Anti-pvrig antibodies and uses thereof

Abstract

The present disclosure provides an antibody or an antigen-binding fragment thereof, which specifically binds to PVRIG, has high binding affinity to human PVRIG protein and cynomolgus PVRIG protein, and has strong activity in both reporter gene level and CD8 T cell activation. Compared with known SRF813 and COM701, the antibodies of the present application exhibit more optimal in vitro activity, and some of the antibodies combined with PD-1 antibodies have obvious anti-tumor effect, making them excellent candidate drug molecules for therapeutic and diagnostic use.

Description

[0001] This application is a divisional application of the invention patent application entitled: Anti-PVRIG antibodies and uses thereof. TECHNICAL FIELD

[0002] The present disclosure relates to the field of antibodies, and more specifically to an antibody of PVRIG. BACKGROUND

[0003] Tumor immunotherapy is one of the main pillars in the field of oncology today, especially for the treatment of unresectable, metastatic and recurrent cancers. The success of immune checkpoint inhibitors (ICIs), and their combination with chemotherapy, immunotherapy and targeted drugs, has changed the mode of cancer treatment. However, PD-1 therapy still only has 10-30% of patients showing long-term, durable response, and most of the population lacks response, and acquired resistance and immune-related adverse events (IRAE) are also great obstacles. One of the mechanisms to overcome the limitations of PD-1 therapy is to develop other ICIs related to the tumor microenvironment, which can help overcome the limitations of previous treatments when used in combination with anti-PD-1 / PD-L1. Studies have shown that in patients resistant to targeted PD-1 immunotherapy, higher PVRL2 (CD112) levels in cancer cells are a potential key biomarker for disease treatment, and are overexpressed in various tumor types, including breast and ovarian cancer. PVRL2 is currently identified as the only functional ligand of PVRIG, which can bind to PVRIG with very high affinity.

[0004] PVRIG, also known as CD112R, was identified as a new inhibitory receptor in 2016, expressed in natural killer cells (NK cells) and T lymphocytes. PVRIG binds to its ligand PVRL2, mediates intracellular signal transduction, leading to reduced T cell cytotoxicity, in addition, it can also inhibit TCR-mediated NFAT activation, further inhibiting T cell activation, and also competes with DNAM-1 for PVRL2 ligand to block the transmitted stimulation signal, thereby achieving double negative regulation of T cell function, to exert the effect of immune suppression.

[0005] Preclinical studies show that blocking PVRIG can inhibit tumor growth in mouse and human solid tumors, and combined with PD-1 / TIGIT treatment can significantly improve the therapeutic effect; anti-PVRIG antibodies enhance NK cell activation through the DNAM-1-PVRL2 signaling pathway, and their anti-tumor activity depends on NK cells and CD8+ T cells. According to the existing clinical data, the single-agent and combination anti-PD-1 antibody treatment of advanced solid tumors has shown good therapeutic effect, especially in patients who have received immunological checkpoint inhibitor treatment, there is still a 72% best response rate (≥SD). In terms of safety, neither single-agent nor combination with Opdivo observed DLT, and there was no increase in toxicity when combined with PD-1 mAb, and no subjects stopped the study treatment due to toxicity of any study drug. Based on the existing clinical data, it has been preliminarily demonstrated that the drugability of this target has been preliminarily demonstrated, and the efficacy is considerable.

[0006] As a new type of NK cell and T cell immune checkpoint inhibitor, PVRIG has a clear target mechanism. On the one hand, the antibody can relieve the immune suppression of NK cells and CD8+ T cells, and on the other hand, it can also mediate the activation signal of NK cells and CD8+ T cells to enhance tumor killing. At present, there is no drug on the market worldwide, and the fastest drug COM701 for tumor indications has just entered clinical phase II. The results of preclinical in vivo efficacy research are significant, and the clinical data show good safety and positive efficacy when combined with PD-1 mAb. SUMMARY

[0007] To address the above problems, the present disclosure provides antibodies, methods of making the same, compositions, and the like. The benefits provided by the present disclosure are widely applicable to the field of antibody therapy and diagnosis, and can be used in combination with antibodies that react with a variety of targets.

[0008] The present disclosure provides an isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to PVRIG and comprises a heavy chain variable region (VH) and a light chain variable region (VL),

[0009] The heavy chain variable region comprises:

[0010] (i) HCDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 35-39 or consists of SEQ ID NOs: 35-39;

[0011] (ii) HCDR2, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 40-45 or consists of SEQ ID NOs: 40-45; and

[0012] (iii) HCDR3, which comprises or consists of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 48-53;

[0013] the light chain variable region comprises:

[0014] (i) LCDR1, which comprises or consists of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 56-62;

[0015] (ii) LCDR2, which comprises or consists of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 63-67; and

[0016] (iii) LCDR3, which comprises or consists of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 68-73.

[0017] In some embodiments of the application, the antibody or fragment comprises:

[0018] (i) HCDR1 as set forth in SEQ ID NO: 35, HCDR2 as set forth in SEQ ID NO: 40, and HCDR3 as set forth in SEQ ID NO: 48, LCDR1 as set forth in SEQ ID NO: 56, LCDR2 as set forth in SEQ ID NO: 63, and LCDR3 as set forth in SEQ ID NO: 68; or

[0019] (ii) HCDR1 as set forth in SEQ ID NO: 35, HCDR2 as set forth in SEQ ID NO: 41, and HCDR3 as set forth in SEQ ID NO: 48, LCDR1 as set forth in SEQ ID NO: 57, LCDR2 as set forth in SEQ ID NO: 63, and LCDR3 as set forth in SEQ ID NO: 68; or

[0020] (iii) HCDR1 as set forth in SEQ ID NO: 36, HCDR2 as set forth in SEQ ID NO: 42, and HCDR3 as set forth in SEQ ID NO: 49, LCDR1 as set forth in SEQ ID NO: 58, LCDR2 as set forth in SEQ ID NO: 64, and LCDR3 as set forth in SEQ ID NO: 69; or

[0021] (iv) HCDR1 as depicted in SEQ ID NO: 37, HCDR2 as depicted in SEQ ID NO: 43, and HCDR3 as depicted in SEQ ID NO: 50, LCDR1 as depicted in SEQ ID NO: 59, LCDR2 as depicted in SEQ ID NO: 65, and LCDR3 as depicted in SEQ ID NO: 70; or

[0022] (v) HCDR1 as depicted in SEQ ID NO: 37, HCDR2 as depicted in SEQ ID NO: 43, and HCDR3 as depicted in SEQ ID NO: 51, LCDR1 as depicted in SEQ ID NO: 60, LCDR2 as depicted in SEQ ID NO: 65, and LCDR3 as depicted in SEQ ID NO: 71; or

[0023] (vi) HCDR1 as depicted in SEQ ID NO: 38, HCDR2 as depicted in SEQ ID NO: 44, and HCDR3 as depicted in SEQ ID NO: 52, LCDR1 as depicted in SEQ ID NO: 61, LCDR2 as depicted in SEQ ID NO: 66, and LCDR3 as depicted in SEQ ID NO: 72; or

[0024] (vii) HCDR1 as depicted in SEQ ID NO: 39, HCDR2 as depicted in SEQ ID NO: 45, and HCDR3 as depicted in SEQ ID NO: 53, LCDR1 as depicted in SEQ ID NO: 62, LCDR2 as depicted in SEQ ID NO: 67, and LCDR3 as depicted in SEQ ID NO: 73.

[0025] In some embodiments of the application, the antibody or fragment, wherein the heavy chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 17-23.

[0026] In some embodiments of the application, the antibody or fragment, wherein the heavy chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 17-23.

[0027] In some embodiments of the application, the antibody or fragment, further comprises a heavy chain constant region comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to, or consisting of, SEQ ID NO: 15; and a light chain constant region comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to, or consisting of, SEQ ID NO: 16.

[0028] In some embodiments of the application, the antibody or fragment, further comprises:

[0029] a light chain comprising a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14; and

[0030] a heavy chain comprising a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13.

[0031] In some embodiments of the application, the antibody or fragment, further comprises:

[0032] (i) a light chain as set forth in SEQ ID NO: 2, and a heavy chain as set forth in SEQ ID NO: 1; or

[0033] (ii) a light chain as set forth in SEQ ID NO: 4, and a heavy chain as set forth in SEQ ID NO: 3; or

[0034] (iii) a light chain as set forth in SEQ ID NO: 6, and a heavy chain as set forth in SEQ ID NO: 5; or

[0035] (iv) a light chain as set forth in SEQ ID NO: 8, and a heavy chain as set forth in SEQ ID NO: 7; or

[0036] (v) a light chain as set forth in SEQ ID NO: 10, and a heavy chain as set forth in SEQ ID NO: 9; or (vi) a light chain as set forth in SEQ ID NO: 12, and a heavy chain as set forth in SEQ ID NO: 11; or (vii) a light chain as set forth in SEQ ID NO: 14, and a heavy chain as set forth in SEQ ID NO: 13.

[0037] 8. In some embodiments of the present application, the antibody or fragment is engineered to avoid the impact of aspartate isomerization on the structure and function of the antibody by hotspot removal; wherein preferably,

[0038] the heavy chain variable region comprises a HCDR2 as set forth in SEQ ID NO: 46 or 47, and / or,

[0039] a HCDR3 as set forth in SEQ ID NO: 54 or 55.

[0040] In some embodiments of the present application, the antibody or fragment, wherein the heavy chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 24-27.

[0041] In some embodiments of the present application, the antibody or fragment, wherein the antibody is a whole antibody, a monoclonal antibody, a chimeric antibody, or a humanized antibody, or an improved antibody thereof such as an improved chimeric antibody.

[0042] wherein the fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, or a single chain Fv fragment (scFv).

[0043] In another aspect, the present application also provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or fragment.

[0044] In another aspect, the present application also provides a vector comprising the nucleic acid molecule.

[0045] In another aspect, the present application also provides a host cell comprising the nucleic acid molecule or the vector.

[0046] In another aspect, the present application also provides a conjugate comprising the antibody or fragment conjugated to at least one detectable label.

[0047] In another aspect, the present application also provides an antibody drug conjugate comprising an antibody, wherein one or more drug moieties are covalently attached to the antibody or fragment, directly or via a linker.

[0048] In another aspect, the present application also provides a multispecific molecule comprising the antibody or antigen binding fragment; preferably, the multispecific molecule specifically binds to PVRIG and additionally specifically binds to one or more other targets; further preferably, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.

[0049] In another aspect, the present application also provides a pharmaceutical composition or kit comprising the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody drug conjugate, or the multispecific molecule, and a pharmaceutically acceptable carrier.

