Anti-CD112R antibodies and uses thereof

Abstract

A CD112R binding protein, in particular an anti-CD112R antibody and an antigen binding fragment thereof, is provided. Also provided are compositions comprising the CD112R binding proteins, nucleic acid molecules encoding the antigen binding proteins, and uses thereof.

Description

Technical Field

[0001] The present disclosure relates to antibodies and fragments thereof against CD112R, and their uses. Background Art

[0002] Immunotherapy has become a powerful means for treating various cancers. One of the most influential methods is based on blocking immune checkpoint receptors or ligands.

[0003] CD112R (also known as PVRIG, i.e., protein containing poliovirus receptor-related immunoglobulin domains) is preferentially expressed on T cells and NK cells, and its expression is upregulated after cell activation. CD112R competes with CD226 (a co-stimulatory receptor on CD8 + T cells and NK cells) for binding to its common ligand CD112 with higher affinity, and then transduces inhibitory signals through its intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM)-like motif, thereby inhibiting the immune functions of T cells and NK cells.

[0004] In preclinical tumor models, the expression of CD112R and the interaction between CD112R-CD112 are closely related to tumor growth. Gene knockout (KO) or CD112R antibody blockade can inhibit tumor growth by enhancing the cytotoxic functions of tumor-infiltrating CD8 + T cells and NK cells, indicating its potential as a cancer immunotherapy target.

[0005] In cancer patients, it is known that the expression of CD112R is upregulated and co-expressed with programmed cell death protein 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on tumor-infiltrating lymphocytes (TIL). Given that CD112 is highly expressed in tumor cells, epithelial cells, and myeloid cells in the tumor microenvironment (TME), it has been suggested that CD112R may inhibit the anti-tumor immune response through CD112R-CD112 interaction. In addition, CD112R has a unique dominant expression in early memory (stem cell-like) T cell subsets, which can self-renew and differentiate into effector cells, while CD112 is widely expressed in dendritic cells (DC), resulting in the interaction of CD112R-CD112 may inhibit the initiation and expansion of T cells. Therefore, blocking CD112R can enhance the activation of memory T cells by DC, promoting their increased amplification and differentiation. In addition, dual blockade of CD112R and PD-1 or dual blockade of CD112R and TIGIT further increases T cell activation and improves the clinical treatment effect. These all indicate that the CD112R-CD112 signaling axis plays an important role in cancer immunity, and targeting this signaling axis may help overcome the clinical limitations of immune checkpoint blockade (ICB) therapy. SUMMARY OF THE INVENTION

[0006] To solve the above-mentioned at least one technical problem, the present disclosure provides monoclonal antibodies (mAbs) that target hCD112R and block its inhibitory function. These antibodies (Abs) are generated by immunizing mice with the extracellular domain (ECD) of hCD112R (hCD112R-ECD) using hybridoma technology.

[0007] According to one aspect of the present disclosure, there is provided an antigen-binding protein that specifically binds to CD112R. In some embodiments, the antigen-binding protein may specifically bind to the extracellular domain (ECD) of CD112R.

[0008] In some embodiments, the antigen-binding protein may be an antibody or an antigen-binding fragment thereof.

[0009] In some embodiments, the antigen-binding protein may comprise: (1) an immunoglobulin heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:1 or an HCDR1 having one or more amino acid additions, deletions, or substitutions on the amino acid sequence shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2 or an HCDR2 having one or more amino acid additions, deletions, or substitutions on the amino acid sequence shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3 or an HCDR3 having one or more amino acid additions, deletions, or substitutions on the amino acid sequence shown in SEQ ID NO:3; and (2) an immunoglobulin light chain variable region comprising LCDR1 as shown in SEQ ID NO:4 or an LCDR1 having one or more amino acid additions, deletions, or substitutions on the amino acid sequence shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5 or an LCDR2 having one or more amino acid additions, deletions, or substitutions on the amino acid sequence shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6 or an LCDR3 having one or more amino acid additions, deletions, or substitutions on the amino acid sequence shown in SEQ ID NO:6.

[0010] It should be understood that the above-mentioned one or more amino acid additions, deletions, or substitutions do not cause the antigen-binding protein to lose its binding ability to CD112R.

[0011] In some embodiments, the antigen-binding protein may comprise: (1) an immunoglobulin heavy chain variable region (VH) comprising HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; and (2) an immunoglobulin light chain variable region comprising LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6.

[0012] In some embodiments, the antigen-binding protein may comprise an immunoglobulin heavy chain variable region (VH) having the amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence as shown in SEQ ID NO:9. In some embodiments, the antigen-binding protein may comprise an immunoglobulin heavy chain variable region (VH) having an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:9.

[0013] In some embodiments, the antigen-binding protein may comprise an immunoglobulin light chain variable region (VL) having the amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence as shown in SEQ ID NO:10. In some embodiments, the antigen-binding protein may comprise an immunoglobulin light chain variable region (VL) having an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:10.

[0014] In some embodiments, the antigen-binding protein may be a humanized antibody or an antigen-binding fragment thereof. In some embodiments, the humanized antibody or an antigen-binding fragment thereof may comprise: (1) a heavy-chain variable region (VH) comprising an HCDR1 having the amino acid sequence as shown in NYLIE, an HCDR2 having the amino acid sequence as shown in VINPGHGFTNYX1X2KFX3G, and an HCDR3 having the amino acid sequence as shown in GEWDWYFDV; and / or (2) a light-chain variable region (VL) comprising an LCDR1 having the amino acid sequence as shown in KASQNVGTAVA, an LCDR2 having the amino acid sequence as shown in STSNRYT, and an LCDR3 having the amino acid sequence as shown in QQX4SSYPFT. In some specific embodiments, X1 may be selected from A (Ala) or N (Asn). In some specific embodiments, X2 may be selected from E (Glu) or Q (Gln). In some specific embodiments, X3 may be selected from K (Lys) or Q. In some specific embodiments, X4 may be selected from C (Cys) or S (Ser).

[0015] In some specific embodiments, the humanized antibody or an antigen-binding fragment thereof may comprise a heavy-chain variable region (VH) comprising an HCDR1 having the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 having the amino acid sequence as set forth in SEQ ID NO:2, and an HCDR3 having the amino acid sequence as set forth in SEQ ID NO:3. In some specific embodiments, the humanized antibody or an antigen-binding fragment thereof may comprise a heavy-chain variable region (VH) comprising an HCDR1 having the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 having the amino acid sequence as set forth in SEQ ID NO:7, and an HCDR3 having the amino acid sequence as set forth in SEQ ID NO:3.

[0016] In some specific embodiments, the humanized antibody or an antigen-binding fragment thereof may comprise a light-chain variable region (VL) comprising an LCDR1 having the amino acid sequence as set forth in SEQ ID NO:4, an LCDR2 having the amino acid sequence as set forth in SEQ ID NO:5, and an LCDR3 having the amino acid sequence as set forth in SEQ ID NO:6. In some specific embodiments, the humanized antibody or an antigen-binding fragment thereof may comprise a light-chain variable region (VL) comprising an LCDR1 having the amino acid sequence as set forth in SEQ ID NO:4, an LCDR2 having the amino acid sequence as set forth in SEQ ID NO:5, and an LCDR3 having the amino acid sequence as set forth in SEQ ID NO:8.

[0017] In some specific embodiments, the humanized antibody or its antigen-binding fragment may comprise (1) a heavy chain variable region (VH) that comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO:1, an HCDR2 having the amino acid sequence shown in SEQ ID NO:2, and an HCDR3 having the amino acid sequence shown in SEQ ID NO:3; and (2) a light chain variable region (VL) that comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO:4, an LCDR2 having the amino acid sequence shown in SEQ ID NO:5, and an LCDR3 having the amino acid sequence shown in SEQ ID NO:6.

