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] The application in this case was filed on December 22, 2023; the application number is 202311790630.9; the invention title is: A divisional application of a patent application for an invention relating to anti-PVRIG antibodies and their uses. Technical Field

[0002] This disclosure relates to the field of antibodies, and more specifically to an antibody against PVRIG. Background Technology

[0003] Tumor immunotherapy is one of the main pillars of oncology today, especially for treating unresectable, metastatic, and recurrent cancers. The success of immune checkpoint inhibitors (ICIs), and their combination with chemotherapy, immunotherapy, and targeted drugs, has transformed cancer treatment paradigms. However, PD-1 therapy still only yields long-term, durable responses in 10-30% of patients, with most patients lacking response. Acquired resistance and immune-related adverse events (IRAEs) also pose significant obstacles. One mechanism to overcome the limitations of PD-1 therapy is the development of other ICIs related to the tumor microenvironment, which, when used in combination with anti-PD-1 / PD-L1, may help overcome the limitations of previous treatments. Studies have shown that elevated PVRL2 (CD112) levels in cancer cells are a potentially key biomarker for disease treatment in patients resistant to targeted PD-1 immunotherapy, and it is overexpressed in multiple tumor types, including breast and ovarian cancer. PVRL2 is currently identified as the only functional ligand for PVRIG, which binds to PVRIG with high affinity.

[0004] PVRIG, also known as CD112R, was identified as a novel inhibitory receptor in 2016. It is expressed in natural killer (NK) cells and T lymphocytes. PVRIG binds to its ligand PVRL2, mediating intracellular signal transduction, leading to reduced T cell cytotoxicity. In addition, it can inhibit TCR-mediated NFAT activation, further suppressing T cell activation. It also competes with DNAM-1 for the PVRL2 ligand, blocking the stimulation signal it transmits. Thus, it achieves a dual negative regulation of T cell function to exert an immunosuppressive effect.

[0005] Preclinical studies have shown that blocking PVRIG can inhibit tumor growth in both mice and human solid tumors, and combining it with PD-1 / TIGIT significantly improves treatment efficacy. 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. Based on existing clinical data, both monotherapy and combination therapy with anti-PD-1 antibodies have shown promising therapeutic effects in advanced solid tumors, especially in patients who have previously received immune checkpoint inhibitor therapy, where a best response rate (≥SD) of 72% was still achieved. Regarding safety, no drug-induced tumor thrombosis (DLT) was observed with monotherapy or in combination with Opdivo, and no increased toxicity was observed with combination therapy with PD-1 monoclonal antibodies. No subjects discontinued study treatment due to toxicity of any investigational drug. Based on existing clinical data, the druggability of this target has been preliminarily demonstrated, exhibiting promising efficacy.

[0006] PVRIG, a novel immune checkpoint inhibitor of NK cells and T cells, has a well-defined target mechanism. The antibody can relieve immunosuppression of NK cells and CD8+ T cells, and also mediate activation signals of NK cells and CD8+ T cells, enhancing tumor killing. Currently, no drugs targeting this indication are available globally. COM701, the fastest-developing drug for oncology, has just entered Phase II clinical trials. Preclinical in vivo efficacy studies have yielded significant results, and clinical data show good safety and positive efficacy when used in combination with PD-1 monoclonal antibodies. Summary of the Invention

[0007] To address the aforementioned problems, this disclosure provides antibodies, methods for their preparation, compositions, etc. The benefits provided by this disclosure are broadly applicable to the fields of antibody therapy and diagnostics, and can be used in combination with antibodies that respond to various targets.

[0008] This invention discloses an isolated antibody or its antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment 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 includes:

[0010] (i)HCDR1, comprising a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 35-39 or consisting of SEQ ID NO: 35-39;

[0011] (ii) HCDR2, comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 40-45; and

[0012] (iii)HCDR3, comprising a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 48-53, or consisting of SEQ ID NO: 48-53;

[0013] The light chain variable region includes:

[0014] (i) LCDR1, comprising a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 56-62, or consisting of SEQ ID NO: 56-62;

[0015] (ii) LCDR2, comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 63-67; and

[0016] (iii) LCDR3, which contains a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 68-73 or is composed of SEQ ID NO: 68-73.

[0017] In some embodiments of the present invention, the antibody or fragment includes:

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

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

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

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

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

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

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

[0025] In some embodiments of the present invention, the antibody or fragment wherein the heavy chain variable region comprises 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 with one of SEQ ID NO: 17-23, or is composed of one of SEQ ID NO: 17-23.

[0026] In some embodiments of the present invention, the antibody or fragment wherein the light chain variable region comprises 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 with one of SEQ ID NO: 28-34, or is composed of one of SEQ ID NO: 28-34.

[0027] In some embodiments of the present invention, the antibody or fragment further comprises a heavy chain constant region and a light chain constant region. The heavy chain constant region comprises 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 with SEQ ID NO: 15, or is composed of SEQ ID NO: 15. The light chain constant region comprises 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 with SEQ ID NO: 16, or is composed of SEQ ID NO: 16.

[0028] In some embodiments of the present invention, the antibody or fragment includes:

[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 with one of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or consisting of one of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14;

[0030] The heavy chain comprises 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 with one of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13, or is composed of one of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13.

[0031] In some embodiments of the present invention, the antibody or fragment includes:

[0032] (i) the light chain shown in SEQ ID NO: 2 and the heavy chain shown in SEQ ID NO: 1; or

[0033] (ii) the light chain shown in SEQ ID NO: 4 and the heavy chain shown in SEQ ID NO: 3; or

[0034] (iii) the light chain shown in SEQ ID NO: 6 and the heavy chain shown in SEQ ID NO: 5; or

[0035] (iv) the light chain shown in SEQ ID NO: 8 and the heavy chain shown in SEQ ID NO: 7; or

[0036] (v) the light chain shown in SEQ ID NO: 10 and the heavy chain shown in SEQ ID NO: 9; or (vi) the light chain shown in SEQ ID NO: 12 and the heavy chain shown in SEQ ID NO: 11; or (vii) the light chain shown in SEQ ID NO: 14 and the heavy chain shown in SEQ ID NO: 13.

[0037] In some embodiments of the present invention, the antibody or fragment is modified by hotspot removal to avoid the impact of aspartic acid isomerization on the antibody structure and function; wherein, preferably,

[0038] The heavy chain variable region includes HCDR2 as shown in SEQ ID NO: 46 or 47, and / or,

[0039] HCDR3 as shown in SEQ ID NO: 54 or 55.

[0040] In some embodiments of the present invention, the antibody or fragment wherein the heavy chain variable region comprises 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 with one of SEQ ID NO: 24-27, or is composed of one of SEQ ID NO: 24-27.

[0041] In some embodiments of the present invention, the antibody or fragment 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] The fragments referred to therein are Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, or single-stranded Fv fragments (scFv).

[0043] On the other hand, the present invention also provides an isolated nucleic acid molecule containing a nucleic acid sequence encoding the antibody or fragment described above.

[0044] On the other hand, the present invention also provides a carrier containing the aforementioned nucleic acid molecules.

[0045] On the other hand, the present invention also provides a host cell containing the aforementioned nucleic acid molecule or the aforementioned vector.

[0046] On the other hand, the present invention also provides a conjugate comprising the antibody or fragment conjugated to at least one detectable marker.

[0047] On the other hand, the present invention also provides an antibody-drug conjugate comprising an antibody, including one or more drug portions which are directly or covalently linked to the antibody or fragment via a linker.

[0048] On the other hand, the present invention also provides a multispecific molecule comprising the antibody or antigen-binding fragment described above; preferably, the multispecific molecule specifically binds to PVRIG and additionally specifically binds to one or more other targets; more preferably, the multispecific molecule further comprises at least one molecule having a second binding specificity against a second target.

[0049] On the other hand, the present invention also provides a pharmaceutical composition or kit comprising the antibody or fragment, or the nucleic acid molecule, or the carrier, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule, and a pharmaceutically acceptable carrier.

[0050] On the other hand, the present invention also provides the 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 preparation of a kit for diagnosing, detecting or monitoring diseases related to PVRIG expression.

[0051] On the other hand, the present invention also provides the 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 preparation of a medicament for treating diseases associated with PVRIG expression or determining their prognosis.

[0052] On the other hand, the diseases associated with PVRIG expression mentioned above are cancers selected from the following group: squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous 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, gastric cancer, melanoma or various types of head and neck cancer (including squamous cell carcinoma of the head and neck), hematologic malignancies, or any other disease or condition characterized by uncontrolled cell growth.

