Anti-pvrig antibodies and uses thereof

Abstract

Provided in the present disclosure are anti-PVRIG antibodies, nucleic acid molecules encoding the anti-PVRIG antibodies, expression vectors and host cells for expressing the anti-PVRIG antibodies. The present disclosure further provides methods for the prevention and treatment of cancer and immune disorders by administering the anti-PVRIG antibody.

Description

[0001] Cross-references

[0002] This application claims the benefit of international patent application PCT / CN2023 / 098601, filed on June 6, 2023, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list, which is incorporated herein by reference in its entirety. Technical Field

[0005] This application generally relates to antibodies. More specifically, this application relates to monoclonal antibodies against PVRIG, methods for their preparation, and uses of said antibodies. Background Technology

[0006] Cancer cells can evade or suppress immune responses by utilizing natural control mechanisms that limit T cell activation to prevent uncontrolled T cell activity. Restoring the ability of immune effector cells, particularly T cells, to recognize and eliminate cancer is a goal of immunotherapy. In the tumor microenvironment, continuous antigen stimulation can lead to T cell exhaustion, T cell dysfunction, and overexpression of co-inhibitory receptors, including PD-1, LAG-3, TIM3, and TIGIT. Currently, various strategies are being explored to revitalize exhausted T cells using single or combined small molecule or therapeutic antibody approaches.

[0007] Poliovirus receptor-associated Ig domain (PVRIG, also known as CD112R) is a co-inhibitory immune checkpoint protein. It plays an important role in reversing T cell exhaustion and increasing NK cell activation. PVRIG belongs to the connexin and connexin-like family, whose members also include TIGIT, DNAM-1 (CD226), and CD96. PVRIG is expressed on NK cells and T cells, and is further upregulated in T cells after activation. The interaction between PVRIG and its ligand PVRL2 (CD112), which is expressed on APCs and various tumor cells, leads to the inhibition of T cell and NK cell activation. PVRL2 is also a ligand of CD226, which activates human T cells and NK cells after ligand interaction.

[0008] PVRIG blockade was shown to be a promising strategy for treating cancer in several preclinical mouse tumor models. COM-701, developed by Compugen, is the first anti-PVRIG antagonistic antibody to enter clinical development. Phase 1 clinical results showed that COM-701 was well tolerated as monotherapy and in combination with nivolumab, with a manageable safety profile. Best responses of complete response (CR), partial response (PR), or stable disease (SD) were observed in 11 / 21 (52%) patients with previously treatment-refractory disease and 13 / 18 (52%) patients previously treated with immune checkpoint inhibitors. GSK4381562, co-developed by GSK and Surface Oncology, is another anti-PVRIG antibody that is in clinical trials. GSK4381562 binds to a unique epitope on PVRIG and blocks the interaction of PVRIG with CD112, and it promotes activation of both NK cells and T cells, with the potential to elicit strong anti-tumor responses and promote immune memory.

[0009] PVRIG, as a single agent or in combination with other immunomodulatory agents, can be a promising therapeutic target for tumor immunotherapy. SUMMARY

[0010] The present disclosure provides these and other objects, and broadly relates to compounds, methods, compositions, and articles of manufacture that provide antibodies with improved efficacy. The benefits provided by the present disclosure are widely applicable to the field of antibody therapy and diagnostics, and can be used in conjunction with other antibodies that react with a variety of targets.

[0011] The present disclosure provides antagonistic antibodies against PVRIG. The antibodies disclosed herein can inhibit the signaling pathway triggered by the binding interaction of PVRIG and PRLR2, which leads to increased T cell and NK cell activation, among others. Further provided are methods of treating a subject having a cancer or an immune disorder or a pathogen infection by administering an anti-PVRIG antibody as disclosed herein. The present disclosure also provides methods for verifying antibody function in vitro and in vivo.

[0012] In some aspects, the present disclosure provides an isolated antibody, or an antigen binding portion thereof, comprising:

[0013] a heavy chain CDR (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1;

[0014] a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2;

[0015] a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3;

[0016] a heavy chain CDR (HCDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 1 ;

[0017] a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and

[0018] a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3.

[0019] In some embodiments, the isolated antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL),

[0020] wherein the VH comprises or consists of:

[0021] (i) the amino acid sequence set forth in SEQ ID NO: 7;

[0022] (ii) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 7; or

[0023] (iii) an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additions, deletions, and / or substitutions of amino acids compared to SEQ ID NO: 7; and / or

[0024] the VL comprises or consists of:

[0025] (i) the amino acid sequence set forth in any one of SEQ ID NOs: 8-9;

[0026] (ii) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 8-9; or

[0027] (iii) an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additions, deletions, and / or substitutions of amino acids compared to any one of SEQ ID NOs: 8-9.

[0028] In some embodiments, the isolated antibody or antigen-binding portion thereof comprises a HCDR1, a HCDR2, and a HCDR3 of a VH region set forth in SEQ ID NO: 7, and a LCDR1, a LCDR2, and a LCDR3 of a VL region set forth in any one of SEQ ID NOs: 8-9.

[0029] In some embodiments, the isolated antibody or its antigen-binding portion comprises: HCDR1 shown in SEQ ID NO: 1; HCDR2 shown in SEQ ID NO: 2; HCDR3 shown in SEQ ID NO: 3; LCDR1 shown in SEQ ID NO: 4; LCDR2 shown in SEQ ID NO: 5; and LCDR3 shown in SEQ ID NO: 6.

[0030] In some embodiments, the isolated antibody or its antigen-binding portion includes a VH region containing the amino acid sequence of SEQ ID NO: 7 and a VL region containing the amino acid sequence of SEQ ID NO: 8.

[0031] In some embodiments, the isolated antibody or its antigen-binding portion further comprises a human IgG constant region, such as a human IgG1, IgG4, IgG2, or IgG3 constant region, which may be natural or a variant thereof. Specifically, the antibody may comprise a human IgG1 Fc region or a human IgG4 Fc region.

[0032] In some embodiments, the isolated antibody or its antigen-binding portion comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 12, 15 or 17 and a light chain containing the amino acid sequence of SEQ ID NO: 13, 16 or 18.

[0033] In some embodiments, the anti-PVRIG antibodies disclosed herein are human antibodies or humanized antibodies. In some embodiments, the antibodies described herein are anti-PVRIG antagonist antibodies.

[0034] In some aspects, this disclosure provides isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of isolated antibodies or their antigen-binding moieties as disclosed herein. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 10 and / or the nucleic acid sequence shown in SEQ ID NO: 11.

[0035] In some respects, this disclosure provides expression vectors comprising the nucleic acid molecules disclosed herein.

[0036] In some respects, this disclosure provides a host cell containing expression vectors as disclosed herein.

[0037] In some aspects, this disclosure provides pharmaceutical compositions comprising an antibody or antigen-binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.

[0038] In some aspects, this disclosure provides methods for preparing antibodies or antigen-binding moieties thereof, comprising expressing the antibody or antigen-binding moieties thereof in host cells and isolating the antibody or antigen-binding moieties thereof from the host cells. In some embodiments, the host cells have been transfected or transformed with expression vectors encoding the heavy and light chains of the antibodies disclosed herein. The heavy chain encoding nucleic acid sequences and the light chain encoding nucleic acid sequences may be in the same vector or in separate vectors.

[0039] In some aspects, this disclosure provides methods for modulating an immune response in a subject, comprising administering to the subject an antibody or its antigen-binding portion as disclosed herein. The immune response may be PVRIG-related, T-cell-related, and / or NK-cell-related.

[0040] In some aspects, this disclosure provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject alone or in combination with another anticancer agent an effective amount of an antibody or antigen-binding portion thereof, as disclosed herein, or a pharmaceutical composition thereof.

[0041] In some aspects, this disclosure provides methods for treating or preventing cancer or immune-related conditions in a subject, comprising administering to the subject an effective amount of an antibody or its antigen-binding portion as disclosed herein. The method may further include administering additional anticancer agents, such as chemotherapeutic agents, monoclonal antibodies, antibody-drug conjugates, etc. In some embodiments, the anticancer agent is an antiPD-1 antibody, an antiPD-L1 antibody, or an antiCTLA-4 antibody.

[0042] Cancers can include colon cancer, lung cancer (such as NSCLC), breast cancer, prostate cancer, bladder cancer, ovarian cancer, stomach cancer, colorectal cancer, esophageal cancer, head and neck cancer, cervical cancer, pancreatic cancer, testicular cancer, lymphoma, leukemia, malignant melanoma, and soft tissue cancer. Immune-related conditions can be T-cell dysfunction disorders, infections, or inflammatory diseases.

[0043] In some respects, this disclosure provides the use of the antibodies or antigen-binding portions thereof disclosed herein (alone or in combination with another anticancer agent) in the preparation of medicaments for the treatment or prevention of diseases such as cancer and immune-related conditions.

[0044] In some respects, this disclosure provides the use of antibodies or antigen-binding portions thereof as disclosed herein in the preparation of diagnostic agents for diagnosing diseases associated with PVRIG overexpression.

[0045] In some respects, this disclosure provides antibodies or antigen-binding portions thereof as disclosed herein for use in the treatment or prevention of cancer and immune-related conditions.

[0046] In some aspects, this disclosure provides methods for detecting the presence of PVRIG antigens in a sample or measuring the amount of PVRIG antigens, comprising contacting the sample with an anti-PVRIG antibody or its antigen-binding portion disclosed herein.

[0047] In some respects, this disclosure provides kits or devices containing an antibody or antigen-binding portion thereof as disclosed herein in one or more containers.

[0048] The foregoing is an overview and therefore necessarily contains simplifications, generalizations, and omissions of details; thus, those skilled in the art will understand that this overview is merely illustrative and not intended to be limiting in any way. This overview is not intended to identify key or essential features of the claimed subject matter, nor is it intended to serve as an aid in determining the scope of the claimed subject matter. Attached Figure Description

[0049] Figure 1-2 The antibody and human PVRIG-engineered cells, as determined by FACS, were shown to be effective. Figure 1 ) and people CD8 + T cells ( Figure 2 The combination of ).

[0050] Figure 3 The binding of antibodies, as determined by FACS, to cynomolgus monkey PVRIG-engineered cells was demonstrated.

[0051] Figure 4 The results show the binding of the antibody to mouse PVRIG, as determined by ELISA.

[0052] Figure 5 The affinity of the anti-PVRIG antibody for human PVRIG was demonstrated in the FACS affinity assay.

[0053] Figure 6 The binding of the antibody to the PVRIG paralog protein, as determined by ELISA, is shown.

[0054] Figure 7 The results show the effects of antibodies that block the binding of PVRL2 to PVRIG, as determined by ELISA.

[0055] Figure 8 This shows the results of the antibody assay in the NFAT (Nuclear Factor of Activated T-cells) reporter gene assay.

[0056] Figure 9 The effects of the antibody on human CD8+ T cells were demonstrated.

[0057] Figure 10The stability of the antibody in human serum was demonstrated.

[0058] Figure 11 The binding of antibodies in the forms of IgG1 and IgG4, as determined by FACS, to human PVRIG-engineered cells is shown.

