Anti-KIR3DL3 antibodies and uses thereof

Abstract

To provide anti-KIR3DL3 antibodies and uses thereof.SOLUTION: The disclosure is based, in part, on the discovery of the monoclonal antibodies that specifically bind to KIR3DL3 and antigen binding fragments thereof, bispecific antibodies that bind to KIR3DL3 and PD-1 and antigen binding fragments thereof, immunoglobulins, polypeptides, nucleic acids thereof, as well as methods for using such antibodies for prognostic, immunomodulatory and therapeutic purposes. The disclosure is based, in part, on the discovery that agents targeting KIR3DL3 (e.g., antibodies) can specifically block the HHLA2-KIR3DL3 interaction, thus usable in methods for modulating immune response.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 910,594, filed October 4, 2019, the entire contents of which are incorporated herein by reference in their entirety.

[0002] STATEMENT OF GOVERNMENT RIGHTS This invention was made with government support under Grant No. P50CA101942 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Immune checkpoints, such as CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, butyrophilin, and A2aR, and many more, negatively regulate the progression of immune responses based on complex combinatorial interactions between multiple inputs. Immune checkpoint inhibitors can modulate immune responses in some subjects, but immune checkpoint expression and interactions with natural binding partners vary between subjects and within a subject's tissues. A significant proportion of patients do not respond to this treatment, and many who respond eventually develop resistance. Thus, there is a significant unmet need to identify additional immune pathways that are not redundant with the PD-1 pathway.

[0004] HERV-H LTR-associated 2 (HHLA2), also known as B7-H5 or B7-H7, is a B7 family member that regulates T cell function. HHLA2 is widely expressed in various tumors (e.g., solid and hematological cancers, including primary human renal cell carcinoma (RCC)) and antigen-presenting cells, and is involved as both an activating and inhibitory ligand for T cells. HHLA2 has been identified as a specific ligand for TMIGD2 (CD28H, IGPR-1), and HHLA2 / TMIGD2 interaction selectively co-stimulates human T cell proliferation and cytokine production through an AKT-dependent signaling cascade (Zhu et al. (2013) Nat. Comm. 4:2043, Janakiram et al. (2015) Clin. Cancer Res. 21:2359-2366). TMIGD2, expressed on naive T cells, is an activating receptor for HHLA2 and transmits costimulatory signals after T cell antigen receptor (TCR) ligation. TMIGD2 is downregulated after repeated TCR stimulation. It is possible that putative inhibitory receptors for HHLA2 are upregulated on activated T cells to regulate T cell activation. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Zhu et al.(2013)Nat.Comm.4:2043 [Non-patent document 2] Janakiram et al.(2015)Clin.Cancer Res.21:2359-2366 Summary of the Invention

[0006] Prior to the present disclosure, several studies suggested the existence of an uncharacterized receptor for HHLA2 on activated T cells that exerts a co-inhibitory function (Zhao et al. (2013) Proc. Natl. Acad. Sci. USA 110:9879-9884; Xiao and Freeman et al. (2015) Clin. Cancer Res. 21:2201-2203; Wang et al. (2014) J. Immunol. 192:126.11). It was discovered that HHLA2 binds to KIR3DL3, a receptor on T cells and NK cells, and that the result of HHLA2-KIR3DL3 interaction is inhibition of T cell and NK cell activation (PCT / US2019 / 026034). Accordingly, the present disclosure encompasses the recognition that the KIR3DL3 receptor is a candidate for cancer immunotherapy, and provides herein compositions and methods for targeting KIR3DL3 to modulate immune responses.

[0007] The present disclosure is based, at least in part, on the discovery that agents (e.g., antibodies) targeting KIR3DL3 can specifically block HHLA2-KIR3DL3 interaction and can be used in methods for regulating immune responses. Importantly, we present here that targeting KIR3DL3 does not disrupt the overall function of HHLA2, including activating immune responses through its interaction with TMIGD2. Thus, the present disclosure provides the important and surprising finding that targeting KIR3DL3 provides the specificity of blocking only the immunoinhibitory function of HHLA2, thereby inducing effective immune responses (e.g., against cancer cells) without downregulating the immunostimulatory function of HHLA2. The development of agents that specifically block the immunoinhibitory activity of the HHLA2 pathway while maintaining its stimulatory function represents a new approach to immune checkpoint blockade in patients with cancer (e.g., hematological cancers and solid tumors, including clear cell renal cell carcinoma (ccRCC)).

[0008] The present disclosure is also based, at least in part, on the discovery that agents targeting both KIR3DL3 and PD-1 can be used to modulate immune responses and / or treat cancer. In some embodiments, the KIR3DL3xPD-1 bispecific antibodies described herein are useful as checkpoint immunotherapies, such as activating T cells and NK cells in tumors. In some embodiments, the KIR3DL3xPD-1 bispecific antibodies are additive or synergistic with PD-1 or PD-L1, or other checkpoint immunotherapies. Furthermore, HHLA2 and / or KIR3DL3 expression in tumors is a useful biomarker for determining responsiveness to KIR3DL3 mAb and / or KIR3DL3xPD-1 bispecific antibody checkpoint blockade.

[0009] A panel of exemplary, representative anti-KIR3DL3 human monoclonal antibodies (mAbs) are described herein as immune checkpoint inhibitor agents. Blocking and non-blocking anti-KIR3DL3 mAbs were identified, and anti-KIR3DL3 mAbs that block HHLA2 binding to KIR3DL3 were shown to be checkpoint inhibitor antibodies in T cell and NK cell assays.

[0010] In one aspect, a monoclonal antibody or antigen-binding fragment thereof is provided, comprising: a) a heavy chain sequence having at least about 95% identity to a heavy chain sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8; and / or b) a light chain sequence having at least about 95% identity to a light chain sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8.

[0011] In another aspect, a monoclonal antibody or antigen-binding fragment thereof is provided, comprising: a) one, two, or three heavy chain CDR sequences, each having at least about 95% identity to a heavy chain CDR sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8; and / or b) one, two, or three light chain CDR sequences, each having at least about 95% identity to a light chain CDR sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8.

[0012] In yet another aspect, a monoclonal antibody or antigen-binding fragment thereof is provided, comprising: a) a heavy chain sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8; and / or b) a light chain sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8.

[0013] In yet another aspect, a monoclonal antibody or antigen-binding fragment thereof is provided, comprising: a) one, two, or three heavy chain CDR sequences selected from the group consisting of the sequences listed in Tables 2, 7, and 8, respectively; and / or b) one, two, or three light chain CDR sequences selected from the group consisting of the sequences listed in Tables 2, 7, and 8, respectively.

[0014] Numerous embodiments are further provided that can be applied to any aspect encompassed by the present disclosure described herein. For example, in one embodiment, the monoclonal antibody or antigen-binding fragment thereof is chimeric, humanized, composite, murine, or human. In another embodiment, the monoclonal antibody or antigen-binding fragment thereof (a) is detectably labeled, (b) is conjugated to a cytotoxic agent, optionally a chemotherapeutic agent, a biological agent, a toxin, and / or a radioisotope, (c) comprises an effector domain, (d) comprises an Fc domain, and / or (e) is selected from the group consisting of Fv, Fav, F(ab')2), Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. In yet another embodiment, the monoclonal antibody or antigen-binding fragment thereof can be obtained from hybridoma ______ deposited under deposit accession number ______. In yet another embodiment, the monoclonal antibody or antigen-binding fragment thereof inhibits binding of HHLA2 to KIR3DL3. KIR3DL3 mAbs that block HHLA2 binding to KIR3DL3 in T cell activation assays have been shown to be checkpoint blockers. In another embodiment, the monoclonal antibody or antigen-binding fragment thereof specifically binds to KIR3DL3.

[0015] A panel of exemplary representative bispecific antibodies that bind to KIR3DL3 and PD-1 are described herein as immune checkpoint inhibitor agents.

[0016] In one aspect, provided herein is a bispecific antibody or antigen-binding fragment thereof, comprising a) a heavy chain sequence having at least about 95% identity to a heavy chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9, and / or b) a light chain sequence having at least about 95% identity to a light chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9.

[0017] In another aspect, a bispecific antibody or antigen-binding fragment thereof is provided, comprising: a) one, two, or three heavy chain CDR sequences, each having at least about 95% identity to a heavy chain CDR sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9; and / or b) one, two, or three light chain CDR sequences, each having at least about 95% identity to a light chain CDR sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9.

[0018] In yet another aspect, there is provided a bispecific antibody or antigen-binding fragment thereof, comprising: a) a heavy chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9; and / or b) a light chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9.

[0019] In yet another aspect, there is provided a bispecific antibody or antigen-binding fragment thereof comprising a) one, two or three heavy chain CDR sequences selected from the group consisting of the sequences listed in Tables 2 and 7-9, respectively, and / or b) one, two or three light chain CDR sequences selected from the group consisting of the sequences listed in Tables 2 and 7-9, respectively.

[0020] Numerous embodiments are provided that can be applied to any aspect encompassed by the present disclosure described herein. For example, in one embodiment, the bispecific antibody or antigen-binding fragment thereof is chimeric, humanized, composite, murine, or human. In another embodiment, the bispecific antibody or antigen-binding fragment thereof (a) is detectably labeled, (b) is conjugated to a cytotoxic agent, optionally a chemotherapeutic agent, a biologic agent, a toxin, and / or a radioisotope, (c) comprises an effector domain, (d) comprises an Fc domain, and / or (e) is selected from the group consisting of Fv, Fav, F(ab')2), Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. In yet another embodiment, the bispecific antibody or antigen-binding fragment thereof can be obtained from hybridoma ______ deposited under deposit accession number ______. In yet another embodiment, the bispecific antibody or antigen-binding fragment thereof inhibits (a) binding of HHLA2 to KIR3DL3 and (b) binding of PD-1 to PD-L1 and / or PD-L2. Bispecific antibodies that bind to both KIR3DL3 and PD-1 have been shown to be checkpoint blockers. In another embodiment, the bispecific antibody or antigen-binding fragment thereof specifically binds to KIR3DL3 and PD-1. In yet another embodiment, the bispecific antibody or antigen-binding fragment thereof comprises a) a heavy chain sequence listed in Table 9, and / or b) a light chain sequence listed in Table 9.

[0021] In another embodiment, an immunoglobulin heavy and / or light chain selected from the group consisting of the immunoglobulin heavy and light chain sequences listed in Tables 2 and 7-9 is provided.

[0022] In yet another aspect, provided is an isolated nucleic acid molecule that hybridizes under stringent conditions to (a) an immunoglobulin heavy chain, an immunoglobulin light chain, and / or a monoclonal antibody or antigen-binding fragment thereof encompassed by the disclosure described herein, and / or (b) the complement of a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 2 and 7-9, or a sequence having at least about 95% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 2 and 7-9.

[0023] In yet another aspect, there is provided a vector comprising the isolated nucleic acid described herein.

[0024] In another aspect, a host cell is provided that comprises an isolated nucleic acid described herein, that comprises a vector described herein, that expresses an antibody or antigen-binding fragment thereof described herein, or that is available under deposit accession number ______.

[0025] In yet another aspect, a device or kit is provided that includes at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) described herein, optionally including a label for detecting the at least one antibody or antigen-binding fragment thereof, or a complex comprising the antibody or antigen-binding fragment thereof.

[0026] In yet another aspect, provided is a method of producing at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) described herein, comprising: (i) culturing a transformed host cell transformed with a nucleic acid comprising at least one encoding sequence according to the present disclosure under conditions suitable to allow expression of the antibody or antigen-binding fragment thereof; and (ii) recovering the expressed antibody or antigen-binding fragment thereof.

[0027] In another aspect, a method for detecting the presence or level of a KIR3DL3 polypeptide comprises detecting the polypeptide in a sample using at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) described herein. In one embodiment, the at least one antibody or antigen-binding fragment thereof forms a complex with the KIR3DL3 polypeptide, and the complex is detected in an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), immunochemically, in a Western blot, or using an intracellular flow assay.

[0028] In yet another aspect, a method for predicting responsiveness to a therapy targeting KIR3DL3 is provided, the method comprising: a) determining the level of KIR3DL3 and / or HHLA2 in a subject sample using at least one antibody or antigen-binding fragment thereof described herein (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof); b) determining the level of KIR3DL3 and / or HHLA2 in a sample from at least one control subject who has a good response to a therapy targeting KIR3DL3 using at least one antibody or antigen-binding fragment thereof described herein; and c) comparing the level of KIR3DL3 and / or HHLA2 in the subject sample with the level of KIR3DL3 and / or HHLA2 in a sample from the control subject, wherein the same or higher level of KIR3DL3 and / or HHLA2 in the subject sample compared to the level in the sample from the at least one control subject indicates that the subject will respond to the therapy. In one embodiment, the therapy targets KIR3DL3 using at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) described herein.

[0029] In yet another aspect, there is provided a method for predicting responsiveness to a therapy targeting KIR3DL3 using at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) described herein, the method comprising: a) determining the level of KIR3DL3 and / or HHLA2 in a subject sample; b) determining the level of KIR3DL3 and / or HHLA2 in a sample from at least one control subject who has a good response to therapy targeting KIR3DL3; and c) comparing the level of KIR3DL3 and / or HHLA2 in the subject sample with the level of KIR3DL3 and / or HHLA2 in a sample from the control subject, wherein the same or higher level of KIR3DL3 and / or HHLA2 in the subject sample compared to the level in the sample from the at least one control subject indicates that the subject will respond to the therapy.

[0030] As mentioned above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, the sample is part of a single sample obtained from at least one subject, or part of pooled samples obtained from at least one subject. In another embodiment, the therapy blocks (a) the interaction and / or signaling between HHLA2 and KIR3DL3, and / or (b) the interaction and / or signaling between PD-1 and PD-L1 and / or PD-L2. In yet another embodiment, the sample comprises cells (e.g., T cells or natural killer (NK) cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from a subject).

[0031] In yet another aspect, provided is a method of treating a subject suffering from cancer, the method comprising administering to the subject at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) described herein.

[0032] As mentioned above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) described herein (a) reduces the number of proliferating cancer cells in a cancer, (b) reduces the volume or size of a cancer tumor, and / or (c) activates T cells and / or NK cells. In another embodiment, at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) described herein is administered in a pharmaceutically acceptable formulation. In yet another embodiment, the method described herein further comprises administering to the subject a therapeutic agent or regimen for treating cancer. In yet another embodiment, the method described herein further comprises administering to the subject an additional therapy selected from the group consisting of immunotherapy, checkpoint blockade, a cancer vaccine, a chimeric antigen receptor (e.g., a CAR targeting CD19), chemotherapy, radiation, targeted therapy, and surgery. In another embodiment, the cancer cells and / or tumor immune infiltrating cells in the subject express HHLA2. In yet another embodiment, the cancer is selected from the group consisting of adenocarcinoma, chronic myeloid leukemia (CML), lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancer infiltrated with immune cells expressing receptors for HHLA2. In yet another embodiment, the cancer is selected from the group consisting of lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), head and neck cancer, liver cancer, ovarian cancer, prostate cancer, and uterine cancer. In another embodiment, the subject is an animal model of cancer. In yet another embodiment, the animal model is a mouse model, and optionally, the mouse model is a humanized mouse model. In yet another embodiment, the subject is a mammal, such as a humanized mouse or a human.

[0033] In another aspect, a method for regulating an immune response using at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein is provided. For example, in one embodiment, at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein inhibits or disrupts the interaction between HHLA2 and its binding inhibitor receptor, KIR3DL3. In another embodiment, at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein is conjugated to a cytotoxic agent (e.g., a chemotherapeutic agent, a biological agent, a toxin, and / or a radioisotope). In yet another embodiment, the immune response is downregulated. In another embodiment, the immune response is upregulated. In yet another embodiment, (a) the interaction between HHLA2 and KIR3DL3 and / or (b) the interaction between PD-1 and PD-L1 and / or PD-L2 is blocked. In another embodiment, the anti-KIR3DL3 antibody or antigen-binding fragment thereof is a checkpoint inhibitor of T cell activation for cancer immunotherapy. In yet another embodiment, modulating the immune response comprises modulating T cell function or NK cell function (e.g., cytotoxicity, such as against cancer cells, such as cancer cells that express HHLA2). In yet another embodiment, the cancer is selected from the group consisting of adenocarcinoma, chronic myeloid leukemia (CML), lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancer infiltrated with immune cells that express receptors for HHLA2. In another embodiment, the cancer is selected from the group consisting of lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), head and neck cancer, liver cancer, ovarian cancer, prostate cancer, and uterine cancer. In yet another embodiment, the method further comprises administering to the subject an additional therapy selected from the group consisting of immunotherapy, checkpoint blockade, a cancer vaccine, a chimeric antigen receptor (e.g., a CAR targeting CD19), chemotherapy, radiation, targeted therapy, and surgery.In yet another embodiment, the immune response is modulated in an animal model of cancer (e.g., a mouse model and / or a humanized animal model). In another embodiment, the immune response is modulated in a mammal, such as a humanized mouse or a human.