[0053] The PVRIG antibody provided by this invention exhibits high binding affinity to both human and cynomolgus monkey PVRIG proteins; and demonstrates potent activity at both the reporter gene level and CD8T cell activation. Compared to known SRF813 and COM701, the antibody of this invention comprehensively exhibits superior in vitro activity, and some antibodies, when combined with PD-1 antibodies, show significant antitumor effects, making them excellent candidate drug molecules for therapeutic and diagnostic applications. Attached Figure Description

[0054] The accompanying drawings are provided to further understand the present disclosure and form part of the specification. They are used together with the embodiments of the present disclosure to explain the present disclosure and do not constitute a limitation thereof.

[0055] Figure 1A The results were obtained by FACS detection of the binding of antibody 1 to CHOK1 cells overexpressing different species of PVRIG. 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 that does not express PVRIG antigen, and MFI represents the mean fluorescence intensity.

[0056] Figure 1B The results were obtained by FACS detection of the binding of antibody 6 to CHOK1 cells overexpressing different species of PVRIG. 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 that does not express PVRIG antigen, and MFI represents the mean fluorescence intensity.

[0057] Figure 1C The results were obtained by FACS detection of the binding of antibody 7 to CHOK1 cells overexpressing different species of PVRIG. 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 that does not express PVRIG antigen, and MFI represents the mean fluorescence intensity.

[0058] Figure 1DThe results were obtained by FACS detection of the binding of antibody 16 to CHOK1 cells overexpressing different species of PVRIG. 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 that does not express PVRIG antigen, and MFI represents the mean fluorescence intensity.

[0059] Figure 1E The results were obtained by FACS detection of the binding of antibody 27 to CHOK1 cells overexpressing different species of PVRIG. 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 that does not express PVRIG antigen, and MFI represents the mean fluorescence intensity.

[0060] Figure 1F The results were obtained by FACS detection of the binding of antibody 52 to CHOK1 cells overexpressing different species of PVRIG. 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 that does not express PVRIG antigen, and MFI represents the mean fluorescence intensity.

[0061] Figure 1G The results were obtained by FACS detection of the binding of antibody 55 to CHOK1 cells overexpressing different species of PVRIG. 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 that does not express PVRIG antigen, and MFI represents the mean fluorescence intensity.

[0062] Figure 2The graphs show the CHOK1 / Human PVRIG cell binding curves of antibodies 1, 6, 7, 16, 27, 52, and 55 with reference antibodies SRF813 and COM701; where Conc. on the x-axis represents antibody concentration in nM, Geom.Mean on the y-axis represents average fluorescence intensity, and Human lgG1 represents human lgG1.

[0063] Figure 3 The graphs show the CHOK1 / Cyno PVRIG cell binding curves of antibodies 1, 6, 7, 16, 27, 52, and 55 with reference antibodies SRF813 and COM701. The x-axis represents the antibody concentration in nM, the y-axis represents the average fluorescence intensity, and Human lgG1 represents human lgG1.

[0064] Figure 4 The graphs show the binding curves of antibodies 1, 6, 7, 16, 27, 52, and 55 to reference antibodies SRF813 and COM701 for Human PVRIG protein. The horizontal axis Conc. represents the antibody concentration in nM, and the vertical axis represents the microplate reader reading at OD450nm. Human lgG1 represents human lgG1.

[0065] Figure 5 The graphs show the binding curves of antibodies 1, 6, 7, 16, 27, 52, and 55 to the reference antibodies SRF813 and COM701 for Cyno PVRIG protein (monkey PVRIG protein). The horizontal axis Conc. represents the antibody concentration in nM, the vertical axis represents the microplate reader reading at OD450nm, and Human lgG1 represents human lgG1.

[0066] Figure 6 The results show the protein level blocking activities of antibodies 1, 6, 7, 16, 27, 52, and 55 against reference antibodies SRF813 and COM701. The horizontal axis Conc. represents the antibody concentration in nM, the vertical axis represents the microplate reader reading at OD450 nm, and Human lgG1 represents human lgG1.

[0067] Figure 7 The graphs show the reporter gene activity detection curves of antibodies 1, 6, 7, 16, 27, 52, and 55 with reference antibodies SRF813 and COM701. The horizontal axis Conc. represents the antibody concentration in μg / ml, and the vertical axis Luminescence (RLU) represents the relative luminescence unit. Human lgG1 represents human lgG1.

[0068] Figure 8A , Figure 8B , Figure 8CThis is the result of the detection of cytokine hIFNγ (human interferon γ). The horizontal axis represents the antibody concentration in μg / ml, and the vertical axis represents the hIFNγ concentration in pg / ml. Human lgG4 refers to human lgG4. The CD8+T+Pretreated CTV-Colo205 at the position of the dashed line in the figure refers to the cytokine hIFNγ detection value of CTV-Colo205 cells treated with 5 μg / ml pp65 and incubated with CD8+T cells.

[0069] Figure 8D , Figure 8E , Figure 8F This is the result of the level of target cell killing. The horizontal axis represents the antibody concentration in μg / ml, and the vertical axis represents the target cell killing ability. Human lgG4 refers to human lgG4. The CD8+T+Pretreated CTV-Colo205 at the dotted line in the figure refers to the hIFNγ value of cytokine after incubation of CTV-Colo205 cells treated with 5 μg / ml pp65 and CD8+T cells.

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

[0071] Figure 10 This is the in vivo pharmacodynamic activity assay result of antibodies 1, 6, 7, 16, 27, 52, and 55 in combination with PD-1 antibody. The horizontal axis, Days Post Treatment, represents the number of days after treatment, in days. The vertical axis, Tumor Volume (mm) 3 This refers to the tumor volume, measured in cubic millimeters (mm). 3 Vehicle represents 10mM phosphate-buffered saline (PBS), ip represents intraperitoneal administration, BIW represents twice a week, and BIW*3 represents twice a week for three consecutive weeks.

[0072] Figure 11 The graphs show the binding curves of antibody 52 and its variants 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) to the Human PVRIG protein. The horizontal axis, Conc., represents the antibody concentration in nM, and the vertical axis represents the reading of the microplate reader at OD450 nm.

[0073] Figure 12The graphs show the binding curves of antibodies 52, 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) to Cyno PVRIG protein (monkey PVRIG protein). The horizontal axis, Conc., represents the antibody concentration in nM, and the vertical axis represents the reading of the microplate reader at OD450 nm.

[0074] Figure 13 The results show the protein-level blocking activity of antibody 52 and its variants 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). The horizontal axis, Conc., represents the antibody concentration in nM, and the vertical axis represents the ELISA reader reading at OD450 nm.

[0075] Figure 14 The graph shows the CHOK1 / Human PVRIG cell binding curves for antibody 52 and its variants 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); where the x-axis Conc. represents antibody concentration in nM, and the y-axis Geom.Mean represents average fluorescence intensity.

[0076] Figure 15 The graphs show CHOK1 / Cyno PVRIG cell binding curves for antibody 52 and its variants 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). The x-axis, Conc., represents the antibody concentration in nM, and the y-axis, Geom.Mean, represents the average fluorescence intensity.

[0077] Figure 16 The graphs show the reporter gene activity detection curves for antibody 52 and its variants 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). The horizontal axis, Conc., represents the antibody concentration in μg / ml, and the vertical axis, Luminescence (RLU), represents the relative luminescence unit. Detailed Implementation

[0078] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are merely illustrative examples of some implementations of the present invention, and not all embodiments. Therefore, the present invention is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are not to be construed as limiting the described subject matter.

[0079] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention will have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise clearly indicates, the singular forms “a,” “an,” and “the” include plural indicators. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not limiting. Moreover, the scope provided in the specification and the appended claims includes all values ​​between endpoints.

[0080] definition

[0081] To better understand this invention, the definitions and explanations of relevant terms are provided below.

[0082] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified as κ or λ light chains. The heavy chains can be classified as μ, δ, γ, α, or ε, which define the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity-determining regions, or CDRs) separated by relatively conserved regions (called framework regions, or FRs). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The CDRs on VH are HCDR1, HCDR2, and HCDR3; the CDRs on VL are LCDR1, LCDR2, and LCDR3. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites / parts, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT, or Chothia. In the specific embodiments of this disclosure, the CDR sequence is determined using the numbering definition in the Kabat system.

[0083] In this disclosure, antibodies also include antigen-binding moieties (used interchangeably with the term "antigen-binding fragment"). An antigen-binding moiety is a polypeptide containing a fragment of a complete antibody that retains the ability to specifically bind to an antigen that binds specifically to a full-length or complete antibody, and / or competes with a full-length antibody for binding to the same antigen. Under certain conditions, antigen-binding moieties include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and antibodies containing at least a portion sufficient to confer specific antigen-binding ability to the polypeptide. The antigen-binding moieties of antibodies can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and specificity can be screened in the same manner as for complete antibodies.