[0059] Figure 12 The activity of antibodies in IgG1 and IgG4 forms in the NFAT reporter gene assay was demonstrated.

[0060] Figure 13 The activity of antibodies in the forms of IgG1 and IgG4 was demonstrated in a T-cell activation assay.

[0061] Figure 14 The pharmacokinetic results of the anti-PVRIG antibody in rats are shown.

[0062] Figure 15 The binding of WT1175-1.158.12-m1-uIgG1L to human PVRIG-engineered cells was demonstrated by FACS.

[0063] Figure 16 The activity of WT1175-1.158.12-m1-uIgG1L in the NFAT reporter gene assay was demonstrated. Invention Details

[0065] While the invention can be embodied in many different forms, the embodiments disclosed herein are specific illustrative schemes that illustrate the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments shown. Furthermore, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter.

[0066] Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context explicitly specifies otherwise. Thus, for example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a mixture of cells, etc. In this application, the use of “or” means “and / or” unless otherwise stated. Furthermore, the use of the term “comprising” and other forms such as “comprises” and “comprised” is not limiting. Moreover, the scope provided in the specification and appended claims includes both endpoints and all points in between.

[0067] Generally, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques disclosed herein are generally performed according to conventional methods well-known in the art and described in the various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6 th ed., WB Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubelet et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Colligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature, laboratory procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well-known and commonly used in the art.

[0068] definition

[0069] To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0070] As used herein, the term "antibody" or "Ab" typically refers to a Y-tetrameric protein, which comprises two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be divided into κ and λ light chains. The heavy chains can be divided into μ, δ, γ, α, and ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H 1. C H 2 and C H 3) Composition. Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L Composed of ) V H and V L The region can be further divided into high-variance regions (called complementary determinant regions (CDRs)), which are separated by relatively conservative regions (called framing regions (FRs)). Each V H and V L It consists of 3 CDRs and 4 FRs, in the following order: from the N-terminus to the C-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V) of each heavy / light chain pair... H and V L Each antibody forms an antigen-binding site. Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0071] In the context of this application, the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody may be used interchangeably to refer to a polypeptide containing a fragment of a full-length antibody that retains the ability to bind specifically to an antigen that binds specifically to the full-length antibody, and / or the ability to compete with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Under certain conditions, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and peptides containing at least a portion of the antibody sufficient to confer specific antigen-binding ability to the peptide. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., the monoclonal anti-human PVRIG antibody provided herein) by conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) and specificity can be screened in the same manner as for intact antibodies.

[0072] As used herein, the term "monoclonal antibody" or "mAb" refers to a formulation of an antibody molecule consisting of a single molecular unit. Monoclonal antibodies exhibit single binding specificity and affinity for a specific epitope.

[0073] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0074] As used herein, the term "humanized antibody" refers to an antibody in which a CDR sequence derived from a lineage of another mammalian species (such as rat or mouse) has been grafted onto a human frame sequence. Additional frame region modifications may be made within the human frame sequence. Humanized antibodies may optionally also contain at least a portion of an immunoglobulin constant region (e.g., Fc), typically at least a portion of the constant region of human immunoglobulins.

[0075] As used herein, the term "human antibody" or "fully human antibody" is intended to include antibodies with variable regions, where both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, then that constant region is also derived from a human germline immunoglobulin sequence.

[0076] The term “PVRIG” or “Poliovirus Receptor Related Immunoglobulin Domain Containing Protein” includes known or wild-type PVRIG, or variants, conjugates, or fragments thereof (particularly ECD fragments). PVRIG is a transmembrane domain protein possessing a signal peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. PVRIG is expressed on the cell surface of NK cells and T cells and shares several similarities with other known immune checkpoints. Identification and methods for demonstrating that PVRIG is a checkpoint receptor can be found in WO2016 / 134333, which is incorporated herein by reference. An example of a human PVRIG amino acid sequence is shown in SEQ ID NO:14 (MRTEAQVPALQPPEPGLEGAMGHRTLVLPWVLLTLCVTAGTPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDPGLSAPPTPAPILRADLAGILGVSGVLLFGCVYLLHLLRRHKHRPAPRLQPSRTSPQAPRARAWAPSQASQAALHVPYATINTSCRPATLDTAHPHGGPSWWASLPTHAAHRPQGPAAWASTPIPARGSFVSVENGLYAQAGERPPHTGPGLTLFPDPRGPRAMEGPLGVR). The term “binding affinity” is used herein as a measure of the strength of the non-covalent interaction between two molecules (e.g., an antibody or its antigenic moiety with an antigen). The binding affinity between two molecules can be quantified using various assays, including surface plasmon resonance (SPR), flow cytometry (FACS), and kinetic exclusion assay (KinExA). Flow cytometry is a common technique for analyzing the binding of ligands to proteins presented on cell surfaces. In flow cytometry affinity assays, the equilibrium association constant (Ka, k) can be determined. on / k off ) and equilibrium dissociation constant (KD, i.e., K off / K on To evaluate protein binding affinity. Antibody binding kinetics and binding affinity can be assessed using standard assays known in the art or as described in Example 3.4 below.

[0077] As used in this article, the term "EC" 50 The term "half-maximum effective concentration" (MCI) refers to the concentration of a drug, antibody, or toxin that induces a response at half the baseline and maximum value after a specified exposure time.

[0078] As used in this article, the term "IC" 50 Also known as the "half-maximal inhibitory concentration," it refers to the half-maximal inhibitory concentration of a drug, antibody, or other substance. It is a measure of a substance's efficacy in inhibiting specific biological or biochemical functions.

[0079] As used herein, the term "separated" refers to a state obtained artificially from the natural state. If a "separated" substance or component exists in nature, 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, and a high-purity copy of the same polynucleotide or polypeptide isolated from such a natural state is called a separated polynucleotide or polypeptide. The term "separated" does not exclude the presence of mixed artificial or synthetic substances, nor does it exclude other impurities that do not affect the activity of the separated substance.

[0080] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to a PVRIG protein is substantially free of antibodies that specifically bind to antigens other than PVRIG proteins). However, isolated antibodies that specifically bind to human PVRIG proteins may be cross-reactive with other antigens, such as PVRIG proteins from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0081] As used herein, the term "vector" refers to a nucleic acid vehicle that may have intercalated polynucleotides. When a vector allows the expression of a protein encoded by an intercalated polynucleotide, it is called an expression vector. Vectors may have elements of carried genetic material that are expressed in a host cell through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, kinases, 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. Furthermore, vectors may contain an origin of replication.

[0082] As used herein, the term "host cell" refers to a cellular system that can be engineered to produce proteins, protein fragments, or peptides of interest. Host cells include, but are not limited to, cultured cells, such as mammalian cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term covers not only the specific subject cells but also the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may differ from the parent cells but are still included within the scope of the term "host cell."

[0083] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence alignment and comparison. "Identity percentage" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably resolved using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in: Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, 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, SIAMJ. Applied Math. 48:1073.

[0084] As used herein, 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 antigens stimulating the activation, proliferation, and differentiation of specific immune cells to ultimately generate immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response that antibodies or sensitized T lymphocytes can form in the organism's immune system after antigen stimulation. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce an immune response in a host depends on three factors: the characteristics of the antigen, the host's reactivity, and the immunization method.

[0085] As used herein, the term "transfection" refers to a process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. In a particular embodiment of the invention, the human PVRIG gene is transfected into 293F cells.

[0086] As used herein, the term “SPR” or “surface plasmon resonance” refers to and includes optical phenomena that allow for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see Example 5 and Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0087] As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". Retrieved 2017-11-09). Instruments used to perform FACS are known to those skilled in the art and are commercially available. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, Calif.), the Epics C from Coulter Epics Division (Hialeah, Fla.), and the MoFlo from Cytomation (Colorado Springs, Colo.).

[0088] As used herein, the term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. Antibodies “arm” cytotoxic cells and are absolutely necessary for this type of killing. Primary NK cells used to mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, such as those described in US Patent Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in animal models, such as those disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0089] The terms “subject” and “patient” are used interchangeably and include mammals, such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammal species. The term does not necessarily mean that a subject has been diagnosed with a specific disease, but generally refers to an individual under medical supervision.

[0090] As used in this article, the terms “prevent,” “prevention,” or “preventing” in relation to a disease condition in mammals refer to the prevention or delay of the onset of a disease, or the prevention of its clinical or subclinical manifestations.

[0091] As used in this article in the context of treating a condition, the terms “treatment,” “treating,” or “treated” generally refer to treatment and therapies, whether in humans or animals, in which some desired therapeutic effect is achieved, such as inhibiting the progression of the condition, and including a reduction in the rate of progression, cessation of the rate of progression, regression of the condition, improvement of the condition, and cure of the condition. In the context of cancer, “treatment” can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof.

[0092] As used herein, the term "effective amount" refers to an amount of an active compound or a material, composition, or dosage form containing an active compound that, when administered according to a desired treatment regimen, effectively produces some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, when used in connection with the treatment of a disease or condition, "effective amount" refers to an amount or concentration of an antibody or its antigen-binding portion that is effective in treating said disease or condition.

[0093] As used herein, the term "pharmaceutically acceptable" means that the medium, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other components in the formulation and physiologically compatible with the recipient.

[0094] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Gennaro AR, ed., 19th 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.

[0095] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with or before an antigen, can enhance the 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 adjuvants are more commonly used in clinical trials.

[0096] Anti-PVRIG antibody

[0097] In some aspects, this disclosure provides antibodies against PVRIGs (such as human, mouse, or cynomolgus monkey PVRIGs) or their antigen-binding moieties. Preferably, the antibodies are capable of binding to PVRIGs with sufficient affinity such that they substantially or completely block the interaction and / or binding of PVRIGs to PVLR2. When PVRIGs are bound by their ligand PVRL2, an inhibitory signal is triggered, which attenuates the immune response of NK cells and T cells against target cells. Blocking the binding of PVRL2 to PVRIGs shuts off this inhibitory signaling of PVRIGs and thus modulates the immune response of NK cells and T cells.

[0098] In some embodiments, the anti-PVRIG antibodies disclosed herein are fully human antibodies. In some embodiments, the anti-PVRIG antibodies disclosed herein are humanized antibodies. The antigen-binding moiety of the antibody may be Fab, Fab', F(ab')2, single-chain variable fragment (scFv), or a biantibody, etc. In some embodiments, the human antibody may contain further modifications to CDR residues and framework residues to remove potential post-translational modifications or improve antibody properties, such as binding affinity.

[0099] The antibodies disclosed herein can bind with high affinity to at least one of human, mouse, and cynomolgus monkey PVRIG. The binding of the disclosed antibodies to PVRIG can be assessed using one or more well-established techniques in the art, such as ELISA. The binding specificity of the disclosed antibodies can also be determined by monitoring the binding of the antibody to cells expressing the PVRIG protein (e.g., flow cytometry). For example, the antibody can be tested by flow cytometry in which the antibody reacts with a cell line expressing human PVRIG (such as HEK293 cells that have been transfected to express PVRIG on their cell surface). Additionally or alternatively, antibody binding, including binding kinetics (e.g., K... D The value can be tested in conjunction with BIAcore assays.