[0018] In some specific embodiments, the humanized antibody or its antigen-binding fragment may comprise (1) a heavy chain variable region (VH) that comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO:1, an HCDR2 having the amino acid sequence shown in SEQ ID NO:7, and an HCDR3 having the amino acid sequence shown in SEQ ID NO:3; and (2) a light chain variable region (VL) that comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO:4, an LCDR2 having the amino acid sequence shown in SEQ ID NO:5, and an LCDR3 having the amino acid sequence shown in SEQ ID NO:8.

[0019] In some specific embodiments, the humanized antibody or its antigen-binding fragment may comprise a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO:11. In some specific embodiments, the humanized antibody or its antigen-binding fragment may comprise a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:13, or an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO:13. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) having an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:13.

[0020] In some specific embodiments, the humanized antibody or its antigen-binding fragment may comprise a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:12. In some specific embodiments, the humanized antibody or its antigen-binding fragment may comprise a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the antibody or its antigen-binding fragment comprises a light chain variable region (VL) having an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:14.

[0021] In some embodiments, the antigen-binding protein of the present disclosure can specifically bind to CD112R, particularly the extracellular domain (ECD) of CD112R. In some embodiments, the antigen-binding protein can specifically bind to amino acids 90-150 of CD112R, particularly amino acids 95-145 of CD112R. In some embodiments, the antigen-binding protein can specifically bind to R95, V90, W100 and / or E145 of CD112R. In some embodiments, CD112R can be human CD112R, and the above amino acid positions are based on human CD112R shown in UniProt accession number #Q6DKI7.

[0022] In a further embodiment, the antigen-binding protein may comprise a heavy chain constant region of the IgG1 or IgG4 subtype, preferably the IgG4 subtype.

[0023] In some embodiments, the antigen-binding fragment of the antibody can be a Fab, F(ab')2, Fv or single-chain Fv fragment (scFv).

[0024] According to another aspect of the present disclosure, there is provided a composition comprising the antigen-binding protein of the present disclosure. In some embodiments, the composition may further comprise other therapeutic agents. In some embodiments, the composition may further comprise a pharmaceutically acceptable carrier.

[0025] According to another aspect of the present disclosure, there is provided a nucleic acid molecule encoding the antigen-binding protein of the present invention.

[0026] According to another aspect of the present disclosure, there is provided a host cell comprising the antigen-binding protein or nucleic acid molecule of the present disclosure. In some embodiments, the host cell can be a prokaryotic cell or a eukaryotic cell. The host cell can be any cell system capable of being engineered to produce the antibody or fragment thereof described in the present disclosure. For example, the host cell can be an animal cell, particularly a mammalian cell. In a specific embodiment, HEK293 cells (human embryonic kidney cells), CHO cells (Chinese hamster ovary cells) or Vero cells can be used as host cells. In another embodiment, the host cell can be a non-human animal or mammalian cell, such as Escherichia coli (E. coli) cells, Pichia cells.

[0027] According to another aspect of the present disclosure, there is provided a method for preventing or treating a subject suffering from a CD112R-related disease, which comprises administering to the subject a therapeutically effective amount of the antigen-binding protein or composition of the present disclosure.

[0028] According to another aspect of the present disclosure, there is provided the use of the antigen-binding protein or composition of the present disclosure in the preparation of a drug for preventing or treating a CD112R-related disease.

[0029] According to another aspect of the present disclosure, there is provided the antigen-binding protein or composition of the present disclosure for preventing or treating a disease associated with upregulation of CD112R expression.

[0030] In some embodiments, the disease may include cancer, infectious diseases, sepsis, autoimmune diseases and / or adverse immune activation after gene therapy. In some embodiments, the disease may have an upregulation of CD112R levels in T cells and NK cells.

[0031] In some embodiments, the cancer may include, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric or abdominal cancer (including gastrointestinal cancer), melanoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular tumor, and various types of head and neck cancers, as well as 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, large mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, multiple myeloma, and post-transplant lymphoproliferative disorder (PTLD).

[0032] In some embodiments, the infectious disease may be a chronic infection characterized by varying degrees of impaired function of virus-specific T cell responses, and this defect is the main reason for the host's inability to clear persistent pathogens. Although functional effector T cells are initially generated in the early stage of infection, these cells gradually lose their function during chronic infection due to continuous exposure to foreign antigens, leading to T cell exhaustion.

[0033] In some embodiments, the infectious disease may include infectious disorders, diseases, and / or conditions caused by bacterial infection, viral infection, fungal infection, and / or parasitic infection.

[0034] According to another aspect of the present disclosure, there is provided the use of the antibody or its antigen-binding fragment as described in the present disclosure in the preparation of a medicament for preventing or treating a subject suffering from a CD112R-related disease.

[0035] According to another aspect of the present disclosure, there is provided the use of the antibody or its antigen-binding fragment of the present disclosure for preventing or treating a subject suffering from a CD112R-related disease.

[0036] The anti-CD112R antibody or its antigen-binding fragment provided by the present disclosure can effectively block the interaction between CD112R and CD112, and reverse lymphocyte function (such as cytotoxicity), without generating complement-dependent cytotoxicity (CDC) effector function, and having a weak antibody-dependent cell-mediated cytotoxicity (ADCC) effector function. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 . FACS analysis of the binding affinity and blocking activity of anti-hCD112R mAb (in the form of mIgG2a). A) Binding analysis of anti-hCD112R mAb or hCD112-ECD-mFc to YTS-hCD112R cells. The binding of mouse anti-hCD112R mAb at the indicated concentrations to YTS-hCD112R cells expressing hCD112R was tested. B) Ligand competition analysis of mAb. The blocking effect of mouse anti-hCD112R mAb or hCD112-ECD-mFc at the indicated concentrations on the binding of hCD112-ECD-hFc (0.6 μg / ml) to YTS-hCD112R cells was tested by a FACS-based competition assay. The hCD112-ECD-mFc protein was used as a control.

[0038] Figure 2 . Anti-hCD112R mAb 733 binds to hCD112R with high affinity. Multicycle kinetic analysis of the interaction between 733-mIgG2a and hCD112R was performed using SPR.

[0039] Figure 3 . Anti-hCD112R mAb 733 effectively blocks the human CD112R-CD112 interaction. The blocking activity of mAb 733 was analyzed by a FACS-based competition assay. The blocking effect of serially diluted concentrations of 733-mIgG2a on the binding of hCD112-ECD-hFc to YTS-hCD112R cells was tested.

[0040] Figure 4 . mAb 733 effectively reverses the NK cell cytotoxicity inhibited by the human CD112R-CD112 interaction. A - B) The expression of hCD112R in the stable cell line YTS-hCD112R (A) or the expression of hCD112 in the stable cell line 721.221-hCD112 (B) was evaluated by flow cytometry using anti-hCD112R Ab (clone W16216D, Biolegend) or anti-hCD112 Ab (clone TX31, Biolegend). C) The CD112R-CD112 interaction disrupted the cytotoxicity of YTS-hCD112R cells (left), while the presence of 733 effectively reversed the cytotoxicity of YTS-hCD112R cells inhibited by the CD112R-CD112 interaction (right). In the killing experiments of YTS cells against 721.221 target cells and YTS-hCD112R cells against 721.221-hCD112 cells, the E:T ratio was 2:1.

[0041] Figure 5 . Amino acid sequence alignment of VH and VL of.mAb 733 and its humanized variants. A) VH amino acid sequence alignment. B) VL amino acid sequence alignment. Dots indicate identical amino acids. Amino acid numbering is based on the mAb 733 sequence. CDRs are determined according to the Kabat numbering system.

[0042] Figure 6 . SPR assay of the binding kinetics of mAb 733 and mAb 733-derived humanized antibodies. A) Single-cycle kinetic analysis of the interaction between full-length mAb 733 or mAb 733-derived humanized antibody and hCD112R using SPR. B) Multi-cycle kinetic analysis of the interaction between full-length H733 Ab and hCD112R using SPR.