[0050] In another aspect, the present application also provides use of the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody drug conjugate, or the multispecific molecule, or the pharmaceutical composition or kit in the manufacture of a kit for diagnosing, detecting or monitoring a disease associated with PVRIG expression.

[0051] In another aspect, the present application also provides use of the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody drug conjugate, or the multispecific molecule, or the pharmaceutical composition or kit in the manufacture of a medicament for treating or determining the prognosis of a disease associated with PVRIG expression.

[0052] In another aspect, the above-mentioned disease associated with PVRIG expression is a cancer selected from the group consisting of squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, cervical cancer, uterine cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, brain cancer, endometrial cancer, testicular cancer, biliary tract cancer, gallbladder cancer, stomach cancer, melanoma or various types of head and neck cancer (including head and neck squamous cell carcinoma), hematological cancer or any other disease or disorder characterized by uncontrolled cell growth.

[0053] The PVRIG antibody provided by the application has high binding affinity to human PVRIG protein and cynomolgus PVRIG protein, and has strong activity in terms of reporter gene level and CD8 T cell activation. Compared with known SRF813 and COM701, the antibody of the application shows more excellent in vitro activity, and some antibodies combined with PD-1 antibodies have obvious anti-tumor effect, so that it becomes an excellent candidate drug molecule for therapeutic and diagnostic use. BRIEF DESCRIPTION OF DRAWINGS

[0054] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of the specification, illustrate embodiments of the present disclosure and are used to explain the present disclosure, and do not constitute a limitation on the present disclosure.

[0055] Figure 1A is the detection result of antibody 1 combined with CHOK1 overexpressing different species PVRIG detected by FACS, wherein CHOK1 / Human PVRIG represents a stable cell line overexpressing human PVRIG antigen, CHOK1 / cyno PVRIG represents a stable cell line overexpressing monkey PVRIG antigen, CHOK1 / mouse PVRIG represents a stable cell line overexpressing mouse PVRIG antigen, CHOK1 is a normal cell line without expressing PVRIG antigen, and MFI represents mean fluorescence intensity;

[0056] Figure 1B is the detection result of antibody 6 combined with CHOK1 overexpressing different species PVRIG detected by FACS, wherein CHOK1 / Human PVRIG represents a stable cell line overexpressing human PVRIG antigen, CHOK1 / cyno PVRIG represents a stable cell line overexpressing monkey PVRIG antigen, CHOK1 / mouse PVRIG represents a stable cell line overexpressing mouse PVRIG antigen, CHOK1 is a normal cell line without expressing PVRIG antigen, and MFI represents mean fluorescence intensity;

[0057] Figure 1C is the detection result of antibody 7 combined with CHOK1 overexpressing different species PVRIG detected by FACS, wherein CHOK1 / Human PVRIG represents a stable cell line overexpressing human PVRIG antigen, CHOK1 / cyno PVRIG represents a stable cell line overexpressing monkey PVRIG antigen, CHOK1 / mouse PVRIG represents a stable cell line overexpressing mouse PVRIG antigen, CHOK1 is a normal cell line without expressing PVRIG antigen, and MFI represents mean fluorescence intensity;

[0058] Figure 1Dis the detection result of antibody 16 binding to CHOK1 overexpressing different species PVRIG by FACS detection, wherein CHOK1 / Human PVRIG represents a stable cell line overexpressing human PVRIG antigen, CHOK1 / cyno PVRIG represents a stable cell line overexpressing monkey PVRIG antigen, CHOK1 / mouse PVRIG represents a stable cell line overexpressing mouse PVRIG antigen, CHOK1 is a normal cell line without expressing PVRIG antigen, and MFI represents the mean fluorescence intensity;

[0059] Figure 1E is the detection result of antibody 27 binding to CHOK1 overexpressing different species PVRIG by FACS detection, wherein CHOK1 / Human PVRIG represents a stable cell line overexpressing human PVRIG antigen, CHOK1 / cyno PVRIG represents a stable cell line overexpressing monkey PVRIG antigen, CHOK1 / mouse PVRIG represents a stable cell line overexpressing mouse PVRIG antigen, CHOK1 is a normal cell line without expressing PVRIG antigen, and MFI represents the mean fluorescence intensity;

[0060] Figure 1F is the detection result of antibody 52 binding to CHOK1 overexpressing different species PVRIG by FACS detection, wherein CHOK1 / Human PVRIG represents a stable cell line overexpressing human PVRIG antigen, CHOK1 / cyno PVRIG represents a stable cell line overexpressing monkey PVRIG antigen, CHOK1 / mouse PVRIG represents a stable cell line overexpressing mouse PVRIG antigen, CHOK1 is a normal cell line without expressing PVRIG antigen, and MFI represents the mean fluorescence intensity;

[0061] Figure 1G is the detection result of antibody 55 binding to CHOK1 overexpressing different species PVRIG by FACS detection, wherein CHOK1 / Human PVRIG represents a stable cell line overexpressing human PVRIG antigen, CHOK1 / cyno PVRIG represents a stable cell line overexpressing monkey PVRIG antigen, CHOK1 / mouse PVRIG represents a stable cell line overexpressing mouse PVRIG antigen, CHOK1 is a normal cell line without expressing PVRIG antigen, and MFI represents the mean fluorescence intensity;

[0062] Figure 2is the CHOK1 / Human PVRIG cell binding curve of antibody 1, 6, 7, 16, 27, 52, 55 vs. reference antibodies SRF813 and COM701, where the abscissa Conc. is the antibody concentration in nM, the ordinate Geom. Mean is the mean fluorescence intensity, Human lgG1 is human lgG1;

[0063] Figure 3 is the CHOK1 / Cyno PVRIG cell binding curve of antibody 1, 6, 7, 16, 27, 52, 55 vs. reference antibodies SRF813 and COM701, where the abscissa Conc. is the antibody concentration in nM, the ordinate Geom. Mean is the mean fluorescence intensity, Human lgG1 is human lgG1;

[0064] Figure 4 is the Human PVRIG protein (human PVRIG protein) binding curve of antibody 1, 6, 7, 16, 27, 52, 55 vs. reference antibodies SRF813 and COM701, where the abscissa Conc. is the antibody concentration in nM, the ordinate is the reading of the microplate reader at OD450 nm, Human lgG1 is human lgG1;

[0065] Figure 5 is the Cyno PVRIG protein (monkey PVRIG protein) binding curve of antibody 1, 6, 7, 16, 27, 52, 55 vs. reference antibodies SRF813 and COM701, where the abscissa Conc. is the antibody concentration in nM, the ordinate is the reading of the microplate reader at OD450 nm, Human lgG1 is human lgG1;

[0066] Figure 6 is the protein level blocking activity result of antibody 1, 6, 7, 16, 27, 52, 55 vs. reference antibodies SRF813 and COM701, where the abscissa Conc. is the antibody concentration in nM, the ordinate is the reading of the microplate reader at OD450 nm, Human lgG1 is human lgG1;

[0067] Figure 7 is the reporter gene activity assay curve of antibody 1, 6, 7, 16, 27, 52, 55 vs. reference antibodies SRF813 and COM701, where the abscissa Conc. is the antibody concentration in ug / ml, the ordinate Luminescence (RLU) is the relative luminescence units, Human lgG1 is human lgG1;

[0068] Figure 8A , Figure 8B , Figure 8Cis the result of cytokine hIFNγ (human interferon gamma) secretion detection, the horizontal coordinate is the antibody concentration, in units of ug / ml, and the vertical coordinate is the hIFNγ concentration, in units of pg / ml, Human lgG4 is human lgG4, and the CD8+T+Pretreated CTV-Colo205 at the position of the dotted line in the figure refers to the cytokine hIFNγ detection value after incubation of CTV-Colo205 cells treated with 5 ug / ml pp65 with CD8+T cells;

[0069] Figure 8D 、 Figure 8E 、 Figure 8F is the result of target cell killing level detection, the horizontal coordinate is the antibody concentration, in units of ug / ml, and the vertical coordinate Target cell killing is the target cell killing ability; Human lgG4 is human lgG4, and the CD8+T+Pretreated CTV-Colo205 at the position of the dotted line in the figure refers to the cytokine hIFNγ detection value after incubation of CTV-Colo205 cells treated with 5 ug / ml pp65 with CD8+T cells;

[0070] Figure 9 is the epitope classification of antibodies 1, 6, 7, 16, 27, 52, 55 and reference antibodies SRF813 and COM701 binding to PVRIG;

[0071] Figure 10 is the result of in vivo pharmacodynamic activity detection of antibodies 1, 6, 7, 16, 27, 52, 55 combined with PD-1 antibodies, the horizontal coordinate Days Post Treatment is the number of days after treatment, in units of days, and the vertical coordinate Tumor Volume (mm 3 ) is the tumor volume, in units of cubic millimeters (mm 3 ), Vehicle represents 10 mM phosphate buffer (PBS), ip represents intraperitoneal administration, BIW represents twice a week, and BIW*3 represents twice a week for three consecutive weeks;

[0072] Figure 11 is the Human PVRIG protein (human PVRIG protein) binding curve of antibodies 52, and variants 52_vH(DG / EG-DS / ES), 52_vH(DG / EG-DS / DA), 52_vH(DG / DA-DS / ES), 52_vH(DG / DA-DS / DA), wherein the horizontal coordinate Conc. is the antibody concentration, in units of nM, and the vertical coordinate is the OD450 nm reading of the microplate reader;

[0073] Figure 12Cyno PVRIG protein (monkey PVRIG protein) binding curves for antibody 52, 52_vH(DG / EG-DS / ES), 52_vH(DG / EG-DS / DA), 52_vH(DG / DA-DS / ES), 52_vH(DG / DA-DS / DA), where the abscissa Conc. is the antibody concentration in nM and the ordinate is the reading of the microplate reader at OD450 nm;

[0074] Figure 13 Protein level blocking activity results for antibody 52, and variants 52_vH(DG / EG-DS / ES), 52_vH(DG / EG-DS / DA), 52_vH(DG / DA-DS / ES), 52_vH(DG / DA-DS / DA), where the abscissa Conc. is the antibody concentration in nM and the ordinate is the reading of the microplate reader at OD450 nm;

[0075] Figure 14 CHOK1 / Human PVRIG cell binding curves for antibody 52, and variants 52_vH(DG / EG-DS / ES), 52_vH(DG / EG-DS / DA), 52_vH(DG / DA-DS / ES), 52_vH(DG / DA-DS / DA), where the abscissa Conc. is the antibody concentration in nM and the ordinate Geom. Mean is the mean fluorescence intensity;

[0076] Figure 15 CHOK1 / Cyno PVRIG cell binding curves for antibody 52, and variants 52_vH(DG / EG-DS / ES), 52_vH(DG / EG-DS / DA), 52_vH(DG / DA-DS / ES), 52_vH(DG / DA-DS / DA), where the abscissa Conc. is the antibody concentration in nM and the ordinate Geom. Mean is the mean fluorescence intensity;

[0077] Figure 16 Reporter gene activity assay curves for antibody 52, and variants 52_vH(DG / EG-DS / ES), 52_vH(DG / EG-DS / DA), 52_vH(DG / DA-DS / ES), 52_vH(DG / DA-DS / DA), where the abscissa Conc. is the antibody concentration in ug / ml and the ordinate Luminescence (RLU) is the relative luminescence units. DETAILED DESCRIPTION

[0078] For a better understanding of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only illustrative embodiments of a part of the present application, rather than all the embodiments. Therefore, the present application is not limited to the specific illustrative embodiments. In addition, any chapter title used herein is not interpreted as limiting the described subject matter.