[0100] In some embodiments, the antibody or its antigen-binding portion is specifically capable of binding to human PVRIG and cynomolgus monkey PVRIG. For example, the antibody or its antigen-binding portion can bind to cells expressing human PVRIG at an EC50 of no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 0.1 nM, no more than 0.09 nM, or no more than 0.08 nM; and to cells expressing cynomolgus monkey PVRIG at an EC50 of no more than 0.5 nM, no more than 0.3 nM, no more than 0.1 nM, no more than 0.08 nM, no more than 0.06 nM, or no more than 0.04 nM, as measured by FACS. In some embodiments, the antibody or its antigen-binding portion of this disclosure is expressed at a concentration of 1 x 10⁻⁶. -9 M or smaller, 5 x 10 -10 M or smaller, 1 x 10 -10 M or smaller, 5 x 10 -11 M or smaller, 4 x 10 -11 M or smaller, 3 x 10 -11 M or smaller or 2 x 10 -11 M or smaller KD binds to human PVRIG, as measured in the FACS affinity test.

[0101] The anti-PVRIG antibody disclosed herein inhibits the interaction between PVRIG and PVRL2 (CD112). Blocking signaling via PVRL2 can restore functional T cell responses to antigen stimulation (e.g., proliferation, cytokine production, target cell killing) from a dysfunctional state. The ability of the anti-PVRIG antibody to inhibit such interactions can be assessed by measuring whether the physical interaction between PVRIG and CD112 is reduced in a binding assay. Binding assays are typically competitive binding assays. These assays can be performed in various forms, such as, but not limited to, ELISA assays, flow cytometry, and surface plasmon resonance (SPR) assays (e.g., Biacore). TM ) or biomembrane interference (e.g., ForteBio Octet) TM In some implementations, the anti-PVRIG antibody described herein can block the binding of human PVRL2 to PVRIG with an IC50 of no more than 0.5 nM, no more than 0.4 nM, or no more than 0.3 nM, as measured by ELISA.

[0102] Anti-PVRIG antibodies containing CDR

[0103] In some embodiments, this disclosure provides isolated antibodies or antigen-binding portions thereof, comprising:

[0104] A) Select one or more heavy chain CDRs (HCDRs) from the following groups:

[0105] HCDR1, comprising an amino acid sequence that differs from SEQ ID NO: 1 by not more than two amino acid additions, deletions, and / or substitutions; HCDR2, comprising an amino acid sequence that differs from SEQ ID NO: 2 by not more than two amino acid additions, deletions, and / or substitutions; and HCDR3, comprising an amino acid sequence that differs from SEQ ID NO: 3 by not more than two amino acid additions, deletions, and / or substitutions.

[0106] B) Select one or more light chain CDRs (LCDRs) from the group consisting of the following:

[0107] LCDR1, comprising an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence differing from SEQ ID NO: 4 by not more than two amino acid additions, deletions, and / or substitutions; LCDR2, comprising an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence differing from any one of SEQ ID NO: 5 by not more than two amino acid additions, deletions, and / or substitutions; and LCDR3, comprising an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence differing from SEQ ID NO: 6 by not more than two amino acid additions, deletions, and / or substitutions; or

[0108] C) One or more HCDRs of A) and one or more LCDRs of B).

[0109] In some implementations, CDR identification is based on the IMGT / Kabat definition.

[0110] In some embodiments, this disclosure provides isolated antibodies or their antigen-binding portions comprising: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6.

[0111] The scope of the frame region and CDR can be precisely identified using methods known in the art, such as by the Kabat definition, Chothia definition, AbM definition, contact definition, IMGT definition (all of which are well known in the art) and any combination thereof. See, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci US A. 1969 May, 63(1):78-85; and Martin and Allen, in “Handbook of Therapeutic Antibodies”, Chapter 5, 2007. See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. Correspondences or alignments between the numberings according to different definitions can be found, for example, at www.imgt.org / (see also Giudicelli V et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25:206–11; and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev CompImmunol. (2003) 27:55–77).

[0112] As those skilled in the art will understand, the exact numbering and location of CDRs can differ in different numbering systems. However, it should be understood that regardless of the numbering method or definition scheme used, the disclosure of variable heavy chain sequences and / or variable light chain sequences includes the disclosure of the associated (inherent) CDR. Therefore, the disclosure of each variable region is a disclosure of a CDR (e.g., HCDR1, HCDR2, and HCDR3). Two antibodies having the same VH and VL mean that their CDRs are identical when determined by the same method (e.g., as defined by Kabat, AbM, Chothia, Contact, and IMGT as known in the art). The same antibodies disclosed herein can have different sets of CDRs when determined by different definition schemes.

[0113] In some embodiments, this disclosure provides isolated antibodies or antigen-binding portions thereof comprising one, two, or all three HCDRs of the amino acid sequence shown in SEQ ID NO: 7, and one, two, or all three LCDRs of the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, this document provides anti-PVRIG antibodies comprising one, two, or all three HCDRs of the amino acid sequence shown in SEQ ID NO: 7, and one, two, or all three LCDRs of the amino acid sequence shown in SEQ ID NO: 9.

[0114] In some embodiments, this disclosure provides isolated antibodies or their antigen-binding portions comprising HCDR1, HCDR2, and HCDR3 of the VH sequence shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 of the VL sequence shown in SEQ ID NO: 8. In some embodiments, this document provides anti-PVRIG antibodies comprising HCDR1, HCDR2, and HCDR3 of the VH sequence shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 of the VL sequence shown in SEQ ID NO: 9.

[0115] Variable regions and CDRs in antibody sequences can be identified according to general rules developed in the art or by comparing the sequence with a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described below and can be accessed through the following: the “Abysis” website at www.bioinf.org.uk / abs (maintained by AC Martin of the Department of Biochemistry & Molecular Biology University College London, London, England) and the VBASE2 website at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Sequences can be analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Database (PDB) with structural data from the PDB. See the chapter "Protein Sequence and Structure Analysis of Antibody Variable Domains" in Dr. Andrew CR Martin's book. See also: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available at bioinforg.uk / abs). The Abysis database website also includes general rules that have been developed for identifying CDRs that can be used according to the teachings herein.

[0116] In some embodiments, the anti-PVRIG antibody disclosed herein comprises the following VH and VL regions, wherein the VH region comprises FRW1-HCDR1-FRW2-HCDR2-FRW3-HCDR3-FRW4, and wherein HCDR1 has the amino acid sequence shown in SEQ ID NO: 1, HCDR2 has the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 has the amino acid sequence shown in SEQ ID NO: 3, and / or wherein the VL region comprises FRW1-LCDR1-FRW2-LCDR2-FRW3-LCDR3-FRW4, and wherein LCDR1 has the amino acid sequence shown in SEQ ID NO: 4, LCDR2 has the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 6.

[0117] In some embodiments, the framework region is derived from human germline, such as human immunoglobulin. In some embodiments, certain residues in the framework region are mutated to improve antibody performance, such as stability, binding affinity, isomerization, immunogenicity, etc. In some embodiments, S7 in FRW1 of the VL region and / or T43 (according to Kabat number) in FRW2 of the VL region are mutated. In some specific embodiments, the VL region of the antibody contains an S7P / T43A substitution. In some embodiments, FRW1 and FRW4 at the N-terminus and C-terminus of the VH region and / or VL region may be truncated, such that they contain only a portion of FRW1 and / or FRW4. In some embodiments, the CDR and FR regions have undergone PTM removal optimization.

[0118] In some embodiments, this document provides an anti-PVRIG antibody comprising at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 7, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 8. In some embodiments, this document provides an anti-PVRIG antibody comprising at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 7, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 9.

[0119] Anti-PVRIG antibodies containing heavy chain variable regions and light chain variable regions

[0120] In some implementations, the isolated antibody or its antigen-binding portion comprises:

[0121] (A) Heavy chain variable region (VH):

[0122] (i) Contains the amino acid sequence of SEQ ID NO: 7;

[0123] (ii) Contains an amino acid sequence having the same CDR set as SEQ ID NO: 7 and having at least 85%, 90%, or 95% identity in the frame region; or

[0124] (iii) An amino acid sequence comprising one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acids added, deleted, and / or substituted amino acids in the frame region compared to the amino acid sequence of SEQ ID NO: 7; and / or

[0125] (B) Light chain variable region (VL):

[0126] (i) Contains an amino acid sequence of SEQ ID NO: 8 or 9;

[0127] (ii) Contains an amino acid sequence having the same CDR set as SEQ ID NO: 8 or 9 and having at least 85%, 90%, or 95% identity in the frame region; or

[0128] (iii) An amino acid sequence comprising one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acids added, deleted, and / or substituted in the frame region compared to the amino acid sequence of SEQ ID NO: 8 or 9.

[0129] The percentage identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) in the ALIGN program (version 2.0), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) in the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum62 matrix or a PAM250 matrix, and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0130] Alternatively or additionally, the protein sequences disclosed herein can be further used as “query sequences” to perform searches against public databases to, for example, identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) as described in Altschul, et al. (1990) J.MoI. Biol. 215:403-10. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. For obtaining gap-bound alignments for comparative purposes, Gapped BLAST, as described in Altschul et al. (1997) Nucleic AcidsRes. 25(17):3389-3402, can be used. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0131] In some further embodiments, the isolated antibody or its antigen-binding moiety may contain conserved substitutions or modifications of amino acids in the variable regions of the heavy and / or light chains. It will be understood in the art that certain conserved sequence modifications that do not remove antigen binding can be performed. See, for example, Brummell et al. (1993) Biochem 32:1180-8; de Wildtet al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem.272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol.10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0132] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein / peptide comprising an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which one amino acid residue is replaced by another amino acid residue having a similar side chain, such that the other amino acid residue is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc.Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference). In some further embodiments, antibodies or their antigen-binding moieties, as disclosed herein, have conserved substitutions at the S7 and / or T43 positions (according to Kabat numbering) in the VL region.

[0133] In some embodiments, the isolated antibody or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or thereof, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or 9 or thereof.

[0134] In other embodiments, the amino acid sequences of the heavy chain variable region and / or the light chain variable region may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequences described above.

[0135] Fc area

[0136] The anti-PVRIG antibody and antigen-binding portion provided herein further includes an immunoglobulin constant region, such as the human IgG1, IgG2, IgG3, or IgG4 immunoglobulin constant region (natural or a variant thereof), which includes an Fc region and an optional hinge region. The anti-PVRIG antibody of this document may be an IgG1 or IgG4 isotype. In some embodiments, the Fc region is a natural Fc region or an Fc variant. The Fc variant may have at least about 80% homology with the natural sequence Fc region, or at least about 90% homology with it, for example, at least about 95% homology. In some embodiments, the Fc region is a human IgG4 Fc region, such as a wild-type Fc region or an Fc variant containing an S228P substitution. In some embodiments, the antibody disclosed herein includes a wild-type human IgG1 Fc region.

