising: (1) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3; (2) HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7 and HCDR3 of SEQ ID NO: 8; (3) HCDR1 of SEQ ID NO: 11, HCDR2 of SEQ ID NO: 12 and HCDR3 of SEQ ID NO: 13; (4) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 17 and HCDR3 of SEQ ID NO: 18; (5) HCDR1 of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22 and HCDR3 of SEQ ID NO: 23; (6) HCDR1 of SEQ ID NO: 26, HCDR2 of SEQ ID NO: 27 and HCDR3 of SEQ ID NO: 28; (7) HCDR1 of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32 and HCDR3 of SEQ ID NO: 33; (8) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 36 and HCDR3 of SEQ ID NO: 18; (9) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 559 and HCDR3 of SEQ ID NO: 18; (10) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 560, and HCDR3 of SEQ ID NO: 18; or (11) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 561 and HCDR3 of SEQ ID NO:

18.

5. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable (VH) region, said heavy chain variable region comprising: (1) HCDR1, HCDR2 and HCDR3 of an antibody containing a heavy chain, wherein the heavy chain contains the amino acid sequence of SEQ ID NO: 4, and is substantially composed of or consists of the heavy chain; (2) HCDR1, HCDR2 and HCDR3 of antibodies containing heavy chains, said heavy chains containing the amino acid sequence of SEQ ID NO: 9, substantially consisting of or composed of therein; (3) HCDR1, HCDR2 and HCDR3 of antibodies containing heavy chains, wherein the heavy chains contain the amino acid sequence of SEQ ID NO: 14, and are substantially composed of or constitute of therein; (4) HCDR1, HCDR2 and HCDR3 of antibodies containing heavy chains, said heavy chains containing the amino acid sequence of SEQ ID NO: 19, substantially consisting of or composed of thereof; (5) HCDR1, HCDR2 and HCDR3 of antibodies containing heavy chains, wherein the heavy chains contain the amino acid sequence of SEQ ID NO: 24, and are substantially composed of or constitute of therein; (6) HCDR1, HCDR2 and HCDR3 of antibodies comprising heavy chains, wherein the heavy chains comprise the amino acid sequence of SEQ ID NO: 29, and are substantially composed of or constitute thereof; (7) HCDR1, HCDR2 and HCDR3 of antibodies comprising heavy chains, wherein the heavy chains comprise the amino acid sequence of SEQ ID NO: 34, and are substantially composed of or constitute thereof; (8) One or more CDR variants that are otherwise identical to one of the CDRs in (1)-(7) except for up to 5 (e.g., up to 5, 4, 3, 2, 1 or 0) amino acid substitutions; (9) A CDR of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(7) above; or (10) A CDR of an anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1)-(7) above for binding to PVRIG.

6. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable (VH) region, said heavy chain variable region comprising: (1) HCDR1 of SEQ ID NO: 11, HCDR2 of SEQ ID NO: 12 and HCDR3 of SEQ ID NO: 13; (2) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 17 and HCDR3 of SEQ ID NO: 18; (3) HCDR1, HCDR2 and HCDR3 of antibodies comprising heavy chains, wherein the heavy chains comprise, are substantially composed of or consist of the amino acid sequence of SEQ ID NO: 14; (4) HCDR1, HCDR2 and HCDR3 of antibodies comprising heavy chains, wherein the heavy chains comprise, are substantially composed of or consist of the amino acid sequence of SEQ ID NO: 19; (5) HCDR1, HCDR2 and HCDR3 of antibodies P-AIL-A15 or P-AIL-A32 as shown in Table 2. (6) One or more CDR variants that are otherwise identical to one of the CDRs (1)-(5) above, except for up to 5 (e.g., up to 5, 4, 3, 2, 1 or 0) amino acid substitutions; (7) A CDR of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(5) above; or (8) A CDR of an anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1)-(5) above for binding to PVRIG.

7. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable region, said heavy chain variable region comprising: (1) The amino acid sequences selected from the following groups: SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29 or SEQ ID NO: 34; (2) An amino acid sequence identical to the amino acid sequence of the heavy chain variable region of the antibody selected from the antibodies listed in Table 2; (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of (1) or (2) above; (4) Having one or more amino acids compared to the amino acid sequence of (1) or (2) above, preferably an amino acid sequence with the addition, deletion and / or substitution of 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids. (5) An amino acid sequence identical to the amino acid sequence of the VH of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1) or (2) above; or (6) An amino acid sequence identical to the amino acid sequence of the VH of the anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1)-(2) above for binding to PVRIG.

8. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, wherein the antibody comprises a heavy chain, the heavy chain comprising, substantially comprising, or comprising of the following amino acid sequence: (1) The amino acid sequences selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35; (2) An amino acid sequence identical to the amino acid sequence of the heavy chain of an antibody selected from the antibodies listed in Table 3; (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of (1) or (2) above; (4) Compared with the amino acid sequence of (1) or (2) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids added, deleted and / or substituted amino acid sequences. (5) An amino acid sequence identical to the amino acid sequence of the anti-PVRIG antibody heavy chain, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1) or (2) above; or (6) An amino acid sequence identical to the amino acid sequence of the anti-PVRIG antibody heavy chain, wherein the antibody competes with one of the antibodies defined in (1)-(2) above for binding to PVRIG.

9. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, wherein it is humanized and optionally has mature affinity.

10. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable (VH) region, said heavy chain variable region comprising: (1) HCDR1 of SEQ ID NO: 11, HCDR2 of SEQ ID NO: 12 and HCDR3 of SEQ ID NO: 13; (2) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 17 and HCDR3 of SEQ ID NO: 18; (3) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 36 and HCDR3 of SEQ ID NO: 18; (4) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 559 and HCDR3 of SEQ ID NO: 18; (5) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 560 and HCDR3 of SEQ ID NO: 18; (6) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 561 and HCDR3 of SEQ ID NO: 18; (7) HCDR1, HCDR2 and HCDR3 of antibodies containing VH, wherein the VH contains, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 14, 19 or 48; (8) HCDR1, HCDR2 and HCDR3 of antibodies P-AIL-A15, P-AIL-A32, P-AIL-A32-Hu10 as shown in Table 2 or Table 4b; (9) HCDR1, HCDR2 and HCDR3 are otherwise identical to HCDR1, HCDR2 and HCDR3 listed in one of (1)-(8) above, except that there are at most 5 (e.g., at most 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more HCDRs. (10) A CDR of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(8) above; or (11) A CDR of an anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1)-(8) above for binding to PVRIG.

11. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable (VH) region, said heavy chain variable region comprising, substantially comprising, or comprising of the following amino acid sequence: (1) The amino acid sequence of SEQ ID NO: 14 or 19; (2) The amino acid sequence of VH of antibody P-AIL-A15 or P-AIL-A32 as shown in Table 2; (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence defined in (1) or (2) above; (4) Compared with the amino acid sequence defined in (1) or (2) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acid additions, deletions and / or substitutions. (5) An amino acid sequence identical to the amino acid sequence of the anti-PVRIG antibody heavy chain, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1) or (2) above; or (6) An amino acid sequence identical to the amino acid sequence of the heavy chain of an anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1) or (2) above for binding to PVRIG.

12. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 9, comprising a heavy chain variable region, said heavy chain variable region comprising: (1) An amino acid sequence selected from the amino acid sequences listed in Tables 4b and 4c; or (2) Select the amino acid sequence of the group consisting of SEQ ID NO: 37-58; (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to (1) or (2) above, or (4) Compared with the amino acid sequence of (1) or (2) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids added, deleted and / or substituted amino acid sequences.

13. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 12, wherein the isolated antibody comprises a heavy chain, the heavy chain comprising, substantially comprising, or comprising of the following amino acid sequence: (1) Select the amino acid sequence from the group consisting of SEQ ID NO: 59-80; (2) An amino acid sequence identical to the amino acid sequence of the heavy chain of an antibody selected from the antibodies listed in Tables 4d and 4e; (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences listed in (1) or (2) above. (4) Compared with one of the amino acid sequences listed in (1) or (2) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acid sequences with addition, deletion and / or substitution. (5) An amino acid sequence identical to the amino acid sequence of the heavy chain of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1) or (2) above; or (6) An amino acid sequence identical to the amino acid sequence of the heavy chain of an anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1) or (2) above for binding to PVRIG.

14. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 1 or 9, comprising a heavy chain variable region, said heavy chain variable region comprising: (1) HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 82 and HCDR3 of SEQ ID NO: 18; (2) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 83 and HCDR3 of SEQ ID NO: 18; (3) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 84 and HCDR3 of SEQ ID NO: 18; (4) HCDR1 of SEQ ID NO: 85, HCDR2 of SEQ ID NO: 86 and HCDR3 of SEQ ID NO: 18; (5) HCDR1 of SEQ ID NO: 87, HCDR2 of SEQ ID NO: 88 and HCDR3 of SEQ ID NO: 18; (6) HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 89 and HCDR3 of SEQ ID NO: 18; (7) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 90 and HCDR3 of SEQ ID NO: 18; (8) HCDR1 of SEQ ID NO: 91, HCDR2 of SEQ ID NO: 92 and HCDR3 of SEQ ID NO: 18; (9) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 93 and HCDR3 of SEQ ID NO: 18; (10) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 82 and HCDR3 of SEQ ID NO: 18; (11) HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 84 and HCDR3 of SEQ ID NO: 18; (12) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 96 and HCDR3 of SEQ ID NO: 18; (13) HCDR1 of SEQ ID NO: 97, HCDR2 of SEQ ID NO: 98 and HCDR3 of SEQ ID NO: 18; (14) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 99 and HCDR3 of SEQ ID NO: 18; (15) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 100 and HCDR3 of SEQ ID NO: 18; (16) HCDR1 of SEQ ID NO: 101, HCDR2 of SEQ ID NO: 99 and HCDR3 of SEQ ID NO: 18; (17) HCDR1 of SEQ ID NO: 102, HCDR2 of SEQ ID NO: 103 and HCDR3 of SEQ ID NO: 18; (18) HCDR1 of SEQ ID NO: 104, HCDR2 of SEQ ID NO: 105 and HCDR3 of SEQ ID NO: 18; (19) HCDR1 of SEQ ID NO: 106, HCDR2 of SEQ ID NO: 105 and HCDR3 of SEQ ID NO: 18; (20) HCDR1 of SEQ ID NO: 97, HCDR2 of SEQ ID NO: 107 and HCDR3 of SEQ ID NO: 18; (21) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 108 and HCDR3 of SEQ ID NO: 18; (22) HCDR1 of SEQ ID NO: 94, HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 18; (23) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 110 and HCDR3 of SEQ ID NO: 18; (24) HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 98 and HCDR3 of SEQ ID NO: 18; (25) HCDR1 of SEQ ID NO: 97, HCDR2 of SEQ ID NO: 100 and HCDR3 of SEQ ID NO: 18; (26) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 559 and HCDR3 of SEQ ID NO: 18; (27) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 560 and HCDR3 of SEQ ID NO: 18; (28) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 561 and HCDR3 of SEQ ID NO: 18; (29) Contains the same heavy chain CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, HCDR3) as the heavy chain CDR1, CDR2, and CDR3 regions of the antibodies selected from those listed in Tables 6a and 6b; (30) HCDR1, HCDR2 and HCDR3 are otherwise identical to HCDR1, HCDR2 and HCDR3 listed in one of (1)-(29) above, except that they have at most 5 (e.g., at most 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more HCDRs. (31) HCDR1, HCDR2, and HCDR3 of anti-PVRIG antibodies, said antibodies binding to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1)-(29) above; or (32) HCDR1, HCDR2 and HCDR3 of anti-PVRIG antibodies, said antibodies competing with one of the antibodies defined in (1)-(29) above for binding to PVRIG.

15. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 14, comprising a heavy chain variable region, said heavy chain variable region comprising, substantially comprising, or comprising of the following amino acid sequences: (1) The amino acid sequences of the antibodies listed in Table 6a are selected; (2) Select the amino acid sequence of the group consisting of SEQ ID NO: 111-123; (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to (1) or (2) above. (4) An amino acid sequence having one or more added, deleted and / or substituted amino acids compared to the amino acid sequence of (1) or (2) above; (5) The amino acid sequence of the VH of the anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on the PVRIG with one of the antibodies defined in (1) or (2) above; or (6) The amino acid sequence of the VH of the anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1) or (2) above for binding to PVRIG.

16. The isolated anti-PVRIG antibody or its antigen-binding fragment according to claim 14, wherein the isolated antibody comprises a heavy chain, the heavy chain comprising, substantially comprising, or comprising of the following amino acid sequence: (1) The amino acid sequences of the antibodies listed in Table 6b are selected; (2) Select the amino acid sequence of the group consisting of SEQ ID NO: 124-136; (3) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to (1) or (2) above. (4) An amino acid sequence having one or more added, deleted and / or substituted amino acids compared to the amino acid sequence of (1) or (2) above; (5) The amino acid sequence of the heavy chain of an anti-PVRIG antibody, wherein the antibody binds to the same or overlapping epitopes on PVRIG with one of the antibodies defined in (1) or (2) above; or (6) The amino acid sequence of the heavy chain of an anti-PVRIG antibody, wherein the antibody competes with one of the antibodies defined in (1) or (2) above for binding to PVRIG.

17. The isolated anti-PVRIG antibody or antigen-binding fragment thereof according to claim 1, comprising a VH, said VH comprising, substantially comprising, or comprising of the amino acid sequence of a reference antibody selected from the antibodies listed in Table 4c or Table 4e, except that the three CDRs in said reference antibody are replaced by a different set of three CDRs contained in the antibodies listed in Table 5a or Table 5b, or in Table 6a, or as defined in claim 2 or 3.