[0034] For any figure showing bar histograms, curves, or other data related to the legend, the bars, curves, or other data presented from left to right for each indicator correspond directly and sequentially from top to bottom or left to right in the legend box. In an embodiment of the present invention, for example, the following items are provided: (Item 1) A monoclonal antibody or antigen-binding fragment thereof, a) a heavy chain sequence having at least about 95% identity to a heavy chain sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8, and / or b) A monoclonal antibody or antigen-binding fragment thereof comprising a light chain sequence having at least about 95% identity to a light chain sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8. (Item 2) A monoclonal antibody or antigen-binding fragment thereof, a) one, two, or three heavy chain CDR sequences each having at least about 95% identity to a heavy chain CDR sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8, and / or b) A monoclonal antibody or antigen-binding fragment thereof comprising one, two, or three light chain CDR sequences each having at least about 95% identity to a light chain CDR sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8. (Item 3) A monoclonal antibody or antigen-binding fragment thereof, a) a heavy chain sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8, and / or b) A monoclonal antibody or antigen-binding fragment thereof, comprising a light chain sequence selected from the group consisting of the sequences listed in Tables 2, 7, and 8. (Item 4) A monoclonal antibody or antigen-binding fragment thereof, a) one, two, or three heavy chain CDR sequences selected from the group consisting of the sequences listed in Tables 2, 7, and 8, respectively; and / or b) A monoclonal antibody or antigen-binding fragment thereof comprising one, two, or three light chain CDR sequences selected from the group consisting of the sequences listed in Tables 2, 7, and 8, respectively. (Item 5) 5. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof is chimeric, humanized, composite, murine, or human. (Item 6) 6. The monoclonal antibody or antigen-binding fragment thereof of any one of items 1 to 5, wherein the monoclonal antibody or antigen-binding fragment thereof (a) is detectably labeled, (b) is conjugated to a cytotoxic agent, optionally a chemotherapeutic agent, a biological agent, a toxin, and / or a radioisotope, (c) comprises an effector domain, (d) comprises an Fc domain, and / or (e) is selected from the group consisting of Fv, Fav, F(ab')2), Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. (Item 7) Any one of items 1 to 6, wherein the monoclonal antibody or antigen-binding fragment thereof is obtainable from hybridoma ______ deposited under deposit accession number ______. The monoclonal antibody or antigen-binding fragment thereof described herein. (Item 8) 8. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the monoclonal antibody or antigen-binding fragment thereof inhibits binding of HHLA2 to KIR3DL3. (Item 9) 9. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8, wherein the monoclonal antibody or antigen-binding fragment thereof specifically binds to KIR3DL3. (Item 10) A bispecific antibody or antigen-binding fragment thereof, a) a heavy chain sequence having at least about 95% identity to a heavy chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9, and / or b) A bispecific antibody or antigen-binding fragment thereof, comprising a light chain sequence having at least about 95% identity to a light chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9. (Item 11) A bispecific antibody or antigen-binding fragment thereof, a) one, two, or three heavy chain CDR sequences each having at least about 95% identity to a heavy chain CDR sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9, and / or b) A bispecific antibody or antigen-binding fragment thereof comprising one, two, or three light chain CDR sequences each having at least about 95% identity to a light chain CDR sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9. (Item 12) A bispecific antibody or antigen-binding fragment thereof, a) a heavy chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9, and / or b) A bispecific antibody or antigen-binding fragment thereof, comprising a light chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7-9. (Item 13) A bispecific antibody or antigen-binding fragment thereof, a) one, two, or three heavy chain CDR sequences selected from the group consisting of the sequences listed in Tables 2 and 7-9, respectively; and / or b) A bispecific antibody or antigen-binding fragment thereof comprising one, two, or three light chain CDR sequences selected from the group consisting of the sequences listed in Tables 2 and 7-9, respectively. (Item 14) 14. The bispecific antibody or antigen-binding fragment thereof according to any one of items 10 to 13, wherein the bispecific antibody or antigen-binding fragment thereof is chimeric, humanized, composite, murine, or human. (Item 15) 15. The bispecific antibody or antigen-binding fragment thereof of any one of Items 10 to 14, wherein the bispecific antibody or antigen-binding fragment thereof (a) is detectably labeled; (b) is conjugated to a cytotoxic agent, optionally a chemotherapeutic agent, a biological agent, a toxin, and / or a radioisotope; (c) comprises an effector domain; (d) comprises an Fc domain; and / or (e) is selected from the group consisting of Fv, Fav, F(ab')2), Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. (Item 16) 16. The bispecific antibody or antigen-binding fragment thereof according to any one of items 10 to 15, wherein the bispecific antibody or antigen-binding fragment thereof is obtainable from hybridoma ______ deposited under deposit accession number ______. (Item 17) 17. The bispecific antibody or antigen-binding fragment thereof of any one of items 10 to 16, wherein the bispecific antibody or antigen-binding fragment thereof inhibits (a) binding of HHLA2 to KIR3DL3 and (b) binding of PD-1 to PD-L1 and / or PD-L2. (Item 18) 18. The bispecific antibody or antigen-binding fragment thereof according to any one of items 10 to 17, wherein the bispecific antibody or antigen-binding fragment thereof specifically binds to KIR3DL3 and PD-1. (Item 19) the bispecific antibody or antigen-binding fragment thereof a) a heavy chain sequence listed in Table 9, and / or b) The bispecific antibody or antigen-binding fragment thereof according to any one of items 10 to 18, comprising a light chain sequence listed in Table 9. (Item 20) An immunoglobulin heavy and / or light chain selected from the group consisting of the immunoglobulin heavy and light chain sequences listed in Tables 2 and 7-9. (Item 21) 21. An isolated nucleic acid molecule that hybridizes under stringent conditions to the complement of a nucleic acid encoding (a) an immunoglobulin heavy chain, an immunoglobulin light chain, and / or an antibody or antigen-binding fragment thereof according to any one of items 1 to 20, and / or (b) a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 2 and 7 to 9, or a sequence having at least about 95% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 2 and 7 to 9. (Item 22) A vector comprising the isolated nucleic acid of Item 21. (Item 23) A host cell comprising the isolated nucleic acid of Item 21, comprising the vector of Item 22, expressing the antibody or antigen-binding fragment thereof of any one of Items 1 to 19, or available under deposit accession number ______. (Item 24) 20. A device or kit comprising at least one antibody or antigen-binding fragment thereof according to any one of items 1 to 19, wherein the device or kit optionally comprises a label for detecting the at least one antibody or antigen-binding fragment thereof, or a complex comprising the antibody or antigen-binding fragment thereof. (Item 25) 20. A method for producing at least one antibody or antigen-binding fragment thereof according to any one of items 1 to 19, the method comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding at least one antibody according to any one of items 1 to 19 under conditions suitable to allow expression of the antibody or antigen-binding fragment thereof; and (ii) recovering the expressed antibody or antigen-binding fragment thereof. (Item 26) 20. A method for detecting the presence or level of a KIR3DL3 polypeptide, comprising detecting said polypeptide in a sample by use of at least one antibody or antigen-binding fragment thereof according to any one of items 1 to 19. (Item 27) the at least one antibody or antigen-binding fragment thereof forms a complex with a KIR3DL3 polypeptide, and the complex is detected by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical analysis, Western blot analysis, or the like. 27. The method of claim 26, wherein the is detected using an intracellular flow assay. (Item 28) 1. A method for predicting responsiveness to a therapy targeting KIR3DL3, said method comprising: a) determining the level of KIR3DL3 and / or HHLA2 in a subject sample using at least one antibody or antigen-binding fragment thereof according to any one of items 1 to 19; b) determining the level of KIR3DL3 and / or HHLA2 in a sample from at least one control subject who has a good response to a therapy targeting KIR3DL3 using the at least one antibody or antigen-binding fragment thereof; c) comparing the level of KIR3DL3 and / or HHLA2 in the subject sample with the level of KIR3DL3 and / or HHLA2 in the sample from the control subject; A method wherein the same or higher level of KIR3DL3 and / or HHLA2 in the subject sample compared to the level in the sample from the at least one control subject indicates that the subject will respond to the therapy. (Item 29) 29. The method of claim 28, wherein the therapy targets KIR3DL3 using at least one antibody or antigen-binding fragment thereof described in any one of items 1 to 19. (Item 30) 20. A method for predicting responsiveness to a therapy targeting KIR3DL3 using at least one antibody or antigen-binding fragment thereof according to any one of items 1 to 19, the method comprising: a) determining the level of KIR3DL3 and / or HHLA2 in a subject sample; b) determining the level of KIR3DL3 and / or HHLA2 in a sample from at least one control subject with a good response to a therapy targeting KIR3DL3; c) comparing the level of KIR3DL3 and / or HHLA2 in the subject sample with the level of KIR3DL3 and / or HHLA2 in the sample from the control subject; A method wherein the same or higher level of KIR3DL3 and / or HHLA2 in the subject sample compared to the level in the sample from the at least one control subject indicates that the subject will respond to the therapy. (Item 31) 31. The method of any one of items 28 to 30, wherein the sample is part of a single sample obtained from at least one subject or part of pooled samples obtained from at least one subject. (Item 32) 32. The method of any one of items 28 to 31, wherein the therapy blocks (a) the interaction and / or signaling between HHLA2 and KIR3DL3, and / or (b) the interaction and / or signaling between PD-1 and PD-L1 and / or PD-L2. (Item 33) 33. The method of any one of items 26 to 32, wherein the sample comprises cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject. (Item 34) 34. The method of claim 33, wherein the cell is a T cell or a natural killer (NK) cell. (Item 35) 20. A method of treating a subject suffering from cancer, comprising administering to the subject at least one antibody or antigen-binding fragment thereof according to any one of items 1 to 19. (Item 36) 36. The method of claim 35, wherein the at least one antibody or antigen-binding fragment thereof (a) reduces the number of proliferating cancer cells in the cancer, (b) reduces tumor volume or size in the cancer, and / or (c) activates T cells and / or NK cells. (Item 37) 37. The method of claim 35 or 36, wherein the at least one antibody or antigen-binding fragment thereof is administered in a pharmaceutically acceptable formulation. (Item 38) 38. The method of any one of items 35 to 37, further comprising administering to the subject a therapeutic agent or regimen for treating cancer. (Item 39) 39. The method of any one of items 35 to 38, further comprising administering to the subject an additional therapy selected from the group consisting of immunotherapy, checkpoint blockade, cancer vaccine, chimeric antigen receptor, chemotherapy, radiation, targeted therapy, and surgery. (Item 40) 40. The method of claim 39, wherein the chimeric antigen receptor targets CD19. (Item 41) 41. The method according to any one of items 35 to 40, wherein cancer cells and / or tumor immune infiltrating cells in the subject express HHLA2. (Item 42) 42. The method of any one of items 35 to 41, wherein the cancer is selected from the group consisting of adenocarcinoma, chronic myeloid leukemia (CML), lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancer infiltrated by immune cells expressing receptors for HHLA2. (Item 43) 43. The method of claim 42, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), head and neck cancer, liver cancer, ovarian cancer, prostate cancer, and uterine cancer. (Item 44) 44. The method according to any one of items 35 to 43, wherein the subject is an animal model of cancer. (Item 45) 45. The method of claim 44, wherein the animal model is a mouse model, and optionally, the mouse model is a humanized mouse model. (Item 46) 46. ​​The method according to any one of items 35 to 45, wherein the subject is a mammal. (Item 47) 47. The method of claim 46, wherein the mammal is a humanized mouse or a human. (Item 48) 48. The method of claim 47, wherein the mammal is a human. (Item 49) A method of modulating an immune response using at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof. (Item 50) 50. The method of item 49, wherein the at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof inhibits or disrupts the interaction between HHLA2 and its binding inhibitor receptor, KIR3DL3. (Item 51) 51. The method of item 49 or 50, wherein at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent. (Item 52) 52. The method of claim 51, wherein the cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, a biological agent, a toxin, and a radioisotope. (Item 53) 53. The method of any one of items 49 to 52, wherein the immune response is downregulated or upregulated. (Item 54) 54. The method of any one of items 49 to 53, wherein the at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof is described in any one of items 1 to 19. (Item 55) 55. The method of any one of items 49 to 54, wherein (a) the interaction between HHLA2 and KIR3DL3, and / or (b) the interaction between PD-1 and PD-L1 and / or PD-L2 is blocked. (Item 56) 56. The method of any one of items 49 to 55, wherein the anti-KIR3DL3 antibody or antigen-binding fragment thereof is a checkpoint inhibitor of T cell activation for cancer immunotherapy. (Item 57) 57. The method of any one of items 49 to 56, wherein said modulating an immune response comprises modulating T cell function or NK cell function. (Item 58) 58. The method of item 57, wherein the T cell function or NK cell function comprises cytotoxic activity. (Item 59) 59. The method of claim 58, wherein the cytotoxic activity is directed against cancer cells. (Item 60) 60. The method of claim 59, wherein the cancer cells express HHLA2. (Item 61) 61. The method of any one of items 56 to 60, wherein the cancer is selected from the group consisting of adenocarcinoma, chronic myeloid leukemia (CML), lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancer infiltrated by immune cells expressing receptors for HHLA2. (Item 62) 62. The method of item 61, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), head and neck cancer, liver cancer, ovarian cancer, prostate cancer, and uterine cancer. (Item 63) 63. The method of any one of items 49 to 62, further comprising an additional therapy selected from the group consisting of immunotherapy, checkpoint blockade, cancer vaccine, chimeric antigen receptor, chemotherapy, radiation, targeted therapy, and surgery. (Item 64) 64. The method of claim 63, wherein the chimeric antigen receptor targets CD19. (Item 65) 65. The method of any one of items 49 to 64, wherein the immune response is modulated in an animal model of cancer. (Item 66) 66. The method of claim 65, wherein the animal model is a mouse model, and optionally, the mouse model is a humanized mouse model. (Item 67) 67. The method of any one of items 49 to 66, wherein the immune response is modulated in a mammal. (Item 68) 68. The method of claim 67, wherein the mammal is a humanized mouse or a human. (Item 69) Item 69. The method of item 68, wherein the mammal is a human. [Brief explanation of the drawings]