[0137] In some implementations, the Fc region contains one or more amino acid variations (e.g., insertions, deletions, or substitutions) that result in a modified binding interaction between Fc and FcRn or FcγR.

[0138] In some embodiments, the Fc region is the IgG4 Fc region containing the S228P mutation (according to the EU numbering in Kabat et al.) that prevents Fab arm exchange and stabilizes the IgG4 molecule. In some embodiments, the Fc region is the IgG1 Fc region and contains the LALA mutation, namely the L234A and L235A mutations. The LALA mutation is perhaps the most commonly used mutation to disrupt antibody effector function, such as eliminating the binding of Fc to specific FcγR and reducing ADCC activity mediated by PBMCs and monocytes. When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., the EU index reported in Kabat et al., ibid.). “EU number in Kabat” or “EU index in Kabat” refers to the residue number of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the antibody constant domain refer to residue numbers obtained through the EU numbering system.

[0139] In some implementations, the hinge region may be derived from the same human IgG immunoglobulin as the Fc region.

[0140] Anti-PVRIG antibodies with certain properties

[0141] The antibodies disclosed herein are characterized by specific functional features or properties. Based on their target-target mechanism of action, the in vitro functional properties and pharmacological activity of the antibodies have been fully evaluated at both the molecular and cellular levels. In some embodiments, the isolated antibody or its antigen-binding moiety possesses one or more of the following properties:

[0142] (a) Specifically binds to at least one of human PVRIG protein and cynomolgus monkey PVRIG protein;

[0143] (b) It does not cross-bind with PVRIG paralogous proteins;

[0144] (c) Blocking the binding between PVRIG and its ligand PVRL2 (CD112);

[0145] (d) Activation of immune cells such as T cells and NK cells;

[0146] (e) It exhibits good serum stability and thermal stability; and

[0147] (f) It showed significantly better efficacy than the baseline antibody in the treatment of cancer.

[0148] As illustrated in the examples, CTLs (cytotoxic T lymphocytes, such as CD8+ T cells) and NK cells can be used in functional assays to assess antibody function. CTLs express T cell receptors (TCRs) that recognize specific antigens (Ag) presented on MHC molecules. Upon TCR Ag binding, CTLs undergo activation, as indicated by cell proliferation, upregulation of activation markers (e.g., CD35, CD137), cytokine secretion, and cytotoxic activity. Upon contact with PVRIG expressed on cancer cells or antigen-presenting cells, PVRIG mediates negative signaling in CTLs, leading to downregulation of CTL activation. Therefore, contacting CTLs with anti-PVRIG antibodies that exhibit binding and / or inhibition of receptor-ligand binding will interrupt the PVRIG-PVRL2 interaction, thereby releasing the PVRIG-mediated negative signaling and enhancing antigen-specific CTL activation, as indicated by cell proliferation, upregulation of activation markers (e.g., CD25, CD137, etc.), and cytokine secretion (e.g., interferon-γ, IL2, TNF-α, etc.). In some implementations, human CD8+ T cells treated with anti-PVRIG antibodies resulted in greater IFN-γ secretion compared to IFN-γ secretion observed with isotype controls and reference antibodies.

[0149] NK cells express various activating and inhibitory receptors. Upon contact with PVRL2 expressed on cancer target cells, PVRIG mediates a negative signal to NK cells, thereby causing a downregulation of NK cell activation. Contacting NK cells with anti-PVRIG antibodies will interrupt the PVRIG-PVRL2 interaction, thereby releasing the PVRIG-mediated negative signal and enhancing NK cell activation, as demonstrated by cell proliferation, cytokine (e.g., interferon-γ, IL2, TNF-α, etc.) secretion, and / or cytotoxic activity.

[0150] Methods of generating antibodies

[0151] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, and transgenic animals (e.g., XenoMouse). ® (or some combination thereof.) For example, monoclonal antibodies can be produced using hybridomas and well-established biochemical and genetic engineering techniques, such as those described in more detail below: An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1 st ed. 2009; Shire et. al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science +Business Media LLC, 1 st The following are cited in reference to: 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. It should be understood that the selected binding sequence can be further modified, for example, to improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing modified target binding sequences are also antibodies of the present invention.

[0152] To obtain human antibodies with minimal immunogenicity, transgenic mouse technologies have been developed that allow the generation of fully human therapeutic mAbs. OmniRat (Open Monoclonal Technology Company) is a transgenic mouse carrying a chimeric human / rat IgH locus (containing 22 human V... H With natural configuration and rat C H All D and J people connected by the gene locus HTransgenic rats containing the human IgL locus (12 Vκ linked to Jκ-Cκ and 16 Vλ linked to Jλ-Cλ) were used. Endogenous Ig loci were silenced using designer zinc finger nucleases. OmniRat rats can produce antibodies with human-specific characteristics as efficiently as wild-type animals.

[0153] Nucleic acid molecules encoding the antibodies disclosed herein

[0154] In some respects, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of isolated antibodies as disclosed herein.

[0155] The nucleic acids disclosed herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), the cDNA encoding the light and heavy chains of the antibody prepared from the 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 acids encoding such antibodies can be recovered from the gene library.

[0156] The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant domains (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), ibid.), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. 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.

[0157] By operatively linking the DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into full-length light chain genes (and Fab light chain genes). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be either a κ or λ constant region.

[0158] Once the DNA fragments encoding the VH and VL regions are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. As used herein, the term "operatively linked" is intended to mean the joining of two DNA fragments such that the amino acid sequences encoded by both fragments remain within a frame.

[0159] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions of isolated antibodies as disclosed herein. In some specific embodiments, the isolated nucleic acid molecules encode heavy chain variable regions of isolated antibodies and comprise nucleic acid sequences selected from the group consisting of:

[0160] (A) The nucleic acid sequence encoding the heavy chain variable region shown in SEQ ID NO: 7;

[0161] (B) The nucleic acid sequence shown in SEQ ID NO: 10; or

[0162] (C) Nucleic acid sequences that hybridize with the complementary strand of (A) or (B) nucleic acid sequences under highly stringent conditions.

[0163] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding a light chain variable region of an isolated antibody as disclosed herein.

[0164] In some specific implementations, the isolated nucleic acid molecule encoding the light chain variable region of the isolated antibody comprises a nucleic acid sequence selected from the group consisting of:

[0165] (A) A nucleic acid sequence encoding the light chain variable region shown in SEQ ID NO: 8 or 9;

[0166] (B) The nucleic acid sequence shown in SEQ ID NO: 11; or

[0167] (C) Nucleic acid sequences that hybridize with the complementary strand of (A) or (B) nucleic acid sequences under highly stringent conditions.

[0168] For example, the nucleic acid molecule contains SEQ ID NO: 10 and 11. In some other embodiments, the nucleic acid molecule shares at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 10 or 11. In some specific embodiments, the identity percentage is derived from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0169] Exemplary high-strict conditions include hybridization at 45°C in 5X SSPE and 45% formamide, followed by a final wash at 65°C in 0.1X SSC. As described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10, it will be understood in the art that conditions of equivalent strictness can be achieved through variations in temperature and buffer or salt concentration. Modifications to hybridization conditions can be determined empirically or precisely calculated based on the length and percentage of guanosine / cytosine (GC) base pairings of the probe. Hybridization conditions can be calculated as described in Sambrook, et al. (Eds.), Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.

[0170] host cells

[0171] The host cells disclosed in this disclosure can be any cells suitable for expressing the antibodies of this disclosure, such as yeast, bacteria, fungi, plant and animal cells, preferably mammalian cells. Mammalian host cells for expressing the antibodies of this disclosure include Chinese hamster ovary (CHO) cells (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, which are used with DHFR selective markers, for example, as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621), 293F cells, NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841.This also includes monkey kidney CV1 line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or subclones used for 293 cells grown in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); and human cervical cancer cells (HELA, ATCC). CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; mouse myeloma cells, such as NSO (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL 1442); 1581); rat myeloma cells, such as YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL 1662); PER.C6 cells; and the human hepatocellular carcinoma line (Hep G2). CHO cells are one of the cell lines that can be used in this study, of which CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)) and Lec13 are exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44, or CHO-DP12 host cells, these host cells can be altered to lack the ability to fucosylate proteins expressed therein.In some implementations, the host cells used in this paper are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C 6, or NSO cells or lymphocytes.

[0172] Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans) and Shigella, and Bacilli (e.g., B. subtilis and B. licheniformis), and Pseudomonas (e.g., Pseudomonas aeruginosa). aeruginosa and Streptomyces.

[0173] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or Bacillus oryzae are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and can be used in this paper, such as *Schizosaccharomyces pombe*; hosts of the genus *Kluyveromyces*, such as, for example, *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wickeramii* (ATCC 24,178), *Kluyveromyces waltii* (ATCC 56,500), *Kluyveromyces drosophilarum* (ATCC 36,906), *Kluyveromyces thermomotolerans*, and *Kluyveromyces marxianus*; *Yarrowia* (EP 402,226); and *Pichia pastoris* (EP 402,226). 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as hosts of Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as Aspergillus nidulans and Aspergillus niger.

[0174] When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow the antibody to be expressed within the host cells or by secreting the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0175] Pharmaceutical Composition

[0176] In some aspects, this disclosure relates to pharmaceutical compositions comprising at least one antibody or antigen-binding moiety thereof as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising a nucleic acid encoding an antibody or antigen-binding moiety thereof as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising cells expressing an antibody or antigen-binding moiety thereof as disclosed herein and a pharmaceutically acceptable carrier.

[0177] Components of the composition

[0178] The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions disclosed herein may also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent, or 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, other components known in the art, and various combinations thereof.

[0179] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoglycolic acid, mercaptosorbitol, butylated anisole, butylated hydroxytoluene, and / or propyl gallate. For example, compositions containing antibodies or antigen-binding fragments of this disclosure may include one or more antioxidants, such as methionine, as reducing antibodies or their antigen-binding fragments may be oxidized. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this disclosure provides compositions comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. This disclosure also provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, thereby preventing oxidation of the antibody or its antigen-binding fragment to extend its shelf life and / or increase its activity.

[0180] 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 lactate Ringer's injection; non-aqueous media such as plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or dextran; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (Tween-80); sequestering agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers. These antimicrobial agents include phenols or cresols, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride. 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 pharmaceutical agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0181] Application, preparation and dosage

[0182] The pharmaceutical compositions disclosed herein can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal administration, or otherwise via implantation or inhalation. The subject compositions can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on the intended application and therapeutic regimen.

[0183] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalers, and their controlled-release forms.

[0184] 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). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of suitable isotonic carriers for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific dosing regimen, including dose, time, and repetition, will depend on the individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0185] The frequency of administration can be determined and adjusted during the course of therapy, and the frequency of administration is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such proliferative cells, reducing the proliferation of proliferative cells, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the subject therapeutic composition may be suitable.