[0043] Figure 7 . ELISA analysis of the ligand competition activity of H733. Serial dilution concentrations of H733 (in hIgG4 form) were tested for competitive binding to biotinylated hCD112R-ECD-His6-Avi with hCD112-ECD-mFc, and biotinylated hCD112R-ECD-His6-Avi was captured by streptavidin immobilized on the ELISA plate.

[0044] Figure 8 . Amino acid sequence alignment of the N-terminal IgV domain of hCD112R and cynoCD112R. Amino acid numbering is based on the hCD112R sequence. Dots indicate identical amino acids.

[0045] Figure 9 . Mapping the binding epitope of H733 on hCD112R by human-to-cynomolgus monkey mutagenesis. A-B) Single-cycle kinetic analysis of the interaction between H733-hIgG4 (A) or hCD112-ECD-hFc (B) and hCD112R-ECD-mFc wild-type (WT) or mutants using SPR.

[0046] Figure 10 . Mapping the binding epitope of H733 on hCD112R by alanine-scanning mutagenesis. A) Sites for alanine-scanning mutagenesis on hCD112R were selected based on AlphaFold structure prediction. B-C) Multi-cycle kinetic analysis of the interaction between H733-hIgG4 (B) or hCD112-ECD-hFc (C) and hCD112R-ECD-mFc WT or mutants using SPR.

[0047] Figure 11.H733 effectively reverses CD112R-mediated NK cell dysfunction. YTS or YTS-hCD112R cells were co-cultured with 721.221-hCD112 cells at a ratio of 2:1, and the indicated concentrations of H733 or COM701 were added. Cytotoxicity was evaluated by detecting LDH release. Here, COM701 mAb is a humanized anti-hCD112R antibody developed by Compugen and used as a control. Both H733 and COM701 are hIgG4 antibodies.

[0048] Figure 12 .H733 enhances human T cell function. A) The expression of the OKT3 Ab scFv fragment (upper panel) and hCD112 (lower panel) in the CHO-OKT3scFv and CHO-OKT3scFv_hCD112 cell lines was evaluated by flow cytometry using anti-mIgG Ab (ThermoFisher) and anti-hCD112 Ab (clone TX31, Biolegend), respectively. B) The expression of hCD112R in Jurkat reporter cells was evaluated by flow cytometry using anti-hCD112R Ab (clone W16216D, Biolegend). C) Jurkat-NFAT-luc-hCD112R reporter cells were co-cultured with CHO-OKT3-hCD112 cells. Serial dilutions of H733 or COM701 Ab (both in hIgG4 form) were added. Then, relative luciferase units (RLU) were recorded using a microplate reader.

[0049] Figure 13 .H733 promotes T cell proliferation. Purified human T cells were labeled with CFSE and co-cultured with CHO-OKT3 cells or CHO-OKT3_CD112 cells. H733 mAb (in hIgG4 form) was added at the beginning of the culture. T cell proliferation was determined by CFSE dilution. Data represent the results of three replicate experiments.

[0050] Figure 14. ADCC effect function induced by anti-hCD112R Ab. A) Generation of Raji-hCD112R cell line for in vitro Fc-mediated effector function analysis. The expression of hCD112R in RajiWT and Raji-hCD112R cells was analyzed by FACS using a specific anti-hCD112R Ab (clone W16216D, Biolegend). B) ADCC effect function induced by H733 Ab. The ADCC activity was measured using a reporter gene assay system. Jurkat-NFAT-Luc2p / hFcγRIIIa (F158) was used as effector cells and Raji-hCD112R cells were used as target cells. The Ab was tested at the indicated concentrations in triplicate. The E:T ratio was 6:1. The relative light unit (RLU) was plotted against the Log of the antibody concentration. The luciferase activity was shown in the figure.

[0051] Figure 15 . CDC effect function induced by H733 Ab. Raji-hCD112R target cells were incubated with H733 Ab in the presence of 5% rabbit complement serum. The CDC activity was measured using lactate dehydrogenase (LDH) release in triplicate to quadruplicate. The Ab was tested at a concentration of 5 μg / ml.

[0052] Figure 16 . H733 exhibited anti-tumor activity in a xenograft mouse tumor model. A) Expression of hCD112 in MDA-MB-231, A375, and A549 tumor cell lines. The expression of hCD112 in tumor cells in cell culture was analyzed by FACS using an hCD112-specific antibody (clone TX31, Biolegend). B) Anti-tumor activity of H733 in a xenograft mouse tumor model. The black arrow indicates H733-hIgG4 treatment (10 mg / kg). Data are presented as mean ± SEM. (****P < 0.0001). Detailed Description

[0053] Before describing the methods and compositions of the present invention, it is to be understood that the invention is not limited to the specific methods or compositions described, and thus may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described below. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications. It should be understood that if there is a conflict between the cited publication and the present disclosure, the present disclosure shall prevail.

[0055] Cancer can be regarded as a state in which a patient is unable to recognize and eliminate cancer cells. In many cases, these transformed (such as cancerous) cells act against immune surveillance. There are some natural control mechanisms in the body to limit T cell activation to prevent excessive T cell activity, and cancer cells may utilize these mechanisms to evade or suppress the immune response. Restoring the ability of immune effector cells (especially T cells) to recognize and eliminate cancer is the goal of immunotherapy.

[0056] The field of immuno - oncology (sometimes referred to as "immunotherapy") is rapidly evolving, and in recent years, a variety of T cell checkpoint inhibitory antibodies have been approved. These antibodies are commonly referred to as "checkpoint inhibitors" because they block negative regulators of T cell immunity. By inhibiting checkpoint proteins, for example, by using antibodies that bind to these proteins, enhanced T cell responses to tumors can be achieved. That is to say, these cancer checkpoint proteins inhibit the immune response; when these proteins are blocked, such as by using antibodies against checkpoint proteins, the immune system is activated, thereby triggering immune stimulation and treating diseases such as cancer and infectious diseases.

[0057] The protein containing the poliovirus receptor - related immunoglobulin domain (PVRIG / CD112R) has recently been identified as an immune checkpoint molecule with potential for therapeutic development. PVRIG / CD112R is a single - transmembrane protein consisting of a single extracellular IgV domain. In humans, PVRIG is expressed on T cells (mainly CD8 + T cells) and natural killer (NK) cells, but not on B cells, monocytes or neutrophils. PVRIG binds to a single ligand - poliovirus receptor - related protein 2 (PVRL2, also known as CD112 or Nectin - 2), and may inhibit the activity of cytotoxic lymphocytes through an ITIM - like motif in its intracellular domain. PVRL2 is an adhesion molecule involved in cell - cell junction formation and is overexpressed in a variety of cancers. Since PVRIG is present on both T cells and NK cells, blocking PVRIG offers the possibility of enhancing the functions of these two major types of cytotoxic effector cells.

[0058] The present invention aims to provide monoclonal antibodies specific for human CD112R or proteins containing poliovirus receptor-related immunoglobulin domains (PVRIG). The monoclonal antibodies (mAbs) provided herein are capable of effectively blocking the inhibitory function of CD112R. These antibodies are specific for the extracellular domain of CD112R.

[0059] It should be noted that, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents. Thus, for example, "a recombinant AAV viral particle" includes a plurality of such viral particles, "microglia" includes one or more microglia and their equivalents, which are known to those skilled in the art, and so on.

[0060] As used herein, the terms "inhibit", "inhibitor", or "antagonist" include reducing a particular parameter, such as the activity of a given molecule, such as the activity of an immune checkpoint inhibitor. For example, inhibition of at least 5%, 10%, 20%, 30%, 40% or more of the activity (such as CD112R activity) is included within the scope of this term.