[0035] [Figure 1A] Figures 1A-1B show the results of an expression screen identifying KIR3DL3 as the receptor for HHLA2. Figure 1A shows the results of a cell microarray analysis using soluble HHLA2-mIgG2a (HHLA2-Ig) that binds to the indicated cell surface receptors expressed individually in HEK293 cells. HHLA2-Ig is shown to bind to TMIGD2, KIR3DL3, and a control (FCGR2A), but not to other members of the KIR family, PD-1, PD-L1, or HHLA2. Figure 1B shows flow cytometry analysis of HHLA2-Ig or control Ig binding to control 300.19 cells or 300.19 cells stably expressing KIR3DL3, TMIGD2, or HHLA2, using the indicated concentrations of HHLA2-Ig or isotype control (0.1 μg / mL to 160 μg / mL). [Figure 1B]Figures 1A-1B show the results of an expression screen identifying KIR3DL3 as the receptor for HHLA2. Figure 1A shows the results of a cell microarray analysis using soluble HHLA2-mIgG2a (HHLA2-Ig) that binds to the indicated cell surface receptors expressed individually in HEK293 cells. HHLA2-Ig is shown to bind to TMIGD2, KIR3DL3, and a control (FCGR2A), but not to other members of the KIR family, PD-1, PD-L1, or HHLA2. Figure 1B shows flow cytometry analysis of HHLA2-Ig or control Ig binding to control 300.19 cells or 300.19 cells stably expressing KIR3DL3, TMIGD2, or HHLA2, using the indicated concentrations of HHLA2-Ig or isotype control (0.1 μg / mL to 160 μg / mL). [Figure 2A]Figures 2A-2D show the identification and characterization of KIR3DL3 as a second receptor for HHLA2. Figure 2A shows a replicate microarray slide of cells expressing 384 human receptors and cells coexpressing GFP, identifying KIR3DL3 as the receptor for HHLA2-Ig (upper panel) and showing GFP expression as a control for transfection and spot localization (lower panel). Figure 2B shows the results of the cell microarray analysis shown in Figure 1A using soluble HHLA2-mIgG2a (HHLA2-Ig) that binds to the indicated cell surface receptors expressed individually in HEK293 cells. HHLA2-Ig is shown to bind to TMIGD2, KIR3DL3, and a control (FCGR2A), but not to other members of the KIR family, PD-1, PD-L1, or HHLA2. Figure 2C shows the transfection control (GFP expression) results for the receptor array shown in Figure 2B (and Figure 1A). Figure 2D shows the results of a positive control treatment of the receptor array. Cell microarray analysis using soluble PD-1-Ig and PD-L1-Ig incubated with the same panel of overexpressed receptors as in Figure 2B and Figure 1A shows binding to FCGR2A, PD-1, and PD-L1, but not to any of the KIRs. The PD-L1 spots that do not bind to PD-1-Ig are alternatively spliced ​​isoforms. [Figure 2B]Figures 2A-2D show the identification and characterization of KIR3DL3 as a second receptor for HHLA2. Figure 2A shows a replicate microarray slide of cells expressing 384 human receptors and cells coexpressing GFP, identifying KIR3DL3 as the receptor for HHLA2-Ig (upper panel) and showing GFP expression as a control for transfection and spot localization (lower panel). Figure 2B shows the results of the cell microarray analysis shown in Figure 1A using soluble HHLA2-mIgG2a (HHLA2-Ig) that binds to the indicated cell surface receptors expressed individually in HEK293 cells. HHLA2-Ig is shown to bind to TMIGD2, KIR3DL3, and a control (FCGR2A), but not to other members of the KIR family, PD-1, PD-L1, or HHLA2. Figure 2C shows the transfection control (GFP expression) results for the receptor array shown in Figure 2B (and Figure 1A). Figure 2D shows the results of a positive control treatment of the receptor array. Cell microarray analysis using soluble PD-1-Ig and PD-L1-Ig incubated with the same panel of overexpressed receptors as in Figure 2B and Figure 1A shows binding to FCGR2A, PD-1, and PD-L1, but not to any of the KIRs. The PD-L1 spots that do not bind to PD-1-Ig are alternatively spliced ​​isoforms. [Figure 2C]Figures 2A-2D show the identification and characterization of KIR3DL3 as a second receptor for HHLA2. Figure 2A shows a replicate microarray slide of cells expressing 384 human receptors and cells coexpressing GFP, identifying KIR3DL3 as the receptor for HHLA2-Ig (upper panel) and showing GFP expression as a control for transfection and spot localization (lower panel). Figure 2B shows the results of the cell microarray analysis shown in Figure 1A using soluble HHLA2-mIgG2a (HHLA2-Ig) that binds to the indicated cell surface receptors expressed individually in HEK293 cells. HHLA2-Ig is shown to bind to TMIGD2, KIR3DL3, and a control (FCGR2A), but not to other members of the KIR family, PD-1, PD-L1, or HHLA2. Figure 2C shows the transfection control (GFP expression) results for the receptor array shown in Figure 2B (and Figure 1A). Figure 2D shows the results of a positive control treatment of the receptor array. Cell microarray analysis using soluble PD-1-Ig and PD-L1-Ig incubated with the same panel of overexpressed receptors as in Figure 2B and Figure 1A shows binding to FCGR2A, PD-1, and PD-L1, but not to any of the KIRs. The PD-L1 spots that do not bind to PD-1-Ig are alternatively spliced ​​isoforms. [Figure 2D]Figures 2A-2D show the identification and characterization of KIR3DL3 as a second receptor for HHLA2. Figure 2A shows a replicate microarray slide of cells expressing 384 human receptors and cells coexpressing GFP, identifying KIR3DL3 as the receptor for HHLA2-Ig (upper panel) and showing GFP expression as a control for transfection and spot localization (lower panel). Figure 2B shows the results of the cell microarray analysis shown in Figure 1A using soluble HHLA2-mIgG2a (HHLA2-Ig) that binds to the indicated cell surface receptors expressed individually in HEK293 cells. HHLA2-Ig is shown to bind to TMIGD2, KIR3DL3, and a control (FCGR2A), but not to other members of the KIR family, PD-1, PD-L1, or HHLA2. Figure 2C shows the transfection control (GFP expression) results for the receptor array shown in Figure 2B (and Figure 1A). Figure 2D shows the results of a positive control treatment of the receptor array. Cell microarray analysis using soluble PD-1-Ig and PD-L1-Ig incubated with the same panel of overexpressed receptors as in Figure 2B and Figure 1A shows binding to FCGR2A, PD-1, and PD-L1, but not to any of the KIRs. The PD-L1 spots that do not bind to PD-1-Ig are alternatively spliced ​​isoforms. [Figure 3A]Figures 3A-3E show the characterization of a panel of KIR3DL3 and HHLA2 mAbs. (Figure 3A) Flow cytometry analysis of KIR3DL3 mAb binding to KIR3DL3-expressing 300.19 cells. Figure 3B shows the ability of KIR3DL3 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells. Figure 3C shows HHLA2 mAb binding to HHLA2-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding, and 6D10, which showed the weakest binding. Figure 3D shows the ability of HHLA2 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding. FIG. 3E shows the ability of HHLA2 mAbs 2G2 and 6F10 to block binding of HHLA2-Ig to 300.19 cells expressing TMIGD2. [Figure 3B] Figures 3A-3E show the characterization of a panel of KIR3DL3 and HHLA2 mAbs. (Figure 3A) Flow cytometry analysis of KIR3DL3 mAb binding to KIR3DL3-expressing 300.19 cells. Figure 3B shows the ability of KIR3DL3 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells. Figure 3C shows HHLA2 mAb binding to HHLA2-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding, and 6D10, which showed the weakest binding. Figure 3D shows the ability of HHLA2 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding. FIG. 3E shows the ability of HHLA2 mAbs 2G2 and 6F10 to block binding of HHLA2-Ig to 300.19 cells expressing TMIGD2. [Figure 3C]Figures 3A-3E show the characterization of a panel of KIR3DL3 and HHLA2 mAbs. (Figure 3A) Flow cytometry analysis of KIR3DL3 mAb binding to KIR3DL3-expressing 300.19 cells. Figure 3B shows the ability of KIR3DL3 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells. Figure 3C shows HHLA2 mAb binding to HHLA2-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding, and 6D10, which showed the weakest binding. Figure 3D shows the ability of HHLA2 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding. FIG. 3E shows the ability of HHLA2 mAbs 2G2 and 6F10 to block binding of HHLA2-Ig to 300.19 cells expressing TMIGD2. [Figure 3D] Figures 3A-3E show the characterization of a panel of KIR3DL3 and HHLA2 mAbs. (Figure 3A) Flow cytometry analysis of KIR3DL3 mAb binding to KIR3DL3-expressing 300.19 cells. Figure 3B shows the ability of KIR3DL3 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells. Figure 3C shows HHLA2 mAb binding to HHLA2-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding, and 6D10, which showed the weakest binding. Figure 3D shows the ability of HHLA2 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding. FIG. 3E shows the ability of HHLA2 mAbs 2G2 and 6F10 to block binding of HHLA2-Ig to 300.19 cells expressing TMIGD2. [Figure 3E]Figures 3A-3E show the characterization of a panel of KIR3DL3 and HHLA2 mAbs. (Figure 3A) Flow cytometry analysis of KIR3DL3 mAb binding to KIR3DL3-expressing 300.19 cells. Figure 3B shows the ability of KIR3DL3 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells. Figure 3C shows HHLA2 mAb binding to HHLA2-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding, and 6D10, which showed the weakest binding. Figure 3D shows the ability of HHLA2 mAb to block HHLA2-Ig binding to KIR3DL3-expressing 300.19 cells using 2C4, 2G2, and 6F10, which showed the strongest binding. FIG. 3E shows the ability of HHLA2 mAbs 2G2 and 6F10 to block binding of HHLA2-Ig to 300.19 cells expressing TMIGD2. [Figure 4A] Figures 4A-4C show HHLA2-mIgG2a binding to KIR3DL3 and TMIGD2. Figure 4A shows the normalized binding data from Figure 1B. HHLA2-mIgG2a binding to KIR3DL3 (blue) or TMIGD2 (cyan) or control HHLA2 (red) transfected or parental 300.19 cells (green). Figures 4B and 4C show HHLA2-mIgG2a or isotype control (10 μg / ml) binding to KIR3DL3-transfected 293T cells (Figure 4B) or TMIGD2-transfected 293T cells (Figure 4C) by flow cytometry. [Figure 4B] Figures 4A-4C show HHLA2-mIgG2a binding to KIR3DL3 and TMIGD2. Figure 4A shows the normalized binding data from Figure 1B. HHLA2-mIgG2a binding to KIR3DL3 (blue) or TMIGD2 (cyan) or control HHLA2 (red) transfected or parental 300.19 cells (green). Figures 4B and 4C show HHLA2-mIgG2a or isotype control (10 μg / ml) binding to KIR3DL3-transfected 293T cells (Figure 4B) or TMIGD2-transfected 293T cells (Figure 4C) by flow cytometry. [Figure 4C] Figures 4A-4C show HHLA2-mIgG2a binding to KIR3DL3 and TMIGD2. Figure 4A shows the normalized binding data from Figure 1B. HHLA2-mIgG2a binding to KIR3DL3 (blue) or TMIGD2 (cyan) or control HHLA2 (red) transfected or parental 300.19 cells (green). Figures 4B and 4C show HHLA2-mIgG2a or isotype control (10 μg / ml) binding to KIR3DL3-transfected 293T cells (Figure 4B) or TMIGD2-transfected 293T cells (Figure 4C) by flow cytometry. [Figure 5] FIG. 5 shows binding data for anti-KIR3DL3 mAbs to the KIR3DL3-transfected 300.19 murine pre-B cell leukemia cell line by flow cytometry. [Figure 6] 6 shows binding data for anti-KIR3DL3 mAb to KIR3DL3 by Western blotting, specifically showing the results of Western blot analysis of KIR3DL3 mAb using Jurkat cells transfected with KIR3DL3. [Figure 7] Figure 7 shows KIR3DL3 expression in parental Jurkat cells, KIR3DL3-transfected Jurkat, NK-92 cells, and NK-92-MI cells. Lysates were blotted with anti-KIR3DL3 mAb 574.1F12 at 5ug / ml. [Figure 8]Figure 8 shows single-cell RNA-sequencing analysis of KIR3DL3 expression as assessed in public databases (see the EMBL-EBI database, available on the World Wide Web at ebi.ac.uk / gxa / sc / home, and the corresponding publication entitled "Reconstructing the human first trimester fetal-maternal interface using single cell transcriptomics," available on the World Wide Web at biorxiv.org / content / 10.1101 / 429589v1). KIR3DL3 expression is shown as blue dots in the right panel. The black box highlights decidual NK cells, where most KIR3DL3 expression is observed. [Figure 9] FIG. 9 shows anti-KIR3DL3 mAb blocking of HHLA2 binding to KIR3DL3. [Figure 10A] Figures 10A-10D show KIR3DL3 expression on activated human T cells and NK92-MI cells. Figure 10A shows the results of T cells purified from whole blood of four normal donors, activated with CD3 / CD28 antibody tetramer, and subjected to FACS analysis performed in duplicate on the indicated days to evaluate KIR3DL3 expression in gated CD3+CD4+ and CD3+CD8+ T cells. Representative FACS plots showing KIR3DL3 expression on day 0 (unactivated) (Figure 10B) and day 21 after activation (Figure 10C). Figure 10D shows KIR3DL3 expression on NK92-MI (left panel), while KIR3DL3 expression on NK-92 cells (right panel) is minimal. [Figure 10B]Figures 10A-10D show KIR3DL3 expression on activated human T cells and NK92-MI cells. Figure 10A shows the results of T cells purified from whole blood of four normal donors, activated with CD3 / CD28 antibody tetramer, and subjected to FACS analysis performed in duplicate on the indicated days to evaluate KIR3DL3 expression in gated CD3+CD4+ and CD3+CD8+ T cells. Representative FACS plots showing KIR3DL3 expression on day 0 (unactivated) (Figure 10B) and day 21 after activation (Figure 10C). Figure 10D shows KIR3DL3 expression on NK92-MI (left panel), while KIR3DL3 expression on NK-92 cells (right panel) is minimal. [Figure 10C] Figures 10A-10D show KIR3DL3 expression on activated human T cells and NK92-MI cells. Figure 10A shows the results of T cells purified from whole blood of four normal donors, activated with CD3 / CD28 antibody tetramer, and subjected to FACS analysis performed in duplicate on the indicated days to evaluate KIR3DL3 expression in gated CD3+CD4+ and CD3+CD8+ T cells. Representative FACS plots showing KIR3DL3 expression on day 0 (unactivated) (Figure 10B) and day 21 after activation (Figure 10C). Figure 10D shows KIR3DL3 expression on NK92-MI (left panel), while KIR3DL3 expression on NK-92 cells (right panel) is minimal. [Figure 10D] Figures 10A-10D show KIR3DL3 expression on activated human T cells and NK92-MI cells. Figure 10A shows the results of T cells purified from whole blood of four normal donors, activated with CD3 / CD28 antibody tetramer, and subjected to FACS analysis performed in duplicate on the indicated days to evaluate KIR3DL3 expression in gated CD3+CD4+ and CD3+CD8+ T cells. Representative FACS plots showing KIR3DL3 expression on day 0 (unactivated) (Figure 10B) and day 21 after activation (Figure 10C). Figure 10D shows KIR3DL3 expression on NK92-MI (left panel), while KIR3DL3 expression on NK-92 cells (right panel) is minimal. [Figure 11A]Figures 11A-11C show that KIR3DL3 is an inhibitory receptor in T cells and that T cell activation is enhanced by HHLA2 / KIR3DL3 blockade. Figure 11A shows the results of KIR3DL3-expressing Jurkat IL-2-reporter T cells cocultured with CHO cells expressing anti-CD3 scFV, CHO cells coexpressing anti-CD3 scFV and HHLA2, or untransfected CHO cells in the presence or absence of CD28 mAb as indicated. Luciferase activity is expressed as relative light units (RLU). Figures 11B and 11C show the results of KIR3DL3-expressing Jurkat IL-2-reporter T cells cocultured with CHO cells coexpressing anti-CD3 scFV and HHLA2 in the presence of CD28 mAb and HHLA2 mAb (Figure 11B) or KIR3DL3 mAb (Figure 11C). Fold activation of IL-2 reporter luciferase activity is presented as the mean ± SD (n≧3; ****P≦0.0001). [Figure 11B] Figures 11A-11C show that KIR3DL3 is an inhibitory receptor in T cells and that T cell activation is enhanced by HHLA2 / KIR3DL3 blockade. Figure 11A shows the results of KIR3DL3-expressing Jurkat IL-2-reporter T cells cocultured with CHO cells expressing anti-CD3 scFV, CHO cells coexpressing anti-CD3 scFV and HHLA2, or untransfected CHO cells in the presence or absence of CD28 mAb as indicated. Luciferase activity is expressed as relative light units (RLU). Figures 11B and 11C show the results of KIR3DL3-expressing Jurkat IL-2-reporter T cells cocultured with CHO cells coexpressing anti-CD3 scFV and HHLA2 in the presence of CD28 mAb and HHLA2 mAb (Figure 11B) or KIR3DL3 mAb (Figure 11C). Fold activation of IL-2 reporter luciferase activity is presented as the mean ± SD (n≧3; ****P≦0.0001). [Figure 11C]Figures 11A-11C show that KIR3DL3 is an inhibitory receptor in T cells and that T cell activation is enhanced by HHLA2 / KIR3DL3 blockade. Figure 11A shows the results of KIR3DL3-expressing Jurkat IL-2-reporter T cells cocultured with CHO cells expressing anti-CD3 scFV, CHO cells coexpressing anti-CD3 scFV and HHLA2, or untransfected CHO cells in the presence or absence of CD28 mAb as indicated. Luciferase activity is expressed as relative light units (RLU). Figures 11B and 11C show the results of KIR3DL3-expressing Jurkat IL-2-reporter T cells cocultured with CHO cells coexpressing anti-CD3 scFV and HHLA2 in the presence of CD28 mAb and HHLA2 mAb (Figure 11B) or KIR3DL3 mAb (Figure 11C). Fold activation of IL-2 reporter luciferase activity is presented as the mean ± SD (n≧3; ****P≦0.0001). [Figure 12] Figure 12 shows that HHLA2 / TMIGD2 interaction enhances T cell activation. Jurkat T cells expressing TMIGD2 and carrying the NFAT promoter linked to luciferase were cocultured with anti-CD3 scFV CHO cells or HHLA2-anti-CD3 scFV CHO cells, and luciferase activity (RLU) was assayed. Quantitation is presented as mean ± SD (n≧3; ***P≦0.001). [Figure 13] FIG. 13 shows anti-KIR3DL3 mAb enhancement of IL-2 promoter-driven luciferase expression in Jurkat-KIR3DL3 T cells in response to anti-CD3-scFV and HHLA2-mediated signals. [Figure 14] FIG. 14 shows anti-HHLA2 mAb enhancement of IL-2 promoter-driven luciferase expression in Jurkat-KIR3DL3 T cells in response to anti-CD3-scFV and HHLA2-mediated signals. [Figure 15A]Figures 15A-15D show the cytotoxicity of KIR3DL3-CD19-CAR-T cells against HeLa tumors expressing CD19 and HHLA2. Figure 15A shows the KIR3DL3 / CAR-19 expression plasmid and lentivirus production. In particular, Figure 15A shows a schematic diagram of the PMC456-Ef1a expression plasmid. Figure 15B shows the generation and proliferation of KIR3DL3 / CD19-CAR-T cells. In particular, Figure 15B shows the FACS profile of KIR3DL3 / CAR-19 T cells (PMC456 cells). Figure 15C shows the generation of stable HeLa-CD19 and HeLa-CD19+KIR3DL3-expressing cells. In particular, Figure 15C shows the FACS profile of HeLa-CD19 and HeLa-CD19-KIR3DL3 tumor cells. Figure 15D shows that HHLA2 mAb enhances the cytotoxicity of KIR3DL3 CD19-CAR-T cells against HHLA2+CD19-transfected HeLa tumor cells. [Figure 15B] Figures 15A-15D show the cytotoxicity of KIR3DL3-CD19-CAR-T cells against HeLa tumors expressing CD19 and HHLA2. Figure 15A shows the KIR3DL3 / CAR-19 expression plasmid and lentivirus production. In particular, Figure 15A shows a schematic diagram of the PMC456-Ef1a expression plasmid. Figure 15B shows the generation and proliferation of KIR3DL3 / CD19-CAR-T cells. In particular, Figure 15B shows the FACS profile of KIR3DL3 / CAR-19 T cells (PMC456 cells). Figure 15C shows the generation of stable HeLa-CD19 and HeLa-CD19+KIR3DL3-expressing cells. In particular, Figure 15C shows the FACS profile of HeLa-CD19 and HeLa-CD19-KIR3DL3 tumor cells. Figure 15D shows that HHLA2 mAb enhances the cytotoxicity of KIR3DL3 CD19-CAR-T cells against HHLA2+CD19-transfected HeLa tumor cells. [Figure 15C]Figures 15A-15D show the cytotoxicity of KIR3DL3-CD19-CAR-T cells against HeLa tumors expressing CD19 and HHLA2. Figure 15A shows the KIR3DL3 / CAR-19 expression plasmid and lentivirus production. In particular, Figure 15A shows a schematic diagram of the PMC456-Ef1a expression plasmid. Figure 15B shows the generation and proliferation of KIR3DL3 / CD19-CAR-T cells. In particular, Figure 15B shows the FACS profile of KIR3DL3 / CAR-19 T cells (PMC456 cells). Figure 15C shows the generation of stable HeLa-CD19 and HeLa-CD19+KIR3DL3-expressing cells. In particular, Figure 15C shows the FACS profile of HeLa-CD19 and HeLa-CD19-KIR3DL3 tumor cells. Figure 15D shows that HHLA2 mAb enhances the cytotoxicity of KIR3DL3 CD19-CAR-T cells against HHLA2+CD19-transfected HeLa tumor cells. [Figure 15D] Figures 15A-15D show the cytotoxicity of KIR3DL3-CD19-CAR-T cells against HeLa tumors expressing CD19 and HHLA2. Figure 15A shows the KIR3DL3 / CAR-19 expression plasmid and lentivirus production. In particular, Figure 15A shows a schematic diagram of the PMC456-Ef1a expression plasmid. Figure 15B shows the generation and proliferation of KIR3DL3 / CD19-CAR-T cells. In particular, Figure 15B shows the FACS profile of KIR3DL3 / CAR-19 T cells (PMC456 cells). Figure 15C shows the generation of stable HeLa-CD19 and HeLa-CD19+KIR3DL3-expressing cells. In particular, Figure 15C shows the FACS profile of HeLa-CD19 and HeLa-CD19-KIR3DL3 tumor cells. Figure 15D shows that HHLA2 mAb enhances the cytotoxicity of KIR3DL3 CD19-CAR-T cells against HHLA2+CD19-transfected HeLa tumor cells. [Figure 16A]Figures 16A-16C show cytotoxicity assays of KIR3DL3-expressing NK92 cells against HeLa tumor target cells expressing or not expressing HHLA2. Figure 16A shows the KIR3DL3 expression plasmid and lentivirus production. In particular, Figure 16A shows a schematic diagram of the PMC579 KIR3DL3 expression plasmid. Figure 16B shows the induction of KIR3DL3-transduced NK92 cells. In particular, Figure 16B shows the KIR3DL3 / NK92 FACS profile. Figure 16C shows the induction of K562 and HeLa cells transfected or transduced with HHLA2, respectively. In particular, Figure 16C shows the FACS profile of K562 cells transfected with HHLA2 and HeLa tumor cells transduced with HHLA2. [Figure 16B] Figures 16A-16C show cytotoxicity assays of KIR3DL3-expressing NK92 cells against HeLa tumor target cells expressing or not expressing HHLA2. Figure 16A shows the KIR3DL3 expression plasmid and lentivirus production. In particular, Figure 16A shows a schematic diagram of the PMC579 KIR3DL3 expression plasmid. Figure 16B shows the induction of KIR3DL3-transduced NK92 cells. In particular, Figure 16B shows the KIR3DL3 / NK92 FACS profile. Figure 16C shows the induction of K562 and HeLa cells transfected or transduced with HHLA2, respectively. In particular, Figure 16C shows the FACS profile of K562 cells transfected with HHLA2 and HeLa tumor cells transduced with HHLA2. [Figure 16C]Figures 16A-16C show cytotoxicity assays of KIR3DL3-expressing NK92 cells against HeLa tumor target cells expressing or not expressing HHLA2. Figure 16A shows the KIR3DL3 expression plasmid and lentivirus production. In particular, Figure 16A shows a schematic diagram of the PMC579 KIR3DL3 expression plasmid. Figure 16B shows the induction of KIR3DL3-transduced NK92 cells. In particular, Figure 16B shows the KIR3DL3 / NK92 FACS profile. Figure 16C shows the induction of K562 and HeLa cells transfected or transduced with HHLA2, respectively. In particular, Figure 16C shows the FACS profile of K562 cells transfected with HHLA2 and HeLa tumor cells transduced with HHLA2. [Figure 17A] Figures 17A-17C show the cytotoxicity of NK92 against HeLa alone and HeLa-transduced HHLA2-expressing tumor target cells. Figure 17A shows the inhibition of NK92 cytotoxicity by KIR3DL3-HHLA2 interaction / pathway. Figure 17B shows the enhancement of NK92-KIR3DL3 cytotoxicity by HHLA2 mAb and KIR3DL3 mAb. Figure 17C shows a schematic diagram of a specific cytotoxicity assay. [Figure 17B] Figures 17A-17C show the cytotoxicity of NK92 against HeLa alone and HeLa-transduced HHLA2-expressing tumor target cells. Figure 17A shows the inhibition of NK92 cytotoxicity by KIR3DL3-HHLA2 interaction / pathway. Figure 17B shows the enhancement of NK92-KIR3DL3 cytotoxicity by HHLA2 mAb and KIR3DL3 mAb. Figure 17C shows a schematic diagram of a specific cytotoxicity assay. [Figure 17C] Figures 17A-17C show the cytotoxicity of NK92 against HeLa alone and HeLa-transduced HHLA2-expressing tumor target cells. Figure 17A shows the inhibition of NK92 cytotoxicity by KIR3DL3-HHLA2 interaction / pathway. Figure 17B shows the enhancement of NK92-KIR3DL3 cytotoxicity by HHLA2 mAb and KIR3DL3 mAb. Figure 17C shows a schematic diagram of a specific cytotoxicity assay. [Figure 18] FIG. 18 shows β2-microglobulin and HHLA2 expression in Raji-B2M KO and HHLA2-transfected Raji-B2M KO by flow cytometry. [Figure 19A] Figures 19A-19E show that KIR3DL3 is an inhibitory receptor on NK cells and that NK cytotoxicity is enhanced by HHLA2 / KIR3DL3 blockade. Figure 19A shows the cytotoxicity of NK92-MI against Raji cells with B2M deletion (Raji-B2M KO cells) and Raji-B2M KO cells expressing HHLA2. Figures 19B and 19C show the cytotoxicity of NK92-MI against Raji-B2M KO cells expressing HHLA2 at the indicated E:T ratios in the presence of 10 μg / ml of KIR3DL3 antibody (Figure 19B) or HHLA2 antibody (Figure 19C) and an isotype control. Figure 19D shows the results of NK92-MI cells incubated with Raji B2M KO cells or Raji B2M KO cells overexpressing HHLA2 at the indicated E:T ratios. Degranulation was measured as the percentage of CD107a-positive cells in the CD56+ population. Controls were effector cells alone or effector cells with PMA / ION, which resulted in total degranulation. Figure 19E shows enhanced degranulation of NK92-MI cells targeting HHLA2-overexpressing Raji B2M KO cells in the presence of KIR3DL3 mAb (1G7) compared to the isotype control. Quantitation is presented as mean ± SD (N≧3; P≧0.05; *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001). [Figure 19B]Figures 19A-19E show that KIR3DL3 is an inhibitory receptor on NK cells and that NK cytotoxicity is enhanced by HHLA2 / KIR3DL3 blockade. Figure 19A shows the cytotoxicity of NK92-MI against Raji cells with B2M deletion (Raji-B2M KO cells) and Raji-B2M KO cells expressing HHLA2. Figures 19B and 19C show the cytotoxicity of NK92-MI against Raji-B2M KO cells expressing HHLA2 at the indicated E:T ratios in the presence of 10 μg / ml of KIR3DL3 antibody (Figure 19B) or HHLA2 antibody (Figure 19C) and an isotype control. Figure 19D shows the results of NK92-MI cells incubated with Raji B2M KO cells or Raji B2M KO cells overexpressing HHLA2 at the indicated E:T ratios. Degranulation was measured as the percentage of CD107a-positive cells in the CD56+ population. Controls were effector cells alone or effector cells with PMA / ION, which resulted in total degranulation. Figure 19E shows enhanced degranulation of NK92-MI cells targeting HHLA2-overexpressing Raji B2M KO cells in the presence of KIR3DL3 mAb (1G7) compared to the isotype control. Quantitation is presented as mean ± SD (N≧3; P≧0.05; *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001). [Figure 19C]Figures 19A-19E show that KIR3DL3 is an inhibitory receptor on NK cells and that NK cytotoxicity is enhanced by HHLA2 / KIR3DL3 blockade. Figure 19A shows the cytotoxicity of NK92-MI against Raji cells with B2M deletion (Raji-B2M KO cells) and Raji-B2M KO cells expressing HHLA2. Figures 19B and 19C show the cytotoxicity of NK92-MI against Raji-B2M KO cells expressing HHLA2 at the indicated E:T ratios in the presence of 10 μg / ml of KIR3DL3 antibody (Figure 19B) or HHLA2 antibody (Figure 19C) and an isotype control. Figure 19D shows the results of NK92-MI cells incubated with Raji B2M KO cells or Raji B2M KO cells overexpressing HHLA2 at the indicated E:T ratios. Degranulation was measured as the percentage of CD107a-positive cells in the CD56+ population. Controls were effector cells alone or effector cells with PMA / ION, which resulted in total degranulation. Figure 19E shows enhanced degranulation of NK92-MI cells targeting HHLA2-overexpressing Raji B2M KO cells in the presence of KIR3DL3 mAb (1G7) compared to the isotype control. Quantitation is presented as mean ± SD (N≧3; P≧0.05; *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001). [Figure 19D]Figures 19A-19E show that KIR3DL3 is an inhibitory receptor on NK cells and that NK cytotoxicity is enhanced by HHLA2 / KIR3DL3 blockade. Figure 19A shows the cytotoxicity of NK92-MI against Raji cells with B2M deletion (Raji-B2M KO cells) and Raji-B2M KO cells expressing HHLA2. Figures 19B and 19C show the cytotoxicity of NK92-MI against Raji-B2M KO cells expressing HHLA2 at the indicated E:T ratios in the presence of 10 μg / ml of KIR3DL3 antibody (Figure 19B) or HHLA2 antibody (Figure 19C) and an isotype control. Figure 19D shows the results of NK92-MI cells incubated with Raji B2M KO cells or Raji B2M KO cells overexpressing HHLA2 at the indicated E:T ratios. Degranulation was measured as the percentage of CD107a-positive cells in the CD56+ population. Controls were effector cells alone or effector cells with PMA / ION, which resulted in total degranulation. Figure 19E shows enhanced degranulation of NK92-MI cells targeting HHLA2-overexpressing Raji B2M KO cells in the presence of KIR3DL3 mAb (1G7) compared to the isotype control. Quantitation is presented as mean ± SD (N≧3; P≧0.05; *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001). [Figure 19E]Figures 19A-19E show that KIR3DL3 is an inhibitory receptor on NK cells and that NK cytotoxicity is enhanced by HHLA2 / KIR3DL3 blockade. Figure 19A shows the cytotoxicity of NK92-MI against Raji cells with B2M deletion (Raji-B2M KO cells) and Raji-B2M KO cells expressing HHLA2. Figures 19B and 19C show the cytotoxicity of NK92-MI against Raji-B2M KO cells expressing HHLA2 at the indicated E:T ratios in the presence of 10 μg / ml of KIR3DL3 antibody (Figure 19B) or HHLA2 antibody (Figure 19C) and an isotype control. Figure 19D shows the results of NK92-MI cells incubated with Raji B2M KO cells or Raji B2M KO cells overexpressing HHLA2 at the indicated E:T ratios. Degranulation was measured as the percentage of CD107a-positive cells in the CD56+ population. Controls were effector cells alone or effector cells with PMA / ION, which resulted in total degranulation. Figure 19E shows enhanced degranulation of NK92-MI cells targeting HHLA2-overexpressing Raji B2M KO cells in the presence of KIR3DL3 mAb (1G7) compared to the isotype control. Quantitation is presented as mean ± SD (N≧3; P≧0.05; *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001). [Figure 20] Figure 20 shows that HHLA2 expression differs from PD-L1 expression. Figure 20 shows the expression levels of B7 gene family members in RCC compared to normal kidney from The Cancer Genome Atlas (TCGA) samples. [Figure 21A]Figures 21A-B show a model of the HHLA2 pathway. HHLA2 delivers immunostimulatory signals via TMIGD2 in naive T cells or NK cells. Figure 21A shows that T cell activation results in the loss of TMIGD2 expression and the acquisition of KIR3DL3. HHLA2 delivers immunoinhibitory signals via KIR3DL3 in activated T cells. Figure 21B shows NK cell lytic activity regulated by inhibitory and activating receptors. Inhibitory receptors include most KIRs, CD94 / NKG2A, and LILRBI, which recognize MHC class I, E, and G, respectively. Activating receptors include NKG2D, NKp30, NKp44, NKp46, CD94 / NKG2C, and TMIGD2, which recognize ULBP-1, MICA, MICB, B7-H6, HLA-E, HHLA2, and others. When tumors lose MHC expression (loss of self), inhibitory signals are reduced and activating signals prevail, leading to tumor lysis by NK cells. HHLA2 on tumors is an inhibitory signal that inhibits lysis by KIR3DL3-positive NK cells, independently of MHC. [Figure 21B]Figures 21A-B show a model of the HHLA2 pathway. HHLA2 delivers immunostimulatory signals via TMIGD2 in naive T cells or NK cells. Figure 21A shows that T cell activation results in the loss of TMIGD2 expression and the acquisition of KIR3DL3. HHLA2 delivers immunoinhibitory signals via KIR3DL3 in activated T cells. Figure 21B shows NK cell lytic activity regulated by inhibitory and activating receptors. Inhibitory receptors include most KIRs, CD94 / NKG2A, and LILRBI, which recognize MHC class I, E, and G, respectively. Activating receptors include NKG2D, NKp30, NKp44, NKp46, CD94 / NKG2C, and TMIGD2, which recognize ULBP-1, MICA, MICB, B7-H6, HLA-E, HHLA2, and others. When tumors lose MHC expression (loss of self), inhibitory signals are reduced and activating signals prevail, leading to tumor lysis by NK cells. HHLA2 on tumors is an inhibitory signal that inhibits lysis by KIR3DL3-positive NK cells, independently of MHC. [Figure 22] FIG. 22 shows a schematic diagram for the construction of a KIR3DL3×PD-1 bispecific antibody. [Figure 23] Figure 23 shows binding sensograms of KIR3DL3 and PD-1 human IgG4 and scFV antibodies in an Octet assay. DETAILED DESCRIPTION OF THE INVENTION

[0036] HHLA2, a member of the B7 gene family, is widely expressed in various tumors and antigen-presenting cells and is involved as both an activating and inhibitory ligand for T cells. TMIGD2, expressed in naive T cells, is an activating receptor for HHLA2 and transmits costimulatory signals after T cell antigen receptor (TCR) ligation. TMIGD2 is downregulated after repeated TCR stimulation. HHLA2 binds to KIR3DL3, another receptor expressed in T cells and NK cells. As described herein, the present disclosure encompasses the recognition that, unlike the immune activation function of HHLA2-TMIGD2 interaction, HHLA2-KIR3DL3 interaction can inhibit immune responses and provide an attractive target for modulation in various diseases, disorders, or conditions, including, for example, cancer.

[0037] The present disclosure is based at least in part on the discovery that targeting KIR3DL3 can specifically block the HHLA2-KIR3DL3 interaction that inhibits immune response.Importantly, targeting KIR3DL3 does not disrupt the overall function of HHLA2, including the activation of immune response through interaction with TMIGD2.Therefore, precisely targeting KIR3DL3 provides the specificity to block only the immune inhibitory function of HHLA2, thereby inducing effective immune response against, for example, cancer cells, without downregulating the immune activation function of HHLA2.

[0038] The present disclosure is also based, at least in part, on the discovery that agents targeting both KIR3DL3 and PD-1 can be used to modulate immune responses and / or treat cancer. In some embodiments, the KIR3DL3xPD-1 bispecific antibodies described herein are checkpoint immunotherapies for activating T cells and NK cells in tumors. In some embodiments, the KIR3DL3xPD-1 bispecific antibodies are additive or synergistic with PD-1 or PD-L1 or other checkpoint immunotherapies. Furthermore, HHLA2 and / or KIR3DL3 expression in tumors is a useful biomarker for determining responsiveness to KIR3DL3 mAb and / or KIR3DL3xPD-1 bispecific antibody checkpoint blockade.

[0039] A panel of exemplary, representative anti-KIR3DL3 human monoclonal antibodies (mAbs) is described herein as immune checkpoint inhibitor drugs. Blocking and non-blocking anti-KIR3DL3 mAbs have been identified, and anti-KIR3DL3 mAbs that block HHLA2 binding to KIR3DL3 have been shown to be checkpoint inhibitor antibodies in T cell and NK cell assays. The binding characteristics and variable region heavy and light chain gene sequences for these candidate therapeutic anti-KIR3DL3 antibodies are described herein.

[0040] A panel of exemplary, representative bispecific antibodies or antigen-binding fragments thereof that bind to both KIR3DL3 and PD-1 are also described herein as immune checkpoint inhibitor drugs. Targeting two immune checkpoints with non-overlapping expression provides combination therapies with additive or synergistic anti-tumor activity.

[0041] Thus, the present disclosure provides monoclonal antibodies and antigen-binding fragments thereof that specifically bind to KIR3DL3, bispecific antibodies and antigen-binding fragments thereof that bind to KIR3DL3 and PD-1, as well as immunoglobulins, polypeptides, nucleic acids thereof, and methods of using such antibodies for immunomodulatory and therapeutic purposes, etc.

[0042] I. Definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. As an example, "an element" is used. "element" means one element or more than one element.

[0043] The term "altered amount" of a marker refers to an increased or decreased copy number of the marker and / or an increased or decreased nucleic acid level of a particular marker gene(s) in a sample compared to that of the marker in a control sample. The term "altered amount" of a marker also includes an increased or decreased protein level of the marker in a sample compared to the protein level of the marker in a normal control sample.