[0084] The term "isotype" refers to an antibody class (e.g., IgM or IgG1) encoded by a gene in the heavy chain constant region.

[0085] The term "monoclonal antibody" or "mAb" refers to an antibody molecule / formulation consisting of a single molecule. Monoclonal antibodies exhibit single binding specificity and affinity for a specific epitope. The antibodies of this invention can be derived from various species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.

[0086] The term "epitope" refers to an antigenic determinant in a molecule, specifically a portion of the molecule that is recognized by the immune system (e.g., by antibodies), such as a discontinuous three-dimensional site on an antigen recognized by the immune system. In this invention, the epitope shown is, for example, a PVRIG protein.

[0087] As used herein, the term "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, such as an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody.

[0088] The term "humanized antibody" refers to an antibody in which a CDR sequence / antigen-binding portion or site derived from another mammalian species, such as a mouse, has been transplanted onto a human frame sequence. Furthermore, additional frame region modifications can be performed within the human frame sequence.

[0089] The term "KD value" refers to the equilibrium dissociation constant between an antibody and its antigen, specifically the ratio of koff / kon or kd / ka (which can be measured using SPR technology). Therefore, a lower KD value (lower concentration) indicates higher antibody affinity. Thus, the "KD value" can be used to measure the binding affinity between an antibody and its antigen. The kd dissociation constant reflects the affinity of a compound for its target; a smaller value indicates stronger affinity. The ka association constant, conversely, is the opposite of kd; a larger value indicates stronger affinity.

[0090] The terms “PVRIG” and “PVRIG antigen” are used interchangeably herein and include any variant, isotype, and species homologue of human PVRIG expressed naturally in cells or on cells transfected with the PVRIG gene. In some embodiments, the binding of the antibody of this disclosure to the PVRIG antigen mediates the killing of PVRIG-expressing cells (e.g., tumor cells) by inactivating PVRIG. The killing of PVRIG-expressing cells can occur through one or more of the following mechanisms: cell death / apoptosis induction, ADCC, and CDC.

[0091] The term "anti-PVRIG antibody" or "PVRIG antibody" refers to an antibody that, as defined herein, is capable of binding to PVRIG antigens or to cells expressing PVRIG. Based on their binding characteristics to PVRIG antigens and their biological activity, two types of anti-PVRIG antibodies (type I and type II anti-PVRIG antibodies) can be distinguished according to Cragg, MS et al., Blood 103 (2004) 2738-2743; and Cragg, MS et al., Blood 101 (2003) 1045-1052.

[0092] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.

[0093] The term "separated" refers to a state obtained artificially from the natural state. If a "separated" substance or component exists naturally, it may be due to changes in its natural environment, separation of the substance from its natural environment, or both. For example, an unseparated polynucleotide or polypeptide naturally exists within a living organism; a high-purity copy of the same polynucleotide or polypeptide separated from that natural state is called a separated polynucleotide or polypeptide. The term "separated" does not exclude the presence of artificial or synthetic substances, nor does it exclude other impurities that do not affect the activity of the separated substance. For example, a separated antibody may be substantially free of other cellular material and / or chemicals.

[0094] The term "vector" refers to a nucleic acid medium in which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. This vector can be used to express the carried genetic material elements in host cells through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (such as SV40). Vectors may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication. For the vector expressing the antibody, a vector type in which the antibody heavy chain and light chain exist in different vectors or a vector type in which the heavy chain and light chain exist in the same vector can be used.

[0095] The term "host cell" refers to a cellular system that can be engineered to produce a target protein, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, CHOK1, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term covers not only the specific test cell but also its progeny. Because certain modifications can occur in subsequent generations due to mutations or environmental influences, such progeny may differ from 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 (or protein) molecules or two or more nucleic acid molecules, determined by alignment and comparison of sequences. "Percentage identity" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules, calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably addressed using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in Computational Molecular Biology (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, 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 of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately produce immune effector substances 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 organism's immune system after stimulation with an antigen. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the host's reactivity, and the immunization method.

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

[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 hybridoma.

[0100] The term "immune effector function" includes any function mediated by components of the immune system that results in the inhibition of tumor growth and / or tumorigenesis, as well as the inhibition of tumor dissemination and metastasis. Preferably, the immune effector function results in the killing of tumor cells. Preferably, the immune effector function in this invention is an antibody-mediated effector function. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), induction of apoptosis in cells carrying tumor-associated antigens (e.g., by binding of antibodies to surface antigens), and / or inhibition of the proliferation of cells carrying tumor-associated antigens, preferably ADCC and / or CDC. Antibodies can also exert their effects simply by binding to tumor-associated antigens on the surface of tumor cells. For example, antibodies can block the function of tumor-associated antigens or induce apoptosis simply by binding to tumor-associated antigens on the surface of tumor cells.

[0101] The term "cancer" refers to any tumor or malignant cell growth, proliferation, or metastasis that causes a medical condition, including solid tumors and non-solid tumors such as leukemia. For example, cancers associated with or caused by abnormal PVRIG expression include, but are not limited to: B-cell lymphomas, including NHL; pre-B-cell lymphocytic leukemia / lymphoma; and mature B-cell tumors 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 follicular center lymphoma; marginal zone B-cell lymphoma (MALT type, intranodal, and splenic type); hairy cell leukemia; diffuse large B-cell lymphoma; Burkitt's lymphoma; plasmacytoma; plasmacytoma myeloma; post-transplant lymphoproliferative disorders; Waldenström macroglobulinemia; and anaplastic large cell lymphoma (ALCL).

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

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

[0104] The term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with or before an antigen, can enhance the organism's immune response to the antigen or alter the type of immune response. Various adjuvants exist, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0105] Anti-PVRIG antibody

[0106] In some aspects, the present invention includes isolated antibodies or antigen-binding fragments thereof.

[0107] In the context of this application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primate-derived antibodies, CDR transplantation antibodies, human antibodies, recombinant antibodies, intracellular antibodies, bifunctional antibodies, multispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotype antibodies, synthetic antibodies, including mutant proteins and variants thereof, modified 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 proteins. Furthermore, unless the context otherwise requires, the term also includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, 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 a modified chimeric monoclonal antibody.

[0108] Variable regions and CDRs in antibody sequences can be identified according to a numbering system based on general rules already developed in the art (as described above, such as Kabat) or by comparing the sequence with a database of known variable regions.

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

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

[0111] The PVRIG specificity described in this invention refers to the ability to bind to one or more PVRIG epitopes, especially those in their natural conformation, particularly human PVRIG specificity.

[0112] In this field, various methods are employed to modify antibodies without altering their desired properties, such as the recombination of the light and heavy chains of the antibody or the substitution of amino acids, as used in this disclosure. For example, the sequences in this invention, including chimeric antibody sequences or humanized antibody sequences, can be modified by making conserved amino acid substitutions.

[0113] In some implementations, the antibody is a bifunctional antibody 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] The fragments referred to therein are Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, or single-stranded Fv fragments (scFv).

[0116] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a constant region of IgG. The constant region of IgG is preferably selected from the constant regions of IgG1, IgG2, IgG3, or IgG4. More preferably, the constant region of IgG is selected from the constant region of IgG1.

[0117] Antibodies primarily interact with target antigens through amino acid residues located in the complementarity-determining regions (CDRs) of the six heavy and light chains. For this reason, the amino acid sequences of the CDRs are more diverse among antibodies than other sequences. Since the CDR sequence is responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody by constructing expression vectors containing the CDR sequence from that specific naturally occurring antibody, which are then grafted onto frame sequences from different antibodies with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such frame sequences are available from public DNA databases that include germline antibody gene sequences. These germline sequences differ from the mature antibody gene sequences because they do not contain the fully assembled variable gene, which is formed during B cell maturation via V(D)J linkage. The germline gene sequences will also have sequences that differ from those of the high-affinity secondary repertoire antibody at individual locations that uniformly traverse the variable region.

[0118] Mouse antibodies exhibit high immunogenicity in humans, leading to decreased therapeutic efficacy with repeated administration. The primary immunogenicity is mediated by the heavy chain constant region. However, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided by chimeric or humanized versions of the antibodies.

[0119] Chimeric antibodies are antibodies whose different parts originate from different animal species, for example, antibodies having a variable region derived from a mouse antibody and a constant region from a human immunoglobulin. Chimeric antibodies are obtained by linking the variable regions of the mouse antibody heavy and light chains to the constant regions of the human heavy and light chains (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are generated by linking the constant region of the human κ light chain to the variable region of the mouse light chain. In another preferred embodiment, chimeric antibodies are generated by linking the constant region of the human λ light chain to the variable region of the mouse light chain.