[0061] The terms "anticancer effect" and "antitumor effect" are used interchangeably herein. Both terms refer to biological effects that can be manifested in various ways, including but not limited to, for example, reduction in tumor volume, reduction in the number of cancer cells, reduction in the number of metastases, extension of life expectancy, reduction in cancer cell proliferation, reduction in cancer cell survival rate, or improvement in various physiological symptoms associated with the cancer condition. The "anticancer effect" can also be manifested by the prevention of cancer occurrence by the peptides, polynucleotides, cells, and antibodies of the first aspect.

[0062] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or can spread to other parts of the body through the blood and lymphatic systems. Examples of various cancers are described herein, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, for example, both encompass solid tumors and liquid (such as diffuse or circulating) tumors. The term "cancer" or "tumor" as used herein includes pre-cancerous lesions as well as malignant cancers and tumors.

[0063] The functional effects of CD112R blocking antibodies on NK cells and T cells can be evaluated in vitro (and in some cases in vivo) by measuring changes in the following parameters: proliferation, cytokine release, and cell surface markers. For NK cells, increased cell proliferation, cytotoxicity (the ability to kill target cells), cytokine production (such as IFN-γ and TNF), or increased cell surface receptor expression (such as CD25) may indicate immunomodulation, for example, enhanced ability to kill cancer cells. For T cells, increased proliferation, cytotoxicity (the ability to kill target cells), or cytokine production (such as IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-10, IL-17A) may indicate immunomodulation, for example, enhanced ability to kill cancer cells.

[0064] Accordingly, the present disclosure provides antibodies (including antigen-binding fragments) that bind to human CD112R, and methods of activating T cells and / or NK cells for treating diseases such as cancer, infectious diseases, and other conditions where a therapeutic effect is achieved due to increased immune activity.

[0065] In the present disclosure, the numbering of the complementarity determining regions (CDRs) is determined according to the Kabat numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., United States Public Health Service, National Institutes of Health, Bethesda), the entire text of which is incorporated herein by reference.

[0066] The term "percent (%) sequence identity" with respect to an amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific (parental) sequence, calculated after aligning the sequences and introducing gaps as necessary to achieve maximum sequence identity, with conservative substitutions not counted towards sequence identity. The alignment used to determine the percentage of amino acid sequence identity can be achieved in various ways known to those skilled in the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve optimal alignment of the full-length sequences being compared.

[0067] As used herein, the term "humanized antibody" encompasses one or more human framework regions in the variable region and complementarity determining regions (CDRs) of a non-human (such as murine, rat or hamster) heavy and / or light chain. In some embodiments, the humanized antibody is entirely derived from humans except for the CDR regions. Relative to non-humanized antibodies, humanized antibodies generally have lower immunogenicity in humans and thus have therapeutic advantages in certain situations.

[0068] The anti-CD112R antibodies or antigen-binding fragments of the present disclosure can be used to generally treat patients (such as human subjects) who generally suffer from diseases associated with CD112R. As used herein, the term "treatment" refers to therapeutic treatment and prophylactic or preventive measures, and in the examples involves the treatment of cancer as well as infectious diseases, sepsis and / or autoimmune diseases, and / or the inhibition of adverse immune activation that occurs after gene therapy. Subjects in need of treatment include individuals who already have cancer and those who need to prevent cancer. Thus, the mammalian to be treated herein may have been diagnosed with cancer, or may be susceptible or predisposed to cancer. As used herein, the terms "treating, treat, treatment" refer to preventing, delaying the onset, curing, reversing, alleviating, relieving, minimizing, inhibiting, preventing the harmful effects of the symptoms of the above-mentioned cancer diseases, disorders or conditions or stabilizing their recognizable symptoms. It also includes the management of the above-mentioned cancer. "Management" refers to reducing the severity of the disease, reducing the frequency of disease attacks, shortening the duration of such attacks, alleviating the severity of such attacks, slowing / reducing the growth or proliferation of cancer cells, delaying the progression of at least one symptom, improving at least one measurable physiological parameter, etc.

[0069] As used herein, the term "Fab" or "Fab region" refers to a polypeptide comprising the VH, CH1, VL and CL immunoglobulin domains. Fab can refer to the independent form of this region or to this region in a full-length antibody, antibody fragment or Fab fusion protein.

[0070] As used herein, the terms "infectious disorder and / or disease" and / or "infection" are used interchangeably and include any disorder, disease and / or condition caused by the presence and / or growth of a pathogenic biological agent in an individual host organism. As used herein, the term "infection" includes the above-mentioned disorders, diseases and / or conditions, manifested as clinically apparent disease (i.e., the characteristic medical signs and / or symptoms of the disease) and / or in most or all asymptomatic cases. As used herein, the term "infection" also includes disorders, diseases and / or conditions caused by the persistent presence of foreign antigens, which result in T cell phenotype exhaustion, characterized by impaired function manifested by reduced proliferation and reduced cytokine production.

[0071] As used herein, the term "sepsis" encompasses sepsis, severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, septicemia, toxemia, and septic syndrome.

[0072] As used herein, the term "CDC" or "complement dependent cytotoxicity" refers to the mechanism by which antibodies mediate the lysis of specific target cells by activating the complement system of an organism. The term "ADCC" or "antibody dependent cellular cytotoxicity" refers to the killing of antibody-coated target cells by effector cells of the immune system. Both CDC and ADCC can promote antibody-induced cell lysis.

[0073] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0074] The following examples are intended to provide a complete disclosure and description to those of ordinary skill in the art to guide how to produce the antibodies and fragments disclosed herein, and are not intended to limit the scope of the invention disclosed. In addition, the following examples do not represent all or the only experimental protocols.

[0075] Examples

[0076] Example 1. Generation of hCD112R-blocking mAb Using Hybridoma Technology

[0077] 1.1 Method

[0078] Recombinant human CD112R protein for mouse immunization and ELISA-based binding and competition assays

[0079] The extracellular domain (ECD) of human CD112R (hCD112R, Uniprot accession number #Q6DKI7) consisting of amino acids (AA) 42-172 (hCD112R-ECD) was amplified by PCR and cloned into an expression vector that was fused with a His6-Avi tag at the C-terminus (hCD112R-ECD-His6-Avi) or the Fc domain of murine IgG2a (hCD112R-ECD-mFc). These fusion proteins were expressed in HEK 293F cells by transient transfection and then purified by affinity chromatography.

[0080] Mouse immunization and hybridoma fusion

[0081] Six-week-old BALB / c mice were immunized subcutaneously with 100 μl of adjuvant (Sigma-Aldrich) containing 50 μg of hCD112R-ECD-mFc. Immunization was carried out by injecting the above immunogen two to three times at three-week intervals. One week after each immunization, blood samples were collected by tail bleeding. The reactivity of mouse sera to hCD112R-ECD was determined by an ELISA (enzyme-linked immunosorbent assay)-based binding assay. Animals with the highest anti-hCD112R Ab titers in their sera were selected and boosted intraperitoneally with 50 μg of hCD112R-ECD-mFc without adjuvant. Three days after boosting, splenocytes were isolated and fused with mouse myeloma cell line SP2 / 0 cells using conventional techniques. Supernatants of hybridoma clones were screened by ELISA-based binding and competition assays.

[0082] Amplification of functional immunoglobulin variable genes from hybridomas and construction of full-length murine IgG2a (mIgG2a) Ab

[0083] The VH and VL sequences of mAb 733 were amplified from the cDNA of hybridomas with binding and competition activities in ELISA-based assays. The PCR products of the VH and VL genes were directly cloned into a TA cloning vector using the pMD TM 18-T Vector Cloning Kit (Takara). The TA plasmids containing the inserted VH and VL genes were sequenced and analyzed using the IgBLAST tool. Then, the PCR products of these VH and VL were separately cloned into expression vectors containing the constant regions of the heavy chain (CH) and light chain (CL) of the mIgG2a molecule. The VH and VL sequences of mAb 733 encode the amino acid sequences shown in SEQ ID NO:9 and SEQ ID NO:10, respectively.