18. The isolated anti-PVRIG antibody or its antigen-binding fragment according to any one of claims 1-17, wherein it is modified to remove various post-translational modifications (PTMs), such as oxidation, deamidation, isomerization, glycosylation, etc., preferably modified to deglycosylate.

19. An isolated anti-TIGIT antibody or an antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: (1) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 137-139, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 140-142, substantially composed of or composed of therein; (2) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 147-149, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 150-152, substantially composed of or composed of therein; (3) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 157-159, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 160-162, substantially composed of or composed of therein; (4) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 167-169, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 170-172, substantially composed of or composed of therein; (5) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 177-179, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 180-182, substantially composed of or composed of therein; (6) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 187-189, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 190-192, substantially composed of or composed of therein; (7) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 197-199, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 200-202, substantially composed of or composed of therein; (8) The VH region comprises HCDR1, HCDR2 and HCDR3, each of which contains the amino acid sequence of SEQ ID NO: 207-209, is substantially composed of or is composed of; and the VL region comprises LCDR1, LCDR2 and LCDR3, each of which contains the amino acid sequence of SEQ ID NO: 210-212, is substantially composed of or is composed of; (9) The VH region comprises HCDR1, HCDR2 and HCDR3, each of which contains the amino acid sequence of SEQ ID NO: 217-219, is substantially composed of or is composed of; and the VL region comprises LCDR1, LCDR2 and LCDR3, each of which contains the amino acid sequence of SEQ ID NO: 220-222, is substantially composed of or is composed of; (10) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 227-229; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 230-232; (11) The VH region comprises HCDR1, HCDR2 and HCDR3, each of which contains the amino acid sequence of SEQ ID NO: 237-239, is substantially composed of or is composed of; and the VL region comprises LCDR1, LCDR2 and LCDR3, each of which contains the amino acid sequence of SEQ ID NO: 240-242, is substantially composed of or is composed of; (12) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 247-249; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 250-252; (13) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 257-259; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 260-262; (14) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 267-269; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 270-272; (15) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 277-279; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 280-282; (16) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 287-289; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 290-292; (17) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 297-299; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 300-302; (18) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 307-309; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 310-312; (19) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 317-319; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 320-322; (20) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 327-329; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or comprising the amino acid sequence of SEQ ID NO: 330-332; (21) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 337-339; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 340-342; (22) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 347-349; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 350-352; (23) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 357-359; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 360-362; (24) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 367-369; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 370-372; (25) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 377-379; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 380-382; (26) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 387-389; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 390-392; (27) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 397-399; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 400-402; (28) The VH region comprises HCDR1, HCDR2 and HCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 407-409; and the VL region comprises LCDR1, LCDR2 and LCDR3, each comprising, substantially comprising or consisting of the amino acid sequence of SEQ ID NO: 410-412; (29) The VH region contains the same HCDR1, HCDR2 and HCDR3 as the reference antibody selected from Table 7 or Table 8a; and the VL region contains the same LCDR1, LCDR2 and LCDR3 as the reference antibody; (30) Except for the presence of up to 5 (e.g., up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the VH region contains the same HCDR1, HCDR2 and HCDR3 as the reference antibody selected from Table 7 or Table 8a, and the VL region contains the same LCDR1, LCDR2 and LCDR3 as the reference antibody; (31) The VH region and the VL region contain the same HCDR and LCDR of the anti-TIGIT antibody, the anti-TIGIT antibody binding to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(29) above; or (32) The heavy chain variable region and the light chain variable region contain the same HCDR and LCDR of the anti-TIGIT antibody, which competes with one of the antibodies defined in (1)-(29) above for binding to TIGIT.

20. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, wherein: The VH includes: (1) Contains the amino acid sequence NYIMS (SEQ ID NO: 217), and is substantially composed of or composed of HCDR1; (2) Contains the amino acid sequence TITSGGGNAYYX 12 DSVX 16 G (SEQ ID NO: 544), HCDR2 which is essentially composed of or consists of, wherein: X 12 It is P or A; and X 16 It is R or K; and (3) Contains the amino acid sequence WLISFYAMDY (SEQ ID NO: 219), substantially composed of or composed of HCDR3; and The VL includes: (1) Contains the amino acid sequence X1ASQNINVWLS (SEQ ID NO: 545), is substantially composed of or is composed of the amino acid sequence X1ASQNINVWLS (SEQ ID NO: 545), and is LCDR1, wherein X1 is H or R; (2) Contains the amino acid sequence KASNLX6X7 (SEQ ID NO: 546), is substantially composed of or is composed of LCDR2, wherein X6X7 is HT or ES; and (3) Contains the amino acid sequence QQGQSYPLT (SEQ ID NO: 222), and is substantially composed of or composed of LCDR3; The amino acids are numbered according to the Kabat numbering system.

21. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, wherein: The VH includes: (1) Contains the amino acid sequence NTYMH (SEQ ID NO: 347), which is substantially composed of or composed of NTYMH; (2) Contains the amino acid sequence RIDPANGNTKY X 12 X 13 KFQG (SEQ ID NO: 547), HCDR2 which is essentially composed of or consists of, wherein X 12 X 13 Is it AP or SQ? and (3) Contains the amino acid sequence EGDDGYYEDY (SEQ ID NO: 349), substantially composed of or composed of HCDR3; and The VL includes: (1) Contains the amino acid sequence RASENIYSYLA (SEQ ID NO: 350), and LCDR1 which is substantially composed of or composed of the amino acid sequence RASENIYSYLA; (2) Containing the amino acid sequence NAKTLX6X7 (SEQ ID NO: 548), substantially composed of or composed of the amino acid sequence LCDR2, wherein X6X7 is AE or QS; and (3) Contains the amino acid sequence QHHYGNPFT (SEQ ID NO: 352), and is substantially composed of or composed of LCDR3; The amino acids are numbered according to the Kabat numbering system.

22. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, wherein: (1) The VH contains, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 143, and The VL contains, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 144; (2) The VH contains the amino acid sequence of SEQ ID NO: 153, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 154, is substantially composed of or is composed of therein; (3) The VH contains the amino acid sequence of SEQ ID NO: 163, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 164, is substantially composed of or is composed of therein; (4) The VH contains the amino acid sequence of SEQ ID NO: 173, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 174, is substantially composed of or is composed of therein; (5) The VH contains the amino acid sequence of SEQ ID NO: 183, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 184, is substantially composed of or is composed of therein; (6) The VH contains the amino acid sequence of SEQ ID NO: 193, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 194, is substantially composed of or is composed of therein; (7) The VH contains the amino acid sequence of SEQ ID NO: 203, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 204, is substantially composed of or is composed of therein; (8) The VH contains the amino acid sequence of SEQ ID NO: 213, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 214, is substantially composed of or is composed of therein; (9) The VH contains the amino acid sequence of SEQ ID NO: 223, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 224, is substantially composed of or is composed of therein; (10) The VH contains the amino acid sequence of SEQ ID NO: 233, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 234, is substantially composed of or is composed of therein; (11) The VH contains the amino acid sequence of SEQ ID NO: 243, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 244, is substantially composed of or is composed of therein; (12) The VH contains the amino acid sequence of SEQ ID NO: 253, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 254, is substantially composed of or is composed of therein; (13) The VH contains the amino acid sequence of SEQ ID NO: 263, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 264, is substantially composed of or is composed of therein; (14) The VH contains the amino acid sequence of SEQ ID NO: 273, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 274, is substantially composed of or is composed of therein; (15) The VH contains the amino acid sequence of SEQ ID NO: 283, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 284, is substantially composed of or is composed of therein; (16) The VH contains the amino acid sequence of SEQ ID NO: 293, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 294, is substantially composed of or is composed of therein; (17) The VH contains the amino acid sequence of SEQ ID NO: 303, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 304, is substantially composed of or is composed of therein; (18) The VH contains the amino acid sequence of SEQ ID NO: 313, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 314, is substantially composed of or is composed of therein; (19) The VH contains the amino acid sequence of SEQ ID NO: 323, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 324, is substantially composed of or is composed of therein; (20) The VH contains the amino acid sequence of SEQ ID NO: 333, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 334, is substantially composed of or is composed of therein; (21) The VH contains the amino acid sequence of SEQ ID NO: 343, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 344, is substantially composed of or is composed of therein; (22) The VH contains the amino acid sequence of SEQ ID NO: 353, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 354, is substantially composed of or is composed of therein; (23) The VH contains the amino acid sequence of SEQ ID NO: 363, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 364, is substantially composed of or is composed of therein; (24) The VH contains the amino acid sequence of SEQ ID NO: 373, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 374, is substantially composed of or is composed of therein; (25) The VH contains the amino acid sequence of SEQ ID NO: 383, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 384, is substantially composed of or is composed of therein; (26) The VH contains the amino acid sequence of SEQ ID NO: 393, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 394, is substantially composed of or is composed of therein; (27) The VH contains the amino acid sequence of SEQ ID NO: 403, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 404, is substantially composed of or is composed of therein; (28) The VH contains the amino acid sequence of SEQ ID NO: 413, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 414, is substantially composed of or is composed of therein; (29) The VH and the VL contain the same amino acid sequences as the VH and VL of the antibodies selected from those listed in Table 8a; (30) The VH and the VL contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the same amino acid sequence as the VH and VL listed in any one of (1)-(29) above. (31) Compared with the amino acid sequences of VH and VL listed in any one of (1)-(29) above, the VH and VL contain one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids added, deleted and / or substituted. (32) The VH and VL contain the same amino acid sequences as the VH and VL of the anti-TIGIT antibody, and the antibody binds to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(29) above; or (33) The VH and the VL contain the same amino acid sequences as the VH and VL of the anti-TIGIT antibody, which competes with one of the antibodies defined in (1)-(29) above for binding to TIGIT.

23. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, wherein the anti-TIGIT antibody comprises a heavy chain and a light chain, wherein: (1) The heavy chain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 145, and The light chain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 146; (2) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 155, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO:

156. (3) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 165, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO:

166. (4) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 175, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO:

176. (5) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 185, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO:

186. (6) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 195, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 196; (7) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 205, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 206; (8) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 215, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 216; (9) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 225, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 226; (10) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 235, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 236; (11) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 245, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 246; (12) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 255, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 256; (13) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 265, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 266; (14) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 275, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 276; (15) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 285, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 286; (16) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 295, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 296; (17) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 305, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 306; (18) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 315, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 316; (19) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 325, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 326; (20) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 335, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 336; (21) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 345, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 346; (22) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 355, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 356; (23) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 365, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 366; (24) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 375, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 376; (25) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 385, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 386; (26) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 395, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 396; (27) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 405, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 406; (28) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 415, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 416; (29) The heavy chain and the light chain contain the same amino acid sequences as the heavy chain and light chain of the antibodies selected from those listed in Table 8b. (30) The heavy chain and the light chain contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the same amino acid sequence as the corresponding heavy chain and light chain listed in any one of (1)-(29) above. (31) Compared with the amino acid sequences of the corresponding heavy and light chains listed in any one of (1)-(29) above, the heavy chain and the light chain contain amino acid sequences having one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids with addition, deletion and / or substitution. (32) The heavy chain and the light chain contain the same amino acid sequences as the corresponding heavy chain and light chain of the anti-TIGIT antibody, and the antibody binds to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(29) above; or (33) The heavy chain and the light chain contain the same amino acid sequences as the corresponding heavy chain and light chain of the anti-TIGIT antibody, and the antibody competes with one of the antibodies defined in (1)-(29) above for binding to TIGIT.

24. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, wherein: (1) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 217-219, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 220-222, substantially composed of or composed of therein; (2) The VH region contains HCDR1, HCDR2 and HCDR3, each containing the amino acid sequence of SEQ ID NO: 347-349, substantially composed of or composed of therein; and the VL region contains LCDR1, LCDR2 and LCDR3, each containing the amino acid sequence of SEQ ID NO: 350-352, substantially composed of or composed of therein; (3) The VH region and the VL region comprise HCDR1, HCDR2 and HCDR3 of the following antibodies and LCDR1, LCDR2 and LCDR3, wherein the antibodies comprise a VH containing the amino acid sequence of SEQ ID NO: 223, substantially composed of or composed of therein, and a VL containing the amino acid sequence of SEQ ID NO: 224, substantially composed of or composed of therein. (4) The VH region and the VL region comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the following antibodies, wherein the antibodies comprise a VH containing the amino acid sequence of SEQ ID NO: 353, substantially composed of or composed of therein and a VL containing the amino acid sequence of SEQ ID NO: 354, substantially composed of or composed of therein. (5) The VH region and the VL region contain the same HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as those of the antibodies T-AIL-A60 or T-AIL-A169 listed in Table 8a or Table 8b. (6) The VH region and the VL region contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 that are otherwise identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 listed in one of (1)-(5) above, except that there are at most 5 (e.g., at most 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more CDRs; (7) The VH region and the VL region contain the same corresponding HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as the anti-TIGIT antibody, wherein the antibody binds to the same or overlapping epitopes on the PVRIG with one of the antibodies defined in (1)-(5) above; or (8) The VH region and the VL region contain the same corresponding HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as the anti-TIGIT antibody, which competes with one of the antibodies defined in (1)-(5) above for binding to TIGIT.

25. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 22, wherein: (1) The VH region contains, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 223, and The VL contains, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 224; (2) The VH region contains, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 353, and the VL region contains, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 354; (3) The VH region and the VL region contain the same amino acid sequences as the VH and VL of the antibodies T-AIL-A60 or T-AIL-A169 shown in Table 8a or Table 8b; (4) The VH region and the VL region contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the corresponding amino acid sequence of the antibody as defined in (1)-(3) above; (5) Except for the addition, deletion and / or substitution of one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, in one or both of the VH region and the VL region, the VH region and the VL region contain the same amino acid sequence as the corresponding amino acid sequence of the VH and VL regions of the antibody as defined in (1)-(3) above; (6) The VH region and the VL region contain amino acid sequences identical to the corresponding VH and VL amino acid sequences of the anti-TIGIT antibody described below, and the antibody binds to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(3) above; or (7) The VH region and the VL region contain the same amino acid sequences as the corresponding VH and VL amino acid sequences of the anti-TIGIT antibody described below, which competes with one of the antibodies defined in (1)-(3) above for binding to TIGIT.

26. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, wherein the antibody comprises a heavy chain and a light chain, wherein: (1) The heavy chain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 225, and The light chain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 226; (2) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 355, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO:

356. (3) The heavy chain and the light chain contain the same amino acid sequences as the heavy chain and light chain of the antibody T-AIL-A60 or T-AIL-A169 shown in Table 8b; (4) The heavy chain and the light chain contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the same amino acid sequence as the corresponding amino acid sequence of the antibody as defined in (1)-(3) above; (5) Except for the presence of one or more amino acids in one or both of the heavy chain and the light chain, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, the heavy chain and the light chain contain the same amino acid sequences as the corresponding amino acid sequences of the heavy chain and the light chain of the antibody as defined in (1)-(3) above. (6) The heavy chain and the light chain contain the same amino acid sequences as the corresponding heavy chain and light chain amino acid sequences of the anti-TIGIT antibody, which binds to the same or overlapping epitopes on TIGIT with one of the antibodies defined in (1)-(3) above; or (7) The heavy chain and the light chain contain the same amino acid sequences as the corresponding heavy chain and light chain amino acid sequences of the anti-TIGIT antibody, which competes with one of the antibodies defined in (1)-(3) above for binding to TIGIT.

27. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, wherein it is humanized and optionally affinity-matured.

28. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise: (1) The heavy chain CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, HCDR3) and light chain CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, LCDR3) of the antibodies selected from those listed in Table 7, Table 9a, or Table 9b are identical to those of the antibodies listed in Table 9b. (2) The heavy chain CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, HCDR3) and light chain CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, LCDR3) of the antibodies selected from those listed in Table 8a, Table 10a, or Table 10b are identical to those of the antibodies listed in Table 8a, Table 10a, or Table 10b. (3) The heavy chain CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2, HCDR3) and the light chain CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2, LCDR3) are otherwise identical to (1) or (2) above, except that there are up to 5 (e.g., up to 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more CDRs. (4) The amino acid sequences of the heavy chain variable region and the light chain variable region of antibodies selected from those listed in Tables 10a and 10b; (5) Each amino acid sequence is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the heavy chain variable region and the light chain variable region of the antibody selected from the antibodies listed in Tables 10a and 10b; or (6) Compared with one or both of the amino acid sequences listed in (4) above, it has one or more amino acids, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acid sequences with additions, deletions and / or substitutions.

29. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, comprising VH and VL, wherein: (1) The VH includes HCDR1 of SEQ ID NO: 217, HCDR2 of SEQ ID NO: 218 and HCDR3 of SEQ ID NO: 219, and the VL includes LCDR1 of SEQ ID NO: 220, LCDR2 of SEQ ID NO: 221 and LCDR3 of SEQ ID NO: 222; (2) The VH includes HCDR1 of SEQ ID NO: 347, HCDR2 of SEQ ID NO: 348 and HCDR3 of SEQ ID NO: 349, and the VL includes LCDR1 of SEQ ID NO: 350, LCDR2 of SEQ ID NO: 351 and LCDR3 of SEQ ID NO: 352; (3) The VH includes HCDR1 of SEQ ID NO: 217, HCDR2 of SEQ ID NO: 417 and HCDR3 of SEQ ID NO: 219, and the VL includes LCDR1 of SEQ ID NO: 220, LCDR2 of SEQ ID NO: 221 and LCDR3 of SEQ ID NO: 222; (4) The VH includes HCDR1 of SEQ ID NO: 217, HCDR2 of SEQ ID NO: 218 and HCDR3 of SEQ ID NO: 219, and the VL includes LCDR1 of SEQ ID NO: 418, LCDR2 of SEQ ID NO: 419 and LCDR3 of SEQ ID NO: 222; (5) The VH includes HCDR1 of SEQ ID NO: 217, HCDR2 of SEQ ID NO: 417 and HCDR3 of SEQ ID NO: 219, and the VL includes LCDR1 of SEQ ID NO: 418, LCDR2 of SEQ ID NO: 419 and LCDR3 of SEQ ID NO: 222; (6) The VH includes HCDR1 of SEQ ID NO: 347, HCDR2 of SEQ ID NO: 348 and HCDR3 of SEQ ID NO: 349, and the VL includes LCDR1 of SEQ ID NO: 350, LCDR2 of SEQ ID NO: 420 and LCDR3 of SEQ ID NO: 352; (7) The VH includes HCDR1 of SEQ ID NO: 347, HCDR2 of SEQ ID NO: 421 and HCDR3 of SEQ ID NO: 349, and the VL includes LCDR1 of SEQ ID NO: 350, LCDR2 of SEQ ID NO: 351 and LCDR3 of SEQ ID NO: 352; (8) The VH includes HCDR1 of SEQ ID NO: 347, HCDR2 of SEQ ID NO: 421 and HCDR3 of SEQ ID NO: 349, and the VL includes LCDR1 of SEQ ID NO: 350, LCDR2 of SEQ ID NO: 420 and LCDR3 of SEQ ID NO: 352; (9) The VH and the VL comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of antibodies selected from those listed in Table 10a or Table 10b; or (10) The VH and the VL contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 that are otherwise identical to the antibodies selected from the antibodies defined in (1)-(9) above, except that they have at most 5 (e.g., at most 5, 4, 3, 2, 1 or 0) amino acid substitutions in one or more of the CDRs.

30. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, comprising VH and VL, wherein: (1) The VH contains, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 422, and The VL contains, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 423; (2) The VH contains the amino acid sequence of SEQ ID NO: 424, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 425, is substantially composed of or is composed of therein; (3) The VH contains the amino acid sequence of SEQ ID NO: 426, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 427, is substantially composed of or is composed of therein; (4) The VH contains the amino acid sequence of SEQ ID NO: 428, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 429, is substantially composed of or is composed of therein; (5) The VH contains the amino acid sequence of SEQ ID NO: 430, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 431, is substantially composed of or is composed of therein; (6) The VH contains the amino acid sequence of SEQ ID NO: 432, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 433, is substantially composed of or is composed of therein; (7) The VH contains the amino acid sequence of SEQ ID NO: 434, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 435, is substantially composed of or is composed of therein; (8) The VH contains the amino acid sequence of SEQ ID NO: 436, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 437, is substantially composed of or is composed of therein; (9) The VH contains the amino acid sequence of SEQ ID NO: 438, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 439, is substantially composed of or is composed of therein; (10) The VH contains the amino acid sequence of SEQ ID NO: 440, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 441, is substantially composed of or is composed of therein; (11) The VH contains the amino acid sequence of SEQ ID NO: 442, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 443, is substantially composed of or is composed of therein; (12) The VH contains the amino acid sequence of SEQ ID NO: 444, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 445, is substantially composed of or is composed of therein; (13) The VH contains the amino acid sequence of SEQ ID NO: 446, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 447, is substantially composed of or is composed of therein; (14) The VH contains the amino acid sequence of SEQ ID NO: 448, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 449, is substantially composed of or is composed of therein; (15) The VH comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 450, and the VL comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 451; (16) The VH contains the amino acid sequence of SEQ ID NO: 452, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 453, is substantially composed of or is composed of therein; (17) The VH contains the amino acid sequence of SEQ ID NO: 454, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 455, is substantially composed of or is composed of therein; (18) The VH contains the amino acid sequence of SEQ ID NO: 456, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 457, is substantially composed of or is composed of therein; (19) The VH contains the amino acid sequence of SEQ ID NO: 458, is substantially composed of or is composed of therein, and the VL contains the amino acid sequence of SEQ ID NO: 459, is substantially composed of or is composed of therein; (20) The VH and VL comprise the corresponding amino acid sequences of the VH and VL of antibodies selected from those listed in Table 10a or Table 10b. (21) The VH and the VL contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the corresponding amino acid sequences of the VH and VL of the antibodies selected from those defined in (1)-(20) above. (22) The VH and the VL contain amino acid sequences identical to the corresponding amino acid sequences of the antibodies selected from the antibodies defined in (1)-(20) above, except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, through addition, deletion and / or substitution. (23) The VH and VL comprise the amino acid sequences of the anti-TIGIT antibody VH and VL, wherein the antibody binds to the same or overlapping epitopes on TIGIT with an antibody selected from the antibodies defined in (1)-(20) above; or (24) The VH and VL comprise the amino acid sequences of the anti-TIGIT antibody that competes with the antibodies selected from those defined in (1)-(20) above for binding to TIGIT.

31. The isolated anti-TIGIT antibody or its antigen-binding fragment according to claim 19, wherein the antibody comprises a heavy chain and a light chain, wherein: (1) The heavy chain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 460, and The light chain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 461; (2) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 462, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 463; (3) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 464, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 465; (4) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 466, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 467; (5) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 468, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO:

469. (6) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 470, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 471; (7) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 472, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 473; (8) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 474, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 475; (9) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 476, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 477; (10) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 478, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 479; (11) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 480, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 481; (12) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 482, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 483; (13) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 484, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 485; (14) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 486, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 487; (15) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 488, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 489; (16) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 490, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 491; (17) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 492, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 493; (18) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 494, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 495; (19) The heavy chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 496, and the light chain comprises, is substantially composed of or is composed of the amino acid sequence of SEQ ID NO: 497; (20) The heavy chain and the light chain comprise the corresponding amino acid sequences of the heavy chain and light chain of the antibody selected from the antibodies listed in Table 10c or Table 10d. (21) The heavy chain and the light chain contain at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the corresponding amino acid sequences of the heavy chain and light chain of the antibody selected from the antibodies defined in (1)-(20) above. (22) The heavy chain and the light chain comprise an amino acid sequence identical to the corresponding amino acid sequence of the heavy chain and light chain of the antibody selected from the antibodies defined in (1)-(20) above, except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, through addition, deletion and / or substitution. (23) The heavy chain and the light chain comprise the amino acid sequences of the heavy and light chains of an anti-TIGIT antibody that binds to the same or overlapping epitopes on TIGIT as antibodies selected from those defined in (1)-(20) above; or (24) The heavy chain and the light chain comprise the amino acid sequences of the heavy chain and light chain of an anti-TIGIT antibody that competes with an antibody selected from the antibodies defined in (1)-(20) above for binding to TIGIT.

32. The isolated anti-TIGIT antibody or antigen-binding fragment thereof according to claim 19 or 20, wherein the isolated antibody comprises VH and VL, the VH and VL comprising, substantially comprising, or comprising of the amino acid sequences of the reference antibody selected from the antibodies listed in Table 10a, except that the six CDRs of the reference antibody are replaced by a different set of six CDRs included in the antibodies listed in Table 9a or Table 10a, or as defined in claim 20.

33. The isolated anti-TIGIT antibody or antigen-binding fragment thereof according to claim 19 or 21, comprising VH and VL, said VH and VL comprising, substantially comprising, or comprising of the amino acid sequences of the reference antibody selected from the antibodies listed in Table 10b, except that the six CDRs of said reference antibody are replaced by a different set of six CDRs included in the antibodies listed in Table 9b or Table 10b, or as defined in claim 21.

34. The isolated anti-TIGIT antibody or its antigen-binding fragment according to any one of claims 19-33, wherein it is modified to remove various PTMs, such as oxidation, deamidation, isomerization, glycosylation, etc., preferably modified to deglycosylate.

35. A multispecific antibody against PVRIG and TIGIT, comprising a first antibody against PVRIG or an antigen-binding fragment thereof and a second antibody against TIGIT or an antigen-binding fragment thereof, wherein the first antibody against PVRIG or an antigen-binding fragment thereof is as claimed in any one of claims 1-18, and / or the second antibody against TIGIT or an antigen-binding fragment thereof is as claimed in any one of claims 19-34.

36. The multispecific antibody against PVRIG and TIGIT according to claim 35, wherein the first antibody against PVRIG or its antigen-binding fragment and the second antibody against TIGIT or its antigen-binding fragment are non-covalently or covalently linked, optionally via a linker, preferably a peptide linker.

37. The multispecific antibody against PVRIG and TIGIT according to claim 35, wherein the first antibody against PVRIG or its antigen-binding fragment is linked via its N-terminus to the C-terminus of the VH or VL region of the second antibody against TIGIT or its antigen-binding fragment.

38. The multispecific antibody against PVRIG and TIGIT according to claim 35, wherein the first antibody against PVRIG or its antigen-binding fragment is linked via its C-terminus to the N-terminus of the VH or VL region of the second antibody against TIGIT or its antigen-binding fragment.

39. The multispecific antibody against PVRIG and TIGIT according to claim 35, wherein the adapter is selected from the group consisting of: GGGGS, GGGGSGGGGS, SGGGGSGGGG, (G4S)3 or GGGGSGGGGSGGGGS, GGGGS GGGGSGGGG or G4(SG4)2 and (G4S)4 or GGGGS GGGGS GGGGS GGGGS, GSPGSSSSGS, GSGSGSGS, GSGSGNGS, GGSGSGSG, GGSGSG, GGSG, GGSGNGSG, GGNGSGSG and GGNGSG.

40. The multispecific antibody against PVRIG and TIGIT according to claim 35, wherein the multispecific antibody against PVRIG and TIGIT is in single-chain form.

41. The multispecific antibody against PVRIG and TIGIT according to claim 35, wherein the multispecific antibody is a bispecific antibody.