[0044] The term "altered activity" of a marker refers to the activity of the marker that is increased or decreased in, for example, a biological sample in a certain disease state compared to the activity of the marker in a normal control sample. Altered activity of a marker can be the result of, for example, altered expression of the marker, altered protein levels of the marker, altered structure of the marker, or altered interaction with other proteins involved in the same or different pathways as the marker, or altered interaction with transcriptional activators or inhibitors, for example.

[0045] The term "altered structure" of a marker refers to the presence of a mutation or allelic variant in a marker gene or protein, e.g., a mutation that affects the expression or activity of the marker, compared to a normal or wild-type gene or protein. For example, mutations include, but are not limited to, substitution, deletion, or addition mutations. Mutations can occur in coding or non-coding regions of the marker.

[0046] The term "activating receptor" includes immune cell receptors that bind to antigens, complex antigens (e.g., in the context of an MHC polypeptide), or antibodies. Such activating receptors include T cell receptors (TCRs), B cell receptors (BCRs), cytokine receptors, LPS receptors, complement receptors, and Fc receptors.

[0047] T cell receptors are present on T cells and are associated with CD3 polypeptides. T cell receptors are stimulated by antigens in the context of MHC polypeptides (and by polyclonal T cell activation reagents). TCR-mediated T cell activation leads to numerous changes, including protein phosphorylation, membrane lipid changes, ion efflux, cyclic nucleotide modifications, RNA transcription changes, protein synthesis changes, and cell volume changes.

[0048] The terms "chimeric antigen receptor," "CAR," or "CAR-T" refer to engineered T cell receptors (TCRs) with desired antigen specificity. T lymphocytes recognize specific antigens through the interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, naive T cells rely on professional antigen-presenting cells (APCs) that provide additional costimulatory signals. TCR activation in the absence of costimulation can result in unresponsiveness and clonal anergy. To bypass immunization, different approaches have been developed to induce cytotoxic effector cells with grafted recognition specificities. CARs have been constructed consisting of binding domains derived from natural ligands or antibodies specific for cell surface components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric antigen receptors couple to endogenous signaling pathways in effector cells, generating activation signals similar to those initiated by the TCR complex. For example, CARs targeting CD19, a protein highly expressed on hematologic cancer cells, have shown favorable clinical efficacy. Since the first report of chimeric antigen receptors, this concept has been steadily refined, and the molecular design of chimeric receptors has been optimized, commonly using several well-known binding domains, e.g., scFV, Fav, and other protein-binding fragments described herein.

[0049] Generally, CARs are a type of "cell therapy" (e.g., T cell therapy) contemplated for use in the present disclosure. Numerous representative embodiments of agents and methods for regulating immune cell activity by modulating the KIR3DL3 pathway, for example, by modulating the interaction between KIR3DL3 and a KIR3DL3 natural binding partner such as HHLA2, are encompassed, but immune cell-based therapies and methods are also encompassed. For example, T cells engineered to have knockout, knockdown, or increased expression of KIR3DL3 are contemplated. Similarly, immune cells or other cells engineered to have knockout, knockdown, or increased expression of a ligand for KIR3DL3, HHLA2, are also contemplated.

[0050] The B cell receptor (BCR) is present on B cells. The B cell antigen receptor is a complex between membrane Ig (mIg) and other transmembrane polypeptides (e.g., Igα and Igβ). The signaling function of mIg is triggered by cross-linking of receptor polypeptides by oligomeric or multimeric antigens. B cells can also be activated by anti-immunoglobulin antibodies. Upon BCR activation, a number of changes occur in the B cell, including tyrosine phosphorylation.

[0051] Fc receptors are found on many cells involved in the immune response. Fc receptors (FcRs) are cell surface receptors for the Fc portion of immunoglobulin polypeptides (Ig). Of the human FcRs identified to date, IgG (designated Fcγ R), IgE (designated Fcε R), and IgE (designated Fcε R) are the most common. Some recognize IgA (Fcα), and polymerized IgM / A (FcμαR). FcRs are found on the following cell types: FcεR I (mast cells), FcεR II (many leukocytes), FcαR (neutrophils), and FcμαR (glandular epithelium, hepatocytes) (Hogg, N. (1988) Immunol. Today 9:185-86). The widely studied FcγRs are central to cell-mediated immune defense and are involved in stimulating the release of inflammatory mediators and hydrolases involved in the pathogenesis of autoimmune diseases (Unkeless, JC et al. (1988) Annu. Rev. Immunol. 6:251-81). FcγRs provide an important link between effector cells and Ig-secreting lymphocytes because they confer specific recognition elements for IgG-mediated signaling on macrophages / monocytes, polymorphonuclear leukocytes, and natural killer (NK) cells. Human leukocytes have at least three distinct receptors for IgG: hFcγ RI (found on monocytes / macrophages), hFcγ RII (found on monocytes, neutrophils, eosinophils, platelets, possibly B cells, and the K562 cell line), and Fcγ III (found on NK cells, neutrophils, eosinophils, and macrophages).

[0052] With respect to T cells, the delivery of costimulatory signals to T cells involves signal transduction pathways that are not inhibited by cyclosporine A. In addition, costimulatory signals can induce cytokine secretion (e.g., IL-2 and / or IL-10) in T cells and / or can prevent the induction of unresponsiveness to antigen, the induction of anergy, or the induction of cell death (deletion) in T cells.

[0053] The term "activity" when used with respect to a polypeptide, e.g., KIR3DL3 and / or a KIR3DL3 natural binding partner, e.g., HHLA2, includes activity inherent to the structure of the protein. For example, with respect to an HHLA2 ligand, the term "activity" includes the ability to regulate immune cell inhibition by regulating an inhibitory signal in the immune cell (e.g., by linking a natural receptor on the immune cell). Those skilled in the art will recognize that when an activated form of an HHLA2 ligand polypeptide binds to an inhibitory receptor, such as KIR3DL3, an inhibitory signal is generated in the immune cell.

[0054] The term "inhibitory signal" refers to a signal transmitted through an inhibitory receptor (e.g., KLRB1, CTLA4, PD-1, etc.) to a polypeptide on an immune cell. Such a signal antagonizes a signal through an activating receptor (e.g., through a TCR, CD3, BCR, TMIGD2, or Fc polypeptide) and can result in, for example, inhibition of second messenger production, inhibition of proliferation, inhibition of effector function in the immune cell, e.g., reduced phagocytosis, reduced antibody production, reduced cytotoxicity, inability of the immune cell to produce mediators (such as cytokines (e.g., IL-2) and / or allergic response mediators), or the development of anergy.

[0055] The amount of a biomarker in a subject is "significantly" higher or lower than the normal amount of the biomarker if the amount of the biomarker is higher or lower than the normal or control level, respectively, by an amount that exceeds the standard error of the assay used to assess the amount, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% of that amount. Alternatively, the amount of the biomarker in the subject may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 200%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 300%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%, 360%, 365%, 370%, 375%, 380%, 385%, 390%, 400%, 410%, 410%, 420%, 425%, 430%, 430%, 440%, 445%, 450%, 460%, 470%, 475%, 480%, 485%, 490%, 500%, 510%, 515%, 520%, 525%, 530%, 535%, 540%, 545%, 550%, 555%, 600%, 655%, A quantity may be considered "significantly" higher or lower than the normal and / or control amount if it is 25%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 2-fold, 3-fold, 4-fold, 5-fold, or more, or any range therebetween, such as 5%-100% higher or lower. Such significant modulation may apply to any metric described herein, such as altered expression levels, altered activity, altered cancer cell hyperproliferative growth, altered cancer cell death, altered biomarker inhibition, altered test agent binding, etc.

[0056] The term "altered expression level" of a marker refers to an expression level or copy number of a marker in a test sample, such as a sample from a subject suffering from cancer, that is higher or lower than the standard error of the assay used to assess expression or copy number, and is preferably at least 2-fold, more preferably 3-fold, 4-fold, 5-fold, or 10-fold or more of the expression level or copy number of the marker or chromosomal region in a control sample (e.g., a sample from a healthy subject without an associated disease), preferably the average expression level or copy number of the marker or chromosomal region in several control samples. An altered expression level is higher or lower than the standard error of the assay used to assess expression or copy number, and is preferably at least 2-fold, more preferably 3-fold, 4-fold, 5-fold, or 10-fold or more of the expression level or copy number of the marker in a control sample (e.g., a sample from a healthy subject without an associated disease), preferably the average expression level or copy number of the marker in several control samples.

[0057] Unless otherwise specified herein, the terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives contain at least an antigen-binding site. Antibody derivatives may include proteins or chemical moieties conjugated to the antibody. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H Each light chain consists of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. L The light chain constant region consists of one domain, CL. H Area and V LThe regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L Each of these heavy and light chains consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term "inactivating antibody" refers to an antibody that does not induce the complement system.

[0058] The term "antibody" as used herein also includes an "antigen-binding portion" (or simply "antibody portion") of an antibody. As used herein, the term "antigen-binding portion" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a KIR3DL3 polypeptide or a fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH domain and the CH1 domain; (iv) a Fv fragment consisting of the VL domain and the VH domain of a single arm of an antibody; and (v) a dAb fragment consisting of the VH domain (Ward et al., 2004). (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains, VL and VH, of an Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using recombinant techniques, allowing them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide (known as single-chain Fvs (scFvs), see, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used to generate Fab, Fv, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing them to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0059] Furthermore, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesive polypeptide formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesive polypeptides include the use of a streptavidin core region to generate tetrameric scFv polypeptides (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent biotinylated scFv polypeptides (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab fragments and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesive polypeptides can be obtained using standard recombinant DNA techniques as described herein.

[0060] Antibodies can be polyclonal or monoclonal, xenogeneic, allogeneic, or syngeneic, or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies may also be fully human. In one embodiment, antibodies encompassed by the present disclosure specifically or substantially specifically bind to a KIR3DL3 polypeptide or a fragment thereof. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one species of antigen-binding site capable of immunoreacting with a particular epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple species of antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically exhibits a single binding affinity for the particular antigen with which it immunoreacts.

[0061] The term "body fluid" refers to fluids that are excreted or secreted by the body, as well as fluids that are not normally excreted or secreted by the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, cerumen and earwax, Cowper's fluid or pre-ejaculate, chyle, chyme, feces, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal mucous fluid, vitreous humor, vomit).

[0062] The term "cancer" or "tumor" or "hyperproliferative disorder" refers to the presence of cells that have properties characteristic of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain distinctive morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone in an animal or may be non-tumorigenic cancer cells, such as leukemia cells. Cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematological tissue cancer, and the like.Other non-limiting examples of cancer types applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, These include bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial in nature, including, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In yet other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers may be characterized in a variety of other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0063] The term "CDR" and its plural form "CDRs" refer to complementarity-determining regions (CDRs), three of which constitute the binding characteristics of a light chain variable region (CDR-L1, CDR-L2, and CDR-L3) and three of which constitute the binding characteristics of a heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3), for example, on an antibody. CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definition of CDR boundaries and lengths is subject to different classification and numbering systems. Thus, CDRs may be referred to by Kabat, Chothia, contact, or any other boundary definition. Despite the different boundaries, each of these systems has some overlap in what constitutes the so-called "hypervariable regions" within the variable sequences. Thus, CDR definitions according to these systems may differ in length and boundary regions with respect to the adjacent framework regions. See, e.g., Kabat, Chothia, and / or MacCallum et al. (each entirely incorporated by reference; Kabat et al., "Sequences of Proteins of Immunological Interest," 5 th Edition, US Department of Health and Human Services, 1992; Chothia et al. (1987) J. Mol. Biol. 196, 901; and MacCallum et al., J. Mol. Biol. (1996) 262, 732).

[0064] As used herein, the term "classifying" includes "associating" a sample with a disease state or "categorizing" a sample with a disease state. In certain instances, "classifying" is based on statistical evidence, experimental evidence, or both. In certain embodiments, classification methods and systems use a training set of samples with so-called known disease states. Once established, the training dataset serves as a basis, model, or template against which features of unknown samples are compared in order to classify the sample's unknown disease state. In certain instances, sample classification resembles diagnosing the sample's disease state. In certain other instances, sample classification resembles distinguishing the sample's disease state from another disease state.

[0065] As used herein, the term "coding region" refers to the region of a nucleotide sequence that contains codons that are translated into amino acid residues, and the term "non-coding region" refers to the region of a nucleotide sequence that is not translated into amino acids (e.g., the 5' and 3' untranslated regions).

[0066] "Complement" or "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue in a first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue in a second nucleic acid region antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand can base pair with a residue in a second nucleic acid strand antiparallel to the first strand if the residue is guanine. A first region of nucleic acid is complementary to a second region of the same or different nucleic acid if at least one nucleotide residue in the first region can base pair with a residue in the second region when the two regions are arranged in an antiparallel fashion. In one embodiment, the first region comprises a first portion and the second region comprises a second portion such that when the first and second portions are arranged in an antiparallel manner, at least about 50%, preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion, hi another embodiment, all nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion.

[0067] As used herein, the term "composite antibody" refers to an antibody having a variable region comprising germline or non-germline immunoglobulin sequences derived from two or more unrelated variable regions. Additionally, the term "composite human antibody" refers to an antibody having a constant region derived from human germline or non-germline immunoglobulin sequences and a variable region comprising human germline or non-germline sequences derived from two or more unrelated human variable regions. Composite human antibodies are useful as active ingredients in therapeutic agents according to the present disclosure due to the reduced antigenicity of composite human antibodies in the human body.

[0068] The term "control" refers to any reference standard suitable for providing a comparison with the expression product in a test sample. In one embodiment, controlling involves obtaining a "control sample" in which expression product levels are detected and compared to expression product levels from a test sample. Such a control sample may comprise any suitable sample, including, but not limited to, a sample from a control cancer patient with a known outcome (which may be an archived sample or a prior sample measurement), normal tissue or cells isolated from a subject such as a healthy patient or cancer patient, cultured primary cells / tissues isolated from a subject such as a healthy subject or cancer patient adjacent to normal cells / tissues obtained from the same organ or body location of a cancer patient, a tissue or cell sample isolated from a healthy subject, or primary cells / tissues obtained from a depository. In another preferred embodiment, the control may comprise a reference expression product level from any suitable source, including, but not limited to, a housekeeping gene, a range of expression product levels from normal tissue (or other previously analyzed control sample), or a previously determined range of expression product levels in test samples from a group or set of patients having a particular outcome (e.g., 1-, 2-, 3-, 4-year survival, etc.) or receiving a particular treatment (e.g., standard of care cancer therapy). Those skilled in the art will understand that such control samples and reference expression product levels may be used as controls in conjunction with the methods encompassed by the present disclosure. In one embodiment, the control may comprise a normal or non-cancerous cell / tissue sample. In another preferred embodiment, the control may comprise expression levels from a set of patients, e.g., a set of cancer patients, or a set of cancer patients receiving a particular treatment, or a set of patients with one outcome versus another. In the former case, each patient's specific expression product level may be assigned a percentile level of expression or expressed as either higher or lower than the mean or average of the reference expression level. In another preferred embodiment, the control may include normal cells, cells from patients treated with combination chemotherapy, and cells from patients with benign cancer. In another embodiment, the control may also include a measured value, for example, the average expression level of a particular gene in a population compared to the expression level of a housekeeping gene in the same population.Such populations may include healthy subjects, cancer patients who have not received any treatment (i.e., untreated), cancer patients receiving standard treatment therapy, or patients with benign cancer. In another preferred embodiment, the control involves ratio conversion of expression product levels, including, but not limited to, determining the ratio of expression product levels of two genes in a test sample and comparing it to any suitable ratio of the same two genes in a reference standard, determining the expression product levels of two or more genes in a test sample and determining the difference in expression product levels in any suitable control, and determining the expression product levels of two or more genes in a test sample, normalizing their expression to the expression of housekeeping genes in the test sample, and comparing it to any suitable control. In a particularly preferred embodiment, the control includes a control sample of the same lineage and / or type as the test sample. In another embodiment, the control may include expression product levels grouped as or based on percentiles in a set of patient samples, such as all patients with cancer. In one embodiment, a control expression product level is established, and expression product levels higher or lower than a particular percentile, for example, are used as a basis for predicting outcome. In another preferred embodiment, a control expression product level is established using expression product levels from cancer control patients with known outcomes, and the expression product level from the test sample is compared to the control expression product level as a basis for predicting outcome. As demonstrated by the data below, the methods encompassed by the present disclosure are not limited to the use of a particular cutpoint when comparing expression product levels in a test sample with controls.

[0069] The term "costimulate," when used in reference to activated immune cells, includes the ability of a costimulatory polypeptide to provide a second, non-activating receptor-mediated signal (a "costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion in, for example, a T cell that has received a T cell-receptor-mediated signal. For example, an immune cell that has received a cell-receptor-mediated signal via an activating receptor is referred to herein as an "activated immune cell."

[0070] The term "costimulatory receptor" includes receptors that transmit costimulatory signals to immune cells, e.g., CD28. As used herein, the term "inhibitory receptor" includes receptors that transmit negative signals to immune cells (e.g., CTLA4, KIR3DL3, or PD-1). The inhibitory signal transmitted by an inhibitory receptor can occur even when a costimulatory receptor (such as CD28) is not present on the immune cell and is therefore not simply a function of competition between the inhibitory receptor and the costimulatory receptor for binding of a costimulatory polypeptide (Fallarino et al. (1998) J. Exp. Med. 188:205). Transmission of an inhibitory signal to an immune cell can result in unresponsiveness or anergy or programmed cell death of the immune cell. Preferably, transmission of the inhibitory signal operates by a mechanism that does not involve apoptosis. As used herein, the term "apoptosis" includes programmed cell death, which can be characterized using techniques known in the art. Apoptotic cell death can be characterized by, for example, cell shrinkage, membrane blebbing, and chromatin condensation, which ultimately leads to cell fragmentation.Cells undergoing apoptosis also exhibit characteristic patterns of internucleosomal DNA cleavage.Depending on the form of polypeptide that binds to receptor, for example, by competing with the activated form of HHLA2 and / or KIR3DL3 for binding to one or more natural binding partners, signal can be transmitted (for example, by the multivalent form of HHLA2 and / or KIR3DL3 polypeptide), or signal can be inhibited (for example, by the soluble monovalent form of HHLA2 and / or KIR3DL3).However, there are also cases where soluble polypeptide can be stimulatory.The effect of regulator can be easily demonstrated using the conventional screening assay described herein.

[0071] The term "determining a suitable therapeutic regimen for a subject" is intended to mean determining a therapeutic regimen for a subject (i.e., a single therapy or a combination of different therapies used for preventing and / or treating cancer in a subject) that is initiated, modified, and / or terminated based on, or essentially based on, or at least in part based on, the results of an analysis according to the present disclosure. One example is determining whether to provide targeted therapy for cancer by providing immunomodulatory therapy (e.g., KIR3DL3 pathway modulator therapy (e.g., a modulator of the interaction between KIR3DL3 and one or more natural binding partners, such as KIR3DL3)). Another example would be initiating adjuvant therapy after surgery aimed at reducing the risk of recurrence, and another example would be modifying the dosage of a particular chemotherapy. The decision may be based on the results of an analysis according to the present disclosure as well as the personal characteristics of the subject being treated. In many cases, the actual decision on a suitable therapeutic regimen for a subject will be made by the attending physician or doctor.

[0072] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus. Native-sequence Fc regions suitable for use in antibodies encompassed by the present disclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0073] As used herein, "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Further, preferred FcRs are those that bind IgG antibodies (gamma receptors), and preferred FcRs include receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

[0074] A molecule is "fixed" or "affixed" to a substrate when it is covalently or non-covalently associated with the substrate such that the substrate can be rinsed with a fluid (e.g., standard citrate saline, pH 7.4) without a significant proportion of the molecule dissociating from the substrate.

[0075] As used herein, "framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0076] A "function-conservative variant" is one in which a given amino acid residue in a protein or enzyme is altered without altering the overall conformation and function of the polypeptide, including, but not limited to, substitution of an amino acid with an amino acid having similar properties (e.g., polarity, hydrogen-bonding ability, acidic, basic, hydrophobic, aromatic, etc.). Amino acids other than those designated as conserved may vary within a protein, such that the percent protein or amino acid sequence similarity between any two proteins with similar functions may vary, e.g., 70% to 99% similarity as determined according to an alignment scheme such as a clustering method based on the MEGALIGN algorithm. A "function-conservative variant" also includes a polypeptide that has at least 60%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95% amino acid identity as determined by the BLAST or FASTA algorithm, and that has the same or substantially similar properties or functions as the native or parent protein to which it is compared.

[0077] As used herein, the term "heterologous antibody" is defined in the context of the transgenic non-human organism producing such an antibody. This term refers to an antibody that does not originate from the transgenic non-human animal, but generally has an amino acid sequence or encoding nucleic acid sequence that corresponds to that found in an organism from a species other than the transgenic non-human animal species.

[0078] The terms "high," "low," "intermediate," and "negative," in reference to cellular biomarker expression, refer to the amount of biomarker expressed compared to cellular expression of the biomarker by one or more reference cells. Biomarker expression may be determined according to any method described herein, including, but not limited to, analysis of cellular levels, activity, structure, etc. of one or more biomarker genomic nucleic acids, ribonucleic acids, and / or polypeptides. In one embodiment, these terms refer to a defined percentage of a cell population that expresses the biomarker at the highest, intermediate, or lowest level, respectively. Such a percentage may be defined as the top 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15%, or more of the cell population that express the biomarker either highly or weakly, or any range therebetween (including boundaries). The term "low" excludes cells that do not detectably express the biomarker, since such cells are "negative" for biomarker expression. The term "intermediate" includes cells that express the biomarker but at a lower level than the population that expresses it at a "high" level. In another embodiment, these terms may also or alternatively refer to cell populations of biomarker expression identified by qualitative or statistical plot areas. For example, cell populations sorted using flow cytometry may be differentiated based on biomarker expression levels by identifying distinct plots based on analysis of detectable moieties, e.g., mean fluorescence intensity, etc., according to methods well known in the art. Such plot areas may be refined according to number, shape, overlap, etc., according to methods well known in the art for the biomarker of interest. In yet another embodiment, these terms may also be determined according to the presence or absence of expression of additional biomarkers.

[0079] As used herein, "homology" refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. If a nucleotide residue position in both regions is occupied by the same nucleotide residue, the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position in each region is occupied by the same residue. Homology between two regions is expressed as the percentage of nucleotide residue positions in the two regions that are occupied by the same nucleotide residue. As an example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, such that at least about 50%, preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions in each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions in each of the portions are occupied by the same nucleotide residue.

[0080] As used herein, the term "host cell" is intended to refer to a cell into which a nucleic acid encompassed by the present disclosure, such as a recombinant expression vector encompassed by the present disclosure, has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur over time, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still be within the scope of the term as used herein.

[0081] As used herein, the term "humanized antibody" is intended to include antibodies produced by non-human cells with variable and constant regions modified to more closely resemble antibodies that would be produced by human cells. For example, the amino acid sequence of a non-human antibody is modified to incorporate amino acids found in human germline immunoglobulin sequences. A humanized antibody can include, for example, amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo). As used herein, the term "humanized antibody" also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0082] As used herein, a humanized mouse is a mouse that possesses functional human genes (e.g., HHLA2, and / or KIR3DL3), cells, tissues, and / or organs. Humanized mice are commonly used as small animal models in biological and medical research for human therapeutics. Nude mice and severe combined immunodeficiency (SCID) mice can be used for this purpose. NCG, NOG, and NSG mice can be used to engraft human cells and tissues more efficiently than other models. Such humanized mouse models can be used to model the human immune system in healthy and pathological scenarios, allowing for the evaluation of therapeutic candidates in an in vivo context relevant to human physiology.