[0120] Humanized antibodies are antibodies that have had their CDR sequences / antigen-binding portions or sites derived from another mammalian species, such as mice, transplanted onto a human frame sequence.

[0121] To reduce the immunogenicity of antibodies to humans, humanized anti-PVRIG antibodies are produced using the sequence of the PVRIG antibody disclosed herein. The CDR region of a mouse-derived anti-PVRIG antibody is combined with a human-derived framework region (e.g., human immunoglobulin) to form the humanized anti-PVRIG antibody of this disclosure. The humanized antibody is expected to retain the function of binding to human PVRIG as well as the function of binding to monkey PVRIG.

[0122] Antibody preparation or production

[0123] The antibodies of this invention can be generated using various techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While hybridoma technology is preferred, other techniques for generating monoclonal antibodies can be used in principle, such as viral or oncogene transformation of B lymphocytes or phage display using antibody gene libraries, somatic cell hybridization, and, for example, genetic engineering recombination techniques. For instance, DNA molecules encoding the heavy and light chain genes of the antibodies of this invention can be obtained through chemical synthesis or PCR amplification, the resulting DNA molecules can be inserted into an expression vector, and then transfected into host cells. The transfected host cells can then be cultured under specific conditions to express the antibodies of this invention.

[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. USA 92: 9348 (1995), see also Rossie et al. Am. J. Clin. Pathol. 124: 295 (2005)). Hybridoma production in mice is a well-established method. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion methods are also known.

[0125] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, or combinations thereof. For example, monoclonal antibodies can be produced using hybridomas and well-established 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, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0126] It should be understood that the selected binding sequence can be further modified, for example, to increase the affinity for the target, humanize the target binding sequence, improve its production in cell cultures, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing modified target binding sequences are also antibodies of the present invention.

[0127] In some embodiments, a method for producing the antibodies or fragments described in this disclosure includes the following steps:

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

[0129] (ii) Isolate the antibody or its antigen-binding fragment from the host cell.

[0130] In a preferred embodiment, anti-PVRIG monoclonal antibodies are prepared by using hybridomas.

[0131] To obtain hybridomas that produce the antibodies of the present invention, such as the human monoclonal antibodies of the present invention, spleen cells and / or lymph node cells from immunized mice can be isolated and fused into a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The generation of hybridomas is well known in the art. See, for example, Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0132] The antibodies of the present invention can also be generated in host cells transfected with tumors using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In some embodiments, DNA encoding a portion or the full length of the light and heavy chains, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the gene is operatively linked to transcriptional and translational regulatory sequences. In this context, the term "operatively linked" is intended to mean linking the antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody gene.

[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 generate a full-length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype, such that the VH segment is operatively linked to the CH segment within the vector and the VL segment is operatively linked to the CL segment within the vector. Alternatively or additionally, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked to the N-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 derived from a non-immunoglobulin protein).

[0134] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells (which can assemble and secrete antibodies with appropriate folding and immunological activity).

[0135] Mammalian host cells used to express the recombinant antibodies of the present invention include Chinese hamster ovary cells (CHO cells) (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220) used with DHFR selection markers (e.g., as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When the recombinant expression vector encoding the antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient to allow antibody expression in the host cells or by secreting the antibody into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods.

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

[0137] Another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding gene from the antibody-producing lymphocytes of a defined strategy, for example, see Babcocketal, 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of a defined strategy. For details on recombinant antibody engineering, see Welschof and Krau, Recombinant Antibodies for Cancer Therapy ISBN-0-89603-918-8 and Benny KCLo Antibody Engineering ISBN 1-58829-092-1.

[0138] To prepare chimeric antibodies, the variable region of mouse immunoglobulin can be ligated to the constant region of human immunoglobulin using methods known in the art (see, for example, US Patent 4,816,567, Cabilly et al.). A separate nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably ligating a nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). Sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), Sequences Of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but more preferably an IgG1 or IgG4 constant region. A separate nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably ligating a DNA molecule encoding the light chain constant region CL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.), and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a κ or λ constant region, but is generally preferred to be a κ constant region. Once the DNA fragments encoding the VH and VL regions are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operatively ligated to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term “operatively ligated” as used herein is intended to mean that two DNA fragments are ligated such that the amino acid sequences encoded by both DNA fragments remain within the frame.

[0139] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter's US 5,225,539; Queen et al.'s US 5,530,101; US ​​5,585,089; US 5,693,762; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals that do not produce endogenous immunoglobulins after immunization and can generate a complete human antibody library can be used. For example, it has been reported that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice can completely suppress the production of endogenous antibodies, and then transfer of human germline immunoglobulin gene arrays into said germline mutant mice will cause the mice to produce human antibodies upon encountering antigen stimulation (see, for example, 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 the aforementioned transgenic animals include the HuMAb mouse (Medarex, Inc.) containing a miniloci of the human immunoglobulin gene encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, coupled with targeted mutations that inactivate the endogenous μ and κ chain loci (see, for example, Lonberg et al. (1994) Nature 368(6474):856-859); or the “KM mouse™” carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). 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 the antibodies of this invention

[0141] In some aspects, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding isolated antibodies or fragments thereof as disclosed herein.

[0142] The nucleic acids of this invention 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 further described below), the light and heavy chains of the antibody prepared via hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acid encoding such antibody can be recovered from the gene library.

[0143] To prepare chimeric antibodies, the variable region of a mouse immunoglobulin can be ligated to the constant region of a human immunoglobulin using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). By operatively ligating a nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), isolated nucleic acids encoding the VH region can be converted into a full-length heavy chain gene, and DNA fragments containing these regions can be obtained by standard PCR amplification. By operatively ligating DNA encoding VL to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). Once DNA fragments encoding the VH and VL regions are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these operations, the DNA fragment encoding VL or VH is operatively ligated to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker.

[0144] Conjugate

[0145] On one hand, this disclosure provides a conjugate comprising an antibody or fragment thereof as described above, conjugated to at least one detectable marker. The detectable marker includes, but is not limited to: (i) providing a detectable signal; (ii) interacting with a second marker to modify the detectable signal provided by the first or second marker, such as FRET (Fluorescence Resonance Energy Transfer); (iii) influencing mobility (e.g., electrophoretic mobility) through charge, hydrophobicity, shape, or other physical parameters; or (iv) providing a trapping motif, such as affinity, antibody / antigen, or ion complexation.

[0146] Suitable structures for labeling include fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels, enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), particulate labels (especially metal particulate labels, magnetic particulate labels, polymer particulate labels), and small organic molecules (e.g., biotin, receptor ligands or binding molecules (e.g., cell adhesion proteins or lecithin), which can be bound by... The reagents detect marker sequences containing nucleic acid and / or amino acid residues. The markers, without limitation, include barium sulfate, iodopic acid, iodopic acid, calcium amiodarone propionate, sodium diatrizoate, meglumine diatrizoate, meglumine methyl diatrizoate, sodium caseinate, 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 isotopes (e.g., fluorine and gadolinium)), luminescent substances (e.g., isoluminol and acridine ester), fluorescent substances (e.g., fluorescein and rhodamine), and colored substances (e.g., latex particles and colloidal gold).

[0147] The detectable markers described above can be detected by methods known in the art. For example, fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing a substrate to an enzyme and detecting the reaction product produced by the enzyme's action on the substrate. In some embodiments, such markers can be used for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention using linkers of varying lengths to reduce potential steric hindrance.

[0148] Antibody drug conjugates / immunoconjugates

[0149] On one hand, this disclosure provides an antibody-drug conjugate comprising one or more drug portions / therapeutic agents, said drug portions being directly or via a linker (e.g., covalently linked) to the antibody or fragment thereof as described above. In the antibody-drug conjugates of this application, there are no particular limitations on the linker structure for conjugating the anti-PVRIG antibody to the drug, 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 target tissues (e.g., tumor-associated antigens, such as tumors expressing PVRIG), antibody-drug conjugates can enhance the therapeutic efficacy of the antibodies or antigen-binding fragments of the present invention in treating diseases (e.g., cancer).