[0186] Those skilled in the art will understand that appropriate dosage may vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or harmful side effects. The chosen dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular compound, route of administration, time of administration, rate of excretion of the compound, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and the patient's species, sex, age, weight, condition, general health, and medical history. The amount of the compound and the route of administration will ultimately be determined by a physician, veterinarian, or clinician, although a dosage will generally be chosen to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or detrimental side effects.

[0187] Typically, the antibodies or antigen-binding portions thereof disclosed herein can be administered in a variety of ranges. These include about 5 μg / kg body weight to about 40 mg / kg body weight per dose; about 50 μg / kg body weight to about 5 mg / kg body weight per dose; and about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, and at least about 10 mg / kg body weight.

[0188] In any case, the antibody or its antigen-binding portion thereof disclosed herein is preferably administered to the subject as needed. The frequency of administration can be determined by those skilled in the art, such as attending physicians, based on considerations of the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health status of the subject being treated.

[0189] In some preferred embodiments, a treatment course involving the antibody or its antigen-binding portion of this disclosure will comprise multiple doses of the selected pharmaceutical product over a period of weeks or months. More specifically, the antibody or its antigen-binding portion of this disclosure may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it should be understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

[0190] The dosage and regimen of the disclosed therapeutic composition can also be determined empirically in individuals who have been given one or more doses. For example, individuals can be given incremental doses of the therapeutic composition prepared as described herein. In selected embodiments, the dosage can be gradually increased, decreased, or reduced based on empirically determined or observed side effects or toxicities. To assess the efficacy of the selected composition, biomarkers of a specific disease, symptom, or condition can be tracked as previously described. For cancer, these include direct measurement of tumor size via palpation or visual observation, indirect measurement of tumor size via X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor biomarkers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein; reduction in pain or paralysis; improvement in tumor-related speech, vision, breathing, or other disabilities; increased appetite; or an increase in quality of life or prolonged survival as measured by recognized tests.

[0191] Compatible formulations for parenteral administration (e.g., intravenous injection) may comprise an antibody or its antigen-binding portion at a concentration of about 10 μg / ml to about 100 mg / ml as disclosed herein. It will be apparent to those skilled in the art that the dosage of the antibody or its antigen-binding portion disclosed herein may vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other sites in the individual, past and current concomitant treatments, and the dosage of therapeutic agents used in combination with the antibodies disclosed herein.

[0192] Application of this disclosure

[0193] The antibodies, antibody compositions, and methods disclosed herein have numerous in vitro and in vivo utilities, including, for example, detection of PVRIG or enhancement of immune responses. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects, to enhance immunity in various situations. Immune responses can be modulated, for example, enhanced, stimulated, or upregulated.

[0194] For example, subjects include patients who require enhanced immune responses. The method is particularly suitable for treating patients with conditions that can be treated by enhancing immune responses (e.g., T-cell-mediated immune responses). In a particular embodiment, the method is particularly suitable for treating cancer in vivo, including cancer-induced immunosuppression.

[0195] The disclosed antibody can be combined with additional therapeutic agents, such as anticancer agents, including anticancer antibodies and chemotherapeutic agents. The additional therapeutic agents can also be antagonists or inhibitors of T-cell co-inhibitors, agonists of T-cell co-activating factors, or immunostimulatory cytokines. When the anti-PVRIG antibody is administered with another agent, such as an anti-PD-1 agent, the two can be administered in any order or simultaneously.

[0196] This disclosure also provides a method for detecting the presence of PVRIG antigen in a sample or measuring the amount of PVRIG antigen, comprising contacting a sample and a control sample with an anti-PVRIG antibody or its antigen-binding portion under conditions that allow the formation of a complex between the antibody or a portion thereof and PVRIG. The formation of the complex is then detected, wherein the difference in complex formation between the sample and the control sample indicates the presence of PVRIG antigen in the sample. Furthermore, the anti-PVRIG antibody of this disclosure can be used to purify PVRIG via immunoaffinity purification.

[0197] Treatment of conditions including cancer

[0198] In some aspects, this disclosure provides methods for treating conditions or diseases in mammals, comprising administering to a subject (e.g., a human) a therapeutically effective amount of an anti-PVRIG antibody or its antigen-binding portion as disclosed herein. Conditions or diseases include, but are not limited to, proliferative conditions (such as cancer), immune conditions, inflammatory diseases, or infectious diseases. For example, a condition may be cancer.

[0199] In some implementations, the cancer is a cancer enriched with PRVL2 expression. In some implementations, the cancer is a cancer enriched with T cells or natural killer (NK) cells expressing PVRIG.

[0200] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoma. More specific examples of such cancers include, but are not limited to, lung cancer, such as non-small cell lung cancer (NSCLC), which includes squamous NSCLC or non-squamous NSCLC, including locally advanced unresectable NSCLC (e.g., stage IIIB NSCLC) or recurrent or metastatic NSCLC. (e.g., stage IV NSCLC), lung adenocarcinoma or squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); esophageal cancer; peritoneal cancer; hepatocellular carcinoma; gastric cancer or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer (e.g., urothelial bladder cancer (UBC), muscle-invasive bladder cancer (MIBC), and BCG-refractory non-muscle-invasive bladder cancer (NMIBC)); urinary tract cancer; liver cancer; breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney cancer. Cancer of the kidneys or renal cells (e.g., renal cell carcinoma (RCC)); prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, including superficial diffuse melanoma, malignant lentiginesoid melanoma, acral lentiginesoid melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL. (high-grade small non-cleaved cell NHL); bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myogenic leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); myelodysplastic syndrome (MDS), as well as abnormal angiogenesis, edema (such as edema associated with brain tumors) associated with phakomatoses, Meigs' syndrome, brain cancer, head and neck cancer, and related metastases.

[0201] As a co-inhibitory receptor on various immune cells, PVRIG is involved in a variety of cancers, whether malignant or benign, primary or secondary, which can be treated or prevented by the methods provided in this disclosure. Preferably, the anti-PVRIG antibody disclosed herein is administered in combination with another anticancer agent. The cancer can be a solid tumor or a hematologic malignancy. Examples of such cancers include lung cancer, such as bronchogenic carcinoma (e.g., non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous), and sarcoma (cancerous); cardiac cancer, such as myxoma, fibroma, and rhabdomyosarcoma; bone cancer, such as osteochondroma, chondroma, chondroblastoma, chondromycinoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's disease, etc. Tumors (Ewing sarcoma) and reticulum cell sarcoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymomas, meningiomas, pituitary adenomas, pineal tumors, osteomas, angioblastomas, craniopharyngiomas, chordomas, germ cell tumors, teratomas, dermoid cysts, and hemangiomas; digestive system cancers such as colon cancer, leiomyomas, epidermoid carcinomas, adenocarcinomas, etc. Leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, enterofibroma, intestinal fibroma, colorectal polyps, and colorectal cancer; liver cancer, such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancer, such as renal adenocarcinoma, renal cell carcinoma, hypernephroma, and transitional cell carcinoma of the renal pelvis; bladder cancer; skin cancer, such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancer; eye-related cancers such as retinal tumors. Cancers include: melanoma and intraocular melanoma; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial cancer), cervical cancer, ovarian cancer, vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous growths of the parathyroid glands; and pancreatic cancer. In some implementations, the cancer is colon cancer.

[0202] In some other embodiments, the condition or disease to be treated or prevented is an immune-related disease. Immune-related diseases may be associated with T-cell dysfunction. In some embodiments, T-cell dysfunction is characterized by reduced responsiveness to antigen stimulation. In some embodiments, T-cell dysfunction is characterized by T-cell unresponsiveness or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In some embodiments, T-cell dysfunction is characterized by T-cell exhaustion. In some embodiments, the T cells are CD4+ T cells and CD8+ T cells. In some embodiments, the immune-related disease is selected from the group consisting of: unresolved acute infection, chronic infection, and reduced tumor immunity.

[0203] Stimulating immune response

[0204] In some aspects, this disclosure also provides methods for enhancing (e.g., stimulating) an immune response in a subject, comprising administering to the subject an antibody of this disclosure or an antigen-binding portion thereof, such that an immune response in the subject is enhanced. For example, the subject is a mammal. In a particular embodiment, the subject is a human.

[0205] The term "enhanced immune response" or its grammatical variations refer to any response that stimulates, induces, increases, improves, or enhances the mammalian immune system. An immune response can be a cellular response (i.e., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (i.e., antibody-mediated), and can be a primary or secondary immune response. Examples of enhanced immune responses include increased CD4+. + Helper T cell activity and generation of cytolytic T cells. Enhancement of the immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, tumor-bearing animal survival, antibody production, immune cell proliferation, cell surface marker expression, and cytotoxicity. Generally, the methods of this disclosure enhance the immune response in mammals when compared to the immune response in untreated mammals or mammals not treated with the methods disclosed herein. In one embodiment, the antibody or its antigen-binding portion is used to enhance a human immune response to a microbial pathogen, such as a virus. In another embodiment, the antibody or its antigen-binding portion is used to enhance a human immune response to a vaccine. In one embodiment, the method enhances a cellular immune response, particularly a cytotoxic T cell response. In another embodiment, the cellular immune response is a T helper cell response. In yet another embodiment, the immune response is cytokine production, particularly IFN-γ production or IL-2 production. The antibody or its antigen-binding portion can be used to enhance a human immune response to a microbial pathogen, such as a virus, or a vaccine.

[0206] Antibodies or their antigen-binding portions can be used alone as a monotherapy or in combination with other antibodies (such as anti-PD-1 or anti-PD-L1 antibodies), chemotherapy, radiotherapy, targeted therapy, or cell immunotherapy.

[0207] Used in combination with chemotherapy

[0208] Antibodies or their antigen-binding portions can be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0209] The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat proliferative disorders such as cancer, and include, but are not limited to, cytotoxic agents, cell growth inhibitors, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiation therapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and antimetastatic agents, and immunotherapeutic agents. It should be understood that such anticancer agents may comprise conjugates and may be associated with the disclosed antibody prior to administration. More specifically, in some embodiments, the selected anticancer agent will be linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate. Therefore, such engineered conjugates are explicitly considered to be within the scope of this disclosure. In some other embodiments, the anticancer agent will be administered in combination with an antibody-drug conjugate comprising a different therapeutic agent.

[0210] As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell damage. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, the following small molecule toxins or enzymatically active toxins: bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-ascorbic acid toxin, restrictocin), plants (e.g., acacia toxin, ricin, senna root toxin, mistletoe lectin, American pokeweed antiviral protein, saponin, white jatropha toxin, momoridin, trichosanthes pollen protein, barley toxin, tung oil (Aleurites fordii) protein, carnation toxin, Phytolacca mericana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, jatropha toxin, croton toxin, saponaria toxin, etc. Inhibitors of officinalis, white tree poison protein, mitegellin, localized aspergillin, phenomycin, neomycin and trichothecenes) or animals (e.g. cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including its fragments and / or variants).