[0084] Preparation of full-length mIgG2a Ab

[0085] To produce full-length mIgG2a Ab, two expression plasmids (heavy chain + light chain plasmids) were transiently co-transfected into HEK 293F cells (Life Technologies) at a ratio of 1:1. Three to six days after transfection, the cell culture supernatants were collected and the IgG Ab was purified by protein A affinity chromatography (Protein A Sepharose CL-4B, GE Healthcare).

[0086] ELISA-based binding and competition assays

[0087] For the ELISA-based binding assay, 96-well plates (Nunc, MaxiSorp TM)Capture biotinylated hCD112R-ECD-His6-Avi protein. For ELISA based on cell culture supernatant, add the supernatant diluted with 2% milk / PBS, and then detect it with HRP anti-mouse IgG secondary antibody (Thermo Fisher Scientific). For ELISA based on mouse serum, add mouse serum diluted with 2% milk / PBS, and then detect it with HRP anti-mouse IgG secondary antibody (Thermo Fisher Scientific). For ELISA based on full-length mouse IgG, add IgG Ab diluted with 2% milk / PBS, and detect the bound antibody using HRP anti-mouse IgG secondary antibody (Thermo Fisher Scientific).

[0088] The ELISA-based competition assay is operated in a similar manner to the ELISA-based binding assay, except that the antibody to be tested is incubated with the captured antigen in the presence of the competing ligand. Briefly, different antibodies are serially diluted in a 2% milk / PBS solution containing the extracellular domain of hCD112 fused to the Fc domain of human IgG1 (hCD112-ECD-hFc) at 0.02 μg / ml, and then added to the ELISA plate to test the competitive binding between hCD112R and hCD112. The signal is measured by ligand detection using HRP-anti-human IgG secondary antibody (Therm Fisher Scientific).

[0089] FACS-based binding and competition assays

[0090] This experiment uses the YTS cell line (YTS-hCD112R) stably expressing full-length hCD112R. For cell line construction, an expression plasmid is constructed by inserting the full-length hCD112R cDNA into a vector. Then the expression plasmid is transfected into YTS cells using the Nucleofector transfection system (Lonza, Nucleofector kit V), and then the hCD112-stained positive population is sorted by FACS. The sorted positive cells are cultured under the selection of G418.

[0091] For the FACS-based binding assay, YTS-hCD112R cells are serially diluted with different anti-hCD112R mAbs or hCD112-ECD-mFc fusion proteins in 0.5% BSA / PBS and incubated at 4°C for 1 h. The antibodies or ligands bound to the cells are detected by adding goat anti-mouse IgG-FITC Ab (Pierce-Thermo Fisher Scientific).

[0092] For the FACS-based competition assay, in the presence of the competitive ligand hCD112-ECD-hFc (at a concentration of 0.6 μg / ml), YTS-hCD112R cells were incubated with antibodies in the mIgG2a format (at concentrations of 60 μg / ml or 0.6 μg / ml) at 4 °C for 1 h. Subsequently, the cells were washed three times with PBS containing 0.5% BSA. Ligands bound to the cells were detected by adding goat anti-human IgG-FITC Ab (Pierce-Thermo Fisher Scientific).

[0093] 1.2 Results

[0094] Anti-hCD112R mAbs were generated based on traditional hybridoma fusion technology. mAbs with high binding activity in ELISA-based binding assays and strong competitive activity in ELISA-based competition assays were selected for further characterization. The binding affinities ( Figure 1 A) and blocking activities ( Figure 1 B) of these mAbs were tested by FACS-based binding assays and competition assays, respectively. The results showed that mAb 733 had higher binding affinity and stronger blocking activity compared to other mAbs. Therefore, mAb 733 was selected for further characterization.

[0095] Example 2. Anti-hCD112R mAb 733 binds to hCD112R with high affinity and effectively blocks human CD112R-CD112 interaction

[0096] 2.1 Methods

[0097] Multi-cycle kinetic analysis of the interaction between 733 and hCD112R by SPR.

[0098] The kinetics of the binding of mAb 733 to hCD112R-ECD was analyzed using a Biacore T200 instrument (Biacore, GE Healthcare). Protein A / G (Thermo Fisher) was covalently linked to the surface of a CM5 sensor chip using an amine coupling kit (GE Healthcare). 733-mIgG2a (2 μg / ml) was captured on the chip, and then serially diluted analytes (hCD112R-ECD-His6-Avi) were injected. The binding kinetics was evaluated using a 1:1 Langmuir binding model. The association rate (ka), dissociation rate (kd), and affinity constant (KD) were calculated using the Biacore T200 evaluation software.

[0099] FACS-based competition assay

[0100] For the FACS-based competition assay, in the presence of the competitive ligand (hCD112-ECD-hFc), YTS-hCD112R cells were incubated with serially diluted 733-mIgG2a at 4 °C for 30 min. The cells were then washed three times with PBS containing 0.5% BSA. Ligand bound to the cells was detected by adding goat anti-human IgG-FITC Ab (Pierce-Thermo Fisher Scientific).

[0101] 2.2 Results

[0102] To detect the binding kinetic constants of mAb 733 in real-time binding reactions, surface plasmon resonance (SPR) assays were performed. The results showed that mAb 733 bound to hCD112R with high affinity (K D = 0.2 nM)( Figure 2 ). To determine the competitive activity of mAb733, FACS-based competition assays were performed. mAb 733 effectively blocked the human CD112R-CD112 interaction, with an IC 50 value of 0.29 μg / ml( Figure 3 ).

[0103] Example 3. Anti-hCD112R mAb 733 effectively reversed the cytotoxicity of NK cells inhibited by the human CD112R-CD112 interaction

[0104] 3.1 Methods

[0105] NK cell cytotoxicity assay

[0106] 1) Establish YTS-hCD112R and 721.221-hCD112 stable cell lines.

[0107] The generation of the YTS-hCD112R stable cell line has been described previously. To generate the 721.221-hCD112 stable cell line, the full-length cDNA of hCD112 delta was amplified by PCR and cloned into a mammalian cell expression plasmid. Then, according to the manufacturer's instructions, the full-length hCD112 expression plasmid was transfected into 721.221 cells using the Nucleofector transfection system (Lonza, Nucleofector kit V). One day after transfection, the transfected cells were immunostained with anti-hCD112 Ab (clone TX31, Biolegend), and then the positive cells were enriched by FACS sorting. The sorted positive cells were cultured under the selection of puromycin.

[0108] 2) Cytotoxicity assay

[0109] 721.221 - hCD112 target cells (10,000 cells / well) were incubated with YTS - hCD112R effector cells at an effector - to - target ratio (E:T) of 2:1 for 6 h, with or without the addition of mAb 733 at a final concentration of 10 μg / ml. Then, according to the instructions of the CytoTox

[0110] 3.2 Results

[0111] In this example, it was investigated whether mAb 733 could restore the activity of immune cells by blocking the human CD112R - CD112 interaction. It is known that CD112R is upregulated on T / NK cells in cancer and inhibits T / NK cell - mediated cytotoxicity through interaction with CD112. We then evaluated whether the binding of mAb 733 to hCD112R could interrupt the inhibitory function of the human CD112R - CD112 interaction on NK cells. Previous studies have shown that YTS cells (a NK cell line) achieved restricted killing of 721.221 target cells (a MHC class I - negative human B cell line). On this basis, YTS cells stably expressing hCD112R (YTS - hCD112R) and 721.221 cells stably expressing hCD112 (721.221 - hCD112) were established ( Figure 4 A - B). The human CD112R - CD112 interaction effectively inhibited the cytotoxicity of YTS cells against 721.221 cells, while mAb 733 significantly restored the ability of YTS - hCD112R cells to kill 721.221 - hCD112 cells ( Figure 4 C). This result indicates that mAb 733 effectively blocked the inhibitory function mediated by the human CD112R - CD112 interaction.