[0151] Multispecific molecules

[0152] The antibodies or antigen-binding fragments of the present invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments of the present invention can be part of a multispecific molecule (e.g., a bispecific molecule) comprising 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 of the present invention, thereby enabling binding to at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments of the present invention can be linked to a second antibody or antigen-binding fragment capable of specifically binding to any protein that can be used as a potential target for combination therapy. To generate said bispecific or multispecific molecules, the antibodies or antigen-binding fragments of the present invention can be linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

[0153] Therefore, in some aspects, the present invention provides a multispecific molecule comprising the antibody or antigen-binding fragment of the present invention.

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

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

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

[0157] Pharmaceutical Composition

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

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

[0160] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl arsenate. As disclosed in this invention, antibodies containing compositions disclosed herein can be oxidized in solvents containing one or more antioxidants such as methionine, which are reducing antibodies or antigen-binding fragments thereof. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this invention provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present invention further provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, thereby preventing the antibody or its antigen-binding fragment from oxidation, thereby extending its shelf life and / or increasing its activity.

[0161] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic dextran injection, sterile water injection, or dextran and lactated Ringer's injection; non-aqueous media such as non-volatile plant-derived oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); isolating agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions containing phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride in multi-dose containers. Suitable excipients may include, for example, water, saline, dextran, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitol monolaurate, triethanolamine oleate, or cyclodextrin.

[0162] Application, formulation and dosage

[0163] The pharmaceutical compositions of the present invention can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, or implantation or inhalation. The compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0164] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalers and their controlled-release formulations.

[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 liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Similarly, specific dosing regimens (including dose, time, and repetition) will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetic parameters (e.g., half-life, clearance, etc.).

[0166] The requirements for effective drug carriers for injectable formulations / compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, edited by Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (1986)).

[0167] The frequency of administration can be determined and adjusted during treatment, and is based on reducing the number of proliferating or tumorigenic cells, maintaining this reduction in tumor cells, reducing tumor cell proliferation, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the therapeutic composition of the present invention may be suitable.

[0168] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or adverse side effects. The chosen dosage level will depend on a variety of factors, including, but not limited to, the activity of the specific compound, administration, timing of administration, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, the patient's sex, age, weight, condition, general health status, and medical history. However, a dosage is generally chosen to achieve a local concentration at the site of action to achieve the desired effect without causing substantial harmful or adverse side effects.

[0169] Generally, the antibodies or antigen-binding fragments of the present invention can be applied in a variety of applications.

[0170] In some preferred embodiments, the treatment process involving the antibody or antigen-binding fragment thereof of the present invention 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 invention may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

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

[0172] The antibody of the present invention can be co-administered with one or more therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, antitumor agents, anti-angiogenic agents, or immunosuppressants) to reduce the induction of an immune response against the antibody of the present invention. The antibody can be conjugated to the therapeutic agent (as an immune complex) or administered separately from the therapeutic agent.

[0173] In the context of treatment administration, the terms "combination" or "co-administration" as used herein refer to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order in which treatments or therapeutic agents are administered to the subject. Treatments or therapeutic agents may be administered before, simultaneously with, or after administering a second treatment or therapeutic agent to the patient. Preferably, treatments or therapeutic agents are administered to the subject in a specific order, amount, and / or at specific time intervals so that the treatments or therapeutic agents can work together. In one specific embodiment, treatments or therapeutic agents are administered to the subject in a specific order, amount, and / or at specific time intervals so that they provide an increased benefit compared to if administered in other ways (particularly independently of each other). Preferably, the increased benefit is a synergistic effect.

[0174] Medical Use

[0175] The antibodies, antibody compositions, and methods of the present invention have numerous in vitro and in vivo uses, including, for example, detection of PVRIG or enhancement of immune responses. For instance, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects.

[0176] Preferred subjects include mammals, such as humans / patients. In the context of this invention, mammals include humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, Guinea pigs, etc., and captive animals, such as zoo animals.

[0177] Treatment of conditions related to PVRIG expression

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

[0179] As described herein, the disclosed antibodies possess one or more activities that can be therapeutically applied to kill and / or inhibit cell proliferation. In particular, they can be used to kill cells, inhibit cell proliferation, and / or inhibit cell colony formation for the treatment or prevention of cancer (including cancer metastasis). Inhibition of cell proliferation, colony formation, and / or metastasis can be applied, especially for the treatment or prevention of cancer metastasis and the metastatic spread of cancer cells.

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

[0181] In some aspects, this disclosure provides an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, an antibody-drug conjugate, a multispecific molecule, or a pharmaceutical composition or kit in a method for treating a disease associated with PVRIG expression or determining its prognosis in a subject.

[0182] In some aspects, this disclosure provides the use of the antibody or its antigen-binding fragment, 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 preparation of reagents (or medicines) for treating diseases associated with PVRIG expression or determining their prognosis.

[0183] In one implementation, diseases associated with PVRIG expression include oncological diseases such as cancer.

[0184] Antibodies or their antigen-binding fragments can be used alone as a monotherapy or in combination with chemotherapy or radiotherapy.

[0185] Antibodies or their antigen-binding fragments can be used in combination with anticancer agents, cytotoxic agents, or chemotherapy agents.

[0186] The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat cell-proliferating conditions such as cancer, and include, but are not limited to, cytotoxic agents, cell inhibitors, anti-angiogenic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, antimetastatic agents, and immunotherapy agents. It should be understood that, in selected embodiments as described above, such anticancer agents may comprise conjugates and may be bound to a disclosed site-specific antibody prior to administration. More specifically, in some embodiments, a selected anticancer agent is linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such engineered conjugates are explicitly contemplated within the scope of this invention. In other embodiments, the disclosed anticancer agent is administered in combination with a site-specific conjugate comprising the various therapeutic agents described above.

[0187] diagnosis

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

[0189] In some aspects, this disclosure provides a method for diagnosing, detecting, or monitoring diseases associated with PVRIG expression, comprising administering an effective dose of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a desired subject.

[0190] In some aspects, this disclosure provides an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, an antibody-drug conjugate, a multispecific molecule, or a pharmaceutical composition or kit in a method for diagnosing, detecting, or monitoring diseases associated with PVRIG expression in a subject.

[0191] In another aspect, this disclosure provides the use of the antibody or its antigen-binding fragment, 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 preparation of reagents (or drugs) for diagnosing, detecting or monitoring diseases associated with PVRIG expression.

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

[0193] The method described in this invention for detecting or monitoring PVRIG expression or the level of PVRIG-expressing cells in vitro can also be used for non-diagnostic purposes.

[0194] Preferred subjects include mammals, such as people / patients in need.

[0195] The subject's samples are blood, excrement (urine or feces), oral or nasal secretions, or bronchoalveolar lavage fluid, tissue fluid, sweat, or extracts thereof.

[0196] Drug packaging and reagent kits

[0197] Pharmaceutical packages and kits containing one or more containers of an antibody or its antigen-binding fragment thereof are also provided. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or its antigen-binding fragment, with or without one or more other reagents. For other embodiments, such a unit dose is supplied in a single-use, pre-filled syringe. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, or the like; buffers, such as phosphates; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the conjugate composition may be provided as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). In some preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the packaged conjugate composition is intended for the treatment of selected oncological conditions.

[0198] Such kits typically contain a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally contain one or more anticancer agents or other pharmaceutical agents in the same or different containers. The kits may 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: Production 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_VL

[0209] QIILTQSPAIMSASPGEKVTMTCSASSSVSYIHWYQQKSDTSPKRWIYDTSKLVSGVPPRFSGTGSGTSYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTKLEVK

[0210] The amino acid sequence of the antibody's 6 variable region was analyzed using the Kabat sequence determination method, as shown below.

[0211] 6_VH

[0212] QVQLQQSGAELARPGASVKMSCKASGYTFTDYWMQWIKQRPGQGLEWIGAIFPGNDDIRYTQKFKGRATLTADKSSSTAYMQLSNLTSEDSAVYYCATFRYYLDYWGQGTTLTVSS

[0213] 6_VL

[0214] QIILTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSDTSPKRWIYDTSKLVSGVPPRFSGSGSGTSYSLTISSVEAEDAATYYCQQWSSNPYTFGGGTKLEIK

[0215] The amino acid sequence of the variable region of antibody 7 was analyzed according to the Kabat sequence determination method, as shown below.

[0216] 7_VH

[0217] QVHLQQSGDDLVKPGASVKLSCKASGYTFTNYLINWIKQRPGQGLEWIGIAPGSGSFYSNEV FKGKATLTVDTSSSTAYIQLSGLSSEDSAVYFCAREKNWDHFDFWGQGTTLTVSS

[0218] 7_VL

[0219] DIQMTQSSSSFSVSLGDRVTITCKASEDINNRLAWYQQKPGNAPRVLISGAVSLEAGVPSRFSG SGSGRDYTLTIISLQTEDVATYYCQQYWSTPFTFGGSGTKLEIK

[0220] The amino acid sequence of the variable region of antibody 16 was analyzed according to the Kabat sequence determination method, as shown below.