[0079] Unless otherwise defined herein, scientific and technical terms used in connection with the present application have meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "include", "includes," and "included" is not limiting. Also, ranges provided in the specification are inclusive of the endpoints and all values between the endpoints.

[0080] Definitions

[0081] For a better understanding of the present application, definitions and explanations of relevant terms are provided as follows.

[0082] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and noncovalent interactions. The light chains of an antibody can be classified as kappa or lambda light chains. The heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, which define the isotype of the antibody as IgM, IgD, IgG, IgA, or IgE, respectively. Within the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into variable regions (called complementarity determining regions, CDRs) spaced apart by relatively conserved regions (called framework regions, FRs). Each VH and VL is composed of three CDRs and four FRs, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from N- to C-terminus. The CDRs on VH are HCDR1, HCDR2, HCDR3; the CDRs on VL are LCDR1, LCDR2, LCDR3. The variable regions of each heavy / light chain pair form an antigen binding site / portion, respectively. The distribution of amino acids in the various regions or domains follows the numbering definitions of Kabat, IMGT, or Chothia, etc., and in specific embodiments of the present disclosure, the determination of CDR sequences uses the numbering definition in the Kabat system.

[0083] Antibodies in the present disclosure also include antigen-binding portions (used interchangeably with the term "antigen-binding fragment"). An antigen-binding portion refers to a polypeptide that comprises a fragment of an intact antibody that retains the ability to specifically bind with the antigen to which the full-length or intact antibody specifically binds, and / or that competes with the full-length antibody for binding to the same antigen. In some conditions, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and comprises at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. An antigen-binding portion of an antibody can be obtained from a given antibody by conventional techniques, e.g., recombinant DNA techniques or enzymatic or chemical cleavage, and can be screened for specificity in the same manner as an intact antibody.

[0084] The term "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0085] The term "monoclonal antibody" or "mAb" refers to antibody molecules / preparations of single molecular composition. Monoclonal antibodies display a single binding specificity and affinity for a particular epitope. Antibodies of the present invention can be derived from different species, including but not limited to mouse, rat, rabbit, guinea pig, and human.

[0086] The term "epitope" refers to an antigenic determinant on a molecule, i.e., a portion of a molecule that is recognized by the immune system, e.g., by an antibody, e.g., a three-dimensional site on an antigen that is recognized by the immune system. In the present invention, the epitope is, for example, a PVRIG protein.

[0087] The term "chimeric antibody" as used herein refers to an antibody whose variable region sequences are from one species and the constant region sequences are from another species, e.g., an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0088] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences / antigen binding portions or sites derived from the germline of another mammalian species, like a mouse, have been grafted onto human framework sequences. In addition, additional framework region modifications can also be made within the human framework sequences.

[0089] The term "KD value" is the equilibrium dissociation constant between an antibody and its antigen, i.e., the ratio of koff / kon or kd / ka (can be determined by SPR technology). Thus the lower the KD value (the lower the concentration), the higher the affinity of the antibody. Thus "KD value" can be used to measure the binding affinity between an antibody and its antigen. The kd dissociation constant, reflects how strong the affinity of a compound to a target is, the smaller the value the stronger the affinity. The ka association constant, is the opposite of kd, the larger the value the stronger the affinity.

[0090] The terms "PVRIG" and "PVRIG antigen" are used interchangeably herein and include any variant, isoform, and species homolog of human PVRIG that is naturally expressed by a cell or expressed on a cell transfected with a PVRIG gene. In some embodiments, binding of an antibody of the present disclosure to a PVRIG antigen mediates killing of a cell expressing PVRIG (e.g., a tumor cell) by inactivation of PVRIG. Killing of a cell expressing PVRIG can occur through one or more of the following mechanisms: induction of cell death / apoptosis, ADCC, and CDC.

[0091] The term "anti-PVRIG antibody" or "PVRIG antibody" refers to an antibody as defined herein which is capable of binding to a PVRIG antigen or to a cell expressing PVRIG. Depending on the binding properties and biological activity of the anti-PVRIG antibody to the PVRIG antigen, two types of anti-PVRIG antibodies (type I and type II anti-PVRIG antibodies) can be distinguished according to Cragg, M.S. et al., Blood 103 (2004) 2738-2743; and Cragg, M.S. et al., Blood 101 (2003) 1045-1052.

[0092] The term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.

[0093] The term "isolated" refers to a state of being obtained by artificial means from a natural state. If a certain "isolated" material or component exists naturally, it can be because its natural environment has changed, or the material has been separated from the natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide exists naturally in a certain living organism, and a high-purity version of the same polynucleotide or polypeptide separated from the natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor does it exclude other impurities that do not affect the activity of the isolated substance. For example, an isolated antibody can be substantially free of other cellular material and / or chemical materials.

[0094] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector allows the expression of a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be used to express the genetic material elements it carries in a host cell by transformation, transduction or transfection into the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda 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, papovaviruses (such as SV40). The vector can comprise a plurality of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can comprise an origin of replication. For vectors expressing antibodies, vector types in which the heavy and light chains of the antibody are present in different vectors or vector types in which the heavy and light chains are present in the same vector can be used.

[0095] The term "host cell" refers to a cellular system that can be engineered to produce a protein of interest, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells, for example, mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, CHOK1, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within a transgenic animal or cultured tissue. The term encompasses not only the particular subject cell but also its progeny. Because certain modifications can occur in succeeding generations due to mutations or environmental influences, such progeny can not be identical to the parent cell, but are still included within the scope of the term "host cell."

[0096] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules (or protein molecules) or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percentage of identical residues between 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 addressed by a particular mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acid or polypeptide sequences include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 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., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAM J. Applied Math. 48:1073.

[0097] The term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It refers not only to the property of an antigen to stimulate specific immune cells to activate, proliferate and differentiate to ultimately produce immunologic effectors such as antibodies and sensitized lymphocytes, but also to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the immune system of an organism after the organism has been stimulated with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen is able to successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the means of immunization.

[0098] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, in particular mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.

[0099] 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 hybridomas.

[0100] The term "immune effector function" includes any function mediated by a component of the immune system that results in the inhibition of tumor growth and / or the inhibition of tumorigenesis, but also the inhibition of the dissemination and metastasis of tumors. Preferably, the immune effector function results in the killing of tumor cells. Preferably, the immune effector function in the present application is an antibody-mediated effector function. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), the induction of apoptosis in cells bearing tumor-associated antigens (e.g., by binding of the antibody to the surface antigen) and / or the inhibition of the proliferation of cells bearing tumor-associated antigens, preferably ADCC and / or CDC. The antibody can also simply act by binding to a tumor-associated antigen on the surface of a tumor cell. For example, the antibody can block the function of the tumor-associated antigen or induce apoptosis simply by binding to a tumor-associated antigen on the surface of a tumor cell.

[0101] The term "cancer" refers to any neoplastic or malignant cell growth, proliferation, or metastasis-mediated solid and non-solid tumors such as leukemias that cause a medical condition. For example, cancers caused in association with or due to abnormal expression of PVRIG include, but are not limited to, B-cell lymphomas, including NHL, pre-B-cell lymphoblastic leukemia / lymphoma, and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal, and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large cell lymphoma (ALCL)

[0102] The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, and / or salt is chemically and / or physically compatible with the other ingredients of the formulation and is physiologically compatible with the recipient.

[0103] The term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is compatible, in pharmacology and / or physiology, with the subject and the active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride. th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0104] The term "adjuvant" means a non-specific immune enhancer that can enhance or alter the type of immune response to an antigen in an organism when delivered with the antigen to the organism or when delivered to the organism in advance. There are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments at present. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0105] Anti-PVRIG antibodies

[0106] In some aspects, the present application includes an isolated antibody or antigen binding fragment thereof.

[0107] In the context of the present application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, bifunctional antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof, improved antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecule that exhibits preferential association or binding to PVRIG protein. Furthermore, unless otherwise specified by context, the term also includes antibodies of all classes (i.e., IgA, IgD, IgE, IgG and IgM) and all subclasses (i.e., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a chimeric monoclonal antibody or a humanized monoclonal antibody or an improved chimeric monoclonal antibody.

[0108] The variable regions and CDRs in an antibody sequence can be identified according to the general rules developed in the art (as described above, e.g., Kabat) numbering system or by aligning the sequence with a database of known variable regions.

[0109] Methods of testing the ability of an antibody to bind to an antigen (e.g., PVRIG), regardless of how the antibody is produced, are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266 and the above section on competitive assays).

[0110] According to the present application, an antibody is capable of binding to a predetermined target (e.g., PVRIG protein or a cell expressing PVRIG) if the antibody has significant affinity for the predetermined target, in a standard assay (e.g., an assay described in the present application), to test the binding of a monoclonal antibody to a live cell expressing PVRIG, flow cytometry (FCM) can be used. Preferably, the antibody is assayed for binding to a target expressed on the surface of a cell in a flow cytometric fluorescence sorting technique (FACS) assay, and the antibody is capable of binding to the target if the antibody binds detectably to the target (PVRIG protein or a cell expressing PVRIG) with "affinity."

[0111] The PVRIG specificity described herein refers to the ability to bind to one or more PVRIG epitopes, in particular to a naturally occurring conformation of PVRIG epitopes, in particular human PVRIG.

[0112] In the art, various means are employed to diversify the modification of an antibody without changing the desired properties of the antibody, such as the manner of recombining the light and heavy chains of the antibody, the substitution of amino acids, etc. as employed in the present disclosure. For example, the sequences of the present disclosure, including the chimeric antibody sequences or humanized antibody sequences, can be subjected to conservative amino acid substitutions.