[0083] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops, and include the CDRs.

[0084] As used herein, the term "immune cell" refers to a cell that plays a role in immune response.Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells, natural killer cells, myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0085] As used herein, the term "immune disorder" includes immune diseases, conditions, and predispositions thereto, including, but not limited to, cancer, chronic inflammatory diseases and disorders (such as, for example, Crohn's disease, inflammatory bowel disease, reactive arthritis, and Lyme disease), insulin-dependent diabetes mellitus, organ-specific autoimmunity (such as, for example, multiple sclerosis, Hashimoto's thyroiditis, autoimmune uveitis, and Graves' disease), contact dermatitis, psoriasis, transplant rejection, graft-versus-host disease, sarcoidosis, atopic conditions (such as, for example, asthma and allergies (including, but not limited to, gastrointestinal allergies such as allergic rhinitis and food allergies)), eosinophilia, conjunctivitis, glomerulonephritis, systemic lupus erythematosus, scleroderma, susceptibility to certain pathogens such as helminths (such as, for example, leishmaniasis) and certain viral infections (such as, for example, HIV and bacterial infections such as tuberculosis and leprosy), and malaria.

[0086] As used herein, the term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production, and cytotoxicity. In addition, the term immune response includes immune responses indirectly mediated by T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages.

[0087] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent capable of stimulating the host immune system to produce an immune response against a tumor or cancer in a subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0088] The term "immune checkpoint" refers to a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by downregulating or inhibiting anti-tumor immune responses. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, and A2aR (see, e.g., WO2012 / 177624). The term further encompasses biologically active protein fragments, as well as nucleic acids encoding full-length immune checkpoint proteins and their biologically active protein fragments. In some embodiments, the term further encompasses any fragment that conforms to the homology descriptions provided herein.

[0089] Immune checkpoints and their sequences are well known in the art, and representative embodiments are described below. For example, the term "PD-1" refers to a member of the immunoglobulin gene superfamily that functions as a co-inhibitory receptor with PD-L1 and PD-L2 as known ligands. PD-1 was previously identified using a cloning-based subtraction approach to select genes upregulated during TCR-induced activated T cell death. PD-1 is a member of the CD28 / CTLA-4 family of molecules based on its ability to bind to PD-L1. Like CTLA-4, PD-1 is rapidly induced on the surface of T cells in response to anti-CD3 (Agata et al. (1996) Int. Immunol. 8:765). However, in contrast to CTLA-4, PD-1 is also induced on B cells (in response to anti-IgM). PD-1 is also expressed on a subset of thymocytes and myeloid cells (Agata et al. (1996) (see above), Nishimura et al. (1996) Int. Immunol. 8:773).

[0090] As used herein, the term "inhibiting" and its grammatical equivalents refer to reducing, limiting, and / or blocking a particular action, function, or interaction. In one embodiment, the term refers to reducing the level of a given output or parameter to an amount (e.g., background staining, KIR3DL3 signaling, KIR3DL3 immunoinhibitory function, etc.) that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or less than the amount in a corresponding control. A reduction in the level of a given output or parameter can, but need not, imply the absolute absence of the output or parameter. The present invention does not require, and is not limited to, methods that completely eliminate the output or parameter. A given output or parameter may be determined using methods well known in the art, including, but not limited to, immunohistochemical, molecular biological, cell biological, clinical, and biochemical assays discussed herein and in the Examples. The antonyms "promoting" and "increasing," and their grammatical equivalents, refer to an increase in the level of a given output or parameter, as opposed to what is described as inhibiting or reducing.

[0091] As used herein, the term "interaction," when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules with each other (e.g., binding of HHLA2 to TMIGD2 or binding of HHLA2 to KIR3DL3). Generally, such an interaction results in the activity of one or both of the molecules (thereby producing a biological effect). The activity can be the direct activity (e.g., signal transduction) of one or both of the molecules. Alternatively, one or both molecules in the interaction can be prevented from binding to their ligands and thus remain inactive with respect to ligand binding activity (e.g., binding to the ligand and inducing or inhibiting an immune response). Inhibiting such an interaction results in the destruction of the activity of one or more molecules involved in the interaction. Enhancing such an interaction extends or increases the likelihood of such physical contact, thereby extending or increasing the likelihood of such activity.

[0092] The term "neoadjuvant therapy" refers to treatment administered before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy.

[0093] 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 KIR3DL3 and is substantially free of antibodies that do not bind to KIR3DL3). However, isolated antibodies that specifically bind to KIR3DL3 may each have cross-reactivity to other KIR family proteins from different species. For example, in some embodiments, the antibody maintains specific binding affinity for at least two species, e.g., humans and other animals, such as non-rodents, or other mammalian or non-mammalian species. However, in some embodiments, the antibody maintains higher or actually specific affinity and selectivity for human KIR3DL3. In addition, isolated antibodies are typically substantially free of other cellular material and / or chemicals. In one embodiment encompassed by the present disclosure, a combination of "isolated" monoclonal antibodies with different specificities for human KIR3DL3 are combined in a well-defined composition.

[0094] As used herein, "isolated protein" refers to a protein that is substantially free of other proteins, cellular material, isolation medium, and culture medium (if isolated from cells or produced by recombinant DNA techniques), or chemical precursors or other chemicals (if chemically synthesized). An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide, or fusion protein is derived, or is substantially free of chemical precursors or other chemicals (if chemically synthesized). The language "substantially free of cellular material" includes preparations of a target polypeptide (e.g., an immunoglobulin) or fragment thereof in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of target proteins or fragments thereof having less than about 30% (by dry weight) non-target proteins (also referred to herein as "contaminating proteins"), more preferably less than about 20% non-target proteins, even more preferably less than about 10% non-target proteins, and most preferably less than about 5% non-target proteins. When an antibody, polypeptide, peptide, or fusion protein, or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it is preferably substantially free of culture medium, i.e., culture medium representing less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0095] As used herein, the term "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0096] As used herein, "K DThe term "" is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. The binding affinity of the antibodies of the disclosed invention can be measured or determined by standard antibody-antigen assays, for example, competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA.

[0097] As used herein, a "kit" is any article of manufacture (e.g., a package or container) that contains at least one reagent, e.g., a probe, for specifically detecting or modulating the expression of a marker encompassed by the present disclosure. The kit may be promoted, distributed, or sold as a device for performing the methods encompassed by the present disclosure.

[0098] A "marker" or "biomarker" is a gene or protein whose expression level in a tissue or cell, altered from that in a normal or healthy tissue or cell, is associated with a disease state, such as cancer. A "marker nucleic acid" is a nucleic acid (e.g., mRNA, cDNA) encoded by or corresponding to a marker encompassed by the present disclosure. Such marker nucleic acids include DNA (e.g., cDNA) comprising the entire or partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing, or the complement of such a sequence. Marker nucleic acids also include RNA comprising the entire or partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing, or the complement of such a sequence, in which all thymidine residues are replaced with uridine residues. A "marker protein" is a protein encoded by or corresponding to a marker encompassed by the present disclosure. The marker protein comprises the entire or partial sequence of any of the sequences set forth in the Sequence Listing. In some embodiments, the entire KIR3DL3 or HHLA2 is used as a marker. In other embodiments, a fragment of KIR3DL3 or HHLA2 is used as a marker. The terms "protein" and "polypeptide" are used interchangeably.

[0099] As used herein, the term "modulate" includes upregulation and downregulation, eg, enhancing or inhibiting a response.

[0100] The term "predetermined" biomarker amount and / or activity measurement can refer, by way of example only, to a biomarker amount and / or activity measurement used to evaluate a subject who may be selected for a particular treatment, to evaluate a response to a treatment, such as one or more modulators of the KIR3DL3 pathway, such as KIR3DL3 modulators, and one or more natural binding partners, such as HHLA2, either alone or in combination with one or more immunotherapies, and / or to evaluate a disease state. The predetermined biomarker amount and / or activity measurement can be determined in a patient population with or without cancer. The predetermined biomarker amount and / or activity measurement can be a single number equally applicable to all patients, or the predetermined biomarker amount and / or activity measurement can vary depending on specific patient subpopulations. A subject's age, weight, height, and other factors can affect the predetermined biomarker amount and / or activity measurement for that individual. Furthermore, the predetermined biomarker amount and / or activity can be determined individually for each subject. In one embodiment, the amounts determined and / or compared in the methods described herein are based on absolute measurements. In another embodiment, the amounts determined and / or compared in the methods described herein are based on relative measurements, such as ratios (e.g., cellular ratios, or serum biomarkers normalized to the expression of a housekeeping or otherwise generally constant biomarker). The predetermined biomarker amount and / or activity measurements can be any suitable reference. For example, the predetermined biomarker amount and / or activity measurements can be obtained from the same or different human beings for whom patient selection is being evaluated. In one embodiment, the predetermined biomarker amount and / or activity measurements can be obtained from a previous evaluation of the same patient. In such a manner, the progress of patient selection can be monitored over time. In addition, a control can be obtained from the evaluation of another human or multiple humans, e.g., a selected group of humans if the subject is a human. In such a manner, the degree of selection of the human being for whom selection is being evaluated can be compared to suitable other humans, e.g., other humans in similar circumstances to the human being of interest, such as humans suffering from a similar or identical condition and / or humans of the same ethnic group.

[0101] The term "predictive" includes the use of biomarker nucleic acid and / or protein status, e.g., over- or under-activity, appearance, expression, growth, remission, recurrence, or resistance of a tumor, before, during, or after therapy to determine the likelihood of a cancer's response to immunomodulatory therapy such as KIR3DL3 pathway modulator therapy (e.g., a modulator of the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2, either alone or in combination with one or more additional therapies such as immunotherapy, e.g., immune checkpoint inhibitor therapy). Such predictive uses of biomarkers include, for example, (1) increased or decreased copy number (e.g., by FISH, FISH+SKY, single molecule sequencing, e.g., those described in the art at least in J. Biotechnol., 86:289-301, or qPCR), overexpression or underexpression of biomarker nucleic acids (e.g., by ISH, Northern blot, or qPCR), increased or decreased biomarker protein (e.g., by IHC) and / or biomarker target, or increased or decreased activity (e.g., in about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, %, 60%, 70%, 80%, 90%, 95%, 100% or more), (2) its absolute or relatively modulated presence or absence in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, cheek scraping, saliva, cerebrospinal fluid, urine, feces, or bone marrow from a subject afflicted with cancer, e.g., a human, or (3) its absolute or relatively modulated presence or absence in a clinical subset of patients with cancer (e.g., patients who respond to or develop resistance to a particular immunomodulatory therapy (e.g., KIR3DL3 pathway modulator therapy (e.g., a modulator of the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2, either alone or in combination with immunotherapy)).

[0102] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition, but who is at risk of developing it or is susceptible to it.

[0103] The term "prognosis" includes a prediction of the likely course and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the prognosis of cancer in an individual is provided by using a statistical algorithm. For example, the prognosis may be surgery, the development of a clinical subtype of cancer (e.g., solid tumors such as lung cancer, melanoma, and renal cell carcinoma), the development of one or more clinical factors, the development of intestinal cancer, or recovery from the disease.

[0104] The term "polypeptide fragment" or "fragment," when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted compared to the reference polypeptide itself, but the remaining amino acid sequence is typically identical to the corresponding positions in the reference polypeptide. Such deletions can occur internally at the amino terminus of the reference polypeptide, or at its carboxyl terminus, or alternatively, both. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, at least 14 amino acids in length, at least 20, 30, 40, or 50 amino acids in length, at least 75 amino acids in length, or at least 100, 150, 200, 300, 500, or more amino acids in length. They may be, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 75 ... The polypeptides may be and / or include lengths of 20, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1020, 1040, 1060, 1080, 1100, 1120, 1140, 1160, 1180, 1200, 1220, 1240, 1260, 1280, 1300, 1320, 1340, or more. Alternatively, they may not exceed and / or exclude such ranges, so long as they are less than the length of the full-length polypeptide.

[0105] The term "probe" refers to any molecule that can selectively bind to a specific intended target molecule, such as a nucleotide transcript or protein encoded by or corresponding to a marker. Probes can be synthesized by those skilled in the art or derived from appropriate biological preparations. For the detection of target molecules, probes can be specifically designed to be labeled as described herein. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0106] As used herein, the term "rearranged" refers to a V segment that is rearranged to form a complete V H and V L This refers to the configuration of a heavy or light chain immunoglobulin locus positioned immediately adjacent to a DJ or J segment in a conformation that essentially encodes the domain. Rearranged immunoglobulin loci can be identified by comparison with germline DNA, and rearranged loci have at least one recombined heptamer / nonamer homology element.

[0107] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such term is intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur over time, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0108] The term "resistance" refers to acquired or natural resistance of a cancer sample or mammal to immunomodulatory therapy (i.e., being unresponsive to therapeutic treatment or having a reduced or limited response to therapeutic treatment), for example, having a 5% or more, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more reduced response to therapeutic treatment. The reduced response can be measured by comparing with the same cancer sample or mammal before resistance was acquired, or by comparing with a different cancer sample or mammal known not to have resistance to therapeutic treatment. Typical acquired resistance to chemotherapy is called "multidrug resistance." Multidrug resistance may be mediated by P-glycoprotein or other mechanisms, or may occur when a mammal is infected with a multidrug-resistant microorganism or combination of microorganisms. Determining resistance to therapeutic treatment is routine in the art and within the skill of those skilled in the art, and can be measured, for example, by cell proliferation and cell death assays described herein as "sensitization." In some embodiments, the term "reverse resistance" refers to situations in which the use of a second agent in combination with a primary cancer therapy (e.g., chemotherapy or radiation therapy) can result in a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor, at a statistically significant level (e.g., p<0.05) compared to the tumor volume of an untreated tumor. This generally applies to tumor volume measurements performed when the untreated tumor is growing exponentially.

[0109] As mentioned above, the term "response" generally relates to determining, for example, the impact on the course, efficacy, or outcome of a clinical intervention. For example, response to a therapy (e.g., KIR3DL3 pathway modulator therapy (e.g., a modulator of the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2, either alone or in combination with immunotherapy, such as immune checkpoint inhibitor therapy)) relates to any response to a therapy (e.g., KIR3DL3 pathway modulator therapy (e.g., a modulator of the interaction between KIR3DL3 and one or more natural binding partners such as KIR3DL3, either alone or in combination with immunotherapy, such as immune checkpoint inhibitor therapy), in the case of cancer, preferably relates to changes in cancer cell number, tumor mass, and / or volume after the initiation of neoadjuvant or adjuvant chemotherapy. Hyperproliferative disorder responses can be assessed, for example, for efficacy or in the neoadjuvant or adjuvant setting, and tumor size after systemic intervention can be assessed by initial size as measured by CT, PET, mammogram, ultrasound, or palpation. The size and dimensions of tumors may be compared with those of tumors after biopsy or surgical resection. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded quantitatively, such as by percentage change in tumor volume, or qualitatively, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. Hyperproliferative disorder response assessment may be performed early after initiation of neoadjuvant or adjuvant therapy, e.g., hours, days, weeks, or preferably months. A typical endpoint for response assessment is the end of neoadjuvant chemotherapy or surgical removal of residual tumor cells and / or tumor bed. This is typically three months after initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR).Clinical benefit rates are measured by determining the percentage of patients in complete remission (CR), the number of patients in partial remission (PR), and the total number of patients with stable disease (SD) at least 6 months after the end of therapy. Shorthand for this formula is CBR = CR + PR + SD over 6 months. In some embodiments, the CBR of a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for assessing response to cancer therapy relate to "survival," which includes all of the following: survival until death, also known as overall survival (death can be from any cause or tumor-related), "recurrence-free survival" (the term recurrence is intended to include both local and distant recurrence), metastasis-free survival, and disease-free survival (the term disease is intended to include cancer and related diseases). The length of survival time can be calculated by referencing a defined starting point (e.g., time of diagnosis or initiation of treatment) and end point (e.g., death, recurrence, or metastasis). In addition, criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence. For example, to determine an appropriate threshold, a particular cancer treatment regimen can be administered to a subject population, and the outcome can be correlated with biomarker measurements determined before the administration of any immunomodulatory therapy. The outcome measurement can be a pathological response to a therapy administered in a neoadjuvant setting. Alternatively, for subjects after immunomodulatory therapy for which biomarker measurements are known, outcome measures such as overall survival and disease-free survival can be monitored over a period of time. In certain embodiments, the administered dose is a standard dose of a cancer therapeutic drug known in the art. The period for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.

[0110] As used herein, the term "specific binding" refers to the binding of an antibody to a predetermined antigen. Typically, an antibody binds to a specific antigen with a specific binding activity of approximately 10 as determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using human KIR3DL3 as the analyte and an antibody as the ligand. -7 Less than M, e.g., approximately 10 -8 M, 10 -9 M or 10 -10 Affinity (K D ) and binds to the predetermined antigen with an affinity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 times greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen (e.g., BSA, casein) or a closely related antigen. The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" may be used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0111] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from a condition of interest (e.g., cancer). The term "subject" is synonymous with "patient." In some embodiments, the term is intended to include a living organism in which an immune response can be elicited. Representative, non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

[0112] As used herein, the term "survival" includes all of the following: survival until death, also known as overall survival (death can be either from any cause or tumor-related), "recurrence-free survival" (the term recurrence is intended to include both local and distant recurrence), metastasis-free survival, and disease-free survival (the term disease is intended to include cancer and related diseases). The length of survival can be calculated by reference to a defined starting point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). In addition, criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence.

[0113] The terms "tolerance" or "unresponsiveness" include the refraction of cells, such as immune cells, to stimuli, e.g., stimulation by activating receptors or cytokines. Unresponsiveness can occur, for example, through exposure to immunosuppressants or high doses of antigen. Several independent methods can cause tolerance. One mechanism is called "anergy," defined as a state in which cells persist in vivo as unresponsive cells rather than differentiating into cells with effector function. Such refraction is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy in T cells is characterized by a lack of cytokine production, such as IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, reexposure of the cells to the same antigen (even when reexposure occurs in the presence of a costimulatory polypeptide) results in a failure to produce cytokines and therefore a failure to proliferate. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes, as measured by ELISA or proliferation assays using indicator cell lines. Alternatively, reporter gene constructs can be used. For example, anergic T cells are unable to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by multimers of AP1 sequences that can be found in the enhancer (Kang et al. (1992) Science 257:1134). Another mechanism is termed "exhaustion." T cell exhaustion is a state of T cell dysfunction that occurs during the development of many chronic infections and cancers. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells.

[0114] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or an analog of such RNA or cDNA) that is complementary to or homologous to all or a portion of a mature mRNA produced by transcription of a marker encompassed by this disclosure, normal post-transcriptional processing (e.g., splicing) of the RNA transcript (if present), and reverse transcription of the RNA transcript.

[0115] As used herein, the term "T cells" includes CD4+ T cells and CD8+ T cells. The term T cells also includes both T helper type 1 T cells and T helper type 2 T cells. The term "antigen-presenting cells" includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes). Conventional T cells, also known as Tconv or Teff, have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) to enhance immune responses by expressing one or more T cell receptors. Tcon or Teff is generally defined as any T cell population that is not a Treg, including, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated into, for example, Th1 or Th2 lineages. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teffs are CD4+ Teffs or CD8+ Teffs, e.g., CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17) and CD8+ cytotoxic T lymphocytes. As further described herein, cytotoxic T cells are CD8+ T lymphocytes. "Naive Tcon" refers to CD4 T cells that have differentiated in the bone marrow and successfully passed through positive and negative central selection processes in the thymus, but have not been activated by exposure to antigen. +T cells. Naive T cells are generally characterized by the surface expression of L-selectin (CD62L), the absence of activation markers such as CD25, CD44, or CD69, and the absence of memory markers such as CD45RO. Thus, naive T cells are thought to be quiescent, non-dividing, and require interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see, at least, WO2010 / 101870). The presence and activity of such cells is undesirable in relation to suppression of immune responses. Unlike Tregs, T cells are not anergic and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol. Sci. 356:625-637). In tumors, exhausted cells can exhibit characteristics of anergy.

[0116] As used herein, the term "unrearranged" or "germline configuration" with respect to a V segment refers to a configuration in which the V segment has not recombined directly adjacent to a D or J segment.

[0117] II. Monoclonal Antibodies, Immunoglobulins, and Polypeptides The present disclosure relates, in part, to isolated monoclonal antibodies or fragments thereof (such as the monoclonal and polyclonal antibodies listed herein) raised against KIR3DL3. Such molecules are characterized, in part, by their ability to recognize KIR3DL3 protein in diagnostic assays, e.g., immunohistochemistry (IHC), Western blot, intracellular flow, ELISA, etc. Such molecules are characterized, in part, by their ability to inhibit binding of KIR3DL3 to a binding partner, such as HHLA2.

[0118] The term "HHLA2," also known as human endogenous retrovirus H long terminal repeat-related protein 2, HERV-H LTR-related 2, B7y, B7H7, B7-H5, and B7-H7, refers to a member of the B7 family. HHLA2 protein has limited expression in normal human tissues but is widely expressed in human cancers. While HHLA2 protein is a membrane protein with three Ig-like domains (IgV-IgC-IgV), other members of the B7 family generally have only two Ig domains (IgV-IgC). In normal human tissues, HHLA2 protein is expressed in kidney, intestinal, gallbladder, and breast epithelium, as well as placental trophoblast cells. In the immune system, HHLA2 protein is constitutively expressed on human monocytes / macrophages. HHLA2 regulates human T cell function; for example, HHLA2 inhibits T cell proliferation and cytokine production and increases T cell proliferation and cytokine production. HHLA2 is expressed at higher levels in a wide range of human cancers, including those of colorectal, renal, lung, pancreatic, ovarian, and prostate origin. HHLA2 is also expressed in human cancers of the thyroid, melanoma, liver, bladder, colon, kidney, breast, and esophagus.

[0119] The structure and function of certain HHLA2s are well known in the art (e.g., Xiao et al. (2015) Clin. Cancer Res. 21:2201-2203, Janakiram et al. (2015) Clin. Cancer Res. 21:2359-2366, Mager et al. (1999) Genomics 21:2359-2366, Flajnik et al. (2012) Immunogenet. 64:571-590, Zhao et al. (2013) Proc. Natl. Acad. Sci. USA 110:9879-9884, and Zhu (See et al. (2013) Nat. Commun. 4:2043).

[0120] The term "HHLA2" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human HHLA2 cDNA and human HHLA2 protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI). Human HHLA2 variants include variant 1 (NM_007072.3 and NP_009003.1, representing the longest transcript and encoding the longest isoform), variant 2 (NM_001282556.1 and NP_001269485.1, representing alternative promoter usage and differing in the 5'UTR compared to variant 1), variant 3 (NM_001282557.1 and NP_001269486.1, representing alternative promoter usage and differing in the 5'UTR compared to variant 1), and variant 4 (NM_001282558.1 and NP_001269487.1, representing isoform b). Variants include variant 1 (NM_001282559.1 and NP_001269488.1), which encodes isoform c, represents the use of an alternative promoter, has multiple differences compared to variant 2, resulting in a distinct 5'UTR, initiates translation at an alternative start codon compared to variant 1, and results in a distinct N-terminus and a shorter isoform than isoform a), and variant 5 (NM_001282559.1 and NP_001269488.1), which encodes isoform c, represents the use of an alternative promoter, has multiple differences compared to variant 2, resulting in a distinct 5'UTR, initiates translation at an alternative start codon compared to variant 1, and results in a distinct N-terminus and a shorter isoform than isoform a). Nucleic acid and polypeptide sequences of HHLA2 orthologs in organisms other than humans are well known, including, for example, frog HHLA2 (NM_001128644.1 and NP_001122116.1). Representative sequences of HHLA2 orthologs are presented in Table 1 below.