[0221] 16_VH

[0222] QVQLQQPGADLVRPGASVKMSCKASGYTFTSYLMDWVKQRPGQGFEWIGNIDPNSGSTYYN EKFKDKAKLTVDKSSSTAYMQLSSLTSEDSAVYYCAIPYYSEYFGFDVWGTGTTVTVSS

[0223] 16_VL

[0224] DIVMTQSQKFMSTSVGDRVNITCKASQNVRTAVAWYQQKPGQSPKSLIYLASNRHTGVPDRF TGSGSGTDFTLTISNVQSEDLADYFCLQHWSYPYTFGGGTKLEIK

[0225] The amino acid sequence of the variable region of antibody 27 was analyzed according to the Kabat sequence determination method, as shown below.

[0226] 27_VH

[0227] QVQLQQPGAELVRPGASVKMSCKASGYTFTSYLMDWVKQRPGQGFEWIGNIDPNSGSTYYN EKFKDKAKLTVDKSSSTAYIQLSSLTSEDSAVYYCAIPYYSEYYGFDVWGTGTTVTVSS

[0228] 27_VL

[0229] DIVMTQSQKFMSTSVGDRVNITCKASQNVRSAVAWYQQKPGQSPKTLIYLASNRHTGVPDRF TGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPYTFGGGTKLEIK

[0230] The amino acid sequence of the variable region of antibody 52 was analyzed according to the Kabat sequence determination method, as shown below.

[0231] 52_VH

[0232] QVQLQQPGAEFVKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGEIDPSDGYTNYN RKFKGKATLTIDTSSNTGYMQLSSSLTSEDSAVYYCARGTYYYGSSLMDSWGQGTSVTVSS

[0233] 52_VL

[0234] DIVLTQSPASLAVSLGQRAIISCKASQSVSFADTSLMHWYQQKPGQQPKVLIYRASNLEAEVPT RFSGSGSGTDFTLNIHPVEEDDAATYYCQQSREYPPTFGGGTKLEIK

[0235] The amino acid sequence of the variable region of antibody 55 was analyzed according to the Kabat sequence determination method, as shown below.

[0236] 55_VH

[0237] QVQLQQPGAELVRPGSSVKLSCKASGYTFTSYWMHWVRQRPIQGLEWIGIIDLFDSKIHYSQK FKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARERGLGRAGAMDYWGQGTSVTVSS

[0238] 55_VL

[0239] DTVMTQSQKLMSTSVGDRVSVTCKASQNVDTDVVWYQQKPGQSPKALIYSASYRYSGVPDR FTGSGSGTDFTLTISNVQSEDLTEYFCQQYNSYPLTFGAGTKLELK

[0240] Example 2: Species cross-reactivity of antibodies 1, 6, 7, 16, 27, 52, and 55

[0241] The species cross-reactivity of antibodies 1, 6, 7, 16, 27, 52, and 55 with CHOK1 overexpressing different species of PVRIG was evaluated by FACS detection.

[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] As described above, stable cell lines CHOK1 / Human PVRIG and CHOK1 / Cyno PVRIG expressing human and monkey PVRIG antigens were cultured and processed using flow cytometry. 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. 1E+05 cells / well were placed in a 96-well plate, centrifuged, and the supernatant was discarded. The cells were resuspended in a gradient of antibody solutions (300nM, 3-fold dilution, 12 dilution points), 50 μL / well, mixed well, and incubated at 4°C for 1 hour. After centrifugation at 300g for 5 minutes at 4°C, the supernatant antibody solution was discarded, and the cells were washed twice with 1×PBS. 100 μL / well of the corresponding 1 μg / mL goat anti-human secondary antibody solution (Jackson, 109-605-088) was added, and 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.

[0249] The results are as follows Figure 2 and Figure 3 As shown, molecule-1, molecule-6, molecule-7, molecule-16, molecule-27, molecule-3, and molecule-4 all exhibited strong binding activity to human and monkey PVRIG cells on the cell surface. This indicates that molecule-1, molecule-6, molecule-7, molecule-16, molecule-27, molecule-3, and molecule-4 can effectively bind to the surface of CHOK1 / HumanPVRIG and CHOK1 / cyno PVRIG cells overexpressing human and monkey PVRIG, and this binding activity is dose-dependent. The overall binding ability of these seven candidate molecules to human and monkey PVRIG-overexpressing cell lines is superior to that of the reference antibodies SRF813 and COM701.

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

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

[0252] Dilute the antigen to 0.5 μg / ml with coating buffer, mix well, and add 100 μL / well to the designed ELISA plate. After coating, incubate at 4°C overnight for 16 hours. Discard the coating buffer, wash the plate once, and blot dry. Add 150 μL of blocking buffer (2% BSA, self-prepared) to each well and incubate at 37°C for 1 hour. Remove the ELISA plate and discard the blocking buffer. The initial concentration of the antibody to be tested in the first well is 10 nM, and it is serially diluted 3-fold to the 10th well. The remaining two wells are added with dilution buffer as blank controls. The control well data is represented by the average of two wells, 100 μL / well; incubate at 37℃ for 1 h, remove the microplate, discard the internal solution, and wash the plate 3 times; add 100 μL of diluted enzyme-labeled goat anti-human secondary antibody (Rockland, 609-103-123) to each well and incubate at 37℃ for 1 h; remove the microplate, discard the internal solution, wash the plate 3 times, pat dry, add 100 μL of chromogenic solution (Beyotime, PO209), and react at 25℃ for 15 min; add 50 μL of 1M HCl to stop the reaction; read the microplate at OD450 nm.

[0253] The results are as follows: Figure 4 and Figure 5 As shown, molecule 1, 6, 7, 16, 27, 52, and 55 exhibit strong binding activity to human and monkey PVRIG proteins, and this activity is dose-dependent. The binding ability of these seven candidate molecules to human and monkey PVRIG proteins is comparable 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 candidate antibody molecules was evaluated through blocking experiments. Specifically, the IC50 of different concentrations of 1, 6, 7, 16, 27, 52, and 55 ligands inhibiting the binding of PVRL2 ligands to Human PVRIG protein was measured by ELISA to evaluate their blocking activity.

[0256] Human PVRIG protein (Acro, PVG-H52H4) was diluted to 1.0 μg / ml with coating buffer, mixed well, and added to the designed microplate at 100 μL / well. After coating, the plate was incubated overnight at 4°C for 16 h. The coating buffer was discarded, the plate was washed once, and the plate was blotted dry. 150 μL of blocking buffer (2% BSA, self-prepared) was added to each well, and the plate was incubated at 37°C for 1 h, then the blocking buffer was discarded. 5 μL each of 4.0 nM PVRL2 ligand (Acro, CD2-H82A3) and the diluted antibody to be tested (100 nM, 3-fold dilution, 11 dilution points) were added. Add 0 μL to the microplate. Separately, add 50 μL each of PVRL2 ligand (Acro, CD2-H82A3) and diluent (4 wells) to the microplate as blank values. Incubate at 37℃ for 1 h. Remove the microplate, discard the internal solution, 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℃ for 1 h. Remove the microplate, discard the internal solution, wash the plate 3 times, pat dry, add 100 μL of chromogenic solution, and react at 25℃ for 15 min. Add 50 μL of 1M HCl to stop the reaction and read the OD at 450 nm using a microplate reader.

[0257] The results are as follows: Figure 6 As shown, 1, 6, 7, 16, 27, 52, and 55 all exhibited strong ligand blocking activity with a certain dose dependence. The ligand blocking activity of these seven candidate molecules was 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] Affinities for antibodies 1, 6, 7, 16, 27, 52, and 55 were detected using Biacore. The affinity assay was performed at 25°C using HBS-EP+ (Cytiva, BR-1006-69) as the buffer system. Antibodies were captured onto a Protein A chip (Cytiva, 29-1275-55) as the stationary phase. Diluted human PVRIG antigen (Acro, PVG-H52H4) was sequentially injected onto the chip surface as the binding phase, followed by the injection of running buffer as the dissociation phase, and affinity was then measured. Data processing was performed using Biacore 8K analysis software version 3.0.

[0260] Please see Table 2 below for the experimental parameters:

[0261] Table 2: Key Experimental Parameters

[0262]

[0263] The results are shown in Table 3 below. Antibodies 16, 27, and 52 showed high affinity for the PVRIG target protein, with an affinity reaching 10. -11 The M-level antibody is significantly superior to the reference antibodies SRF813 and COM701, while the affinities at 1, 6, 7 and 55 are comparable to those of the reference antibodies.