[0113] In some embodiments, the antibody is a diabody or a bispecific antibody.

[0114] In some embodiments, the antibody is a whole antibody, a monoclonal antibody, a chimeric antibody, or a humanized antibody, or an improved antibody thereof, such as an improved chimeric antibody.

[0115] In some embodiments, the antibody is a whole antibody, a monoclonal antibody, a chimeric antibody, or a humanized antibody, or an improved antibody thereof, such as an improved chimeric antibody.

[0116] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a constant region of an IgG. The constant region of the IgG is preferably selected from the group consisting of a constant region of IgG1, IgG2, IgG3, or IgG4. The constant region of the IgG is more preferably selected from the group consisting of a constant region of IgG1.

[0117] Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences of the CDRs are more diverse between antibodies than other sequences. Since the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express a recombinant antibody that mimics the properties of a particular naturally occurring antibody by constructing an expression vector that contains the CDR sequences from the particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1988) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germlines are sequences different from mature antibody gene sequences in that they do not contain a complete set of assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also have sequences at individual positions across the variable region that are different from a high affinity secondary repertoire antibody.

[0118] Mouse antibodies are highly immunogenic in humans, leading to a decrease in therapeutic efficacy upon repeated applications, mediated primarily through the heavy chain constant region. If each antibody is chimerized or humanized, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided.

[0119] A chimeric antibody refers to an antibody having different portions from different animal species, e.g., an antibody having variable regions from a mouse antibody and human immunoglobulin constant regions. Chimeric antibodies are obtained by joining together the variable regions of the heavy and light chains of a mouse antibody with the constant regions of a human heavy and light chain (e.g., as described in Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, a chimeric antibody is produced by joining a human kappa light chain constant region to a mouse light chain variable region. In another preferred embodiment, a chimeric antibody can be produced by joining a human lambda light chain constant region to a mouse light chain variable region.

[0120] A humanized antibody refers to an antibody in which CDR sequences / antigen binding portions or sites derived from the germline of another mammalian species, such as a mouse, are grafted onto a human framework sequence.

[0121] To reduce the immunogenicity of the antibodies to humans, humanized anti-PVRIG antibodies are produced using the sequences of the PVRIG antibodies of the disclosure, utilizing the CDR regions of the murine-derived anti-PVRIG antibodies in combination with human-derived framework regions (e.g., human immunoglobulins) to form the humanized anti-PVRIG antibodies of the disclosure, which are expected to retain the functionality of binding to human PVRIG as well as the functionality of binding to monkey PVRIG.

[0122] Preparation or production of antibodies

[0123] Antibodies of the present application can be produced by a variety of techniques, including conventional monoclonal antibody methodology, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although the hybridoma technique is preferred, in principle, other techniques for producing monoclonal antibodies also can be employed, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries, somatic cell hybridization methods, and recombinant techniques, such as those involving the engineering of genes by gene recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present application are obtained, for example, by chemical synthesis or PCR amplification, inserted into expression vectors, and then transfected into host cells, which are then cultured under conditions suitable for the expression of the antibodies of the present application.

[0124] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and rabbit systems (e.g., described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al. Am. J. Clin. Pathol. 124:295 (2005)). Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

[0125] Monoclonal antibodies can be produced using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, or some combination thereof. For example, monoclonal antibodies can be produced using hybridoma and art-recognized biochemical and genetic engineering techniques, as described in detail in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1st ed. 2009; Shire et. al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC, 1st ed. 2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), each of which is hereby incorporated by reference in its entirety.

[0126] It will be appreciated that selected binding sequences can be further altered, for example to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and antibodies comprising altered target binding sequences are also antibodies of the application.

[0127] In some embodiments, the method of producing an antibody or fragment of the present disclosure comprises the steps of:

[0128] (i) expressing the antibody or fragment in a host cell; and optionally

[0129] (ii) isolating the antibody or antigen binding fragment thereof from the host cell.

[0130] In a preferred embodiment, the anti-PVRIG monoclonal antibody is produced by using a hybridoma.

[0131] To obtain hybridomas producing antibodies of the application, e.g., human monoclonal antibodies of the application, spleen and / or lymph node cells from immunized mice can be isolated and fused to suitable immortalized cell lines, e.g., mouse myeloma cell lines. The resulting hybridomas are screened for production of antigen-specific antibodies. The production of hybridomas is well known in the art. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0132] Antibodies of the application can also be produced in host cell transfectomas using, e.g., a combination of recombinant DNA technology and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In some embodiments, DNA encoding the partial or full light and heavy chains will be inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector in such a way that transcriptional and translational control sequences within the vector

[0133] Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors. In some embodiments, the variable region is used to produce a full-length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes a heavy chain constant region and a light chain constant region of the desired isotype, such that the V segment is operably linked to the CH segment within the vector and the VL segment is operably linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain(s) from a host cell. The antibody chain genes can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0134] To express the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfect" are intended to encompass a variety of techniques for getting exogenous DNA into a prokaryotic or eukaryotic host cell such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Antibodies of the application can be expressed in prokaryotic or eukaryotic host cells, e.g., mammalian host cells, which can assemble and secrete properly folded and immunologically active antibodies.

[0135] Mammalian host cells for expressing the recombinant antibodies of the application include Chinese hamster ovary cells (CHO cells) used with a DHFR selectable marker (e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159:601-621) including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or to permit the secretion of the antibody into the culture medium in which the host cells are grown. Antibody can be recovered from the culture medium using standard protein purification methods.

[0136] In another preferred embodiment, transgenic or transchromosomal mice with a partial human immune system (rather than a mouse system) can be used to generate human monoclonal antibodies to PVRIG.

[0137] Another strategy for generating monoclonal antibodies is to isolate the genes encoding the antibodies directly from antibody-producing lymphocytes of defined strategy, e.g., see Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. See also Welschof and Krau, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1 for details of recombinant antibody engineering.

[0138] To create a chimeric antibody, the murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). An isolated nucleic acid encoding a VHand can be converted to a full-length heavy chain gene by operably linking the VHand to another DNA molecule encoding heavy chain constant regions (CHI, CH2, and CH3). Sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991), Sequences Of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably is an IgGl or IgG4 constant region. An isolated nucleic acid encoding a VLregion can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VLto another DNA molecule encoding a light chain constant region, CL. Sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al., supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region, but a kappa constant region is generally preferred. Once DNA fragments encoding VHand VLsegments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes to full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, DNA fragments encoding VLor VHand another protein-encoding DNA fragment, such as an antibody constant region or a flexible linker, are operably linked. The term "operably linked," as used in this document, is intended to mean the joining together of two DNA fragments in such a way that the amino acid sequence encoded by the two DNA fragments remains in frame.

[0139] To prepare humanized antibodies, the murine CDR regions can be inserted into human framework sequences using methods known in the art (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 to Queen et al.; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can also be utilized that are capable of producing a complete repertoire of human antibodies upon immunization without the presence of endogenous immunoglobulin production. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array VDJ and C mu to the mutant mice results in the production of entirely human antibodies in response to antigen challenge (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of such transgenic animals include the HuMAb mouse (Medarex, Inc.) which contains human immunoglobulin genes miniloci that encode unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (see, e.g., Lonberg et al. (1994) Nature 368(6474):856-859); or the "KM mouse" (XenoMouse®, Green et al., 1994, Nature Genetics 7: 13-21; and U.S. Patent No. 6,075,181 to Wood et al.) which also contains human immunoglobulin loci that encode human heavy and light chain proteins. T Other methods of antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0140] Nucleic acid molecules encoding antibodies of the invention

[0141] In some aspects, the present application relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an isolated antibody or fragment thereof as described above in the present disclosure.

[0142] Nucleic acids of the present application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), nucleic acids encoding such antibodies can be recovered from the gene library.

[0143] To make chimeric antibodies, the murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). An isolated nucleic acid encoding a VHand can be converted to a full-length heavy chain gene by operably linking the nucleic acid encoding the VHand another DNA molecule encoding heavy chain constant regions (CHI, CH2, and CH3), and DNA fragments comprising these regions can be obtained by standard PCR amplification. An isolated nucleic acid encoding a VLcan be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VLto another DNA molecule encoding a light chain constant region, CL. Once DNA fragments encoding the VHand VLsegments have been obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as to convert the variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragments encoding the VLor VHand another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker, are operably linked.

[0144] Conjugates

[0145] In one aspect, the disclosure provides a conjugate comprising an antibody or fragment thereof as previously described coupled to at least one detectable label. Detectable labels include, but are not limited to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g., FRET (Fluorescence Resonance Energy Transfer); (iii) affect mobility (e.g., electrophoretic mobility) by charge, hydrophobicity, shape, or other physical parameter, or (iv) provide a capture moiety, e.g., affinity, antibody / antigen, or ionic complexation.

[0146] Suitable structures for labels include, for example, fluorescent labels, luminescent labels, chromophoric labels, radioisotope labels, isotopic labels, preferably stable isotopic labels, isobaric labels, enzymatic labels (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), particulate labels (especially metal, magnetic, and polymeric particles), small organic molecules (e.g., biotin, ligands or binding molecules for receptors (e.g., cell adhesion proteins or lecithin), label sequences comprising nucleic acid and / or amino acid residues that can be detected using binding agents, etc. Labels include, without limitation, barium sulfate, ioxitalamic acid, iodipamide, calcium iodipamide, sodium amidotrizoate, meglumine amidotrizoate, meglumine metrizamide, sodium tyropanoate, and radiodiagnostic agents (including positron emitters (e.g., fluorine-18 and carbon-11), gamma emitters (e.g., iodine-123, iodine-125, technetium-99m, iodine-131, and indium-111), nuclear magnetic resonance nuclides (e.g., fluorine and gadolinium), luminous substances (e.g., isoluminol and acridinium ester), fluorescent substances (e.g., fluorescein and rhodamine), and colored substances (e.g., latex particles and colloidal gold).

[0147] Detectable labels as described above can be detected by methods known in the art. For example, fluorescent labels can be detected using a light detector to detect emitted light. Enzymatic labels are generally detected by providing the enzyme with a substrate and detecting the reaction product produced by action of the enzyme on the substrate. In certain embodiments, such labels can be adapted for use in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In certain embodiments, detectable labels as described above can be attached to antibodies or antigen-binding fragments thereof of the present application via linkers of varying lengths to reduce potential steric hindrance.