[0121] Anti-HHLA2 antibodies suitable for detecting HHLA2 proteins are well known in the art, and include, for example, antibody catalog numbers ab107119 and ab214327 (abcam), antibodies PA5-24146 and PA5-6313 (ThermoFisher Scientific), antibodies MAB80841, AF8084, FAB80841R, FAB80841T, and MAB8084 (R&D systems), antibody AP52042PU-N (Origene), antibodies NBP2-49187, MAB80842, H00011148-B01P, and NBP2-32420 (Novus Biologicals), antibody GTX51981 (GeneTex), antibody HPA055478 (Atlas Antibodies), antibodies LS-C321945, LS-C308228, LS-C246742, LS-C246743, LS-C246744, LS-C236210, and LS-C249186 (LifeSpan Biosiences), and the like.Additionally, several siRNAs, shRNAs, and CRISPR constructs for reducing HHLA2 expression are available, such as shRNA product numbers TL312462, TF312462, TR312462, TG312462, and TL312462V, siRNA product number SR323358 (Origene). Technologies), SiRNA Product Numbers i009616, i009616a, i009616b, i009616c, i009616d, iV009616, iV009616a, iV009616b, iV009616c, iV009616d, iAAV00961600, iAAV00961601, iAAV00961602, iAAV00961603, iAAV00961604, iAAV00961605, iAAV00961606, iAAV00961607, iAAV009616 08, and iAAV00961609, CRISPR product numbers K0950321, K0950301, K0950302, K0950303, K0950304, K0950305, K0950306, K0950307, ​​K0950308, and K0950311 (abm), siRNA product number sc-78498, shRNA product numbers sc-78498-V and sc-78498-SH, CRISPR product numbers sc-411576, sc-411576-HDR, sc-411576-NIC, and sc-411576-NIC-2 (Santa Cruz Biotechnology), and the like, can be found in the commercially available product listings of the companies listed above. It should be noted that this term can also be used to refer to any combination of the characteristics described herein for HHLA2 molecules. For example, any combination of sequence composition, percent identity, sequence length, domain structure, functional activity, etc. can be used to describe the HHLA2 molecules encompassed by the present disclosure.

[0122] The term "HHLA2 pathway" includes HHLA2 and the interaction of HHLA2 with one or more of its natural binding partners, such as TMIGD2 and KIR3DL3.

[0123] The term "KIR3DL3 pathway" includes KIR3DL3 and the interaction of KIR3DL3 with one or more of its natural binding partners, such as HHLA2.

[0124] The term "TMIGD2" refers to the transmembrane and immunoglobulin domain containing 2, CD28H, IGPR1, and IGPR-1, a membrane protein with approximately 10% amino acid identity to CD28, CTLA-4, ICOS, and PD-1. TMIGD2 has one extracellular IgV-like domain, a transmembrane region, and a proline-rich cytoplasmic domain with two tyrosine signaling motifs. TMIGD2 protein is constitutively expressed on all naive T cells and the majority of natural killer (NK) cells, but not on T regulatory cells or B cells. TMIGD2 expression is slowly lost upon repeated stimulation of T cells. Consistent with this, TMIGD2 is expressed on only approximately half of memory T cells, and TMIGD2-negative T cells have a terminally differentiated, senescent phenotype. TMIGD2 has also been shown to be expressed in endothelial and epithelial cells, where it functions to reduce cell migration and promote capillary tube formation during angiogenesis.

[0125] The structure and function of certain TMIGD2s are well known in the art (e.g., Xiao et al. (2015) Clin. Cancer Res. 21:2201-2203, Janakiram et al. (2015) Clin. Cancer Res. 21:2359-2366, Zhu et al. (2013) Nat. Commun. 4:2043, and Rahimi (2012) Cell 23:1646-1656).

[0126] The term "TMIGD2" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human TMIGD2 cDNA and human TMIGD2 protein sequences are well known in the art and are available from National It is publicly available from the National Center for Biotechnology Information (NCBI). Human TMIGD2 isoforms include isoform 1 (NM_144615.2 and NP_653216.2), isoform 2 (NM_001169126.1 and NP_001162597.1, which use an alternative in-frame splice site within the 3' coding region compared to variant 1, resulting in a shorter isoform compared to isoform 1), and isoform 3 (NM_001308232.1 and NP_001295161.1, which lack an alternative in-frame exon within the 5' coding region compared to variant 1, resulting in a shorter isoform compared to isoform 1). The nucleic acid and polypeptide sequences of TMIGD2 orthologs in organisms other than humans are well known, including, for example, chimpanzee TMIGD2 (XM_009434393.2 and XP_009432668.2, and XM_001138228.4 and XP_001138228.3) and bovine TMIGD2 (XM_005208980.3 and XP_005209037.1, XM_005208979.3 and XP_005209036.1, and XM_002688933.5 and XP_002688979.1).Representative sequences of TMIGD2 orthologs are presented in Table 1 below.

[0127] Anti-TMIGD2 antibodies suitable for detecting TMIGD2 protein are well known in the art and include, for example, antibody catalog numbers MAB8316, MAB83162, FAB8316R, FAB83162R, FAB83162G, FAB83162N, FAB83162S, FAB83162T, FAB83162U, and FAB83162V (R&D systems), antibody TA326695 (Origene), antibodies PA5-52787, and PA5-38055 (ThermoFisher Scientific), antibodies MAB83161 and NBP1-81164 (Novus Biologicals), and the like.Additionally, multiple siRNA, shRNA, and CRISPR constructs for reducing TMIGD2 expression are available, such as shRNA product numbers TF317829, TG317829, TL317829, TR317829, and TL317829V, siRNA product number SR314913, and CRISPR product numbers KN204938, KN204938LP, KN204938RB, and KN204938BN (Origene). Technologies), siRNA product numbers i024914, i024914a, i024914b, i024914c, i024914d, iV024914, iV024914a, iV024914b, iV024914c, iV024914d, iAAV02491400, iAAV02491401, iAAV02491402, iAAV02491403, iAAV02491404, iAAV02491405, iAAV02491406, iAAV02491407, iAAV02491408, and iAAV0249 1409, as well as CRISPR product numbers K2409321, K2409301, K2409302, K2409303, K2409304, K2409305, K2409306, K2409307, K2409308, and K2409311 (Abm), siRNA product number sc-97757, shRNA product numbers sc-97757-SH and sc-97757-V, as well as CRISPR product numbers sc-414261, sc-414261-HDR, sc-414261-NIC, and sc-414261-NIC-2 (Santa Cruz Biotechnology), shRNA product numbers SH888208 and SH874720 (Vigene Biosciences), and the like, can be found in the commercially available product listings of the companies listed above.In addition, multiple CRISPR constructs for increasing TMIGD2 expression can be found in the commercial product list of the above-mentioned company, such as CRISPR product numbers K2409378, K2409377, K2409376, K2409375, K2409374, K2409373, K2409372, and K2409371 (Abm), CRISPR product numbers sc-414261-ACT, sc-414261-ACT-2, sc-414261-LAC, and sc-414261-LAC-2 (Santa Cruz Biotechnology). Note that this term can also be used to refer to any combination of the characteristics described herein for TMIGD2 molecules. For example, any combination of sequence composition, identity percentage, sequence length, domain structure, functional activity, etc. can be used to describe the TMIGD2 molecules encompassed by the present disclosure.

[0128] The above-mentioned interaction between TMIGD2 and HHLA2, as well as their functions, are well known in the art (see, for example, Xiao et al. (2015) Clin. Cancer Res. 21:2201-2203, and Janakiram et al. (2015) Clin. Cancer Res. 21:2359-2366).

[0129] The term "KIR3DL3," also known as killer cell immunoglobulin-like receptor 3DL3, CD158Z, KIR3DL7, KIR44, KIRC1, and KIR2DS2, killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 3, refers to a member of a family of transmembrane glycoproteins expressed by natural killer cells and a subset of T cells. Killer cell immunoglobulin-like receptor (KIR) genes are polymorphic and highly homologous; they are found in a cluster on chromosome 19q13.4 within the 1-Mb leukocyte receptor complex (LRC). The gene content of the KIR gene cluster varies between haplotypes, but several "framework" genes are found in all haplotypes (KIR3DL3, KIR3DP1, KIR3DL4, and KIR3DL2). KIR proteins are classified by the number of extracellular immunoglobulin domains (2D or 3D) and whether they have a long (L) or short (S) cytoplasmic domain. While KIR proteins with long cytoplasmic domains transmit inhibitory signals upon ligand binding via immunotyrosine-based inhibitory motifs (ITIMs), KIR proteins with short cytoplasmic domains lack ITIM motifs and instead bind to TYRO protein tyrosine kinase-binding proteins to transmit activating signals. The ligands for some KIR proteins are a subset of HLA class I molecules, and therefore, KIR proteins are thought to play an important role in regulating immune responses. This gene is one of the "framework" loci present in all haplotypes. The KIR3DL3 protein has an N-terminal signal sequence, three Ig domains, a transmembrane region lacking positively charged residues, and a long cytoplasmic tail containing an immunoreceptor tyrosine-based inhibitory motif (ITIM). KIR3DL3 lacks the stalk region found in other KIRs.

[0130] The structure and function of certain KIR3DL3s are well known in the art (see, e.g., Hsu et al. (2002) Immunol Rev. 190:40-52, Trompeter et al. (2005) J. Immunol. 174:4135-4143, Trundley et al. (2006) Immunogenet. 57:904-916, and Jones et al. (2006) Immunogenet. 58:614-627).

[0131] The term "KIR3DL3" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human KIR3DL3 cDNA and human KIR3DL3 protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI). For example, at least one human KIR3DL3 isoform is known, and human KIR3DL3 (NM_153443.4) can be encoded by the transcript (NP_703144.3). The nucleic acid and polypeptide sequences of KIR3DL3 orthologues in organisms other than humans are well known, including, for example, chimpanzee KIR3DL3 (XM_003316679.3 and XP_003316727.3), rhesus macaque KIR3DL3 (NM_001104552.2 and NP_001098022.1), mouse KIR3DL3 (NM_001310690.1 and NP_001297619.1, NM_177749.4 and NP_808417.2, NM_177748.2 and NP_808416.1) and rat KIR3DL3 (NM_181479.2 and NP_852144.1).Representative sequences of KIR3DL3 orthologues are presented in Table 1 below.

[0132] Anti-KIR3DL3 antibodies suitable for detecting KIR3DL3 protein are well known in the art and include, for example, antibody catalog numbers FAB8919R, MAB8919, FAB8919G, FAB8919N, FAB8919S, FAB8919T, FAB8919U, and FAB8919V (R&D systems), antibody AP52374PU-N (Origene), antibody PA5-26178 (ThermoFisher Scientific), antibodies OAAB05761, OAAF08125, OAAN04122, OACA09134, OACA09135, OACD04988, and OASG01190 (Aviva Systems Biology), etc.Additionally, multiple siRNA, shRNA, and CRISPR constructs for reducing KIR3DL3 expression are available, such as shRNA product numbers TF303684, TR303684, TG303684, TL303684, and TL303684V, siRNA product number SR314516, and CRISPR product numbers KN224383, KN224383BN, KN224383RB, and KN224383LP (Origene). Technologies), siRNA product numbers i011627, i011627a, i011627b, i011627c, i011627d, iV011627, iV011627a, iV011627b, iV011627c, iV011627d, iAAV01162700, iAAV01162701, iAAV01162702, iAAV01162703, iAAV01162704, iAAV01162705, iAAV01162706, iAAV01162707, iAAV01162708, and iAAV01162709, and CRISPR product numbers K1151421, K1151401, K1151402, K1151403, K1151404, K1151405, K1151406, K1151407, K1151408, and K1151411 (Abm), siRNA product number sc-60892, shRNA product numbers sc-60892-SH and sc-60892-V, and CRISPR product numbers sc-406227, sc-406227-KO-2, sc-406227-HDR-2, sc-406227-NIC, and sc-406227-NIC-2 (Santa Cruz Biotechnology), etc., can be found in the commercially available product listings of the companies listed above. It should be noted that this term may further be used to refer to any combination of the characteristics described herein for a KIR3DL3 molecule. For example, any combination of sequence composition, percent identity, sequence length, domain structure, functional activity, etc. may be used to describe a KIR3DL3 molecule encompassed by the present disclosure.

[0133] The term "peripheral blood cell subtype" refers to cell types normally found in peripheral blood, including, but not limited to, eosinophils, neutrophils, T cells, monocytes, NK cells, granulocytes, and B cells.

[0134] The term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, e.g., (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells that have been transformed to express the antibody, e.g., from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thus allowing the V and V regions of the recombinant antibody to be further refined. H Area and V L The amino acid sequence of the region is human germline V H Array and V L These are sequences that, while derived from and related to a sequence, may not naturally occur in the human antibody germline repertoire in vivo.

[0135] The term "sample" as used to detect or determine the presence or level of at least one biomarker typically refers to whole blood, plasma, serum, saliva, urine, feces (e.g., feces), tears, and any other bodily fluid (e.g., those described under the definition of "bodily fluid" above), or a tissue sample (e.g., biopsy), such as a small intestine, colon sample, or surgically resected tissue. In certain examples, methods encompassed by the present disclosure further include obtaining a sample from an individual prior to detecting or determining the presence or level of at least one marker in the sample.

[0136] As used herein, an "RNA interfering agent" is defined as any agent that interferes with or inhibits the expression of a target biomarker gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules or fragments thereof comprising RNA molecules homologous to the target biomarker gene encompassed by the present disclosure, short interfering RNAs (siRNAs), and small molecules that interfere with or inhibit the expression of a target biomarker nucleic acid by RNA interference (RNAi).

[0137] "RNA interference (RNAi)" is an evolutionarily conserved process in which the expression or introduction of RNA with a sequence identical or closely similar to that of a target biomarker nucleic acid results in sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from the targeted gene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting the expression of the target biomarker nucleic acid. In one embodiment, the RNA is double-stranded RNA (dsRNA). This process has been described in plants, vertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes the processive cleavage of long dsRNA into double-stranded fragments called siRNAs. The siRNAs are incorporated into a protein complex that recognizes and cleaves the target mRNA. RNAi can also be initiated by the introduction of nucleic acid molecules, such as synthetic siRNA, shRNA, or other RNA interference agents, to inhibit or silence the expression of target biomarker nucleic acids. As used herein, "inhibition of target biomarker nucleic acid expression" or "inhibition of marker gene expression" includes any reduction in the expression or protein activity or level of a target biomarker nucleic acid or a protein encoded by the target biomarker nucleic acid. This reduction is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to the expression of a target biomarker nucleic acid that is not targeted by an RNA interference agent, or the activity or level of a protein encoded by the target biomarker nucleic acid.

[0138] In addition to RNAi, genome editing can be used to regulate the copy number or gene sequence of a biomarker of interest, for example, constitutive or inducible knockout or mutation of a biomarker of interest, such as HHLA2, TMIGD2, and / or KIR3DL3 pathway components such as KIR3DL3. For example, CRISPR-Cas systems can be used to precisely edit genomic nucleic acids (e.g., to create non-functional or null mutations). In such embodiments, CRISPR guide RNA and / or Cas enzyme can be expressed. For example, a vector containing only guide RNA can be administered to animals or cells transgenic for the Cas9 enzyme. Similar strategies can be used (e.g., designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases). Such systems are well known in the art (e.g., U.S. Pat. No. 8,697,359; Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Patent Publication Nos. 2014 / 0087426 and 2012 / 0178169; Boch et al. (2011) Nat. Biotech. 29:135-136; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al. (2011) PLoS One 6:e19722, Li et al. (2011) Nucl. Acids Res. 39:6315-6325, Zhang et al. (2011) Nat. Biotech. 29:149-153, Miller et al. (2011) Nat. Biotech. 29:143-148, Lin et al. (2014) Nucl. Acids Res. 42:e47. Such genetic strategies can use constitutive or inducible expression systems according to methods well known in the art.

[0139] Piwi-interacting RNAs (piRNAs) are the largest class of small non-coding RNAs. piRNAs form RNA-protein complexes through interactions with Piwi proteins. These piRNA complexes are associated with both epigenetic and posttranscriptional gene silencing of retrotransposons and other genetic elements in germline cells, particularly during spermatogenesis. They differ from microRNAs (miRNAs) in their size (26–31 nucleotides instead of 21–24 nucleotides), lack of sequence conservation, and increased complexity. However, like other small RNAs, piRNAs are thought to be involved in gene silencing, particularly transposon silencing. The majority of piRNAs are antisense to transposon sequences, suggesting that transposons are piRNA targets. In mammals, piRNA activity during transposon silencing appears to be most important during embryonic development, and piRNAs are required for spermatogenesis in both C. elegans and humans. piRNAs play a role in RNA silencing through the formation of RNA-induced silencing complexes (RISCs).

[0140] An "aptamer" is an oligonucleotide or peptide molecule that binds to a specific target molecule. A "nucleic acid aptamer" is a nucleic acid species engineered through repeated rounds of in vitro selection or, equivalently, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. A "peptide aptamer" is an artificial protein selected or engineered to bind to a specific target molecule. These proteins consist of one or more peptide loops of variable sequence displayed by a protein scaffold. They are typically isolated from combinatorial libraries and then often improved through directed mutation or variable region mutagenesis and selection rounds. An "affimer protein," an evolutionary evolution of peptide aptamers, is a highly stable, small protein engineered to display peptide loops that provide a high-affinity binding surface for a specific target protein. It is a low molecular weight (12-14 kDa) protein derived from the cystatin family of cysteine ​​protease inhibitors. Aptamers are useful in biotechnological and therapeutic applications because they offer molecular recognition properties that compete with those of antibodies, a commonly used biomolecule. In addition to their differential recognition, aptamers offer advantages over antibodies because they can be fully engineered in vitro, are easily produced by chemical synthesis, have desirable storage properties, and induce little or no immunogenicity in therapeutic applications.

[0141] "Short interfering RNA" (siRNA), also referred to herein as "small interfering RNA," is defined as an agent that functions to inhibit expression of a target biomarker nucleic acid, for example, by RNAi. siRNA can be chemically synthesized, produced by in vitro transcription, or produced in a host cell. In one embodiment, siRNA is a double-stranded RNA (dsRNA) molecule about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides in length, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, or 22 nucleotides in length, and may have 3' and / or 5' overhangs on each strand having lengths of about 0, 1, 2, 3, 4, or 5 nucleotides. The lengths of the overhangs are independent between the two strands; i.e., the length of the overhang on one strand is independent of the length of the overhang on the second strand. Preferably, siRNA can promote RNA interference by degradation of target messenger RNA (mRNA) or specific post-transcriptional gene silencing (PTGS).

[0142] In another embodiment, the siRNA is a small hairpin (also called stem-loop) RNA (shRNA). In one embodiment, these shRNAs consist of a short (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop structure, followed by the antisense strand. These shRNAs can be contained in plasmids, retroviruses, and lentiviruses and expressed, for example, from the polymerase III U6 promoter or another promoter (see, e.g., Stewart, et al. (2003) RNA Apr;9(4):493-501, incorporated herein by reference).

[0143] RNA interfering agents, e.g., siRNA molecules, can be administered to patients with or at risk of having cancer to inhibit the expression of biomarker genes that are overexpressed in cancer, thereby treating, preventing, or suppressing cancer in the subject.

[0144] The term "small molecule" is a term of the art and includes molecules less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not contain exclusively peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. 1998. Science 282:63), and natural product extract libraries. In another embodiment, these compounds are non-peptidic small organic compounds. In a further embodiment, the small molecules are not biosynthetic.

[0145] The terms "selective modulator" or "selectively modulates," as applied to a biologically active agent, refer to the ability of the agent to modulate a target, such as a cell population, signaling activity, etc., relative to an off-target cell population, signaling activity, etc., either directly or through a mutual interaction with the target. For example, an agent that selectively inhibits the interaction between KIR3DL3 and one or more natural binding partners, such as HHLA2, over another interaction between KIR3DL3 and another binding partner, and / or such interaction in a cell population of interest, is referred to as a KIR3DL3 pathway modulator therapy (e.g., a modulator of the interaction between KIR3DL3 and one or more natural binding partners, such as HHLA2, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 2-fold, or more (e.g., at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 105x, 110x, 1 20x, 125x, 150x, 200x, 250x, 300x, 350x, 400x, 450x, 500x, 600x, 700x, 800x, 900x, 1000x, 1500x, 2000x, 2500x, 30 The activity of a therapeutic agent against a target gene (interaction) may be 500-fold, 3500-fold, 4000-fold, 4500-fold, 5000-fold, 5500-fold, 6000-fold, 6500-fold, 7000-fold, 7500-fold, 8000-fold, 8500-fold, 9000-fold, 9500-fold, 10000-fold, or more, or any range therebetween (including boundaries). Such metrics are typically expressed in terms of the relative amount of agent required to reduce the interaction / activity by half.

[0146] More generally, the term "selective" refers to a preferential action or function. The term "selective" can be quantified in terms of a preferential effect on a particular target of interest compared to other targets. For example, a measured variable (e.g., modulation of Tregs / Bregs relative to other cells (e.g., other immune cells such as Tcon)) can be increased or decreased by 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, The fold increase can be 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, or any range therebetween (including boundaries) (e.g., 50% to 16-fold), varying for intended versus unintended or undesired targets. The same fold increase analysis can be used to ascertain the magnitude of the effect in a given tissue, cell population, measured variable, measured effect, etc., e.g., Treg:Tcon ratio, Breg:Tcon ratio, hyperproliferative cell growth rate or volume, Treg / Breg expansion rate or number, etc.

[0147] In contrast, the term "specific" refers to an exclusive action or function. For example, specific modulation of HHLA2-KIR3DL3 interaction refers to exclusive modulation of HHLA2-KIR3DL3 interaction, and not modulation of the interaction between KIR3DL3 and another ligand. In another example, specific binding of an antibody to a predetermined antigen refers to the antibody's ability to bind to the target antigen without binding to other antigens. Typically, an antibody has a specific binding activity of approximately 1 x 10 as determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using the target antigen as the analyte and the antibody as the ligand. -7 Less than M, e.g., approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 Affinity (KD ) and binds to the predetermined antigen with an affinity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 times greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen (e.g., BSA, casein) or a closely related antigen. In addition, D is K A The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" may be used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0148] The term "sensitize" means modifying a cell, such as a cancer cell or tumor cell, in a way that allows for more effective treatment with a therapy (e.g., KIR3DL3 pathway modulator therapy (e.g., a modulator of the interaction between KIR3DL3 and one or more natural binding partners, such as HHLA2), either alone or in combination with an immunotherapy, such as immune checkpoint inhibition therapy). In some embodiments, normal cells are not affected by the therapy (e.g., KIR3DL3 pathway modulator therapy (e.g., a modulator of the interaction between KIR3DL3 and one or more natural binding partners, such as HHLA2), either alone or in combination with an immunotherapy, such as immune checkpoint inhibition therapy) to an extent that would unduly damage normal cells. Increased or decreased sensitivity to therapeutic treatment can be determined by cell proliferation assays (Tanigawa N, Kern DH, Kikasa Y, Morton DL, Cancer Res 1982;42:2159-2164), cell death assays (Weisenthal LM, Shoemaker RH, Marsden JA, Dill PL, Baker JA, Moran EM, Cancer Res 1984;94:161-173, Weisenthal LM, Lippman ME, Cancer Treat Rep Sensitivity or resistance is measured according to methods known in the art for the specific treatments and methods described herein below, including, but not limited to, those described in: Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, PA: Harwood Academic Publishers, 1993:415-432; Weisenthal LM, Contrib Gynecol Obstet 1994;19:82-90). Sensitivity or resistance can also be measured in animals by measuring tumor size reduction over a period of time, e.g., 6 months in humans and 4-6 weeks in mice. A composition or method sensitizes a response to a therapeutic treatment if the increase in therapeutic sensitivity or decrease in resistance is 5% or more, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or more, compared to the therapeutic sensitivity or resistance in the absence of such composition or method. Determining sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of one of ordinary skill in the art. It should be understood that any of the methods described herein for enhancing the effectiveness of immunomodulation can be equally applied to methods of sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to therapy.