[0264] Table 3: Binding dissociation constants of 1, 6, 7, 16, 27, 52, 55 with human PVRIG

[0265]

[0266]

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

[0268] The PVRL2 / PVRIG reporter gene assay is a rapid and reproducible cell-based detection method. This reporter gene assay consists of two genetically engineered cell lines: a GS-J2C / PVRIG effector cell line expressing human PVRIG protein and an NFAT-RE-driven luciferase reporter gene, and a GS-C2 / PVRL2 target cell line expressing human PVRL2 protein and anti-human CD3ScFv. When the GS-J2C / PVRIG effector cell line and the GS-C2 / PVRL2 target cell line are co-cultured, the PVRL2 / PVRIG interaction inhibits the activation of the TCR pathway by anti-CD3, thereby suppressing the expression of NFAT-driven luciferase, resulting in a lower fluorescence signal. The addition of a PVRIG therapeutic antibody blocks the PVRL2 / PVRIG interaction, thereby restoring the TCR signaling pathway and increasing the fluorescence signal value.

[0269] The specific method involved digesting and centrifuging GS-C2 / PVRL2 target cells (Genscript construction) and resuspending them in their corresponding complete medium (F-12K (Gibco, 21127-022) + 10% FBS (Gibco, 10091-148)). The target cell density was adjusted, and the cells were seeded into experimental plates. The plates were then transferred to a cell culture incubator (37℃ / 5% CO2) and incubated for approximately 16 hours. Experimental buffer (RPMI 1640 (Gibco, 22400-089) + 1.25% FBS) was then used. Prepare the reference antibody and test antibody working solutions using FBS (Gibco, 10091-148); collect effector cells by centrifugation, resuspend the cells in experimental buffer and adjust the cell density; remove the experimental plate incubating target cells from the cell culture incubator and remove the culture medium; sequentially transfer the reference antibody, test antibody working solutions, and GS-J2C / PVRIG effector cell (Genscript construction) suspension to the experimental plate; transfer the experimental plate to a cell culture incubator (37℃ / 5% CO2) and incubate for 6 hours; prepare the luciferase detection reagent working solution (GenScript, L00877C); remove the incubated experimental plate from the cell culture incubator, 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 a PHERA Star microplate reader to read the chemiluminescence values ​​and record the data.

[0270] The results are as follows: Figure 7 As shown, the reporter gene activity of the reference antibody SRF813 was weaker than that of COM701 (EC50: 0.142 ug / ml vs 0.087 ug / ml, Span: 168139 vs 238134). Compared with COM701, the reporter gene activities of antibodies 16, 27, and 52 were significantly better than those of the reference antibody COM701, with antibody 52 exhibiting approximately three times the reporter gene activity of COM701. The reporter gene activities of antibodies 1, 6, 7, and 55 were all between 0.9 and 1.1 ug / ml, reaching levels comparable to the reference antibody.

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

[0272] To further detect the activation level of CD8T cells in vitro, antigen-specific CD8+T cell activation experiments were performed on cells 1, 6, 7, 16, 27, 52, and 55.

[0273] PBMCs (Miaoshun Biotechnology, PB100C-W) were resuscitated using RPMI 1640 complete medium (RPMI 1640 + 10% FBS) and induced according to the established induction protocol for 12 days. After 12 days, PBMCs were collected by centrifugation and primary CD8+ T cells were sorted. Simultaneously, flow cytometry analysis was performed on the induced PBMCs or sorted CD8+ T cells to analyze the levels of PVRIG, TIGIT, pp65, and CD226 in CD8+ T cells. Colo205 target cells (ATCC, CCL-222) were digested, centrifuged, and resuspended in experimental buffer (RPMI 1640 + 10% FBS). Pre-incubate a certain amount of Colo205 target cells (ATCC, CCL-222) with a high concentration of pp65 (GenScript, RP11781) working solution (4×) in a cell culture incubator (37℃ / 5% CO2) for 0.5-2 hours; after premixing, centrifuge to remove pp65; adjust the Colo205 target cell (ATCC, CCL-222) density and transfer the cell suspension to 96-well plates. Prepare control / test sample working solutions (4×) using RPMI 1640 complete medium. Transfer the control / test sample working solutions to the corresponding wells of the 96-well experimental plate. Adjust the CD8+ T cell density according to the effector-to-target ratio (1:1) set in the protocol and transfer the cell suspension to the corresponding wells of the 96-well experimental plate. Incubate the experimental plate in a cell culture incubator (37℃ / 5% CO2) for approximately 16-18 hours. After incubation, the supernatant was collected by centrifugation for human hINFγ cytokine detection (Cisbio, 62HIFNGPEH). Cells were digested, washed with FACS buffer, and stained with 7-AAD (Biolegend, 420403). After staining, the supernatant was discarded by centrifugation, and cells were washed and resuspended with FACS buffer. Cells were collected using flow cytometry, and fluorescence signal values ​​were read. Raw data from the cytokine release assay were exported using the Phera Star FSX system and analyzed using Microsoft Office Excel 2016 and GraphPad Prism 6 software. Raw data from the cell flow cytometry analysis were exported using BD FACSDiva Software v8.0.1.1 and analyzed using FlowJo 7.6.1 and Microsoft Office Excel 2016 software. The results were used to assess the level of target cell killing (percentage of 7-AAD-positive cells).

[0274] The results are as follows Figures 8A-8C and 8D- Figure 8FAs shown, the reference antibody COM701 exhibited weak cytokine induction and secretion capacity, as well as weak cell-killing activity. Compared to COM701, antibodies 6, 55, 27, and 52 demonstrated stronger ability to induce human hINFγ cytokine secretion, with antibodies 27 and 52 showing the best performance, significantly stronger than the reference antibody. Antibody 52 showed significantly superior CD8+ T cell-killing activity compared to other antibody molecules, including the reference molecule, exhibiting significant cell-killing activity.

[0275] Example 9: Classification of antigenic epitopes of antibodies 1, 6, 7, 16, 27, 52, and 55

[0276] To further clarify the epitopes of 1, 6, 7, 16, 27, 52, and 55 that bind to PVRIG, the antigen-binding regions of reference antibodies SRF813 and COM701, which are known to bind to different regions of PVRIG epitopes, were determined by competitive ELSA.

[0277] Epitope competition ELISA assay: Human PVRIG protein (Acro, PVG-H52H4) was diluted to 0.5 μg / ml with coating buffer, mixed well, and added to the designed microplate at 100 μL / well; after coating, it was placed in a 4°C refrigerator overnight for 16 h; the coating buffer was discarded, the plate was washed once, and the plate was patted dry; 150 μL of blocking buffer (2% BSA, self-prepared) was added to each well, and the plate was placed in a 37°C incubator for 1 h, then the blocking buffer was discarded; 5 each of biotin-labeled antibodies (self-prepared) and diluted antibody samples (see Table 4) were used as competitors. 0 μL of the reagent was added to the microplate. Separately, 50 μL each of the competitor and diluent (4 wells) were added to the microplate as blanks. The plates were incubated at 37°C for 1 h. The plates were then removed, the internal solution discarded, and the plates were washed 3 times. 100 μL of diluted enzyme-labeled secondary antibody (Genscript, M00091) was added to each well, and the plates were incubated at 37°C for 1 h. The plates were then removed, the internal solution discarded, the plates were washed 3 times, and the plates were patted dry. 100 μL of the chromogenic reagent was added, and the reaction was carried out at 25°C for 15 min. The reaction was terminated by adding 50 μL of 1M HCl, and the OD450 nm reading was recorded using a microplate reader. The results are shown in Table 4.

[0278] Table 4: Classification of antigenic epitopes 1, 6, 7, 16, 27, 52, and 55

[0279]

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

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

[0282] The efficacy of the test drug was evaluated in an animal model in which human melanoma A375 cells (ATCC, CRL-1619) were subepithelially inoculated into PBMC-reconstructed B-NDG mice (Biocytogen).

[0283] Will contain 5×10 7 0.1 mL of A375 cell suspension (at a concentration of 1 / mL) was subcutaneously inoculated above the right scapula of each B-NDG mouse. The following day, the mice were inoculated with PBMCs (Miaoshun Biotechnology, PB100C-W) at a concentration of 2.5 × 10⁶ cells / mL. 7 / mL, 0.2mL was injected into the tail vein of each mouse. When the tumor volume in the mouse reached 50-100mm... 3 Mice were randomly assigned to groups based on tumor volume. The tumor volume CV of enrolled mice was ≤30%. Each group consisted of 8 mice, administered 10 mg / kg PD-1 + 30 mg / kg the test antibody twice weekly for 3 weeks. If weight loss exceeded 15%, administration was immediately discontinued until weight recovered to at least 98% of initial weight before resuming normal administration. If weight loss exceeded 20% and did not recover within 72 hours, the mice were euthanized.