42. The multispecific antibody against PVRIG and TIGIT according to claim 35, wherein the bispecific antibody comprises: (1) Nine CDRs of antibodies selected from those listed in Table 11, including BsAb01, BsAb02, BsAb03, BsAb04, BsAb05, BsAb06, BsAb07, BsAb08, BsAb09, BsAb10, BsAb11, BsAb12, BsAb13, BsAb14, BsAb15, BsAb16, BsAb17, BsAb18, BsAb19, BsAb20, and BsAb21, or (2) Nine CDRs of antibodies selected from those listed in Table 12.

43. The multispecific antibody against PVRIG and TIGIT according to claim 35, wherein the multispecific antibody comprises: (1) The amino acid sequence comprising SEQ ID NO: 498, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 499, the light chain consisting substantially of or consisting of therein; (2) The amino acid sequence comprising SEQ ID NO: 500, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 501, the light chain consisting of or consisting of therein. (3) The amino acid sequence containing SEQ ID NO: 502, the heavy chain consisting of or consisting of therein, and the amino acid sequence containing SEQ ID NO: 503, the light chain consisting of or consisting of therein; (4) The amino acid sequence comprising SEQ ID NO: 504, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 505, the light chain consisting of or consisting of therein. (5) The amino acid sequence comprising SEQ ID NO: 506, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 507, the light chain consisting of or consisting of therein. (6) The amino acid sequence comprising SEQ ID NO: 508, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 509, the light chain consisting of or consisting of therein. (7) The amino acid sequence comprising SEQ ID NO: 510, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 511, the light chain consisting of or consisting of therein. (8) The amino acid sequence comprising SEQ ID NO: 512, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 513, the light chain consisting of or consisting of therein. (9) The amino acid sequence comprising SEQ ID NO: 514, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 515, the light chain consisting substantially of or consisting of therein. (10) The amino acid sequence comprising SEQ ID NO: 516, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 517, the light chain consisting substantially of or consisting of therein. (11) The amino acid sequence comprising SEQ ID NO: 518, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 519, the light chain consisting substantially of or consisting of therein. (12) The amino acid sequence comprising SEQ ID NO: 520, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 521, the light chain consisting substantially of or consisting of therein; (13) The amino acid sequence comprising SEQ ID NO: 522, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 523, the light chain consisting of or consisting of therein; (14) The amino acid sequence comprising SEQ ID NO: 524, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 525, the light chain consisting of or consisting of therein; (15) The amino acid sequence comprising SEQ ID NO: 526, the heavy chain consisting substantially of or composed of therein, and the amino acid sequence comprising SEQ ID NO: 527, the light chain consisting substantially of or composed of therein. (16) The amino acid sequence comprising SEQ ID NO: 528, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 529, the light chain consisting substantially of or consisting of therein; (17) The amino acid sequence comprising SEQ ID NO: 530, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 531, the light chain consisting substantially of or consisting of therein. (18) The amino acid sequence comprising SEQ ID NO: 532, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 533, the light chain consisting substantially of or consisting of therein; (19) The amino acid sequence comprising SEQ ID NO: 534, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 535, the light chain consisting substantially of or consisting of therein. (20) The amino acid sequence comprising SEQ ID NO: 536, the heavy chain consisting substantially of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 537, the light chain consisting substantially of or consisting of therein. (21) The amino acid sequence comprising SEQ ID NO: 538, the heavy chain consisting of or consisting of therein, and the amino acid sequence comprising SEQ ID NO: 539, the light chain consisting of or consisting of therein; (22) Heavy and light chains, comprising, or substantially consisting of, the corresponding amino acid sequences of the heavy and light chains of antibodies selected from those listed in Table 11, or... It is composed of; (23) Heavy and light chains comprising, substantially consisting of, or consisting of at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the corresponding amino acid sequences of the heavy and light chains of antibodies selected from those defined in (1)-(22) above; or (24) Heavy and light chains comprising, substantially consisting of, or consisting of, amino acid sequences identical to, or substantially consisting of, the corresponding amino acid sequences of the heavy and light chains of antibodies selected from those defined in (1)-(22) above, except for the presence of one or more amino acids in one or both of the amino acid sequences, preferably 1-50, 2-40, 3-30, 4-20 or 5-10 amino acids, including additions, deletions and / or substitutions; or (25) Heavy and light chains comprising, substantially composed of or composed of, the corresponding amino acid sequences of the heavy and light chains of the antibodies selected from BsAb01, BsAb02, BsAb03, BsAb04, BsAb05, BsAb06, BsAb07, BsAb08, BsAb09, BsAb10, BsAb11, BsAb12, BsAb13, BsAb14, BsAb15, BsAb16, BsAb17, BsAb18, BsAb19, BsAb20 and BsAb21 listed in Table 11.

44. The isolated antibody or antigen-binding fragment thereof as described in any of the preceding claims, wherein the antibody is fused to a constant region of human IgG.

45. The isolated antibody or antigen-binding fragment thereof as described in any of the preceding claims, wherein the antibody is fused to a constant region of human IgG1 or IgG4.

46. ​​An isolated antibody or an antigen-binding fragment thereof, which competes with the isolated antibody or an antigen-binding fragment thereof of any one of the preceding claims for binding to the same epitope.

47. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and / or a light chain variable region of an isolated antibody as defined in any one of claims 1-46.

48. The isolated nucleic acid molecule according to claim 47, which encodes the heavy chain variable region of the isolated antibody as defined in any one of claims 1-46.

49. The isolated nucleic acid molecule according to claim 48, which encodes the light chain variable region of the isolated antibody as defined in any one of claims 19-46.

50. A vector comprising the nucleic acid molecule of any one of claims 47-49.

51. A host cell comprising the vector of claim 50.

52. An immunoconjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-46, and an additional portion thereof conjugated non-covalently or covalently thereto.

53. The immunoconjugate of claim 52, wherein the additional portion is a chemical portion, such as a therapeutic agent, BSA, Fc fragment, PEG molecule, etc.

54. A pharmaceutical composition comprising at least one antibody as defined in any one of claims 1-46 or an antigen-binding fragment thereof, or an immunoconjugate as described in claim 52 or 53, and a pharmaceutically acceptable carrier.

55. The pharmaceutical composition of claim 54, further comprising one or more additional therapeutic agents.

56. The pharmaceutical composition of claim 55, wherein the one or more additional therapeutic agents are selected from the group consisting of: immunotherapeutic agents, anticancer agents, cytotoxic agents, chemotherapeutic agents, and any combination thereof.

57. The pharmaceutical composition of claim 56, wherein the one or more additional therapeutic agents are selected from PD-L1 inhibitors, ADCC-enabled antibodies, or any combination thereof.

58. The pharmaceutical composition of claim 57, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody, such as LAE005.

59. The pharmaceutical composition of claim 56, wherein the ADCC-enabled antibody is an anti-EGFR antibody, such as cetuximab.

60. The pharmaceutical composition of claim 56, wherein the ADCC-enabled antibody is an anti-EGFR-MET antibody, such as ervantumab.