[0112] Example 4. Humanization of mAb 733

[0113] 4.1 Methods

[0114] Humanization of mAb 733

[0115] For the humanization of mAb 733, the amino acid sequences of human germline immunoglobulin G (IgG) homologous to the amino acid sequence of mAb 733 were searched by aligning with the human immunoglobulin gene database on the NCBI website (http: / / www.ncbi.nlm.nih.gov / igblast / ). The human framework with the highest homology to the mAb 733 framework was selected as the template for CDR grafting. Other mutations were also made to evaluate the impact on antibody binding.

[0116] Kinetics of the interaction between mAb 733 and 733-derived humanized antibodies and hCD112R by SPR Analysis

[0117] The kinetics of the binding of 733 and various 733-derived humanized antibodies to the hCD112R-ECD protein were analyzed using a Biacore T200 instrument (Biacore, GE Healthcare). Anti-hFc Ab (Thermo Fisher) was covalently linked to the surface of a CM5 sensor chip using an amine coupling kit (GE Healthcare). The optimal concentration of the antibody was captured on the chip, and then a single concentration or serial dilutions of the analyte (hCD112R-ECD-His6-Avi) were injected. The binding kinetics were evaluated using a 1:1 Langmuir binding model. The binding rate (ka), dissociation rate (kd), and affinity constant (KD) were calculated using the Biacore T200 evaluation software.

[0118] ELISA-based competition assay

[0119] A 96-well plate (Nunc, MaxiSorp TM ) coated with streptavidin (Sigma) was used to capture the biotinylated hCD112R-ECD-His6-Avi protein antigen. H733 was serially diluted in 2% milk / PBS containing 0.1 μg / ml of hCD112-ECD-mFc protein and added to the ELISA plate to test for competitive binding between hCD112R and hCD112. The signal was determined by ligand detection using an HRP-anti-mouse IgG secondary antibody (Thermo Fisher Scientific).

[0120] 4.2 Results

[0121] For the humanization of mAb 733, we searched for the human germline IgG domain sequences homologous to the mAb 733 amino acid sequence by aligning with the human immunoglobulin database on the NCBI website (http: / / www.ncbi.nlm.nih.gov / igblast / ). The human framework with the highest homology to the mAb 733 framework was selected as the template for CDR grafting.Figure 5 shows the transplantation of the Kabat-defined CDRs of the mAb733 heavy chain (SEQ ID NO:9) and light chain (SEQ ID NO:10) into the closest human germline templates IGHV1-46*01 and IGKV1-9*01, respectively. The transplanted VL and VH chains of mAb 733 were named Ab733-VL-human V1 (SEQ ID NO:12) and Ab 733-VH-human V1 (SEQ ID NO:11). Given that the unpaired cysteine in the VL-CDR3 domain of mAb 733 might affect the protein expression level, the cysteine was further mutated to serine ( Figure 5 ). The humanized VL with one amino acid substitution was named Ab 733-VL-human-C91S (SEQ ID NO:14). In addition, three amino acids in CDR2 of Ab 733-VH-human V1 were further mutated from the original murine residues to human germline residues. The resulting VH was named Ab 733-VH-human V2 (SEQ ID NO:13). Full-length IgG Abs composed of various VH / VL combinations were expressed and their binding affinities to hCD112R were analyzed by SPR.

[0122] The results showed that the binding affinities of different humanized antibody versions were almost the same as those of the parental mAb 733 ( Figure 6 A). The antibody composed of mAb 733-VH-human V2 and 733-VL-human-C91S was named H733. The binding affinity of H733 to hCD112R was further evaluated by multi-cycle kinetic analysis. The results showed that the binding affinity of H733 (KD = 0.65 nM) was comparable to that of 733 ( Figure 6 B). The blocking activity of H733 was tested by an ELISA-based competition experiment. The results showed that H733 could effectively block the CD112R-CD112 interaction, with an IC 50 value of 0.87 μg / ml ( Figure 7 ). Therefore, H733 was used in the following experiments.

[0123] Example 5. Epitope mapping of H733

[0124] 5.1 Method

[0125] Kinetic analysis of the interaction between H733 or hCD112 and mutant hCD112R by SPR.

[0126] The kinetics of the binding of WT or mutant hCD112R-ECD-mFc proteins to H733 and hCD112-ECD were analyzed using a Biacore T200 instrument (Biacore, GE Healthcare). For single-cycle SPR analysis, anti-mFc Ab (Thermo Fisher) was covalently linked to the CM5 sensor chip surface using an amine coupling kit (GE Healthcare). WT or mutant hCD112R-ECD mFc fusion proteins were captured on the chip, and then H733-hIgG4 or hCD112-ECD-hFc was injected. For multi-cycle SPR analysis, anti-hFc Ab (ThermoFisher) was covalently linked to the CM5 sensor chip surface using an amine coupling kit (GE Healthcare). H733-hIgG4 or hCD112-ECD-hFc proteins were captured on the chip, and then two-fold serial dilutions of WT or mutant hCD112R-ECD-mFc proteins (3.125 - 100 nM) were injected. The binding kinetics were evaluated using a 1:1 Langmuir binding model. The binding rate (ka), dissociation rate (kd), and affinity constant (KD) were calculated using Biacore T200 evaluation software.

[0127] 5.2 Results

[0128] To investigate the sites on hCD112R that might contribute to H733 binding, we constructed several hCD112R mutants by replacing residues in the IgV domain of hCD112R with the corresponding residues from cynomolgus monkey CD112R (cynoCD112R). Figure 8 ) These hCD112R mutants and wild-type (WT) hCD112R proteins were expressed and purified, and then their binding to H733 and hCD112 ligand was tested by SPR. The results of single-cycle SPR analysis showed that the R95L human-cynomolgus monkey mutation of CD112R had a negative impact on the binding of H733. Figure 9 )

[0129] Based on the above results, we next selected other residues in the hCD112R-IgV domain for alanine-scanning mutagenesis based on the structure predicted by the AlphaFold Protein Structure Database. These residues were L72, V90, H92, E94, W100, K135, F139, and E145. Figure 10A). After expression and purification, these hCD112R mutants were used to analyze the binding to H733 and hCD112. The multi-cycle SPR results showed that the V90A, W100A, or E145A mutations of hCD112R reduced the binding to H733 (about 4 - 15 times less than that of WT hCD112R). Figure 10 B). Notably, the V90A, W100A, or E145A mutations of hCD112R also significantly reduced the binding to hCD112. Figure 10 C). In summary, by applying site-directed mutagenesis mapping, we found that the R95, V90, W100, and E145 residues of hCD112R affected the binding to H733.

[0130] Example 6. H733 effectively reversed the function of human lymphocytes inhibited by CD112R - CD112 interaction

[0131] 6.1 Method

[0132] Cytotoxicity assay of NK cells

[0133] In the presence of serially diluted anti - hCD112R mAb, 721.221 - hCD112 target cells (10000 cells / well) were co - incubated with YTS - hCD112R effector cells at a specified effector - to - target ratio (E:T) of 2:1 for 6 h. Then, the lactate dehydrogenase (LDH) released by the cells was detected according to the instructions of the non - radioactive cytotoxicity detection kit (Promega). The percentage of cytotoxicity was calculated according to the manufacturer's instructions.

[0134] Human T cell proliferation assay

[0135] 1) Isolation of primary human T cells

[0136] Human peripheral blood mononuclear cells (PBMC) from healthy donors were purchased from ORiCELLS. According to the manufacturer's instructions, human T cells were negatively selected and purified using the EasySep human T cell enrichment kit (STEMCELL, #19051).