[0148] Antibody drug conjugates / immunoconjugates

[0149] In one aspect, the present disclosure provides an antibody drug conjugate comprising an antibody, which includes one or more drug moieties / therapeutic agents, which are linked (e.g., covalently linked) directly or via a linker to an antibody or fragment thereof as previously described. In the antibody-drug conjugates of the present application, the linker structure for conjugating an anti-PVRIG antibody to a drug is not particularly limited as long as the resulting antibody-drug conjugate can be used.

[0150] Because antibody-drug conjugates have the ability to selectively deliver one or more drugs to a target tissue (e.g., an antigen associated with a tumor, such as a tumor expressing PVRIG), antibody-drug conjugates can improve the therapeutic efficacy of antibodies or antigen-binding fragments thereof of the present application in treating a disease (e.g., cancer).

[0151] Multispecific molecules

[0152] The antibodies or antigen-binding fragments thereof of the present application can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments thereof of the present application can be part of a multispecific molecule (e.g., bispecific molecule) that comprises a second functional module (e.g., a second antibody) or a third functional module (e.g., a third antibody) having a binding specificity different from that of the antibodies or antigen-binding fragments thereof of the present application, thereby being able to bind at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present application can be linked to a second antibody or antigen-binding fragment thereof that is capable of specifically binding to any protein that can serve as a potential target for a combination therapy. To generate such bispecific or multispecific molecules, the antibodies or antigen-binding fragments thereof of the present application can be linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimetics).

[0153] Thus, in some aspects, the present application provides a multispecific molecule comprising an antibody or antigen-binding fragment thereof of the present application.

[0154] In certain preferred embodiments, the multispecific molecule specifically binds PVRIG (e.g., human PVRIG or monkey PVRIG), and specifically binds one or more other targets.

[0155] In certain preferred embodiments, the multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity for a second target.

[0156] In certain preferred embodiments, the multispecific molecule is a bispecific antibody.

[0157] Pharmaceutical compositions

[0158] In some aspects, the present application relates to pharmaceutical compositions, the present disclosure provides a pharmaceutical composition or kit comprising an antibody or fragment as previously described, a nucleic acid molecule as previously described, a vector as previously described, a host cell as previously described, a conjugate as previously described, an antibody drug conjugate as previously described, a multispecific molecule as previously described; and a pharmaceutically acceptable carrier.

[0159] The pharmaceutical composition can optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present application can also be administered in combination with, for example, another immunostimulatory agent, anticancer agent, antiviral agent, or vaccine, such that the anti-PVRIG antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffering agents, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or nontoxic, auxiliary substances, combinations of various components known in the art or more.

[0160] Suitable components can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickening agents, colorings, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiothio sulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, mercapto acetic acid, mercapto sorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl arsenic acid salts. As disclosed herein, an antibody or antigen-binding fragment thereof comprising one or more antioxidants such as methionine in a solvent containing the antibody or antigen-binding fragment thereof of the disclosed composition can be oxidized. Oxidation reduction can prevent or reduce reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Accordingly, in some embodiments, the present application provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present application further provides methods in which an antibody or antigen-binding fragment thereof is mixed with one or more antioxidants such as methionine, such that the antibody or antigen-binding fragment thereof can be prevented from oxidizing, to extend its shelf life and / or increase activity.

[0161] For further explanation, the pharmaceutically acceptable carrier can include, for example, aqueous vehicles, such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer injection, non-aqueous vehicles such as fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial or antifungal agents, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydropropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as poly sorbate 80 (TWEEN-80), sequestering 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 added to a pharmaceutical composition in a multi-dose container comprising phenolics or cresylics, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0162] Administration, formulation, and dosage

[0163] The pharmaceutical compositions of the present application can be administered to a subject in need thereof in vivo by a variety of routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or by implantation or inhalation. The compositions of the present application can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. The appropriate preparation and administration route can be selected depending on the intended application and therapeutic regimen.

[0164] Suitable preparations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups, or inhalers, and controlled release dosage forms thereof.

[0165] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). These liquids can additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulations isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, e.g., water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the particular dosage regimen (including dosage, timing, and repetition) will depend on the particular individual and that individual's medical history, and empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0166] The requirements for effective pharmaceutical carriers for injectable formulations / compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers eds., pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986)).

[0167] The frequency of administration can be determined and adjusted over the course of therapy and is based on reducing the number of proliferating or tumorigenic cells, maintaining such reduction in tumor cells, reducing proliferation of tumor cells, or delaying development of metastases. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the application can be appropriate.

[0168] Those of skill will appreciate that an appropriate dosage can vary from patient to patient. Determining the optimal dosage will generally involve balancing the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route, time and

[0169] Generally, the antibodies or antigen-binding fragments thereof of the application can be administered in a variety of ranges.

[0170] In certain preferred embodiments, the course of treatment involving the antibody or antigen-binding fragment thereof of the present application will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibody or antigen-binding fragment thereof of the present application can 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 is understood that the dosage or interval can be altered based on patient response and clinical practice.

[0171] Compatible formulations for parenteral administration (e.g., intravenous injection) will comprise the antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 pg / mL to about 100 mg / mL.

[0172] The antibody of the present application can be co-administered with one or more other therapeutic agents (e.g., a cytotoxic agent, a radiotoxic agent, an anti-neoplastic agent, an anti-angiogenic agent, or an immunosuppressive agent) to reduce the induction of an immune response against the antibody of the present application. The antibody can be linked to the therapeutic agent (as an immunoconjugate) or can be administered separately from the therapeutic agent.

[0173] In the context of administering a treatment, the term "in combination" or "co-administered" as used herein means the use of more than one treatment or therapeutic agent. The use of the term "in combination" does not restrict the order in which treatments or therapeutic agents are administered to a subject. A treatment or therapeutic agent can be administered prior to, simultaneously with, or following the administration of a second treatment or therapeutic agent to a patient. Preferably, the treatments or therapeutic agents are administered to a subject in an order, amount, and / or at an interval of time such that they can act together. In a particular embodiment, the treatments or therapeutic agents are administered to a subject in an order, amount, and / or at an interval of time such that they provide an increased benefit than if administered otherwise, in particular independently of each other. Preferably, the increased benefit is a synergistic effect.

[0174] Medical uses

[0175] The antibodies, antibody compositions, and methods of the present application have a number of in vitro and in vivo uses, including, for example, detection of PVRIG or enhancement of an immune response. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to a human subject.

[0176] Preferred subjects include mammals, e.g., humans / patients. Mammals in the context of the present application are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals, such as animals in zoos.

[0177] Treatment of disorders associated with PVRIG expression

[0178] In some aspects, the present disclosure provides a method of treating a disorder in a mammal comprising administering to a subject (e.g., a human) in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0179] As described herein, the antibodies of the present disclosure have one or more activities that are therapeutically applicable to killing cells and / or inhibiting cells. In particular, the killing of cells, the proliferation of inhibitory cells, and / or the colony formation of cells can be used to treat or prevent cancer (including cancer metastasis). The proliferation, colony formation, and / or metastasis of inhibitory cells can be used, in particular, to treat or prevent cancer metastasis and metastatic spread of cancer cells.

[0180] In some aspects, the present disclosure provides a method for treating or determining the prognosis of a disease associated with PVRIG expression in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.

[0181] In some aspects, the present disclosure provides a method for treating or determining the prognosis of a disease associated with PVRIG expression in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.

[0182] In some aspects, the present disclosure provides the use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the manufacture of a medicament (or drug) for treating or determining the prognosis of a disease associated with PVRIG expression.

[0183] In one embodiment, the disease associated with PVRIG expression comprises a tumor disease, e.g., cancer.

[0184] The antibody or antigen-binding fragment thereof can be used alone as a monotherapy or can be used in combination with chemotherapy or radiotherapy.

[0185] The antibody or antigen-binding fragment thereof can be used in combination with an anti-cancer agent, a cytotoxic agent, or a chemotherapeutic agent.

[0186] The term "anti-cancer agent" or "anti-proliferative agent" means any agent useful in the treatment of a cell proliferative disorder, such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapies, radiotherapy, anti-metastatic agents, and immunotherapeutic agents. It will be appreciated that in selected embodiments as described above, such anti-cancer agents can comprise conjugates and can be combined with the disclosed site-specific antibodies prior to administration. More particularly, in certain embodiments, a selected anti-cancer agent will be linked to an unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Accordingly, such engineered conjugates are expressly contemplated within the scope of the present application. In other embodiments, the disclosed anti-cancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.

[0187] Diagnosis

[0188] The present application provides in vitro and in vivo methods for detecting, diagnosing or monitoring a proliferative disorder and methods of screening cells from a patient to identify tumor cells, including tumorigenic cells. Such methods include identifying an individual having cancer for treatment or monitoring the progression of cancer, including contacting a patient or a sample obtained from a patient (in vivo or in vitro) with an antibody described herein and detecting the presence or absence or level of binding of the antibody to bound or free target molecules in the sample. In some embodiments, the antibody will comprise a detectable label or reporter molecule as described herein.

[0189] In some aspects, the present disclosure provides a method of diagnosing, detecting or monitoring a disease associated with PVRIG expression, comprising administering to a subject in need thereof an effective amount of the antibody or antigen binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.

[0190] In some aspects, the present disclosure provides a method of diagnosing, detecting or monitoring a disease associated with PVRIG expression in a subject, comprising administering to the subject an effective amount of the antibody or antigen binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.

[0191] In another aspect, the present disclosure provides use of the antibody or antigen binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the manufacture of a medicament (or drug) for diagnosing, detecting or monitoring a disease associated with PVRIG expression.

[0192] The sample can be analyzed by a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluorescent immunoassay, immunoblot assay, Western blot analysis, and flow cytometry assay. Compatible in vivo diagnostic or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, and the like, as known to those skilled in the art.

[0193] The methods described in the present application for detecting or monitoring levels of PVRIG expression or PVRIG-expressing cells in vitro can also be used for non-diagnostic purposes.

[0194] Preferred subjects include mammals, such as a human / patient in need.

[0195] The sample of the subject is blood, excretion (urine or feces), oral or nasal secretion, or alveolar lavage fluid, interstitial fluid, sweat, or an extract thereof from the subject.

[0196] Pharmaceutical packaging and kits

[0197] Also provided are pharmaceutical packs and kits comprising one or more containers containing one or more dosages of an antibody or antigen-binding fragment thereof. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or antigen-binding fragment thereof, with or without one or more other agents. For other embodiments, such unit doses are supplied as single-unit dosage preparations in a ready-to-use form in a vial. In other embodiments, the composition contained in the unit dose can include saline, sucrose or similar agents; buffers such as phosphate, and / or be formulated in a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition can be supplied as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid, such as sterile water or a saline solution. In certain preferred embodiments, the composition comprises one or more agents that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the conjugate composition contained therein is used for treating a selected neoplastic disease condition.