[0149] The term "synergistic effect" refers to the fact that the combined effect of two or more therapeutic agents, such as two or more KIR3DL3 pathway modulators, a KIR3DL3 pathway modulator and an immunotherapy, a KIR3DL3 pathway modulator either alone or in combination with an immunotherapy such as an immune checkpoint inhibitor therapy, can be greater than the sum of the individual effects of the anti-cancer agents alone.

[0150] The term "survival" includes all of the following: survival until death, also known as overall survival (death can be from any cause or tumor-related), "recurrence-free survival" (the term recurrence is intended to include both local and distant recurrence), metastasis-free survival, and disease-free survival (the term disease is intended to include cancer and related diseases). The length of survival can be calculated by reference to a defined starting point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). In addition, criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence.

[0151] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or the enhancement of desirable physical or mental development and conditions, in animals or humans.

[0152] As used herein, the terms "therapeutically effective amount" and "effective amount" refer to an amount of a compound, material, or composition comprising a compound encompassed by the present disclosure effective to produce some desired therapeutic effect on at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. Toxicity and therapeutic efficacy of a subject compound can be determined, for example, by the LD 50 and ED 50 The LD can be determined by standard pharmaceutical procedures in cell culture or experimental animals to determine the therapeutic index. Compositions that exhibit a high therapeutic index are preferred. In some embodiments, the LD 50 The lethal dose (ED) can be measured and can be reduced, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, for a drug compared to administration of no drug.50 The IC (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more increase for the drug compared to administration without the drug. Similarly, the IC 50 The concentration (i.e., the concentration that achieves half-maximal cytotoxicity or cytostatic effect on cancer cells) can be measured and can be increased by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more for the agent compared to administration of the agent alone. In some embodiments, cancer cell growth in the assay can be inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. Cancer cell death may be promoted by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, a reduction in cancer cell number and / or solid malignancy by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% may be achieved.

[0153] The term "substantially free of chemical precursors or other chemicals" includes preparations of antibodies, polypeptides, peptides, or fusion proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of antibodies, polypeptides, peptides, or fusion proteins having less than about 30% (by dry weight) chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals, more preferably less than about 20% chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals, even more preferably less than about 10% chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals, and most preferably less than about 5% chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals.

[0154] A "transcribed polynucleotide" or "nucleotide transcript" refers to a polynucleotide (e.g., mRNA, hnRNA, cDNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA) that is complementary to or homologous to all or a portion of a mature mRNA produced by transcription of a marker encompassed by the present disclosure and normal post-transcriptional processing (e.g., splicing) of the RNA transcript, if present, and reverse transcription of the RNA transcript. * , anti-miRNA, or miRNA binding site, or variants thereof, or analogs of such RNA or cDNA).

[0155] The term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0156] There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequence that can encode that protein as defined by the genetic code (shown below).Similarly, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid as defined by the genetic code. [Table 1A]

[0157] An important and well-known feature of the genetic code is its redundancy, whereby more than one coding nucleotide triplet can be used for most of the amino acids used to make proteins (as exemplified above). Thus, several different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than others). Furthermore, methylated variants of purines or pyrimidines can occasionally be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0158] In view of the foregoing, the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) can be used to obtain a polypeptide amino acid sequence using the genetic code to translate DNA or RNA into an amino acid sequence. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence capable of encoding the polypeptide can be deduced from the genetic code (whose redundancy results in multiple nucleic acid sequences for any given amino acid sequence). Thus, any description and / or disclosure herein of a nucleotide sequence encoding a polypeptide should be considered to also include a description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, any description and / or disclosure herein of a polypeptide amino acid sequence should be considered to also include a description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0159] Finally, nucleic acid and amino acid sequence information for nucleic acid and polypeptide molecules useful in the present disclosure is well known in the art and readily available in publicly available databases, such as the National Center for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences from publicly available sequence databases are provided in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0160] The term "KIR3DL3 activity" includes the ability of a KIR3DL3 polypeptide to regulate inhibitory signals in activated immune cells, for example, by ligating natural HHLA2 ligands on cancer cells. Regulation of inhibitory signals in immune cells results in regulation of immune cell proliferation and / or cytokine secretion by immune cells. Thus, the term "KIR3DL3 activity" includes the ability of a KIR3DL3 polypeptide to bind to its natural ligand, regulate immune cell inhibitory signals, and regulate an immune response.

[0161] In some embodiments, the condition, such as cancer, is responsive to KIR3DL3 blockade alone. In other embodiments, the condition, such as cancer, is responsive to KIR3DL3 blockade alone, but is significantly or synergistically more responsive when treated with KIR3DL3 blockade in combination with at least one other therapy. Many conditions that are responsive to KIR3DL3 blockade, alone or in combination, include, but are not limited to, melanoma (e.g., advanced or metastatic melanoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), breast cancer (e.g., HER-2 negative breast cancer, estrogen receptor positive / HER-2 negative breast cancer, and triple negative breast cancer), pancreatic cancer (e.g., pancreatic adenocarcinoma), and Hodgkin's lymphoma, as well as bladder cancer, gastric cancer, head and neck cancer, renal cancer, prostate cancer, gynecological cancer, colorectal cancer, ovarian cancer, adenocarcinoma, adenocarcinoma, chronic myeloid leukemia (CML), and hematological cancers.

[0162] Preferred B7 polypeptides can provide costimulatory or inhibitory signals to immune cells, thereby promoting or inhibiting immune cell responses. For example, B7 family members that bind to costimulatory receptors increase T cell activation and proliferation, while B7 family members that bind to inhibitory receptors decrease costimulation. Furthermore, the same B7 family member can increase or decrease T cell costimulation. For example, when bound to a costimulatory receptor, HHLA2 can induce costimulation of immune cells, and when bound to an inhibitory receptor, HHLA2 can inhibit immune cells. When bound to an inhibitory receptor, HHLA2 can transmit inhibitory signals to immune cells. Preferred B7 family members include HHLA2, B7-1, B7-2, B7h, PD-L1, or PD-L2, and soluble fragments or derivatives thereof. In one embodiment, B7 family members that bind to one or more receptors on immune cells, e.g., TMIGD2, KIR3DL3, CTLA4, CD28, ICOS, PD-1, and / or other receptors, have the ability to deliver inhibitory or costimulatory signals to immune cells, preferably T cells, depending on the receptor.

[0163] Modulation of costimulatory signals leads to modulation of the effector functions of immune cells. Thus, the term "KIR3DL3 activity" includes the ability of a KIR3DL3 ligand polypeptide to bind to its natural receptor (e.g., HHLA2), to modulate costimulatory or inhibitory signals in immune cells, and to modulate immune responses.

[0164] The KIR3DL3 pathway is a negative regulator of immune function, such that immune function can be regulated by modulating the interaction between KIR3DL3 and one or more natural binding partners, such as HHLA2. Thus, agents encompassed by the present disclosure described herein that modulate the interaction between KIR3DL3 and one or more natural binding partners, whether directly or indirectly, can upregulate or downregulate the immune system, thereby upregulating or downregulating the immune response. Agents that modulate such interactions can do so either directly or indirectly.

[0165] Exemplary agents for upregulating immune responses include antibodies against HHLA2 or KIR3DL3 that block the interaction between HHLA2 and KIR3DL3, inactive forms of HHLA2 or KIR3DL3 (e.g., dominant negative polypeptides), small molecules or peptides that block the interaction between HHLA2 and KIR3DL3, fusion proteins (e.g., the extracellular portion of HHLA2 or KIR3DL3 fused to the Fc portion of an antibody or immunoglobulin) that bind to HHLA2 or KIR3DL3, respectively, and inhibit the interaction between HHLA2 and KIR3DL3, nucleic acid molecules and / or genetic modifications that block HHLA2 and / or KIR3DL3 transcription or translation, inactive forms of natural HHLA2 ligands, and soluble forms of natural KIR3DL3 ligands.

[0166] In other exemplary embodiments, agents that promote binding of HHLA2 polypeptides to one or more natural binding partners, such as KIR3DL3 polypeptides, promote inhibitory signals to immune cells. Agents that modulate such interactions can do so either directly or indirectly. Thus, in one embodiment, agents that directly enhance the interaction between HHLA2 and KIR3DL3 (HHLA2 agonists and / or KIR3DL3 agonists) can promote inhibitory signaling and downregulate immune responses. Alternatively, agents that block the binding of KIR3DL3 to other targets increase the effective concentration of KIR3DL3 available for binding to HHLA2. Exemplary agents for downregulating immune responses include antibodies against HHLA2 or KIR3DL3 that activate or promote the interaction between HHLA2 and KIR3DL3, small molecules or peptides that activate or promote the interaction between HHLA2 and KIR3DL3, and blocking antibodies that bind to natural binding partners of HHLA2 and KIR3DL3 other than HHLA2 and KIR3DL3, respectively.

[0167] Additional agents useful in the methods encompassed by the present disclosure include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands that can either bind to and / or activate or inhibit protein biomarkers or fragments thereof encompassed by the present disclosure, including the biomarkers listed in Table 1; RNA interference, antisense, nucleic acid aptamers, and the like that can downregulate the expression and / or activity of biomarkers or fragments thereof encompassed by the present disclosure, including the biomarkers listed in Table 1.

[0168] An isolated monoclonal antibody or fragment thereof produced against KIR3DL3 is provided. In some embodiments, the mAb produced by the hybridoma has been deposited with the American Type Culture Collection (ATCC) under the terms of the Budapest Treaty at ______ under deposit number ______.

[0169] Because it is well known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in determining the binding specificity / affinity of an antibody to an antigen, recombinant monoclonal antibodies encompassed by the present disclosure prepared as described above preferably comprise heavy and light chain CDR3s of variable regions encompassed by the present disclosure (e.g., comprising a sequence in Table 2 or a portion thereof). The present antibody may further comprise a CDR2 of a variable region encompassed by the present disclosure (e.g., comprising a sequence in Table 2 or a portion thereof). The present antibody may further comprise a CDR1 of a variable region encompassed by the present disclosure (e.g., comprising a sequence in Table 2 or a portion thereof). In other embodiments, the present antibody may comprise any combination of these CDRs.

[0170] The CDR1, 2, and / or 3 regions of the engineered antibodies described above may comprise the exact amino acid sequences as those of the variable regions encompassed by the present disclosure (e.g., including the sequences in Table 2 or portions thereof). However, one of skill in the art will understand that some deviation from the exact CDR sequences may be possible while still retaining the ability of the antibody to effectively bind to KIR3DL3 (e.g., conservative sequence modifications). Thus, in another embodiment, the engineered antibody may consist of one or more CDRs that are, for example, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to one or more CDRs encompassed by the present disclosure (e.g., including the sequences in Table 2 or portions thereof).

[0171] Structural features of known non-human or human antibodies (e.g., murine or non-rodent anti-human KIR3DL3 antibodies) can be used to generate structurally related human anti-human KIR3DL3 antibodies that retain at least one functional property of the antibodies encompassed by the present disclosure, such as binding of KIR3DL3. Another functional property includes inhibiting binding of the original known non-human or human antibody in a competitive ELISA assay.

[0172] In some embodiments, a monoclonal antibody capable of binding to human KIR3DL3 is provided, comprising a heavy chain whose variable domain comprises at least one CDR having a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the heavy chain variable domain CDRs presented in Table 2.

[0173] Also provided are monoclonal antibodies capable of binding to human KIR3DL3, comprising a light chain whose variable domain comprises at least one CDR having a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to a set of light chain variable domain CDRs presented in Table 2.

[0174] Also provided is a monoclonal antibody capable of binding to human KIR3DL3, comprising a heavy chain whose variable domain comprises at least one CDR having a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to a heavy chain variable domain CDR set forth in Table 2, and a light chain whose variable domain comprises at least one CDR having a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to a light chain variable domain CDR set forth in Table 2.

[0175] Those skilled in the art will note that such percentages of homology are equivalent to, and are achieved by, introducing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions within a given CDR.

[0176] Monoclonal antibodies encompassed by the present disclosure may comprise a heavy chain whose variable domain comprises at least one CDR having a sequence selected from the group consisting of the heavy chain variable domain CDRs presented in Table 2, and a light chain whose variable domain comprises at least one CDR having a sequence selected from the group consisting of the light chain variable domain CDRs presented in Table 2.

[0177] Such monoclonal antibodies may comprise a light chain whose variable domain comprises at least one CDR having a sequence selected from the group consisting of CDR-L1, CDR-L2, and CDR-L3 described herein, and / or a heavy chain whose variable domain comprises at least one CDR having a sequence selected from the group consisting of CDR-H1, CDR-H2, and CDR-H3 described herein. In some embodiments, monoclonal antibodies capable of binding to human KIR3DL3 comprise or consist of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 described herein.

[0178] The heavy chain variable domains of the monoclonal antibodies encompassed by this disclosure may comprise or consist of the vH amino acid sequences set forth in Table 2, and / or the light chain variable domains of the monoclonal antibodies encompassed by this disclosure may comprise or consist of the vL amino acid sequences set forth in Table 2.

[0179] Monoclonal antibodies encompassed by the present disclosure can be produced and modified by any technique known in the art. For example, such monoclonal antibodies can be murine or non-rodent antibodies, such as those available from hybridomas deposited with the ATCC at ______. Similarly, such monoclonal antibodies can be chimeric antibodies, preferably chimeric mouse / human antibodies. In some embodiments, the monoclonal antibodies are humanized antibodies, such that the variable domains comprise human acceptor framework regions, and optionally human constant domains (if present), and non-human donor CDRs, such as murine or non-rodent CDRs, as defined above.

[0180] The present disclosure further provides fragments of such monoclonal antibodies, including, but not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies, as well as multispecific antibodies formed from the antibody fragments. For example, several immune inhibitory molecules, such as HHLA2, PD-L2, PD-L1, CTLA-4, and KIR3DL3, can be detected in a bispecific or multispecific manner to efficiently characterize the expression of such molecules.

[0181] Other fragments of the monoclonal antibodies encompassed by the present disclosure are also contemplated. For example, individual immunoglobulin heavy and / or light chains are provided, the variable domains of which comprise at least one CDR as presented in Table 2. In one embodiment, the immunoglobulin heavy chain comprises at least one CDR having a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to a set of heavy or light chain variable domain CDRs presented in Table 2. In another embodiment, the immunoglobulin light chain comprises at least one CDR having a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to a set of light or heavy chain variable domain CDRs described herein (e.g., presented in Table 2).

[0182] In some embodiments, the immunoglobulin heavy and / or light chains comprise variable domains that include at least one of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3 described herein. Such immunoglobulin heavy chains may comprise or consist of at least one of CDR-H1, CDR-H2, and CDR-H3. Such immunoglobulin light chains may comprise or consist of at least one of CDR-L1, CDR-L2, and CDR-L3.

[0183] In other embodiments, the immunoglobulin heavy and / or light chains according to the present disclosure comprise or consist of the vH or vL variable domain sequences, respectively, provided in Table 2.

[0184] The present disclosure further provides polypeptides having a sequence selected from the group consisting of a vH variable domain, a vL variable domain, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences described herein.

[0185] The antibodies, immunoglobulins, and polypeptides encompassed by this disclosure may be used in isolated (e.g., purified) form or may be contained within a vector, such as a membrane or lipid vesicle (e.g., liposome). [Table 2-1] 1C7 variable heavy (vH) and variable light (vL) chain DNA and amino acid sequences * [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] 2A3 variable heavy (vH) and variable light (vL) chain DNA and amino acid sequences * [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] 8F7 variable heavy (vH) and variable light (vL) chain DNA and amino acid sequences * [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] 1G7 variable heavy (vH) and variable light (vL) chain DNA and amino acid sequences * [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] 2D8 variable heavy (vH) and variable light (vL) chain DNA and amino acid sequences* [Table 2-18] [Table 2-19] [Table 2-20] [Table 2-21] 2F11 variable heavy (vH) and variable light (vL) chain DNA and amino acid sequences * [Table 2-22] [Table 2-23] [Table 2-24] [Table 2-25] 2H1 variable heavy (vH) and variable light (vL) chain DNA and amino acid sequences * [Table 2-26] [Table 2-27] [Table 2-28] [Table 2-29] 1D12 variable heavy (vH) and variable light (vL) chain DNA and amino acid sequences * [Table 2-30] [Table 2-31] [Table 2-32] [Table 2-33] 8C2 variable heavy (vH) and variable light (vL) chain DNA and amino acid sequences * [Table 2-34] [Table 2-35] [Table 2-36] [Table 2-37] * CDR definition and protein sequence numbering according to Kabat. * Included in Table 2 are RNA nucleic acid molecules (e.g., those in which thymine is replaced with uridine), nucleic acid molecules encoding orthologs of the encoded proteins, as well as DNA, cDNA, or RNA nucleic acid sequences that include a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity over its entire length to a nucleic acid sequence of any SEQ ID NO: listed in Table 2, or a portion thereof. Such nucleic acid molecules can have the function of the full-length nucleic acid as further described herein.

[0186] III. Nucleic Acids, Vectors, and Recombinant Host Cells Further aspects encompassed by the present disclosure relate to nucleic acid sequences encoding the monoclonal antibodies and fragments thereof, immunoglobulins, and polypeptides encompassed by the present disclosure.

[0187] Typically, the nucleic acid is a DNA or RNA molecule that may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0188] The vector may contain control elements, such as a promoter, enhancer, terminator, etc., for causing or directing expression of the polypeptide upon administration to a subject. Examples of promoters and enhancers that can be used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), and the immunoglobulin heavy chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983).

[0189] Any expression vector for animal cells can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), and pSG1 beta d2-4- (Miyaji H et al. 1990). Other representative examples of plasmids include replicative plasmids containing an origin of replication or integrative plasmids, such as pUC, pcDNA, and pBR. Representative examples of viral vectors include adenoviral vectors, retroviral vectors, herpesvirus vectors, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as transfection of packaging cells or transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and WO94 / 19478.

[0190] Further aspects encompassed by the present disclosure relate to cells transfected, infected, or transformed with nucleic acids and / or vectors according to the present disclosure. The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene or DNA or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme, encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0191] Nucleic acids encompassed by the present disclosure can be used to produce recombinant polypeptides encompassed by the present disclosure in a suitable expression system. The term "expression system" refers to, for example, a host cell and a compatible vector under conditions suitable for the expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

[0192] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces, or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.). Examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") (Urlaub G et al; 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"). YB2 / 0 cells are preferred because the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in YB2 / 0 cells.

[0193] The present disclosure also relates to methods for producing recombinant host cells that express an antibody or polypeptide encompassed by the present disclosure in accordance with the present disclosure, comprising the steps of (i) introducing the above-described recombinant nucleic acid or vector into a competent host cell in vitro or ex vivo, (ii) culturing the resulting recombinant host cell in vitro or ex vivo, and (iii) optionally selecting cells that express and / or secrete the antibody or polypeptide. Such recombinant host cells can be used to produce the antibodies and polypeptides described herein.

[0194] In another aspect, the present disclosure provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides encompassed by the present disclosure can be used to identify, isolate, or amplify partial-length or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated from a human or mammalian nucleic acid library or otherwise complementary to cDNAs derived from a human or mammalian nucleic acid library. Preferably, the cDNA library contains at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low or moderate stringency hybridization conditions are typically, but not exclusively, used for sequences with reduced sequence identity compared to the complementary sequence. Moderate and high stringency conditions can optionally be used for sequences with higher identity. Low stringency conditions allow selective hybridization of sequences with about 70% sequence identity and can be used to identify orthologous or paralogous sequences. Optionally, the polynucleotides of the present invention encode at least a portion of an antibody encoded by a polynucleotide described herein. The polynucleotides of the present invention include nucleic acid sequences that can be used for selective hybridization to polynucleotides encoding antibodies encompassed by the present disclosure. See, for example, Ausubel (supra), Colligan (supra), each of which is incorporated herein by reference in its entirety.

[0195] IV. Methods of Producing Antibodies Antibodies and fragments thereof, immunoglobulins, and polypeptides encompassed by the present disclosure may be produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, either alone or in combination.

[0196] Knowing the amino acid sequence of the desired sequence, one of skill in the art can readily produce the antibody or polypeptide using standard techniques for producing polypeptides. For example, they can be synthesized using the well-known solid-phase method, preferably using a commercially available peptide synthesizer (e.g., from Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions. Alternatively, antibodies and other polypeptides encompassed by the present disclosure can be synthesized by recombinant DNA techniques well known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of a DNA sequence encoding the desired (poly)peptide into an expression vector and introduction of such a vector into a suitable eukaryotic or prokaryotic host that expresses the desired polypeptide, and the desired polypeptide can then be isolated using well-known techniques.

[0197] Specifically, the present disclosure further relates to a method of producing an antibody or polypeptide encompassed by the present disclosure, comprising the steps of: (i) culturing a transformed host cell according to the present disclosure under conditions suitable to allow expression of the antibody or polypeptide; and (ii) recovering the expressed antibody or polypeptide.

[0198] Antibodies and other polypeptides encompassed by the present disclosure are suitably separated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety.