[0211] For the purposes of this disclosure, "chemotherapeutic agents" include compounds (e.g., cytotoxic agents or cell growth inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemical agents typically target intracellular processes necessary for cell growth or division and are therefore particularly effective against cancerous cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules and thereby inhibits cells from entering mitosis. Generally, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or generate tumorigenic progeny (e.g., TICs). Such agents are often administered in combination and are often the most effective, for example, in regimens such as CHOP or FOLFIRI.

[0212] Examples of anticancer agents that can be used in combination with the antibodies disclosed herein (as a component of a site-specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridine, ethylenimine and methylamelamine derivatives, acetogenin, camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, and pancrati statin), sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enedyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromogens of enedyne antibiotics, aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, actinomycin C, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN ®Doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin Streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-adrenergics, folic acid supplements (such as frolinic acid) acid), aceglatone, aldehyde phosphoramide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etogluci d) Gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazine, methylbenzylhydrazine, PSK ®Polysaccharide complexes (JHS Natural Products, Eugene, OR), razorcinol; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaminoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine; urethan; vindesine; dacarbazine; mannitol mustard; dibromomannitol; mitolactal; pipebroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids; chloranbucil; GEMZAR ® Gemzar ® Gemcitabine; 6-thioguanine; mercaptopurine; methamidopurine; platinum analogues, vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine ® Changchun Ruibin (NAVELBINE) ®Vinorelbine; Novantrone; Teniposide; Edatraxate; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS2000; Difluoromethylornithine; Retinoids; Capecitabine; Combretastatin; Leucovorin; Oxaliplatin; Inhibitors of PKC-α, Raf, H-Ras, EGFR, and VEGF-A (which reduce cell proliferation) and any of the above in pharmaceutically acceptable salts, acids, or derivatives. This definition also includes antihormonal agents used to regulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors (which inhibit aromatase, an enzyme that regulates estrogen production in the adrenal glands), and anti-androgens; as well as troxacitabine (a 1,3-dioxane cyclopentane nucleoside cytosine analog); antisense oligonucleotides; ribonucleases, such as VEGF expression inhibitors; and the vaccine PROLEUKIN. ® rIL-2; LURTOTECAN ® Topoisomerase 1 inhibitor; ABARELIX ® rmRH; vinorelbine and esperamicin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0213] Used in combination with radiotherapy

[0214] This disclosure also provides combinations of antibodies or their antigen-binding portions with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as gamma radiation, X-rays, UV radiation, microwaves, electron emission, etc.). Combination therapies using the targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed antibodies can be used in conjunction with targeted anticancer agents or other targeted approaches. Typically, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple sequential doses.

[0215] Drug packaging and reagent kits

[0216] Pharmaceutical packages and kits comprising one or more containers containing one or more doses of an antibody or its antigen-binding portion 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 portion thereof, with or without one or more additional pharmaceutical agents. For other embodiments, such unit doses are supplied in single-use pre-filled syringes for injection. In yet another embodiment, the composition contained in the unit dose may comprise saline, sucrose, etc.; buffers, such as phosphates, etc.; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that 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 encapsulated antibody is intended for the treatment of a selected aplastic disease condition.

[0217] This disclosure also provides kits comprising single- or multiple-dose administration units of antibodies and optionally one or more anticancer agents. The kit includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from various materials such as glass or plastic and contains a pharmaceutically effective amount of the disclosed antibody. In some embodiments, the container includes a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the antibody in a suitable container and optionally one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy purposes. For example, in addition to the antibodies or their antigen-binding portions disclosed herein, such kits may contain any one or more of a range of anticancer agents such as chemotherapy or radiotherapy drugs; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0218] More specifically, the kits may have a single container containing an antibody or its antigen-binding moiety, with or without additional components, or they may have different containers for each desired agent. In cases where combination therapy is provided for conjugation, single solutions may be combined in molar equivalents or premixed with one component in greater quantities than another. Alternatively, the antibody and any optional anticancer agent in the kit may be maintained separately in different containers prior to administration to the patient. The kit may also include a second / third container for containing sterile, pharmaceutically acceptable buffers or other diluents, such as antibacterial water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextran solution.

[0219] When the reagent kit components are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, with sterile aqueous solutions or saline solutions being particularly preferred. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent.

[0220] As briefly noted above, the kit may also contain means by which an antibody or its antigen-binding portion and any optional components are administered to a patient, such as one or more needles, IV bags, or syringes, or even eye drops, pipettes, or other similar devices, through which the preparation may be injected or introduced into an animal or applied to a diseased area of ​​the body. The kits disclosed herein will also typically include means for containing vials, etc., and other tightly controlled components for commercial sale, such as injection or blow-molded plastic containers in which the desired vials and other devices are placed and held.

[0221] Summary of sequence lists

[0222] This application includes a sequence listing containing numerous nucleic acid and amino acid sequences. Tables A, B, and C below provide a summary of the included sequences.

[0223] Table A: CDR sequences of antibodies

[0224] HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 WT1175-1.158.12-m1-uIgG1L; WT1175-1.158.12-uIgG4LV1; WT1175-1.158.12-uIgG1L GGTFSSYTIS (SEQ ID NO: 1) GIIPIFGSADYAQKFQG (SEQ ID NO: 2) EGLTGYSSFDY (SEQ ID NO: 3) TGTSSDIGSYKFVS (SEQ ID NO: 4) EGSKRPS (SEQ ID NO: 5) SSYLGSGTVV (SEQ ID NO: 6)

[0225] Table B: Amino acid and nucleic acid sequences of the variable region

[0226]

[0227]

[0228] Table C: Sequences of heavy and light chains

[0229]

[0230] Example

[0231] The present disclosure, which is generally described herein, will be more readily understood by referring to the following embodiments, which are provided by way of illustration and are not intended to limit the present disclosure. The embodiments are not intended to represent all or only the experiments conducted.

[0232] Example 1

[0233] Preparation of antigens, reference antibodies and cell lines

[0234] 1.1 Antigen generation

[0235] WT117-hPro1.ECD.His is the extracellular domain of human PVRIG (NP_076975.2), which has a C-terminal multihistidine tag; WT117-hPro1.ECD.hFc is the extracellular domain of human PVRIG (NP_076975.2), which has a C-terminal Fc region of human IgG1; WT117-mPro1.ECD.His is the extracellular domain of mouse PVRIG (XP_011239268.1), which has a C-terminal multihistidine tag; WT117-mPro1.ECD.hFc is the extracellular domain of mouse PVRIG (XP_011239268.1), which has a C-terminal Fc region of human IgG1; WT117-hPro1L1.ECD.mFc is the extracellular domain of human PVRIG (XP_011239268.1); ... The extracellular domain of (NP_001036189.1) has the Fc region of mouse IgG2a at its C-terminus.

[0236] 1.2 Preparation of the reference antibody (BMK)

[0237] Anti-human PVRIG reference antibodies WT117-BMK1 and WT117-BMK3 were prepared based on the sequences disclosed in their respective patents, and their information is summarized in Table 1.

[0238] Table 1. Reference Antibody Information

[0239] Antibody code Company Patent number Designation WT117-BMK1 Compugen US20180244774 CHA.7.518.1 WT117-BMK3 Surface Oncology US20200040081 Antibody 35

[0240] 1.3 Cell Pool / Cell Line Generation

[0241] The cell line WT117-293F.hPro1.G11, expressing human PVRIG, was generated using 293F cells transfected with full-length human PVRIG (NP_076975.2). The cell pool WT117-Flpin293.cPro1.pool, expressing cynomolgus monkey PVRIG, was generated using Flpin293 cells transfected with full-length cynomolgus monkey PVRIG (XP_005549281.1).

[0242] Example 2

[0243] Generation of human antibodies against PVRIG

[0244] 2.1 Immunization

[0245] Two female OmniRat animals (Open Monoclonal Technology Company), aged 8–13 weeks, were purchased from Charles River and housed in an IACUC-approved animal facility. Both animals were alternately immunized with proteins or plasmids of WT117-hPro1.ECD.hFc and WT117-mPro1.ECD.hFc. Immunization was repeated approximately weekly for a total of 111 days.

[0246] 2.2 Serum titer detection

[0247] The titer of anti-human / mouse PVRIG antibodies in serum samples was determined by ELISA. Microplates were coated with WT117-hPro1.ECD.His at a concentration of 0.5 μg / mL in 100 μL of coating buffer (Na2CO3 / NaHCO3, pH 9.2) per well and incubated overnight at 4°C. On the day of assay, after blocking with 1×PBS / 2% BSA for 1 hour, diluted rat serum samples (first diluted 1:100 in 1×PBS / 2% BSA, then 3-fold diluted) and negative controls were added to the plates, and the plates were incubated at ambient temperature for 1 hour. After washing three times with 1×PBST (PBS containing 0.05% Tween-20), HRP-labeled goat anti-rat IgG Fc (Bethyl, catalog number A110-236P) was added and incubated at ambient temperature for 1 hour. After removing unbound material, TMB (3,3',5,5'-tetramethylbenzidine) substrate was added, and the reaction was terminated with 2M HCl. The absorbance at 450 nm was measured using a microplate spectrophotometer.

[0248] The serum titers of immunized OMT rats are shown in Table 2. Lymph nodes from both animals were collected and used for fusion.

[0249] Table 2. Serum titers of anti-PVRIG antibodies

[0250]

[0251] 2.3 Hybridoma generation, antibody screening, and subcloning

[0252] Lymph nodes and spleens were collected from OMT rats under sterile conditions and dissociated into single-cell suspensions, which were then mixed with myeloma cells SP2 / 0 at a ratio of 1:1.2. Electrofusion was performed using a BTX 2001 electrocytometer according to an optimized electrofusion procedure. After fusion, cells were transferred to 96-well plates (1×10⁶ cells / wells). 4 The plate contains cells per well and is filled with DMEM medium supplemented with 20% FBS and 1% HAT selective reagent. Plates are incubated at 37°C with 5% CO2 and monitored periodically. When clones reach approximately 80% confluence in the wells, 100 μL of supernatant is transferred from the tissue culture plate to a 96-well assay plate for antibody screening.

[0253] Dilute the logarithmically grown positive lines to approximately 200 cells per 1.5 mL of semi-solid HAT medium. Gently mix the cell suspension on a vortex mixer for 5 to 10 seconds, then seed it into 6-well plates. Incubate the plates at 37°C with 5% CO2 for 7–8 days. When cell clusters have grown, pick each visible single colony and seed it into a 96-well plate containing DMEM medium supplemented with 10% fetal bovine serum. After 2–3 days, collect the supernatant from each clone and screen again to obtain positive hybridoma monoclonals.

[0254] 2.4 Hybridoma Sequencing

[0255] RNA was isolated from monoclonal hybridoma cells and used SuperScript. TM III. First-Strand Synthesis SuperMix Kit: Amplification was performed according to the manufacturer's instructions. The resulting cDNA was used as a template for subsequent PCR amplification using primers specific to the gene of interest. The PCR product was inserted into the pMD18-T vector, and the ligation product and PCR product were sent for sequencing.