[0137] 2) Construction of human T cell - stimulating cells

[0138] CHO cells were transfected with an expression plasmid encoding the single-chain variable fragment (OKT3-scFv) sequence of the membrane-bound anti-human CD3 mAb OKT3 (Leitner et al., Journal of immunological methods (2010) 362, 131-141) to obtain a CHO cell pool expressing membrane-bound OKT3-scFv. After 24 h of transfection, the cells were stained with FITC-labeled goat anti-mouse whole IgG Ab (Sigma Aldrich), and the positively stained single cells were isolated by FACS single cell sorting and then cultured in 200 μl of complete medium containing G418. The effect of H733 on human T cell proliferation was tested by transfecting the CHO-OKT3-scFv stable cell line with an expression plasmid encoding the full-length hCD112 delta molecule (CHO-Okt3-scFv-hCD112). After 24 h of transfection, the cells were stained with APC conjugated to anti-hCD112 Ab (clone TX31, Biolegend). Then the positive cells were enriched by FACS sorting technology.

[0139] 3) Human T cell proliferation assay

[0140] Negatively selected and enriched human T cells were labeled with CFSE and stimulated with stimulator cells. The stimulator cells (CHO-OKT3-scFv or CHO-OKT3-scFv-hCD112) were first treated with mitomycin C (10 μg / ml for 10 h) and then co-cultured with CFSE-labeled human T cells at a ratio of 1:100. A control antibody or H733 at 1 μg / ml was added at the start of the culture. After 5 days of culture, T cell proliferation was evaluated by CFSE dilution.

[0141] Luciferase reporter gene assay

[0142] 1) Establishment of the Jurkat-NFAT-luc_hCD112R cell line

[0143] The Jurkat-NFAT-luc-hCD112R cells were generated by electroporating a plasmid encoding the full-length hCD112R cDNA sequence into Jurkat-NFAT-luc cells (purchased from InvivoGen) and screened with G418 antibiotic (500 μg / ml). The surface expression of hCD112R was confirmed by flow cytometry after staining with PE-conjugated anti-hCD112R Ab (clone 301503, Biolegend).

[0144] 2) Luciferase reporter gene assay

[0145] Harvest CHO-OKT3scFv or CHO-OKT3scFv-hCD112 cells and seed them into 96-well plates, 100 μL of medium (DMEM containing 10% FBS) per well with 50,000 cells, and then incubate at 37 °C and 5% CO2 for 2 h. Then remove the medium and add Jurkat-NFAT-luc-hCD112R cells, 100 μL of assay medium (IMDM containing 10% FBS) per well with 50,000 cells. The anti-hCD112R antibody is serially diluted at a ratio of 1:2 in the assay medium starting at a concentration of 5 μg / ml and then added to each well. After that, the plates are incubated at 37 °C and 5% CO2 for 18 h. Subsequently, transfer 20 μL of the sample from each well to a 96-well black plate and add 50 μL of QUANTI-Luc TM detection reagent. Then record the relative luciferase units (RLU) using a microplate reader (BioTek Synergy H1M).

[0146] 6.2 Results

[0147] We evaluated the blocking function of H733 on NK cells using the YTS-hCD112R / 721.221-hCD112 cell killing assay. In the example, an antibody named COM701 developed by Compugen (U.S. Patent No.: US20160244521A1) was used as the anti-hCD112R reference antibody control. The results showed that both H733 and COM701 could effectively reverse CD112R-mediated NK cell dysfunction( Figure 11 ).

[0148] Next, we tested the potential function of H733 on human T cells using the Jurkat luciferase reporter assay and proliferation assay. For the Jurkat luciferase reporter experiment, CHO cells expressing the membrane-bound anti-human CD3 antibody fragment (CHO-OKT3scFv) or expressing both the membrane-bound OKT3scFv fragment and hCD112 (CHO-OKT3scFv_hCD112)( Figure 12 A) were co-incubated with Jurkat-NFAT-luc-hCD112R reporter gene cells( Figure 12 B). The addition of H733 or COM701 increased luciferase production( Figure 12 C), and their EC 50 values were 0.39 μg / ml and 0.69 μg / ml, respectively, indicating that the blockade of CD112R-CD112 interaction by H733 could enhance pro-inflammatory cytokine signal transduction in human T cells.

[0149] To evaluate the potential effect of H733 on human T cell proliferation, we labeled purified human T cells with CFSE and stimulated them with CHO-OKT3scFv or CHO-OKT3scFv-hCD112 cells. By CFSE dye dilution, we observed a significant increase in T cell division in the presence of H733 compared to the control group( Figure 13 ). These data indicate that the blockade of CD112R-CD112 interaction by H733 can promote the proliferation of human T cells.

[0150] Taken together, these results indicate that H733 can effectively reverse the inhibitory function of CD112R-mediated on human NK and T cells, suggesting that H733 may enhance the killing ability of cancer cells by blocking the CD112R-CD112 interaction in vivo.

[0151] Example 7. H733-hIgG4 mAb did not exhibit CDC effector function and had a weak ADCC effector function

[0152] 7.1 Method

[0153] Construction of antibodies in the context of different Fc

[0154] Various H733 antibodies and three Fc mutants of hIgG1 isotype were constructed by site-directed mutagenesis in the context of different human IgG isotypes (hIgG1 and hIgG4): Fc-D265A / N297G (DANG) mutant; Fc-S239D / I332E (DE) mutant and Fc-S239D / A330L / I332E (DLE) mutant, which have eliminated or enhanced complement and FcγR-mediated effector functions respectively, and were expressed in HEK 293F cells by transient transfection.

[0155] Complement-mediated cytotoxicity (CDC) assay

[0156] For the CDC assay, Raji cells stably expressing full-length hCD112R (Raji-hCD112R) were established and used as target cells in the assay. The cells were seeded at 2.5×10 4 cells / well in a U-bottom 96-well plate and incubated with 5 μg / ml antibody in the presence of 5% rabbit serum (Sigma). After 2 h of incubation, the release of LDH in the supernatant of each well was analyzed using the CytoTox Non-Radioactive Cytotoxicity Assay Kit (Promega).

[0157] Antibody-dependent cell-mediated cytotoxicity (ADCC) assay using T cell lines.

[0158] For ADCC assays, Raji-hCD112R cells were used as target cells in this assay. A Jurkat cell line stably expressing human FcγRIIIa (F158) and a nuclear factor of activated T cells (NFAT)-responsive element-driven firefly luciferase reporter gene (named Jurkat-NFAT-Luc2p / hFcγRIIIa (F158)) was generated and used as effector cells. The target cells (15,000 cells / well) were seeded into U-bottom 96-well cell culture plates and briefly incubated with different antibodies. Subsequently, effector cells (90,000 cells / well) were added to the wells and incubated at 37 °C for 5 h in RPMI 1640 medium supplemented with 1% heat-inactivated fetal bovine serum. According to the instructions of the Bright-Glo TM Luciferase Assay Reagent (Promega), the ADCC activity was measured by the expression of luciferase.

[0159] 7.2 Results

[0160] Since CD112R is preferentially expressed on T cells and NK cells, it was expected that CD112R-blocking antibodies conjugated to naturally occurring IgG-Fc proteins would induce Fc-mediated effector cell functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and the degree of induction would vary depending on the IgG isotype, which might lead to the elimination of activated effector cells. Therefore, to prevent CD112R + T cells from being killed, H733 was conjugated to human IgG4 and its ability to induce ADCC and CDC was evaluated by cell-based assays.