[0198] Such kits will typically comprise a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally one or more anti-cancer agents or other agents in the same or different container(s). The kits can also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy.

[0199] More specifically, the kits may have a single container containing the antibody or antigen-binding fragment of the present disclosure, with or without additional components, or they may have different containers for each desired reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution may be premixed in molar equivalents or in a manner where one component is more than another. Alternatively, the conjugates and any optional anticancer agents in the kit may be stored separately in different containers prior to administration to the patient. The kits may also contain 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.

[0200] 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 dry powders. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container.

[0201] Example

[0202] The invention generally described herein will be more readily understood by referring to the following examples, which are provided by way of illustration and are not intended to limit the invention. Furthermore, unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the raw materials, reagents, and other materials used in the following examples are commercially available products.

[0203] Example 1: Generation of antibodies 1, 6, 7, 16, 27, 52, 55

[0204] Various immunization strategies (DNA immunization, protein immunization, and cell immunization) were used to immunize mice of different species (Balb / c, SJL, and A / J strains). Mouse B cells that were positive for human PVRIG were screened using Beacon, or subclones capable of binding to human and monkey PVRIG were obtained via hybridoma assay. Candidate molecules 1, 6, 7, 16, 27, 52, and 55, which specifically recognize human and monkey PVRIG, were isolated through sequence extraction and sequencing.

[0205] The amino acid sequence of the variable region of antibody 1 was analyzed according to the Kabat sequence determination method, as shown below.

[0206] 1_VH

[0207] QVQLQQSGAELARPGASVKMSCKASGYTFTDYWMQWIKQRPGQGLEWIGAIYPGNDDTRYTQKFKGRATLTADKSSSTVYMQLSNLTSEDSAVYYCATFRYYLDYWGQGTTLTVSS

[0208] 1_VH

[0209] QIILTQSPAIMSASPGEKVTMTCSASSSVSYIHWYQQKSDTSPKRWIYDTSKLVSGVPPRFSGTGSGTSYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTKLEVK

[0210] The amino acid sequences of the variable regions of antibody 6 were analyzed according to the Kabat sequence determination method as shown below.

[0211] 1_VL

[0212] QIILTQSPAIMSASPGEKVTMTCSASSSVSYIHWYQQKSDTSPKRWIYDTSKLVSGVPPRFSGTGSGTSYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTKLEVK

[0213] 1_VL

[0214] QIILTQSPAIMSASPGEKVTMTCSASSSVSYIHWYQQKSDTSPKRWIYDTSKLVSGVPPRFSGTGSGTSYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTKLEVK

[0215] The amino acid sequences of the variable regions of antibody 7 were analyzed according to the Kabat sequence determination method as shown below.

[0216] 7_VH

[0217] QVHLQQSGDDLVKPGASVKLSCKASGYTFTNYLINWIKQRPGQGLEWIGHIAPGSGSFYSNEVFKGKATLTVDTSSSTAYIQLSGLSSEDSAVYFCAREKNWDHFDFWGQGTTLTVSS

[0218] 7_VL

[0219] DIQMTQSPSSVSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS GSGTDFTLTISSLQPEDAATYYCAHWVSSGFPYAMDYWGQGTTVTVSS

[0220] The amino acid sequences of the variable regions of antibody 16 were analyzed according to the Kabat sequence determination method as shown below.

[0221] 16_VH

[0222] QVQLQQPGAELVRPGASVKMSCKASGYTFTSYLMDWVKQRPGQGFEWIGNIDPNSGSTYYNEKFKDKAKLTVDKSSSTAYIQLSSLTSEDSAVYYCAIPYYSEYYGFDVWGTGTTVTVSS

[0223] 16_VL

[0224] DIVMTQSQKFMSTSVGDRVNITCKASQNVRSAVAWYQQKPGQSPKTLIYLASNRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPYTFGGGTKLEIK

[0225] The amino acid sequences of the variable regions of antibody 27 were analyzed according to the Kabat sequence determination method as shown below.

[0226] 27_VH

[0227] QVQLQQPGAELVRPGASVKMSCKASGYTFTSYLMDWVKQRPGQGFEWIGNIDPNSGSTYYNEKFKDKAKLTVDKSSSTAYIQLSSLTSEDSAVYYCAIPYYSEYYGFDVWGTGTTVTVSS

[0228] 27_VL

[0229] DIVMTQSQKFMSTSVGDRVNITCKASQNVRSAVAWYQQKPGQSPKTLIYLASNRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPYTFGGGTKLEIK

[0230] The amino acid sequences of the variable regions of antibody 52 were analyzed according to the Kabat sequence determination method as shown below.

[0231] 52_VH

[0232] QVQLQQPGAELVRPGSSVKLSCKASGYTFTSYWMHWVRQRPIQGLEWIGIIDLFDSKIHYSQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARERGLGRAGAMDYWGQGTSVTVSS

[0233] 52_VL

[0234] DIVLTQSPASLAVSLGQRAIISCKASQSVSFADTSLMHWYQQKPGQQPKVLIYRASNLEAEVPTRFSGSGSGTDFTLNIHPVEEDDAATYYCQQSREYPPTFGGGTKLEIK

[0235] The amino acid sequences of the variable regions of antibody 55 were analyzed according to the Kabat sequence determination method as shown below.

[0236] 55_VH

[0237] QVQLQQPGAELVRPGSSVKLSCKASGYTFTSYWMHWVRQRPIQGLEWIGIIDLFDSKIHYSQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARERGLGRAGAMDYWGQGTSVTVSS

[0238] 55_VL

[0239] DIVLTQSPASLAVSLGQRAIISCKASQSVSFADTSLMHWYQQKPGQQPKVLIYRASNLEAEVPTRFSGSGSGTDFTLNIHPVEEDDAATYYCQQSREYPPTFGGGTKLEIK

[0240] Species Cross-reactivity of Antibodies 1, 6, 7, 16, 27, 52, 55

[0241] The species cross-reactivity of antibodies 1, 6, 7, 16, 27, 52, 55 was evaluated by detecting the binding of the antibodies to CHOK1 overexpressing different species of PVRIG by FACS.

[0242] Following standard operating procedures (SOPs) for cell culture, stable cell lines expressing human, monkey, and mouse PVRIG antigens were cultured and processed, including CHOK1 / Human PVRIG (self-made), CHOK1 / cyno PVRIG (self-made), and CHOK1 / mouse PVRIG (self-made). After digestion, the harvested cells were centrifuged at 300g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed twice with 1×PBS (Gibco, 14190144). 1E+05 cells / well were placed in a 96-well plate, centrifuged, and the supernatant was discarded. The cells were resuspended in 20μg / mL antibody solution (50µL / well), mixed, and incubated at 4°C for 1 hour. After centrifugation at 300g for 5 minutes at 4°C, the supernatant antibody solution was discarded, the cells were washed twice with 1×PBS, and 100µL / well of the corresponding 1μg / mL goat anti-human secondary antibody solution (Jackson, 109-605-088) was added. The cells were incubated at 4°C for 30 minutes. Centrifuge at 300g for 5 minutes at 4℃, discard the supernatant antibody solution, wash the cells twice with 1×PBS, and finally resuspend the cells with PBS before detecting the signal value on a BD flow cytometer.

[0243] The results are as follows Figures 1A-1G As shown in Table 1, numbers 1, 6, 7, 16, 27, 52, and 55 can all identify and combine with the PVRIG of primates and cynomolgus monkeys, but none can identify the PVRIG of rodent mice.

[0244] Table 1

[0245]

[0246] Example 3: Cellular binding activity of antibodies 1, 6, 7, 16, 27, 52, and 55

[0247] The binding activity of 1, 6, 7, 16, 27, 52, and 55 at different concentrations with human PVRIG and cynoPVRIG on the cell surface was evaluated by measuring the EC50.

[0248] The stably transfected cell lines CHOK1 / Human PVRIG, CHOK1 / Cyno PVRIG expressing human PVRIG antigen and monkey PVRIG antigen were cultured and treated as described above in the flow cytometry method. The harvested cells after digestion were centrifuged at 300g for 5 minutes at room temperature, and after discarding the supernatant, washed twice with 1xPBS. 1E+05 / well of cells were placed in a 96-well plate, and after centrifugation and discarding the supernatant, the cells were resuspended with different concentration gradient antibody solutions diluted (300nM, 3-fold dilution, 12 dilution points), 50uL / well, mixed, and incubated at 4°C for 1 hour. After centrifugation at 4°C, 300g for 5 minutes, the supernatant antibody solution was discarded, the cells were washed twice with 1xPBS, 1ug / mL of goat anti-human secondary antibody solution (Jackson, 109-605-088) was added, 100uL / well, and incubated at 4°C for 30 minutes. After centrifugation at 4°C, 300g for 5 minutes, the supernatant antibody solution was discarded, the cells were washed twice with 1xPBS, and finally resuspended with PBS, and the signal value was detected on a BD flow cytometer.

[0249] The results are shown in Figure 2 and Figure 3 1, 6, 7, 16, 27, 52, 55 have strong binding activity to human and monkey PVRIG on the cell surface. It is shown that 1, 6, 7, 16, 27, 52, 55 can effectively bind to the CHOK1 / Human PVRIG and CHOK1 / cyno PVRIG cell surface overexpressing human and monkey PVRIG, and have a certain dose-dependent, and the binding ability of the 7 candidate molecules to human and monkey PVRIG overexpressing cell lines is better than the reference antibodies SRF813 and COM701.

[0250] Example 4 Protein level binding activity of antibodies 1, 6, 7, 16, 27, 52, 55

[0251] The EC50 of 1, 6, 7, 16, 27, 52, 55 binding to Human PVRIG protein (Acro, PVG-H52H4) and Cyno PVRIG (Acro, PVG-C5253) protein at different concentrations was detected by ELISA to evaluate the binding activity.