[0284] The results are as follows Figure 10 As shown, based on tumor volume and TGI values ​​at D20, compared to the Vehicle (PBS-treated group), the PD1+6, 7, 10, 16, 27, and 52 combination groups all exhibited certain tumor-suppressive effects (TGI: 16.93%, 17.03%, 10.5%, 18.71%, 25.01%, 16.06%). Antibody 27 showed the best anti-tumor effect. Specific results are shown in Table 5.

[0285] Table 5

[0286]

[0287] Example 11 Hotspot Removal of Antibody 52

[0288] Sequence analysis revealed an aspartic acid isomerization site in CDRH2 at position 52, posing a high risk of PTM (Potentially Transformed Mutation). Therefore, hotspot removal modification was performed to avoid the impact of aspartic acid isomerization on antibody structure and function. The hotspot removal 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 hotspot removal variant was expressed using ExpiCHO-S cells (Thermo) transiently transfected with the antibody. Twenty-four hours prior to transfection, ExpiCHO-S cells were cultured normally in expression medium at a passage density of 3.8 × 10⁻⁶ cells / year. 6 Cells / mL were cultured in a shaker at 37°C with 8% CO2. On the day of transfection, cell density and viability were measured. Cells were then diluted to the density required for transfection. Transfection was performed using liposomes. After transfection, the shake flasks were returned to the shaker at 37°C with 8% CO2 for further culture. 18-22 hours after transfection, feed and additives (Gibco, A29129) were added to the cells. 7-10 days after transfection, samples were taken for SDS-PAGE to confirm sufficient expression levels. The supernatant was collected by centrifugation and purified for antibody preparation.

[0293] The binding activity of the hotspot removal variants was evaluated by measuring the EC50 of their binding to Human PVRIG and Cyno PVRIG proteins at different concentrations using ELISA. The detection procedure is described in Example 4. The properties of the hotspot removal variants were identified as follows: Figure 11 and Figure 12 As shown, the target protein binding activities of the four variants are comparable to those of the original sequence 52. This indicates that the four hotspot removal variants have little impact on the target protein binding activity of the original sequence.

[0294] The inhibition rate of candidate antibody molecules was evaluated through blocking experiments. Specifically, the IC50 of the hotspot removal variants at different concentrations was measured using ELISA to detect their inhibition of PVRL2 ligand binding to Human PVRIG protein, thus evaluating their blocking activity. The detection procedure is described in Example 5. The properties of the hotspot removal variants were identified as follows: Figure 13As shown, the ligand receptor blocking activities of 52_vH(DG / EG-DS / ES), 52_vH(DG / EG-DS / DA), and 52_vH(DG / DA-DS / DA) are comparable to those of the original 52 sequence. The blocking activity of 52_vH(DG / DA-DS / ES) is slightly weaker than that of the original 52 sequence.

[0295] The binding activity of the hotspot removal variants was evaluated by measuring the EC50 of their binding to human PVRIG and cyno PVRIG at different concentrations using FACS. The detection procedure is described in Example 3. The properties of the hotspot removal variants were identified as follows: Figure 14 , Figure 15 As shown, the FACS binding activity of human and monkey cells with the 52_vH(DG / DA-DS / ES) and 52_vH(DG / DA-DS / DA) variants is comparable to that of the original 52 sequence. The FACS binding activity of human and monkey cells with the 52_vH(DG / EG-DS / ES) and 52_vH(DG / EG-DS / DA) variants is weaker than that of the original 52 sequence.

[0296] The affinity of the hotspot removal variants was detected using Biacore. The specific detection method is described in Example 6. The results of the property identification of the hotspot removal variants are shown in Table 7. The affinity of the 52_vH(DG / DA-DS / ES) and 52_vH(DG / DA-DS / DA) variants is comparable to that of the original 52 sequence. The affinity of the 52_vH(DG / EG-DS / ES) and 52_vH(DG / EG-DS / DA) variants decreased compared to the original 52 sequence.

[0297] Table 7

[0298]

[0299] The reporter gene activity of hotspot removal variants was detected by reporter gene assays; specific detection methods are described in Example 7. The results of the characterization of hotspot removal variants are as follows: Figure 16 As shown, the 52_vH(DG / DA-DS / ES) and 52_vH(DG / DA-DS / DA) variants have similar affinity to the original 52 sequence.

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

[0301] By incorporating references

[0302] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0303] Equivalence

[0304] This invention may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases, and not as limiting of the invention described herein. Consequently, the scope of the invention is defined by the appended claims rather than by the foregoing description, and is intended to be encompassed by all variations within the equivalent meaning and scope of the claims.

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 include: HCDR1 shown in SEQ ID NO: 38, HCDR2 shown in SEQ ID NO: 44, and HCDR3 shown in SEQ ID NO: 52, LCDR1 shown in SEQ ID NO: 61, LCDR2 shown in SEQ ID NO: 66, and LCDR3 shown in SEQ ID NO:

72.

2. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the heavy chain variable region is as shown in SEQ ID NO: 22; The light chain variable region is shown in SEQ ID NO:

33.

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

16.

4. The antibody or antigen-binding fragment thereof as described in claim 3, comprising: The light chain shown in SEQ ID NO: 12 and the heavy chain shown in SEQ ID NO:

11.

5. The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, wherein the antibody is a whole antibody; The fragments referred to therein are Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, or single-stranded Fv fragments (scFv).

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

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

8. The antibody or a variant of the antigen-binding fragment thereof according to any one of claims 1-7, comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region and the light chain variable region include: HCDR1 shown in SEQ ID NO: 38, HCDR2 shown in SEQ ID NO: 46 or 47, and HCDR3 shown in SEQ ID NO: 54 or 55; LCDR1 shown in SEQ ID NO: 61, LCDR2 shown in SEQ ID NO: 66, and LCDR3 shown in SEQ ID NO:

72.

9. The variant of claim 8, wherein the heavy chain variable region is as shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27; and wherein the light chain variable region is as shown in SEQ ID NO:

33.

10. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7, or a variant as described in claim 8 or 9.

11. A vector comprising the nucleic acid molecule as described in claim 10.

12. A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11.

13. A conjugate comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-7 conjugated to at least one detectable marker.

14. An antibody-drug conjugate comprising an antibody, including one or more drug portions, said drug portions being directly or covalently linked via a linker to an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-7 or a variant of claim 8 or 9.

15. A multispecific molecule comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-7 and / or a variant as described in claim 8 or 9.

16. A pharmaceutical composition or kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-7, or a variant as claimed in claim 8 or 9, or a nucleic acid molecule as claimed in claim 10, or a vector as claimed in claim 11, or a host cell as claimed in claim 12, or a conjugate as claimed in claim 13, or an antibody-drug conjugate as claimed in claim 14, or a multispecific molecule as claimed in claim 15, and a pharmaceutically acceptable carrier.

17. The use of an antibody or antigen-binding fragment thereof as described in any one of claims 1-7, or a variant as described in claim 8 or 9, or a nucleic acid molecule as described in claim 10, or a vector as described in claim 11, or a host cell as described in claim 12, or a conjugate as described in claim 13, or an antibody-drug conjugate as described in claim 14, or a multispecific molecule as described in claim 15, or a pharmaceutical composition or kit as described in claim 16, in the preparation of a kit for diagnosing, detecting, or monitoring diseases associated with PVRIG expression, wherein the diseases associated with PVRIG expression are cancers selected from squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, peritoneal cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, brain cancer, endometrial cancer, testicular cancer, biliary tract cancer, gallbladder cancer, gastric cancer, melanoma, or head and neck cancer.

18. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-7, or the variant as described in claim 8 or 9, or the nucleic acid molecule as described in claim 10, or the vector as described in claim 11, or the host cell as described in claim 12, or the conjugate as described in claim 13, or the antibody-drug conjugate as described in claim 14, or the multispecific molecule as described in claim 15, or the pharmaceutical composition or kit as described in claim 16 in the preparation of a medicament for treating or determining the prognosis of a disease associated with PVRIG expression, wherein the disease associated with PVRIG expression is cancer selected from squamous cell carcinoma, small cell lung cancer, pituitary carcinoma, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, peritoneal cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, brain cancer, endometrial cancer, testicular cancer, biliary tract cancer, gallbladder cancer, gastric cancer, melanoma, or head and neck cancer.

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