[0256] 2.5 Fully Human Antibody Generation

[0257] The DNA sequences of the VH and VL domains were amplified by PCR and then subcloned with the constant regions of human IgG1 or human IgG4 into a pcDNA expression vector. Plasmids containing the VH and VL genes were co-transfected into Expi293 cells, and the cells were cultured for approximately 5 days until the supernatant was harvested. Antibodies were purified from the supernatant using a protein A column.

[0258] Forty-five positive cell lines were selected for subcloning through primary and secondary binding screening, as well as PVRIG / PVRL2 blockade and TCR / NFAT luciferase activation assays. After confirming the monoclonal antibody, 20 hits were sequenced, and four of them were subsequently transformed with human IgG. Following further in vitro characterization (see below), one clone (named WT1175-1.158.12-uIgG4LV1) was identified, and its sequence is shown in Table AB above.

[0259] Example 3

[0260] In vitro characterization

[0261] 3.1 Human PVRIG Binding Assay

[0262] WT117-293F.hPro1.G11 (1×10 5 (cells / well) or activated human CD8 + T cells (1×10) 5 Cells (1 cell / well) were incubated with different concentrations of anti-PVRIG antibody at 4°C for 1 hour. After washing with 1×PBS / 1% BSA, secondary antibody, namely PE-labeled goat anti-human IgG (Jackson Immuno Research catalog number 109-115-098), was added, and the cells were incubated with the secondary antibody at 4°C in the dark for 1 hour. Anti-human PVRIG antibody WT117-BMK1 was used as a positive control. Human IgG4 isotype antibody was used as an isotype control. The cells were then washed and resuspended in 1×PBS / 1% BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0263] Anti-PVRIG antibody against WT117-293F.hPro1.G11 or human CD8 + The binding of T results in cells such as Figure 1 and Figure 2 As shown, this demonstrates that WT1175-1.158.12-uIgG4LV1 can bind strongly to cells expressing human PVRIG, and the binding potency is significantly higher than that of the reference antibody. A summary of antibody binding is shown in Table 3 below.

[0264] 3.2 Vernig binding assay in cynomolgus monkeys

[0265] WT117-Flpin293F.cPro1.pool (1×10 5Cells (cells / well) were incubated with different concentrations of anti-PVRIG antibody at 4°C for 1 hour. After washing with 1×PBS / 1% BSA, secondary antibody, namely PE-labeled goat anti-human IgG (Jackson Immuno Research catalog number 109-115-098), was added, and the cells were incubated with the secondary antibody at 4°C in the dark for 1 hour. Anti-human PVRIG antibody WT117-BMK1 was used as a positive control. Human IgG4 isotype antibody was used as an isotype control. The cells were then washed and resuspended in 1×PBS / 1% BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0266] The binding results of anti-PVRIG antibody to WT117-Flpin293F.cPro1.pool or human CD8+ T cells were as follows: Figure 3 As shown, this demonstrates that WT1175-1.158.12-uIgG4LV1 can bind strongly to cells expressing cynomolgus monkey PVRIG, and the binding potency is significantly higher than that of the reference antibody. A summary of antibody binding is shown in Table 3 below.

[0267] 3.3 Mouse PVRIG Binding Assay

[0268] Pre-coat plates overnight at 4°C with 0.5 μg / mL WT117-hPro1.ECD.His or WT117-mPro1.ECD.His in 100 μL coating buffer / well. After blocking with 200 μL of 1×PBS / 2% BSA, add 100 μL of anti-PVRIG antibody at a concentration of 6.67 nM to the plate and incubate at ambient temperature for 1 hour. After incubation, wash the plate three times with 1×PBST. Add HRP-labeled goat anti-human IgG antibody (Bethyl catalog number A80-304P) diluted in 1×PBS / 2% BSA and incubate at ambient temperature for 1 hour. After washing with 1×PBST, develop the color by dispensing 100 μL of TMB substrate and then stop the reaction by adding 100 μL of 2M HCl. Read the absorbance at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices).

[0269] The binding results of anti-PVRIG antibody to the extracellular domain of mouse PVRIG cells are as follows: Figure 4 As shown, this demonstrates that WT1175-1.158.12-uIgG4LV1 does not bind to mouse PVRIG. A summary of antibody binding is shown in Table 3 below.

[0270] Table 3. Summary of antibodies that bind to PVIRG

[0271] Assay WT1175-1.158.12-uIgG4LV1 WT117-BMK1 Binding to human PVRIG engineered cells, EC50 (nM) 0.073 0.63 binds to human CD8 + T cell binding, EC50 (nM)] 0.037 0.28 Binding to cynomolgus PVRIG, EC50 (nM) 0.031 0.36 Binding to mouse PVRIG Negative Negative

[0272] 3.4 Human PVRIG Affinity Determination

[0273] The binding affinity of the WT1175 antibody to human PVRIG on the cell surface was measured using FACS. WT117-293F.hPro1.G11 cells were cultured at 5 × 10⁻⁶ cells per cell line. 4 Cells were transferred at a density of 1 cell / well to 96-well round-bottom plates. Anti-PVRIG antibody was serially diluted in 1×PBS / 1% BSA and incubated with cells at 4°C for 1 hour. Secondary antibody, FITC-labeled goat anti-human IgG Fc (Jackson Immuno Research catalog number 109-095-098), was added and incubated in the dark at 4°C for 0.5 hours. Cells were then washed once and resuspended in 1×PBS / 1% BSA for analysis by flow cytometry (BD). Fluorescence intensity was converted to the number of bound molecules per cell based on quantitative beads (Bangs Laboraties catalog number 555pB).

[0274] The binding affinity of anti-PVRIG antibody to WT117-293F.hPro1.G11 cells was as follows: Figure 5 As shown in Table 4. Bmax represents the maximum specific binding, and KD is the ligand concentration required to achieve half-maximal binding under equilibrium conditions. WT1175-1.158.12-uIgG4LV1 binds to human PVRIG on the cell surface with high affinity.

[0275] Table 4. Affinity constants of anti-PVRIG antibodies to human PVRIG

[0276] Parameter WT1175-1.158.12-uIgG4LV1 WT117-BMK1 r 2 ]]> 0.9958 0.9651 Bmax (M) 7.46E-12 7.52E-12 KD (M) 1.33E-11 1.31E-10

[0277] 3.5 PVRIG paralogous protein binding assay

[0278] Pre-coat plates overnight at 4°C with 1 μg / mL of WT117-hPro1.ECD.His, recombinant human TIGIT, CD226, CD96, or PD-1 extracellular domain in 100 μL coating buffer / well. After blocking with 200 μL of 1×PBS / 2% BSA, add 100 μL of test antibody at a concentration of 10 μg / mL to the plate and incubate at ambient temperature for 1 hour. After incubation, wash the plate three times with 1×PBST. Add HRP-labeled goat anti-human IgG antibody (Bethyl catalog number A80-304P) diluted in 1×PBS / 2% BSA and incubate at ambient temperature for 1 hour. After washing six times with 1×PBST, develop the color by dispensing 100 μL of TMB substrate and then stop the reaction by adding 100 μL of 2M HCl. Absorbance was read at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices).

[0279] The binding results of anti-PVRIG antibody to PVRIG paralogous protein are as follows: Figure 6 As shown, this demonstrates that WT1175-1.158.12-uIgG4LV1 specifically binds to PVRIG and has no cross-reactivity with human TIGIT, CD226, CD96, or PD-1.

[0280] 3.6-person PVRIG / PVRL2 blocking assay

[0281] Pre-coat plates overnight at 4°C with 2 μg / mL WT1175-hPro1.ECD.His in 100 μL coating buffer / well. Block with 200 μL of 1×PBS / 2% BSA. Mix serially diluted anti-PVRIG antibody with a constant concentration of WT1175-hPro1L1.ECD.mFc (final concentration 10 μg / mL) at a 1:1 volume ratio. After 1 hour of blocking, add the antibody / ligand mixture to the plate and incubate at ambient temperature for 2 hours. After washing three times with 1×PBST, add HRP-labeled goat anti-mouse IgG (Bethyl catalog number A90-231P) to the plate and incubate at ambient temperature for 1 hour. After washing six times with 1×PBST, add TMB substrate and terminate the interaction with 2M HCl. Read absorbance at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices).

[0282] Human PVRIG / PVRL2 binding blockade results are as follows Figure 7As shown in the figure. The results indicate that WT1175-1.158.12-uIgG4LV1 can effectively block the binding of human PVRL2 to PVRIG. The antibody blocking activity is summarized in Table 5 below. The maximum inhibition rate is calculated as max inhibition% = (OD max - OD 底部 ) / OD max × 100%, of which OD max It is defined as OD450 minus OD540 under the condition of no antibody.

[0283] 3.7 Jurkat PVRIG / NFAT-luciferase reporter gene assay

[0284] Jurkat cells overexpressing human PVRIG and NFAT-luciferase reporter genes were stimulated via T cell receptor binding by co-culturing with CHOK1 cells expressing human PVRL2 and TCR activator. The cells were cultured at 4 × 10⁻⁶ cells per cell line. 4 CHOK1 / PVRL2 / TCR activated cells were seeded in 96-well plates at a cell / well density and incubated overnight at 37°C with 5% CO2. The next day, after removing the supernatant and non-adherent cells, serially diluted anti-PVRIG antibody and Jurkat / PVRIG / NFAT-luciferase cells (2 × 10⁶ cells / well) were seeded. 4 Add cells per well to the plate and co-incubate at 37°C and 5% CO2 for 5–6 hours. After incubation, add the reconstituted luciferase substrate (Promega catalog number E6130) to each well and mix thoroughly. Read the luciferase intensity using an Envision microplate reader (PerkinElmer).

[0285] The result of anti-PVRIG antibody reversing the inhibition of NFAT signaling induced by PVRIG / PVRL2 interaction is as follows: Figure 8 As shown, this demonstrates that WT1175-1.158.12-uIgG4LV1 can enhance TCR / NFAT activation. A summary of antibody RGA activity is shown in Table 5.

[0286] Table 5. Summary of antibody characteristics

[0287] Assay WT1175-1.158.12-uIgG4LV1 WT117-BMK1 Blocking PVRL2 binding to PVRIG, IC50 (nM), max inhibition % 0.30, 97% 0.72, 94% NFAT reporter assay, EC50 (nM) 0.064 1.0

[0288] 3.8 Human primary T cell activation assay

[0289] Original CD8 +T cells were stimulated via T cell receptor binding by co-culturing with CHOK1 cells expressing human PVRL2 and TCR activators. Human CD8 cells were isolated from human peripheral blood mononuclear cells (PBMCs) using human CD8 MicroBeads (Miltenyi Biotec catalog number 130-045-201) via magnetic selection, according to the manufacturer's protocol. + T cells. Isolated human CD8 + T cells (1×10) 5 (each well) and irradiated CHOK1 / PVRL2 / TCR activated cells (2 × 10⁻⁶) 4 Each well was incubated with serially diluted anti-PVRIG antibody at 37°C and 5% CO2 for 5 days. After incubation, the supernatant was collected for IFN-γ measurement using an ELISA (capture antibody Thermo catalog number M700A, detection antibody Thermo catalog number M701B). Absorbance was measured using an M5e microplate reader (Molecule Devices).