[0252] Antigen was diluted to 0.5 μg / ml with coating solution, mixed well and added to the designed enzyme-labeled plate, 100 μL / well; after coating, it was placed in a 4°C refrigerator overnight for 16 h; the coating solution was discarded, and the plate was washed once and dried; 150 μL of blocking solution (2% BSA, self-prepared) was added to each well, and it was placed in a 37°C incubator for 1 h; the enzyme-labeled plate was taken out, and the blocking solution was discarded; the concentration of the antibody to be tested in the first well was 10 nM, which was diluted by 3 times to the 10th well, and the remaining 2 wells were added with diluent as blank controls, and the data of the control wells were represented as the average of two wells, 100 μL / well; 37°C incubation for 1 h, the enzyme-labeled plate was taken out, the inner liquid was discarded, and the plate was washed 3 times; 100 μL of diluted enzyme-labeled goat anti-human secondary antibody (Rockland, 609-103-123) was added to each well, and it was incubated in a 37°C incubator for 1 h; the enzyme-labeled plate was taken out, the inner liquid was discarded, and the plate was washed 3 times, then 100 μL of color developing solution (Biyun Tian, P0209) was added after drying, and it was reacted at 25°C for 15 min; 50 μL of 1M HCl was added to terminate the reaction; and the OD450 nm reading was performed by an enzyme-labeled instrument.

[0253] The results are as follows Figure 4 and Figure 5 As shown in Table 1, 1, 6, 7, 16, 27, 52, and 55 have strong binding activity to human and monkey PVRIG proteins, and have a certain dose-dependent property, and the binding ability of the 7 candidate molecules to human and monkey PVRIG proteins is equivalent to that of the reference antibodies SRF813 and COM701.

[0254] Example 5 Protein level blocking activity of antibodies 1, 6, 7, 16, 27, 52, and 55

[0255] The inhibition rate of the candidate antibody molecules was evaluated by a blocking experiment. Specifically, the IC50 of 1, 6, 7, 16, 27, 52, and 55 in inhibiting the binding of PVRL2 ligand to human PVRIG protein was measured by ELISA, and the blocking activity was evaluated.

[0256] Coat Human PVRIG protein (Acro, PVG-H52H4) with coating solution to 1.0 μg / ml, mix well and add to the designed enzyme-labeled plate, 100 μL / well; after coating, put it in the 4℃ refrigerator overnight for 16h; discard the coating solution, wash the plate once, and pat the enzyme-labeled plate dry; add 150 μL blocking solution (2% BSA, self-prepared) to each well, and put it in a 37℃ constant temperature incubator for 1h, then discard the blocking solution; add 50 μL of 4.0 nM PVRL2 ligand (Acro, CD2-H82A3) and diluted antibody (100 nM, 3-fold dilution, 11 dilution points) to the enzyme-labeled plate, and add 50 μL of PVRL2 ligand (Acro, CD2-H82A3) and diluent (4 wells) to the enzyme-labeled plate as blank values, and incubate at 37℃ for 1h; take out the enzyme-labeled plate, discard the inner liquid, and wash the plate 3 times; add 100 μL of 1:5000 diluted enzyme-labeled secondary antibody (Genscript, M00091) to each well, and incubate at 37℃ in a constant temperature incubator for 1h; take out the enzyme-labeled plate, discard the inner liquid, wash the plate 3 times, pat dry, and add 100 μL of color developing solution, and react at 25℃ for 15min; add 50 μL of 1M HCl to stop the reaction, and read OD450nm on the enzyme-labeled instrument.

[0257] The results are as follows Figure 6 As shown in the table, 1, 6, 7, 16, 27, 52, and 55 all have strong ligand blocking activity, and have a certain dose dependence. The ligand blocking activity of the 7 candidate molecules is comparable to that of the reference antibodies SRF813 and COM701.

[0258] Example 6 Affinity detection of antibodies 1, 6, 7, 16, 27, 52, and 55

[0259] The affinity of 1, 6, 7, 16, 27, 52, and 55 was detected by Biacore. The detection temperature of the affinity determination experiment was 25℃, and the buffer system was HBS-EP+(Cytiva, BR-1006-69). The antibody was captured as the stationary phase on a protein A chip (Cytiva, 29-1275-55). The diluted human PVRIG antigen (Acro, PVG-H52H4) was sequentially injected into the chip surface as the binding stage, followed by injection of running buffer as the dissociation stage, and the affinity was detected. The data processing used Biacore 8K analysis software version 3.0.

[0260] The experimental parameters are shown in Table 2 below:

[0261] Table 2: Key experimental parameters

[0262]

[0263] The results are shown in Table 3 below. Antibodies 16, 27 and 52 have higher affinity to PVRIG target protein, with affinity reaching 10 -11 M level, significantly better than the reference antibodies SRF813 and COM701, while the affinity of 1, 6, 7 and 55 can also reach the level comparable to the reference antibodies.

[0264] Table 3: Dissociation constant of binding of 1, 6, 7, 16, 27, 52, 55 to human PVRIG

[0265]

[0266]

[0267] Example 7 Detection of reporter gene activity of antibodies 1, 6, 7, 16, 27, 52, 55

[0268] The PVRL2 / PVRIG reporter gene experiment is a rapid and repeatable cell-based detection method. The reporter gene method consists of two genetically engineered cell lines, one is GS-J2C / PVRIG effector cell line expressing human PVRIG protein and luciferase reporter gene driven by NFAT-RE, and the other is GS-C2 / PVRL2 target cell line expressing human PVRL2 protein and anti-human CD3 ScFv. When the GS-J2C / PVRIG effector cell line is co-cultured with the GS-C2 / PVRL2 target cell line, due to the PVRL2 / PVRIG interaction, the activation of anti-CD3 to the cell TCR pathway is inhibited, thereby inhibiting the expression of NFAT-driven luciferase, and the corresponding fluorescence signal is low; the addition of PVRIG therapeutic antibodies blocks the PVRL2 / PVRIG interaction, thereby restoring the TCR signaling pathway, and the fluorescence signal value increases.

[0269] The specific method is to digest, centrifuge to collect GS-C2 / PVRL2 target cells (Genscript construction), and resuspend the cells using their corresponding complete medium (F-12K (Gibco, 21127-022) + 10% FBS (Gibco, 10091-148)); adjust the target cell density, inoculate in the experimental plate, and transfer the experimental plate to the cell incubator (37°C / 5% CO2) for incubation for about 16 hours; use the experimental buffer (RPMI 1640 (Gibco, 22400-089) + 1.25% FBS (Gibco, 10091-148)) to configure the reference antibody and the antibody to be tested working solution; centrifuge to collect the effector cells, resuspend the cells using the experimental buffer and adjust the cell density; remove the culture medium from the experimental plate incubating the target cells from the cell incubator; sequentially transfer the reference antibody and the antibody to be tested working solution and the GS-J2C / PVRIG effector cell (Genscript construction) suspension to the experimental plate; transfer the experimental plate to the cell incubator (37°C / 5% CO2) for incubation for 6 hours; prepare the luciferase detection reagent working solution (GenScript, L00877C); remove the experimental plate from the cell incubator after completing the incubation, transfer the luciferase detection reagent working solution to the corresponding wells of the experimental plate, and incubate at room temperature for 3-5 minutes; use the PHERA Star microplate reader to read the chemiluminescence value, and record the data.

[0270] The results are as follows Figure 7 As shown, the reporter gene activity of the reference antibody SRF813 is weaker than COM701 (EC50: 0.142ug / ml vs 0.087ug / ml, Span: 168139 vs 238134). Compared with COM701, the reporter gene activity of 16, 27 and 52 is significantly better than the reference antibody COM701, and the reporter gene activity of the antibody No. 52 is about 3 times that of COM701. The reporter gene activity of 1, 6, 7 and 55 is between 0.9-1.1ug / ml, and can also reach the level comparable to the reference antibody.

[0271] Example 8 Detection of CD8+ T cell activation level of antibodies 1, 6, 7, 16, 27, 52, 55

[0272] In order to further detect the in vitro CD8 T cell activation level, antigen-specific CD8+ T cell activation experiments were performed on 1, 6, 7, 16, 27, 52, 55.

[0273] PBMCs (Mybiosource, PB100C-W) were resuscitated using RPMI 1640 complete medium (RPMI 1640 + 10% FBS) and induced for 12 days according to the established induction protocol. After 12 days, PBMCs were collected by centrifugation and primary CD8+ T cells were sorted. The levels of PVRIG, TIGIT, pp65, CD226 in CD8+ T cells were analyzed by flow cytometry. Colo205 target cells (ATCC, CCL-222) were collected by digestion and centrifugation and resuspended in experimental buffer (RPMI 1640 + 10% FBS). A certain amount of Colo205 target cells (ATCC, CCL-222) were pre-incubated with high concentration of pp65 (GenScript, RP11781) working solution (4x) in a cell incubator (37°C / 5% CO2) for 0.5-2 hours; after pre-mixing, pp65 was removed by centrifugation; the density of Colo205 target cells (ATCC, CCL-222) was adjusted and the cell suspension was transferred to a 96-well plate. The control / test working solution (4x) was prepared using RPMI 1640 complete medium. The control / test working solution was transferred to the corresponding wells of the 96-well experimental plate. According to the effector-to-target ratio (1:1) set in the protocol, the density of CD8+ T cells was adjusted and the cell suspension was transferred to the corresponding wells of the 96-well experimental plate. The experimental plate was incubated in a cell incubator (37°C / 5% CO2) for about 16-18 hours. After incubation, the supernatant was collected by centrifugation for human hINFy cytokine detection (Cisbio, 62HIFNGPEH), and the cells were treated by digestion, washed with FACS buffer, and stained with 7-AAD (Biolegend, 420403). After staining, the supernatant was discarded by centrifugation, the cells were washed and resuspended using FACS buffer. The cells were collected using a flow cytometer and the fluorescence signal values were read. The raw data of the cytokine release experiment were exported via the PHERA Star FSX system and analyzed using Microsoft Office Excel 2016 and GraphPad Prism 6 software. The experimental raw data of the cell flow cytometry analysis were exported via the BD FACSDiva Software v8.0.1.1 system and analyzed using FlowJo 7.6.1 and Microsoft Office Excel 2016 software, and the results were used to evaluate the level of target cell killing (percentage of 7-AAD positive cells).

[0274] Results are shown in Figures Figures 8A-8C and 8D- Figure 8FAs shown, the cytokine-induced secretion ability and cell killing activity of the reference antibody COM701 were weak. Compared with COM701, 6, 55, 27 and 52 had stronger ability to induce human hINFy cytokine secretion, among which antibodies 27 and 52 were optimal, significantly stronger than the reference antibody. 52 was significantly superior to other antibody molecules including the reference molecule in CD8+T killing activity detection, and had obvious cell killing activity.

[0275] Example 9 Epitope classification of antibodies 1, 6, 7, 16, 27, 52, 55

[0276] To further clarify the epitope of 1, 6, 7, 16, 27, 52, 55 binding to PVRIG, the reference antibodies SRF813 and COM701 known to bind to different regions of PVRIG were used to clarify their antigen binding regions by competitive ELSA method.