[0161] To test the ADCC of the antibody, an improved system was used in this example, in which Jurkat T lymphocytes expressing human FcγRIIIa (F158) and the NFAT response element driving firefly luciferase (Jurkat-NFAT-Luc2p / hFcγRIIIa (F158)) were used as effector cells, and Raji-hCD112R was used as target cells ( Figure 14A). Jurkat-NFAT-Luc2p / hFcγRIIIa (F158) cells were co-cultured with Raji-hCD112R cells in a 96-well U-bottom plate in the presence of different forms of H733 for 5 h. Cytotoxicity was detected by LDH release. Consistent with expectations, H733 conjugated with hIgG1 or hIgG1-DE mutant (S239D / I332E mutation with enhanced Fc-mediated effector function) showed significant ADCC. In contrast, H733 conjugated with hIgG4 induced weaker ADCC, while H733 conjugated with hIgG1-DANG mutant (D265A / N297G mutation with abolished Fc-mediated effector function) did not induce ADCC. Figure 14 B).

[0162] For the detection of antibody CDC activity, Raji-hCD112R cells were used as target cells. Figure 14 A), and the amount of lactate dehydrogenase (LDH) release was used as a readout of target cell lysis by complement. The results showed that only H733 hIgG1 and hIgG1-DE exhibited effective CDC function, while H733 hIgG4 and other mutants did not. Figure 15 ).

[0163] In summary, these cell-based assays showed that H733-hIgG4 exhibited weaker Fc-mediated effector function compared to the H733-hIgG1 antibody. Therefore, H733 (in hIgG4 form) can be used to test the anti-tumor effect in vivo.

[0164] Example 8. H733 exerted potent anti-tumor activity in xenograft mouse tumor models

[0165] 8.1 Method

[0166] Xenograft mouse tumor model and treatment

[0167] For the MDA-MB231 tumor model, 1×10 6 MDA-MB-231 cells were mixed with 3×10 5 unstimulated PBMCs and subcutaneously injected (s.c.) into the right flank of 6-8-week-old NSG mice on day 0. For the A375 tumor model, 3×10 6 A375 cells were mixed with 1×10 6 unstimulated PBMCs and subcutaneously injected into the right flank of 6-8-week-old NSG mice on day 0. For the A549 tumor model, 2×10 6 A549 cells were mixed with 6×10 5A mixture of unstimulated PBMCs was subcutaneously injected into the right flank of NCG mice on day 0. Mice with similar tumor volumes (100 - 150 mm 3 ) were randomly divided into two groups (n = 3 - 6 per group): an untreated control group or an H733-hIgG4 treatment group (10 mg / kg), and then antibody treatment was carried out. The treatment was repeated twice a week.

[0168] 8.2 Results

[0169] Since H733 does not bind to mCD112R, we used a humanized xenograft mouse tumor model based on human peripheral blood mononuclear cells (PBMCs) to evaluate the in vivo antitumor effect of H733. Female immunodeficient NSG mice at 6 - 8 weeks of age were subcutaneously injected with a mixture of human PBMCs and tumor cells (MDA-MB-231 human breast cancer cells, A375 human melanoma cells, or A549 human lung cancer cells; note that all of these tumor cells are hCD112 positive) ( Figure 16 A). Compared with the untreated control group, H733 treatment could cause regression of all these tumors ( Figure 16 B). This result indicates that H733 exerted a powerful antitumor activity in vivo.

Claims

1. An antigen-binding protein that specifically binds to CD112R.

2. The antigen-binding protein according to claim 1, which is an antibody or an antigen-binding fragment thereof.

3. The antigen-binding protein according to claim 1 or 2, comprising: (1) An immunoglobulin heavy chain variable region, comprising: HCDR1 as shown in SEQ ID NO:1 or HCDR1 having one or more amino acid additions, deletions or substitutions on the amino acid sequence shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2 or HCDR2 having one or more amino acid additions, deletions or substitutions on the amino acid sequence shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3 or HCDR3 having one or more amino acid additions, deletions or substitutions on the amino acid sequence shown in SEQ ID NO:3; and / or (2) An immunoglobulin light chain variable region, comprising: LCDR1 as shown in SEQ ID NO:4 or LCDR1 having one or more amino acid additions, deletions or substitutions on the amino acid sequence shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5 or LCDR2 having one or more amino acid additions, deletions or substitutions on the amino acid sequence shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6 or LCDR3 having one or more amino acid additions, deletions or substitutions on the amino acid sequence shown in SEQ ID NO:

6.

4. The antigen-binding protein according to any one of claims 1 to 3, comprising: An immunoglobulin heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:9; and / or An immunoglobulin light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:

10.

5. The antigen-binding protein according to any one of claims 1 to 4, which is a humanized antibody or an antigen-binding fragment thereof.

6. The antigen-binding protein according to claim 5, wherein the humanized antibody or its antigen-binding fragment comprises: (1) A heavy chain variable region (VH), which comprises: HCDR1, which has the amino acid sequence as shown by NYLIE, HCDR2, which has the amino acid sequence as shown by VINPGHGFTNYX1X2KFX3G; and HCDR3, which has the amino acid sequence as shown by GEWDWYFDV; and / or (2) A light chain variable region (VL), which comprises: LCDR1, which has the amino acid sequence as shown by KASQNVGTAVA, LCDR2, which has the amino acid sequence as shown by STSNRYT, and LCDR3, which has the amino acid sequence as shown by QQX4SSYPFT; wherein, X1 is selected from A or N, X2 is selected from E or Q, X3 is selected from K or Q, and / or X4 is selected from C or S.

7. The antigen-binding protein according to claim 5 or 6, wherein the humanized antibody or its antigen-binding fragment comprises: (1) A heavy chain variable region (VH), which comprises: HCDR1, which has the amino acid sequence as shown by SEQ ID NO:1, HCDR2, which has the amino acid sequence as shown by SEQ ID NO:2, and HCDR3, which has the amino acid sequence as shown by SEQ ID NO:3; or HCDR1, which has the amino acid sequence as shown by SEQ ID NO:1, HCDR2, which has the amino acid sequence as shown by SEQ ID NO:7, and HCDR3, which has the amino acid sequence as shown by SEQ ID NO:3; and / or (2) A light chain variable region (VL), which comprises: LCDR1, which has the amino acid sequence as shown by SEQ ID NO:4, LCDR2, which has the amino acid sequence as shown by SEQ ID NO:5, and LCDR3, which has the amino acid sequence shown in SEQ ID NO:6; or LCDR1, which has the amino acid sequence shown in SEQ ID NO:4, LCDR2, which has the amino acid sequence shown in SEQ ID NO:5, and LCDR3, which has the amino acid sequence shown in SEQ ID NO:

8.

8. The antigen-binding protein according to any one of claims 5 to 7, wherein the humanized antibody or its antigen-binding fragment comprises: A heavy chain variable region (VH) that has the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:13, or has an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:13; and / or A light chain variable region (VL) that has the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:14, or has an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:

14.

9. The antigen-binding protein according to any one of claims 1 to 8, which specifically binds to CD112R, particularly the extracellular domain (ECD) of CD112R, and more preferably, specifically binds to amino acids 90-150 of CD112R.

10. A composition comprising the antigen-binding protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. The composition according to claim 10, which further comprises an additional therapeutic agent.

12. A nucleic acid molecule encoding the antigen-binding protein according to any one of claims 1 to 9.

13. A host cell comprising the antigen-binding protein according to any one of claims 1 to 9 or the nucleic acid molecule according to claim 12, wherein the host cell includes a prokaryotic cell or a eukaryotic cell.

14. A method for preventing or treating a subject suffering from a CD112R-related disease, which comprises administering to the subject a therapeutically effective amount of the antigen-binding protein according to any one of claims 1 to 9, or the composition according to claim 10 or 11.

15. The method according to claim 14, wherein the disease includes cancer, infectious diseases, sepsis, autoimmune diseases, and / or adverse immune activation after gene therapy.

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