61. A method for producing an antibody or antigen-binding fragment thereof as defined in any one of claims 1-46, comprising the following steps: - Expressing the antibody or antigen-binding fragment thereof as defined in any one of claims 1-46 in the host cell of claim 51; and - Isolate the antibody or its antigen-binding fragment from the host cell.

62. A method for detecting or monitoring the expression level of PVRIG in vitro, comprising contacting a test sample with an antibody or antigen-binding fragment thereof as defined in any one of claims 1-46, an immunoconjugate as described in claim 52 or 53, or a pharmaceutical composition as described in any one of claims 54-60.

63. A method for detecting or monitoring the expression level of TIGIT in vitro, comprising contacting a test sample with an antibody or antigen-binding fragment thereof as defined in any one of claims 19-46, an immunoconjugate as described in claim 52 or 53, or a pharmaceutical composition as described in any one of claims 54-60.

64. A method for preventing or treating a subject with a proliferative condition (e.g., cancer or tumor), infection, or sepsis associated with PVRIG and / or TIGIT overexpression, or for improving symptoms of said proliferative condition, infection, or sepsis, comprising administering to the subject an effective amount of an antibody as defined in any one of claims 1-46 or an antigen-binding fragment thereof, an immunoconjugate as described in claim 52 or 53, or a pharmaceutical composition as described in any one of claims 54-60.

65. The method of claim 64, wherein the cancer or tumor is selected from the group consisting of: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, stomach / gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial carcinoma, lung cancer (small cell lung cancer or non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, and myelodysplastic syndrome (MDS).

66. The method of claim 64, wherein the cancer or tumor is selected from the group consisting of: lung cancer (small cell lung cancer or non-small cell lung cancer), prostate cancer, breast cancer (e.g., triple-negative breast cancer), head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematologic malignancies, Merkel cell carcinoma and MSI-high cancer, and any other disease or condition characterized by uncontrolled cell growth.

67. The method of claim 64, wherein the infection is a pathogen infection characterized by varying degrees of dysfunction of the virus-specific T cell response, such as HIV, HCV, or HBV.

68. The method of claim 64, wherein the sepsis is selected from the group consisting of: severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, and sepsis syndrome.

69. Use of an antibody or antigen-binding fragment thereof as defined in any one of claims 1-18, or an immunoconjugate as described in claim 52 or 53, in the preparation of a formulation for detecting or monitoring PVRIG expression levels.

70. Use of an antibody or antigen-binding fragment thereof as defined in any one of claims 19-34, or an immunoconjugate as described in claim 52 or 53, in the preparation of a formulation for detecting or monitoring TIGIT expression levels.

71. Use of an antibody or antigen-binding fragment thereof as defined in any one of claims 1-18, or an immunoconjugate as described in claim 52 or 53, in the preparation of a formulation for blocking the binding of CD112 to PVRIG or interrupting the signal transduction of CD112 through PVRIG.

72. Use of an antibody or antigen-binding fragment thereof as defined in any one of claims 19-34, or an immunoconjugate as described in claim 52 or 53, in the preparation of a formulation for blocking the binding of PVR to TIGIT or interrupting PVR signaling via TIGIT.

73. Use of an antibody or antigen-binding fragment thereof as defined in any one of claims 1-46, an immunoconjugate as described in claim 52 or 53, or a pharmaceutical composition as described in any one of claims 54-60 in the preparation of a medicament for the prevention or treatment of proliferative disorders (e.g., cancer or tumors), infections, or sepsis associated with PVRIG and / or TIGIT overexpression in a subject, or for the improvement of symptoms of said proliferative disorders, infections, or sepsis.

74. An antibody or antigen-binding fragment thereof as defined in any one of claims 1-46, an immunoconjugate as described in claim 52 or 53, or a pharmaceutical composition as described in any one of claims 54-60, for the treatment or prevention of proliferative disorders (e.g., cancer or tumors), infections, or sepsis associated with PVRIG and / or TIGIT overexpression in a subject, or for the improvement of symptoms of said proliferative disorders, infections, or sepsis.

75. A kit for treating or preventing proliferative disorders (e.g., cancer or tumors), infections, or sepsis associated with overexpression of PVRIG and / or TIGIT, or for improving symptoms of said proliferative disorders, infections, or sepsis, comprising a container containing at least one antibody or antigen-binding fragment thereof as defined in any one of claims 1-46, an immunoconjugate as claimed in claim 52 or 53, or a pharmaceutical composition as claimed in any one of claims 54-60.

76. The use according to claim 73, the antibody or antigen-binding fragment or immunoconjugate thereof according to claim 74, or the kit according to claim 75, wherein the cancer or tumor is selected from the group consisting of: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, stomach / gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial carcinoma, lung cancer (small cell lung cancer or non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, and myelodysplastic syndrome (MDS).

77. The use according to claim 73, the antibody or antigen-binding fragment or immunoconjugate thereof according to claim 74, or the kit according to claim 75, wherein the cancer or tumor is selected from the group consisting of: lung cancer (small cell lung cancer or non-small cell lung cancer), prostate cancer, breast cancer (e.g., triple-negative breast cancer), head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematologic malignancies, Merkel cell carcinoma and MSI-high cancer, and any other disease or condition characterized by uncontrolled cell growth.

78. The use according to claim 73, the antibody or antigen-binding fragment or immunoconjugate thereof according to claim 74, or the kit according to claim 75, wherein the infection is a pathogen infection characterized by varying degrees of dysfunction of the virus-specific T cell response, such as HIV, HCV, or HBV.

79. The use according to claim 73, the antibody or antigen-binding fragment or immunoconjugate thereof according to claim 74, or the kit according to claim 75, wherein the sepsis is selected from the group consisting of: severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, and sepsis syndrome.

80. A method for preventing the suppression of immune cells or the activation of immune cell-mediated responses, comprising contacting the immune cells with an antibody or fragment thereof as claimed in any one of claims 1-46, an immunoconjugate as claimed in claim 52 or 53, or a pharmaceutical composition as claimed in any one of claims 54-60.

81. The method of claim 80, wherein the immune cell is an NK cell or a T cell.

82. The method of claim 80, wherein the T cell is a CD8+ T cell.

83. A method for reducing or eliminating the number and / or activity of at least one type of regulatory T cells (Tregs) in vitro and in vivo, comprising administering to a subject in need an anti-PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1-18, an anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 19-34, a multispecific / bispecific antibody against PVRIG and TIGIT according to any one of claims 35-46, a nucleic acid according to any one of claims 47-49, a vector according to claim 50, a host cell according to claim 51, an immunoconjugate according to claim 52 or 53, and / or a pharmaceutical composition according to any one of claims 54-60.

84. The method according to any one of claims 80-83, wherein the immune cells express PVRIG and / or TIGIT.

85. The method according to any one of claims 80-83, wherein the reaction mediated by the immune cells is an antitumor response, optionally wherein the tumor cells express CD112 and / or CD155.

86. The method according to any one of claims 80-83, wherein the subject is a human subject.

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