[0290] Compared with IFN-γ secretion observed using isotype controls and reference antibodies, human CD8 cells treated with anti-PVRIG antibodies showed significantly higher levels of IFN-γ secretion. + T cells lead to greater IFN-γ secretion. Results showed that WT1175-1.158.12-uIgG4LV1 enhanced CD8+ T cell activation. Data are as follows: Figure 9 As shown.

[0291] 3.9 Antibody Serum Stability Assay

[0292] Fresh human serum was isolated from a healthy donor. Anti-PVRIG antibody was diluted in the serum. Samples were aliquoted into five tubes and incubated at 37°C. Samples were then collected on days 0, 1, 4, 7, and 14, flash-frozen, and stored at -70°C until ready for analysis. Binding activity of the samples was evaluated by FACS according to the method described in Section 3.1.

[0293] The binding of serum-incubated WT1175-1.158.12-uIgG4LV1 to WT117-293F.hPro1.G11 is as follows: Figure 10 As shown, antibodies incubated with serum for up to two weeks maintained similar binding activity and very similar EC50 as fresh antibodies. The results indicate that WT1175-1.158.12-uIgG4LV1 is stable in human serum at 37°C for at least two weeks.

[0294] 3.10 Antibody thermostability assay

[0295] Conformational stability is a crucial characteristic of successful antibodies. Conformational stability can be assessed by measuring thermal stability using differential scanning fluorometry (DSF), which is sensitive to changes in protein folding. DSF measures the temperature (Tm) of protein unfolding transition based on changes in the fluorescence intensity of the environmentally sensitive dye SYPROOrange.

[0296] DSF was performed in the appropriate reagent buffer using a Quant Studio 7 Flex real-time PCR instrument (Applied Biosystems). SYPRO orange dye (Invitrogen catalog number S6651) was added to the antibody, and the mixture was transferred to a 96-well plate. The plate was then placed in the Quant Studio 7 Flex instrument. ® A 7-flex Real-Time PCR system was used, with a temperature range of 26°C to 95°C and a heating rate of 0.9°C / min. The first two temperatures for protein unfolding transitions were recorded as Tm1 and Tm2. QuantStudio was used. ® The Real Time PCR software (v1.3) calculates these two values ​​based on the melt curve.

[0297] The DSF temperature spectrum of the WT1175-1.158.12-uIgG4LV1 antibody showed two transitions: the first with a lower melting temperature (Tm1) and the second with a higher melting temperature (Tm2), at 62.5°C and 64.9°C, respectively. The results are shown in Table 6.

[0298] Table 6. Tm values ​​of antibodies

[0299] Antibody Tm1 (°C) Tm2 (°C) WT1175-1.158.12-uIgG4LV1 62.5 64.9

[0300] 3.11 Comparison of human IgG1 and IgG4 formats

[0301] To compare the effects of different Fc on antibody activity, we also generated the corresponding IgG1 formatted antibody for WT1175-1.158.12-uIgG4LV1, named WT1175-1.158.12-uIgG1L, and also generated a reference antibody WT117-BMK3 with IgG1 Fc and used it as a positive control.

[0302] The binding activity of WT1175-1.158.12-uIgG1L to WT117-293F.hPro1.G11 was evaluated according to the methods described in Section 3.1. The effect of WT1175-1.158.12-uIgG1L in reversing PVRIG-mediated inhibitory signaling was evaluated according to the methods described in Section 3.7. The activity of WT1175-1.158.12-uIgG1L in enhancing CD8+ T cell activation was evaluated according to the methods described in Section 3.8.

[0303] Showing the combination ( Figure 11 ), TCR / NFAT luciferase activation assay ( Figure 12 ) and CD8 + Enhanced T cell activation ( Figure 13 The results demonstrate that WT1175-1.158.12-uIgG4LV1 and WT1175-1.158.12-uIgG1L possess considerable binding and functional activity.

[0304] 3.12 Generation and Characterization of Variant PVRIG Antibodies

[0305] Positions S7 and T43 (according to Kabat numbering) in VL were identified as potentially unstable residues in the antibody WT1175-1.158.12-uIgG1L. Mutations were performed on S7P and T43A to improve stability, and the mutated variant was named WT1175-1.158.12-m1-uIgG1L. The activities of the two antibodies were compared using FACS binding assay (method in 3.1) and NFAT reporter assay (method in 3.7). Thermostability of WT1175-1.158.12-m1-uIgG1L was evaluated according to the method described in Section 3.10 to demonstrate the improvement.

[0306] Combining the results as follows Figure 15 As shown, and the report gene assay results are as follows: Figure 16 As shown in the figure. These results demonstrate that WT1175-1.158.12-uIgG1L and WT1175-1.158.12-m1-uIgG1L have comparable binding and functional activity.

[0307] The results of DSF are shown in Table 7. Compared with WT1175-1.158.12-uIgG4LV1, WT1175-1.158.12-m1-uIgG1L showed improved thermal stability, with Tm1 increasing from 62.5°C to 68.3°C.

[0308] Table 7. Tm values ​​of antibodies

[0309] Antibody Tm1 (°C) Tm2 (°C) WT1175-1.158.12-m1-uIgG1L 68.3 -

[0310] Example 4

[0311] In vivo characterization

[0312] 4.1 Rodent Pharmacokinetic Studies

[0313] This study aimed to determine the pharmacokinetics of WT1175-1.158.12-uIgG1L and WT1175-1.158.12-uIgG4LV1 in untreated (naïve) rats following a single intravenous bolus administration. Eight animals were randomly assigned to two groups and treated with WT1175-1.158.12-uIgG1L (10 mg / kg) and WT1175-1.158.12-uIgG4LV1 (10 mg / kg), respectively.

[0314] Blood samples for PK were collected before administration and at 0.5 h, 4 h, and on days 2, 3, 5, 7, 10, 12, 14, and 21. Serum concentrations of anti-PVRIG antibodies were determined by ELISA. Briefly, goat anti-human IgG Fc was used as the capture reagent, and biotinylated goat anti-human IgG Fc was used as the detection reagent. Streptavidin-HRP and TMB substrates were used for colorimetric development, and the reaction was terminated with 2M HCl. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. Non-compartmental pharmacokinetic analysis of serum concentrations of WT1175-1.158.12-uIgG1L and WT1175-1.158.12-uIgG4LV1 was performed using Phoenix WinNonlin software. PK parameters were obtained by applying linear / logarithmic trapezoidal rules.

[0315] Since neither of these antibodies binds to rat PVRIG, there is no target-mediated drug disposition effect. Linear pharmacokinetics were observed at a single dose of 10 mg / kg. Figure 14 The results of the PK parameters are summarized in Table 8. WT1175-1.158.12-uIgG1L showed a mean serum clearance of 4.89 mL / day / kg, a half-life of 429 hours, a volume of distribution of 123 mL / kg, and an AUC of 0-t 25547 h µg / mL, while WT1175-1.158.12-uIgG4LV1 showed a mean serum clearance of 5.03 mL / day / kg, a half-life of 368 hours, a volume of distribution of 108 mL / kg, and an AUC of 0-t 29589 h µg / mL.

[0316] Table 8. Summary of PK parameters

[0317] Parameter WT1175-1.158.12-uIgG1L WT1175-1.158.12-uIgG4LV1 Dose 10 mg / kg, iv 10 mg / kg, iv T 1 / 2 (h)]]> 429 368 C max (µg / ml)]]> 212 215 AUC 0-t (h µg / ml)]]> 25547 29589 Cl_obs (ml / day / kg) 4.89 5.03 MRTINF_obs (h) 615 516 Vss_obs (mL / kg) 123 108

[0318] Those skilled in the art will further understand that this disclosure may be implemented in other specific forms without departing from the spirit or central attributes of this disclosure. Since the foregoing description of this disclosure only discloses exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of this disclosure. Therefore, the invention is not limited to the specific embodiments already described in detail herein, but should instead be referred to the appended claims, which indicate the scope and content of the invention.

Claims

1. An isolated antibody, or an antigen binding portion thereof, comprising: a heavy chain CDR (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1 ; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain CDR (LCDR) 1 comprising the amino acid sequence of SEQ ID NO: 4; a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

6.

2. The isolated antibody, or antigen binding portion thereof, of claim 1, comprising: (A) a heavy chain variable region (VH): (i) comprising the amino acid sequence set forth in SEQ ID NO: 7; or (ii) comprising an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 7; and / or (B) a light chain variable region (VL): (i) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 8 and 9; or (ii) comprising an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 8 and 9.

3. The isolated antibody, or antigen binding portion thereof, of claim 1 or 2, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

8.

4. The isolated antibody, or antigen binding portion thereof, of any one of the preceding claims, wherein the isolated antibody further comprises a human IgG constant region.

5. The isolated antibody, or antigen binding portion thereof, of claim 4, wherein the human IgG constant region is a human IgG1, IgG4, IgG2, or IgG3 constant region or a variant thereof.

6. The isolated antibody, or antigen binding portion thereof, of any one of the preceding claims, wherein the antibody comprises a human IgG1 Fc region or a human IgG4 Fc region, optionally comprising a S228P mutation.

7. The isolated antibody, or antigen binding portion thereof, of any one of the preceding claims, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

8. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and / or a light chain variable region of the isolated antibody, or antigen binding portion thereof, of any one of claims 1-7.

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

10. A host cell comprising the vector of claim 9.

11. A pharmaceutical composition comprising an isolated antibody, or antigen binding portion thereof, as defined in any one of claims 1-7, and a pharmaceutically acceptable carrier.

12. A method of producing an antibody, or antigen binding portion thereof, as defined in any one of claims 1-7, comprising the steps of: - culturing a host cell comprising an expression vector encoding the antibody, or antigen binding portion thereof, under suitable conditions; and - harvesting the antibody, or antigen binding portion thereof, from the cell culture.

13. A method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject an effective amount of an antibody, or antigen binding portion thereof, as defined in any one of claims 1-7 or a pharmaceutical composition according to claim 11.

14. A method for treating or preventing a cancer or an immune-related disorder in a subject, comprising administering to the subject an effective amount of an antibody, or antigen binding portion thereof, as defined in any one of claims 1-7 or a pharmaceutical composition according to claim 11.

15. The method according to claim 14, wherein the cancer is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell kidney cancer, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, central nervous system tumor, mesothelioma, myeloma, and sarcoma.

16. The method according to claim 14, wherein the immune-related disorder is a T cell dysfunctional disorder or an infection.

17. The method according to any one of claims 14-16, wherein the method further comprises administering an additional therapeutic agent.

18. Use of an antibody, or antigen binding portion thereof, as defined in any one of claims 1-7 for the manufacture of a medicament for the treatment or prevention of a cancer or an immune-related disorder.

19. An antibody, or antigen binding portion thereof, as defined in any one of claims 1-7 for use in the treatment or prevention of a cancer or an immune-related disorder.

20. A kit comprising a container comprising an antibody, or antigen binding portion thereof, as defined in any one of claims 1-7.

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