[0277] Epitope competition ELISA detection: Human PVRIG protein (Acro, PVG-H52H4) was diluted to 0.5 pg / ml with coating solution, mixed and added to the designed enzyme-labeled plate, 100 pL / well; after coating, it was placed in a 4°C refrigerator overnight for 16 h; the coating solution was discarded, and the plate was washed once and dried; 150 pL of blocking solution (2% BSA, self-prepared) was added to each well, and it was placed in a 37°C incubator for 1 h, and then the blocking solution was discarded; biotin-labeled antibodies were used as competitors (self-made) and diluted antibody samples (see Table 4) were added to the enzyme-labeled plate, 50 pL each, and 50 pL of competitors and diluents (4 wells) were added to the enzyme-labeled plate as blank values, and incubated at 37°C for 1 h. The enzyme-labeled plate was removed, the inner liquid was discarded, and the plate was washed 3 times; 100 pL of diluted enzyme-labeled secondary antibody (Genscript, M00091) was added to each well, and incubated at 37°C for 1 h; the enzyme-labeled plate was removed, the inner liquid was discarded, and the plate was washed 3 times, then 100 pL of color developing solution was added, and reacted at 25°C for 15 min; 50 pL of 1M HCl was added to terminate the reaction, and the OD450 nm reading was taken on the enzyme-labeled instrument. The results are shown in Table 4.

[0278] Table 4: Epitope classification of antibodies 1, 6, 7, 16, 27, 52, 55

[0279]

[0280] As shown in the above table, the binding epitopes of COM701 and SRF8131 are different; the binding epitopes of 16, 27 and 52 completely overlap, and the binding epitopes of 3 of them partially overlap with the binding epitopes of COM701 and SRF8131; the binding epitope of 7 partially overlaps with the binding epitope of COM701; the binding epitopes of 1, 6 and 55 completely overlap, and the binding epitopes of 3 of them do not overlap with the binding epitopes of COM701 and SRF8131. The specific results are as follows:Figure 9 Results are shown in Table 5.

[0281] Example 10 In vivo efficacy of antibodies 1, 6, 7, 16, 27, 52, 55

[0282] The efficacy of the test drugs was evaluated in an animal model of subcutaneous inoculation of human melanoma A375 cells (ATCC, CRL-1619) in PBMC-reconstituted B-NDG mice (Bannosetou).

[0283] A 0.1 mL cell suspension containing 5 x 10 7 A375 cells was subcutaneously inoculated on the right scapula of each B-NDG mouse. After inoculation of tumor cells, the next day, PBMC (Miaoshun Biotechnology, PB100C-W) were inoculated at 2.5 x 10 7 / mL, 0.2 mL was inoculated into the tail vein of each mouse. When the tumor volume of the mice reached 50-100 mm 3 , the mice were randomly divided according to the tumor volume. The CV value of the tumor volume of the enrolled mice was ≤30%. Each group had 8 mice, and the dose was 10 mg / kg PD-1 + 30 mg / kg of the test antibody, administered twice a week for 3 weeks. If the body weight decreased by more than 15%, the administration should be stopped immediately, and the normal administration should be resumed after the body weight recovered to more than 98% of the initial body weight. If the body weight decreased by more than 20% and did not recover within 72 h, the mice were euthanized.

[0284] Results are shown in Table 5. Figure 10 As shown in Table 5, compared with Vehicle (PBS administration group), PD1 + 6, 7, 10, 16, 27, 52 combination groups showed certain tumor inhibition effect (TGI: 16.93%, 17.03%, 10.5%, 18.71%, 25.01%, 16.06%). Antibody No. 27 had the best anti-tumor effect. The specific results are shown in Table 5.

[0285] Table 5

[0286]

[0287] Example 11 Hot spot removal of antibody 52

[0288] After sequence analysis, there is an aspartic acid isomerization site in the CDRH2 of 52, and there is a high risk of PTM, so it is removed and modified to avoid the influence of aspartic acid isomerization on the structure and function of the antibody. The hot spot removal modification variants are shown in Table 6. The four variants are 52_vH(DG / EG-DS / ES), 52_vH(DG / EG-DS / DA), 52_vH(DG / DA-DS / ES), and 52_vH(DG / DA-DS / DA).

[0289] Table 6

[0290]

[0291]

[0292] The hot spot removal variants were expressed in ExpiCHO-S cells (Thermo) by transient transfection. 24 hours before transfection, the ExpiCHO-S cells were normally cultured in expression medium at a passage density of 3.8 x 10 6 cells / mL, and then cultured in a shaker at 37°C, 8% CO2; on the transfection day, the viable cell density and cell viability were counted. Then the cells were diluted to the required density for transfection. Lipofectin method was used for transfection, and after transfection, the shake flask was placed back in the shaker at 37°C, 8% CO2 for continuous culture; 18-22 hours after transfection, the cells were supplemented with feed and additives (Gibco, A29129), and 7-10 days after transfection, the expression was confirmed by SDS-PAGE, the supernatant was collected by centrifugation, and the antibody was purified.

[0293] The EC50 of the hot spot removal variants at different concentrations was detected by ELISA to measure the binding of Human PVRIG and Cyno PVRIG protein, and the binding activity was evaluated. The detection process is described in Example 4. The property identification results of the hot spot removal variants are shown in Figure 11 and Figure 12 The target protein binding activity of the four variants is equivalent to that of the original sequence of 52. It can be seen that the four hot spot removal variants have little effect on the target protein binding activity of the original sequence.

[0294] The inhibition rate of the candidate antibody molecule was evaluated by blocking experiment. The specific method is to detect the IC50 of the hot spot removal variants at different concentrations by ELISA to measure the inhibition of PVRL2 ligand binding to Human PVRIG protein, and to evaluate the blocking activity. The detection process is described in Example 5. The property identification results of the hot spot removal variants are shown in Figure 13As shown in Table 6, the ligand receptor blocking activity of 52_vH(DG / EG-DS / ES), 52_vH(DG / EG-DS / DA) and 52_vH(DG / DA-DS / DA) variants were comparable to 52 original sequence. The blocking activity of 52_vH(DG / DA-DS / ES) was slightly weaker than 52 original sequence.

[0295] The EC50 of hotspot removal variants binding to cell surface human PVRIG and cyno PVRIG at different concentrations were measured by FACS to evaluate their binding activity. The detection process was referred to Example 3. The property identification results of hotspot removal variants were shown in Table 7. The FACS binding activity of 52_vH(DG / DA-DS / ES) and 52_vH(DG / DA-DS / DA) variants to human and monkey cells were comparable to 52 original sequence. Figure 14 、 Figure 15 The FACS binding activity of 52_vH(DG / EG-DS / ES) and 52_vH(DG / EG-DS / DA) variants to human and monkey cells were weaker than 52 original sequence.

[0296] The affinity of hotspot removal variants were detected by Biacore. The specific detection method was referred to Example 6. The property identification results of hotspot removal variants were shown in Table 7. The affinity of 52_vH(DG / DA-DS / ES) and 52_vH(DG / DA-DS / DA) variants were comparable to 52 original sequence.

[0297] Table 7

[0298]

[0299] The reporter gene activity of hotspot removal variants were detected by reporter gene experiment. The specific detection method was referred to Example 7. The property identification results of hotspot removal variants were shown in Table 7. The reporter gene activity of 52_vH(DG / DA-DS / ES) and 52_vH(DG / DA-DS / DA) variants were comparable to 52 original sequence. Figure 16

[0300] In summary, the influence of 52_vH(DG / DA-DS / ES) and 52_vH(DG / DA-DS / DA) variants on activity was the weakest, and their ELISA binding activity, blocking activity, FACS binding activity, affinity and reporter gene activity were comparable to 52 original sequence.

[0301] By reference

[0302] ​The entire contents of each of the patent and scientific literature documents mentioned herein are incorporated by reference for all purposes.

[0303] equivalents

[0304] The application can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the above embodiments are merely illustrative, and not restrictive. The scope of the application should be determined by the appended claims and their equivalents, rather than by the description presented above. All changes coming within the meaning and equivalency range of the claims are intended to be embraced therein.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to PVRIG, and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region and the light chain variable region comprise: a HCDR1 as set forth in SEQ ID NO: 35, a HCDR2 as set forth in SEQ ID NO: 41, and a HCDR3 as set forth in SEQ ID NO: 48, a LCDR1 as set forth in SEQ ID NO: 57, a LCDR2 as set forth in SEQ ID NO: 63, and a LCDR3 as set forth in SEQ ID NO:

68.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region is as set forth in SEQ ID NO: 18; wherein the light chain variable region is as set forth in SEQ ID NO:

29.

3. The antibody or antigen-binding fragment thereof of claim 2, further comprising a heavy chain constant region as set forth in SEQ ID NO: 15; and a light chain constant region as set forth in SEQ ID NO:

16.

4. The antibody or antigen-binding fragment thereof of claim 3, comprising: a light chain as set forth in SEQ ID NO: 4, and a heavy chain as set forth in SEQ ID NO:

3.

5. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a whole antibody; wherein the fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, or a single chain Fv fragment (scFv).

6. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody is a chimeric antibody.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody is a monoclonal antibody.

8. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-7.

9. A vector comprising the nucleic acid molecule of claim 8.

10. A host cell comprising the nucleic acid molecule of claim 8 or the vector of claim 9.

11. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7 coupled to at least one detectable label.

12. An antibody drug conjugate comprising an antibody comprising one or more drug moieties covalently attached to the antibody or antigen-binding fragment thereof of any one of claims 1-7, directly or via a linker.

13. A multispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7.

14. A pharmaceutical composition or kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7, or the nucleic acid molecule of claim 8, or the vector of claim 9, or the host cell of claim 10, or the conjugate of claim 11, or the antibody drug conjugate of claim 12, or the multispecific molecule of claim 13, and a pharmaceutically acceptable carrier.

15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7, or the nucleic acid molecule of claim 8, or the vector of claim 9, or the host cell of claim 10, or the conjugate of claim 11, or the antibody drug conjugate of claim 12, or the multispecific molecule of claim 13, or the pharmaceutical composition or kit of claim 14, for the manufacture of a kit for diagnosing, detecting or monitoring a disease associated with PVRIG expression, wherein the disease associated with PVRIG expression is a cancer selected from melanoma.

16. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7, or the nucleic acid molecule of claim 8, or the vector of claim 9, or the host cell of claim 10, or the conjugate of claim 11, or the antibody drug conjugate of claim 12, or the multispecific molecule of claim 13, or the pharmaceutical composition or kit of claim 14, for the manufacture of a medicament for treating or determining the prognosis of a disease associated with PVRIG expression, wherein the disease associated with PVRIG expression is a cancer selected from melanoma.

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