[0199] Chimeric antibodies (e.g., mouse-human chimeras or non-rodent-human chimeras) encompassed by the present disclosure can be produced by obtaining nucleic acid sequences encoding the VL and VH domains as described above, inserting them into an expression vector for animal cells carrying genes encoding human antibody CH and CL, thereby constructing a human chimeric antibody expression vector, and expressing the coding sequences by introducing the expression vector into animal cells. The CH domain of a human chimeric antibody can be any region belonging to human immunoglobulins, such as the IgG class or its subclasses, e.g., IgG1, IgG2, IgG3, and IgG4. Similarly, the CL domain of a human chimeric antibody can be any region belonging to an Ig class, such as the kappa or lambda class. Chimeric and humanized monoclonal antibodies containing both human and non-human portions, which can be produced using standard recombinant DNA techniques, are within the scope encompassed by the present disclosure. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, as described in International Patent Publication No. PCT / US86 / 02269 (Robinson et al.), European Patent Application No. 184,187 (Akira et al.), European Patent Application No. 171,496 (Taniguchi, M.), European Patent Application No. 173,494 (Morrison et al.), PCT Application No. WO86 / 01533 (Neuberger et al.), U.S. Pat. No. 4,816,567 (Cabilly et al.), European Patent Application No. 125,023 (Cabilly et al.), Better et al. (1988) Science 240:1041-1043, Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443, Liu et al. (1987) J. Immunol.139:3521-3526, Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218, Nishimura et al. (1987) Cancer Res. 47:999-1005, Wood et al. al. (1985) Nature 314:446-449, Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559), Morrison, SL (1985) Science 229:1202-1207, Oi et al. (1986) Biotechniques 4:214, U.S. Pat. No. 5,225,539 (Winter), Jones et al. (1986) Nature 321:552-525, Verhoeyan et al. (1988) Science 239:1534, and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[0200] Additionally, humanized antibodies can be made according to standard protocols, such as those disclosed in U.S. Patent No. 5,565,332. In some embodiments, antibody chains or specific binding pair members can be produced by recombination between a vector containing a nucleic acid molecule encoding a fusion of a polypeptide chain of a specific binding pair member with a component of a replicable generic display package and a vector containing a nucleic acid molecule encoding a second polypeptide chain of a single binding pair member, using techniques known in the art, for example, those described in U.S. Patent Nos. 5,565,332, 5,871,907, or 5,733,743. Humanized antibodies encompassed by the present disclosure can be produced by obtaining nucleic acid sequences encoding the CDR domains as described above, inserting them into an expression vector for animal cells containing genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, thereby constructing a humanized antibody expression vector, and expressing the genes by introducing the expression vector into animal cells. The humanized antibody expression vector may be either of a type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are present on separate vectors, or of a type in which both of these genes are present on the same vector (tandem type).

[0201] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988, Neuberger M S. et al. 1985). Antibodies can be produced by, for example, CDR grafting (EP 239,400, PCT Publication No. 91 / 09967, U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106, EP 519,596, Padlan EA (1991), Studnicka G Antibodies may be humanized using a variety of techniques known in the art, including antibody fragmentation (M et al. (1994), Roguska M A. et al. (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application No. EP 125023 and International Patent Application No. WO 96 / 02576).

[0202] Similarly, the bispecific or multispecific antibodies described herein can be produced according to standard procedures. For example, triomas and hybrid hybridomas are two examples of cell lines capable of secreting bispecific or multispecific antibodies. Examples of bispecific and multispecific antibodies produced by hybrid hybridomas or triomas are disclosed in U.S. Patent No. 4,474,893. Such antibodies can also be constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and Staerz and Bevan (1986) Immunol. Today 7:241). Alternatively, such antibodies can be produced by fusing hybridomas or other cells producing different antibodies to create heterohybridomas, followed by identifying clones that produce and coassemble the desired antibodies. They can also be produced by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof, such as Fab and Fv sequences. The antibody component can bind to a polypeptide or fragment thereof of one or more biomarkers encompassed by this disclosure, including one or more immunoinhibitory biomarkers described herein.

[0203] In addition, methods for producing antibody fragments are well known. For example, Fab fragments encompassed by the present disclosure can be obtained by treating an antibody that specifically reacts with human KIR3DL3 with a protease such as papain. Fab can also be produced by inserting DNA encoding the Fab of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote (as appropriate) to express the Fab.

[0204] Similarly, F(ab')2 fragments encompassed by the present disclosure can be obtained by treating an antibody specifically reactive with KIR3DL3 with the protease pepsin, and can also be produced by linking the Fab' fragments described below via a thioether bond or disulfide bond.

[0205] Fab' fragments encompassed by the present disclosure can be obtained by treating F(ab')2 that specifically reacts with human KIR3DL3 with the reducing agent dithiothreitol. Fab' fragments can also be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and expressing the vector in a prokaryote or eukaryote (as appropriate).

[0206] Additionally, scFvs encompassed by the present disclosure can be produced by obtaining cDNAs encoding the VH and VL domains as described above, constructing DNA encoding the scFv, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic organism (as appropriate) to express the scFv. To generate humanized scFv fragments, a well-known technique called CDR grafting can be used, which involves selecting complementarity-determining regions (CDRs) from a donor scFv fragment and grafting them onto a human scFv fragment framework with a known three-dimensional structure (see, e.g., WO98 / 45322, WO87 / 02671, U.S. Pat. Nos. 5,859,205, 5,585,089, 4,816,567, and EP0173494).

[0207] V. Modifications of Antibodies, Immunoglobulins, and Polypeptides Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. When a humanized antibody is produced by simply grafting only the CDRs in the VH and VL of an antibody derived from a non-human animal onto the FRs of the VH and VL of a human antibody, it is known that the antigen-binding activity is reduced compared to the original antibody derived from a non-human animal. Some amino acid residues in the VH and VL of a non-human antibody, not only in the CDRs but also in the FRs, are considered to be directly or indirectly related to the antigen-binding activity. Therefore, replacing these amino acid residues with different amino acid residues derived from the FRs of the VH and VL of a human antibody reduces the binding activity, which can be corrected by replacing the amino acids with amino acid residues from the original antibody derived from a non-human animal.

[0208] Modifications and changes may be made in the structure of the antibodies encompassed by the present disclosure and the DNA sequences encoding them, while still obtaining functional molecules encoding antibodies and polypeptides with desirable characteristics. For example, certain amino acids may be substituted by other amino acids in the protein structure without appreciable loss of activity. Because the interactive capabilities and properties of a protein define its biological functional activity, certain amino acid substitutions may be made in the protein sequence, and thus in its DNA coding sequence, while still obtaining a protein with similar properties. Thus, it is contemplated that various changes may be made in the antibody sequences encompassed by the present disclosure or the corresponding DNA sequences encoding the polypeptides, without appreciable loss of their biological activity.

[0209] In some embodiments, amino acid changes can be achieved by changing codons in a DNA sequence to encode conservative substitutions based on the conservation of the genetic code. Specifically, there is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequence that can encode that protein as defined by the genetic code (shown below). Similarly, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid as defined by the genetic code (see genetic code chart above).

[0210] When altering the amino acid sequence of a polypeptide, the hydropathic index of amino acids can be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art. It is recognized that the relative hydropathic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics, and they are: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), and riboflavin (-3.5). <RTI 3.5)、アスパラギン(-3.5)、リジン(-3.9)、およびアルギニン(-4.5)である。

[0211] It is known in the art that certain amino acids may be substituted with other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., a functionally equivalent biological protein is still obtained.

[0212] Thus, as outlined above, amino acid substitutions are generally based on the relative similarity of the substitute amino acid side chains, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various characteristics mentioned above are well known to those of skill in the art and include arginine and lysine, glutamic acid and aspartic acid, serine and threonine, glutamine and asparagine, and valine, leucine, and isoleucine.

[0213] Another type of amino acid modification of antibodies encompassed by the present disclosure may be useful in altering the original glycosylation pattern of the antibody, for example, to increase stability. By "altering" is meant deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies may typically be N-linked. "N-linked" refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Another type of covalent modification involves chemically or enzymatically coupling glycosides to antibodies. These procedures are advantageous in that they do not require the production of antibodies in host cells with glycosylation capabilities for N-linked or O-linked glycosylation. Depending on the coupling method used, sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO87 / 05330.

[0214] Similarly, removal of any carbohydrate moieties present on an antibody can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described by Sojahr H. et al. (1987) and Edge, A S. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of various endo- and exoglycosidases, as described by Thotakura, N R. et al. (1987).

[0215] Other modifications may include the formation of immunoconjugates. For example, in one type of covalent modification, the antibody or protein is covalently linked to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0216] Conjugation of antibodies or other proteins to heterologous agents encompassed by the present disclosure may be carried out using a variety of bifunctional protein coupling agents, including, but not limited to, N-succinimidyl(2-pyridyldithio)propionate (SPDP), succinimidyl(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6 diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies (WO 94 / 11026).

[0217] In another aspect, the present disclosure features antibodies that specifically bind to KIR3DL3 conjugated to a therapeutic moiety, such as a cytotoxin, a drug, and / or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). The antibodies of the present disclosure can be conjugated to radioisotopes, for example, radioactive iodine, to generate cytotoxic radiopharmaceuticals for treating related disorders such as cancer.

[0218] Conjugated anti-KIR3DL3 antibodies can be used, inter alia, to diagnostically or prognostically monitor polypeptide levels in tissues as part of clinical trial procedures, for example, to determine the effectiveness of a given treatment regimen or to select patients most likely to respond to immunotherapy. For example, cells can be permeabilized in a flow cytometry assay to allow detection of binding by targeting an antibody that binds to KIR3DL3 and its recognized intracellular epitope, and analyzing the signal emitted from the conjugated molecule. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin (PE); an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H. As used herein, the term "labeled" with respect to an antibody is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)), to the antibody, as well as indirect labeling of the antibody by reaction with a detectable substance.

[0219] The antibody conjugates encompassed by the present disclosure can be used to modulate a given biological response. The chemical moieties should not be construed as limited to classical chemical agents. For example, the drug moiety can be a protein or polypeptide possessing a desired biological activity. Such proteins can include, for example, tumor necrosis factor or interferon-gamma, or biological response modifiers (e.g., lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte-colony-stimulating factor ("G-CSF"), or other cytokines or growth factors, etc.).

[0220] Techniques for conjugating such therapeutic moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospects Of Therapeutic Agents In Cancer Therapy," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); See "Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62:119-58 (1982).

[0221] In some embodiments, conjugation can be performed using a "cleavable linker" that facilitates the release of the cytotoxic drug or growth inhibitory agent in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (see, e.g., U.S. Pat. No. 5,208,020) can be used. Alternatively, a fusion protein comprising an antibody and a growth inhibitory agent can be produced by recombinant techniques or peptide synthesis. The length of DNA can include regions encoding the two portions of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

[0222] VI. Uses and Methods The anti-KIR3DL3 antibodies, immunoglobulins, polypeptides, and nucleic acids encompassed by the present disclosure described herein may be useful for a variety of applications, such as KIR3DL3 detection methods, therapeutic purposes (e.g., therapeutic, prophylactic, and immunomodulatory), alone or in combination with other therapeutic agents. Furthermore, the anti-KIR3DL3 antibodies, immunoglobulins, polypeptides, and nucleic acids encompassed by the present disclosure described herein may be used in a number of predictive pharmaceutical assays based on the detection of KIR3DL3 levels. For example, the present disclosure provides prognostic (or predictive) assays for determining whether an individual will respond to a particular therapy (e.g., a therapy targeting KIR3DL3). As described herein, the KIR3DL3 polypeptide or fragment thereof encompassed by the present disclosure has one or more of the following activities: 1) binding to and / or modulating the activity of its natural binding partner, such as HHLA2; 2) modulating intracellular or intercellular signaling, such as co-immunoinhibitory signaling; 3) modulating the activation of T cells or NK cells; 4) modulating the immune response of an organism, e.g., a mammalian organism, such as a mouse, a non-rodent, or a human; and 5) modulating immune cell anergy.

[0223] The present invention also provides detection of KIR3DL3 as a means for identifying agents that transmit KIR3DL3 signals, which may attenuate immune responses and be useful in autoimmune diseases, asthma, and the establishment of tolerance.

[0224] In any of the methods described herein, KIR3DL3 can be detected alone or in combination with the expression of other molecules, such as other immune checkpoints and / or costimulatory molecules. Combinatorial detection of several molecules (e.g., sequentially or simultaneously) can provide useful information regarding the synergistic effects of therapeutic intervention and / or personalized high-resolution diagnosis of disorder subtypes. In some embodiments, KIR3DL3 is detected in combination with another marker.

[0225] 1. Therapeutic Methods and Uses In some embodiments, antibodies, fragments, or immunoconjugates (e.g., anti-KIR3DL3 antibodies) encompassed by the present disclosure are useful for treating any disorder associated with aberrant or undesired KIR3DL3 activation (e.g., cancer). In certain embodiments, the treatment is in a mammal, such as a human. Such antibodies encompassed by the present disclosure can be used alone or in combination with any suitable agent or appropriate therapy to treat the disorder of interest. For example, therapeutic synergy is believed to emerge when cells are treated with an anti-KIR3DL3 mAb and another immune checkpoint inhibitor or cell therapy, such as a therapy including a CAR.

[0226] The antibodies or fragments thereof encompassed by the present disclosure described herein are useful for regulating immune responses by blocking or disrupting the interaction between KIR3DL3 and its natural ligand, HHLA2. Similarly, the antibodies or fragments thereof described herein are useful for treating diseases, such as cancer, by increasing the immune response against cancer cells and T cell and / or NK cell activity. Thus, an object encompassed by the present disclosure relates to a method for regulating immune responses and / or treating disorders associated with abnormal KIR3DL3 activation, comprising administering a therapeutically effective amount of an antibody or fragment thereof encompassed by the present disclosure to a subject in need thereof.

[0227] The upregulation of immune response can be in the form of enhancing existing immune response or inducing early immune response.For example, the enhancement of immune response using the subject compositions and methods is useful for improving immunological defense against cancer and microbial (e.g., bacterial, viral, or parasitic) infection.For example, the upregulation or enhancement of immune response function described herein is useful for inducing tumor immunity.

[0228] In another embodiment, an immune response can be stimulated by the methods described herein such that pre-existing tolerance, clonal deletion, and / or exhaustion (e.g., T cell exhaustion) are overcome. For example, an immune response against an antigen to which a subject is unable to mount a significant immune response, e.g., an autoantigen such as a tumor-specific antigen, can be induced by administering a suitable agent described herein that upregulates the immune response. In one embodiment, an autoantigen such as a tumor-specific antigen can be co-administered. In another embodiment, an immune response can be stimulated against an antigen (e.g., an autoantigen) to treat a neurological disorder. In another embodiment, the subject agent can be used as an adjuvant to enhance responses to foreign antigens during active immunization.

[0229] In certain instances, to further enhance immune response, it may be desirable to further administer other agents that upregulate immune response, such as other forms of B7 family members that signal through costimulatory receptors.Also, agents that upregulate immune response can be used prophylactically in vaccines against various polypeptides (for example, polypeptides derived from pathogens).Immunity against pathogens (for example, viruses) can be induced by vaccinating viral proteins with agents that upregulate immune response in suitable adjuvants.

[0230] Alternatively or additionally, in some embodiments, the antibodies and antigen-binding fragments encompassed by the present disclosure have diagnostic, prognostic, and preventative uses, as well as therapeutic applications for diseases that upregulate immune responses, such as asthma, autoimmune diseases (glomerulonephritis, arthritis, dilated cardiomyopathy-like diseases, ulcerative colitis, Sjogren's syndrome, Crohn's disease, systemic lupus erythematosus, chronic rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, scleroderma, periarteritis nodosa, and periarteritis nodosa. , rheumatic fever, vitiligo vulgaris, insulin-dependent diabetes mellitus, Behçet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, pernicious anemia, Goodpasture's syndrome, infertility, chronic active hepatitis, pemphigus, autoimmune thrombocytopenic purpura, and autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, antiphospholipid syndrome, atopic allergy, autoimmune atrophic gastritis, autoimmune achlorhydria and inflammatory bowel disease (e.g., inflammatory bowel disease), celiac disease, Cushing's syndrome, dermatomyositis, discoid lupus erythematosus, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes mellitus, Lambert-Eaton syndrome, lupoid hepatitis, some cases of lymphocytopenia, mixed connective tissue disease, pemphigoid, pemphigus vulgaris, pernicious anemia, phacogenic uveitis, polyarteritis nodosa, polyglandular autoimmune syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt's syndrome, morphea (or CREST syndrome), sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, hyperthyroidism, type B insulin resistance, ulcerative colitis, and Wegener's granulomatosis.

[0231] Similarly, the antibodies and antigen-binding fragments encompassed by the present disclosure are useful for diagnostic, prognostic, and preventative applications, as well as therapeutic applications (such as treating the disease and delaying its onset or progression) for persistent infectious diseases (e.g., viral infectious diseases including HPV, HBV, Hepatitis C virus (HCV), retroviruses, e.g., human immunodeficiency viruses (HIV-1 and HIV-2), herpes viruses, e.g., Epstein-Barr virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, and influenza viruses). Other pathogen-associated antigens that can be used as described herein include malaria, preferably various antigens including malaria peptides based on NANP repeats. In addition, antigens of bacterial, fungal, and other pathogenic diseases, such as Aspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma, and Vibriocholerae, are included.These include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, B Streptococcus species, Microplasma hominis、Hemophilus ducreyi、Granuloma inguinale、Lymphopathia venereum、Treponema pallidum、Brucella abortus、Brucella melitensis、Brucella suis、Brucella canis、Campylobacter fetus、Campylobacter fetus intestinalis、Leptospira pomona、Listeria monocytogenes、Brucella ovis、Chlamydia psittaci、Trichomonas foetus、Toxoplasma gondii、Escherichia coli、Actinobacillus equuli、Salmonella abortus ovis、Salmonella abortus equi、Pseudomonas aeruginosa、Corynebacterium equi、Corynebacterium pyogenes、Actinobaccilus seminis、Mycoplasma bovigenitalium、Aspergillus fumigatus、Absidia ramosa、Trypanosoma equiperdum、Babesia caballi、Clostridium tetani、Clostridium botulinum in the case of Paracoccidioides brasiliensis and in the case of Plasmodium falciparum and the National Agency for Infectious Diseases (NIAID).These include category A agents such as variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularemia), filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagic fever), arenaviruses (Lassa (Lassa fever), Junin virus (Argentine hemorrhagic fever), and related viruses), category B agents such as Coxiella burnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), alphaviruses (Venezuelan encephalomyelitis, eastern and western equine encephalomyelitis), ricin toxin from Ricinus communis (castor seed), epsilon toxin of Clostridium perfringens, Staphylococcus enterotoxin B, Salmonella species, Shigella dysenteriae, Escherichia coli strain O157:H7, Vibrio cholerae, Cryptosporidium parvum, Category C agents such as Nipah virus, Hantavirus, tick-borne hemorrhagic fever virus, tick-borne encephalitis virus, yellow fever, and multidrug-resistant Mycobacterium tuberculosis, helminths such as Schistosoma and Taenia, and protozoa such as Leishmania (e.g., L. mexicana) and Plasmodium.

[0232] In some embodiments, antibodies or antigen-binding fragments encompassed by the present disclosure are useful for therapeutic applications in relation to the induction of immunological tolerance, organ transplant rejection, graft-versus-host disease (GVHD), allergic diseases, and diseases caused by attenuation of KIR3DL3-mediated immune responses, in addition to prognostic and preventative applications.

[0233] In the context of the present invention, the term "treating" or "treatment" as used herein means reversing, alleviating, or inhibiting the progression of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. As used herein, the term "treating cancer" means inhibiting the growth and / or proliferation of cancer cells. Preferably, such treatment also results in regression of tumor growth (i.e., a decrease in the size of a measurable tumor). Most preferably, such treatment results in complete regression of the tumor.

[0234] Therapeutic formulations comprising one or more antibodies encompassed by the present disclosure are prepared for storage by mixing antibodies of the desired purity, in the form of a lyophilized formulation or aqueous solution, with any physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Antibody compositions may be formulated, dosed, and administered in any manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to physicians.

[0235] The therapeutic dose may be at least about 0.001 μg / kg body weight, 0.005 μg / kg body weight, 0.01 μg / kg body weight, at least about 0.05 μg / kg body weight, at least about 0.1 μg / kg body weight, at least about 0.5 μg / kg body weight, at least about 1 μg / kg body weight, at least about 2.5 μg / kg body weight, at least about 5 μg / kg body weight, at least about 50 μg / kg body weight, or at least about 100 μg / kg body weight. Those skilled in the art will understand that such guidelines will be adjusted depending on the molecular weight of the active agent, for example, when using antibody fragments or antibody conjugates. Dosages may also vary depending on local administration, e.g., intranasal, inhalation, etc., or systemic administration, e.g., intramuscular, intraperitoneal, intravenous, etc.

[0236] The present compositions do not require, but are optionally formulated with, one or more agents that enhance activity or otherwise enhance therapeutic efficacy.

[0237] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; These include proteins such as albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes), and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0238] The active ingredients may also be encapsulated in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0239] The compositions described herein can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the compositions can be suitably administered by pulse infusion, particularly with declining doses of the antibody.

[0240] For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease being treated, as defined above, the severity and course of the disease, whether the antibody is being administered prophylactically, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0241] Agents that directly block the interaction between KIR3DL3 and HHLA2, such as anti-HHLA2 antibodies, anti-KIR3DL3 antibodies, anti-KIR3DL3 / anti-immune checkpoint bispecific antibodies (e.g., anti-KIR3DL3 / PD-1 bispecific antibodies), etc., can inhibit KIR3DL3 signaling and its downstream immune responses. Alternatively, agents that indirectly block the interaction between KIR3DL3 and HHLA2 can inhibit KIR3DL3 signaling and its downstream immune responses. For example, in some embodiments, a soluble form of KIR3DL3, such as the extracellular domain of KIR3DL3, can indirectly reduce the effective concentration of HHLA2 available for binding to KIR3DL3 on the cell surface by binding to HHLA2. Exemplary agents include monospecific or bispecific blocking antibodies against KIR3DL3 and / or HHLA2 that block the interaction between the receptor and ligand, inactive forms of HHLA2 and / or KIR3DL3 (e.g., dominant negative or soluble polypeptides), small molecules or peptides that block the interaction between KIR3DL3 and HHLA2, fusion proteins (e.g., the extracellular portion of HHLA2 and / or KIR3DL3 fused to the Fc portion of an antibody or immunoglobulin) that bind to KIR3DL3 and / or HHLA2 and inhibit the interaction between its receptor and ligand, inactive forms of native KIR3DL3 and / or HHLA2, and soluble forms of native KIR3DL3 and / or HHLA2.

[0242] In some embodiments, an anti-KIR3DL3 antibody therapy or combination of therapies (e.g., one or more anti-KIR3DL3 antibody therapies in combination with one or more additional anti-cancer therapies, such as another immune checkpoint inhibitor) may be administered. The combination therapy may include, for example, one or more chemotherapeutic agents and radiation, one or more chemotherapeutic agents and immunotherapy, or one or more chemotherapeutic agents, radiation, and chemotherapy, each of which may be used in conjunction with an anti-immune checkpoint therapy. In addition, any of the representative embodiments of agents for modulating a specific target may be adapted by one skilled in the art to any other target described herein and below (e.g., the direct and indirect KIR3DL3 inhibitors described herein may be applied to other immune checkpoint inhibitors and / or monospecific antibodies, bispecific antibodies, inactive forms, small molecules, peptides, interfering nucleic acids, etc.).

[0243] Thus, therapeutic agents encompassed by the present disclosure may be used alone or in combination with, for example, chemotherapeutic agents, hormones, antiangiogenic agents, CARs, radiolabeled compounds, or surgery, cryotherapy, and / or radiation therapy. The aforementioned therapies may be administered either sequentially with, prior to, or after conventional therapy, or in conjunction with other forms of conventional therapy (e.g., standard of care cancer treatments known to those skilled in the art). For example, agents encompassed by the present disclosure may be administered with a therapeutically effective dose of a chemotherapeutic agent. In another embodiment, agents encompassed by the present disclosure are administered in combination with chemotherapy to enhance the activity and effectiveness of the chemotherapeutic agent. Physician's Package Inserts (PDRs) disclose the dosages of chemotherapeutic agents used in the treatment of various cancers. The therapeutically effective dosage regimens and dosages of these aforementioned chemotherapeutic agents depend on the particular cancer being treated, the extent of the disease, and other factors familiar to physicians in the art, and can be determined by a physician.

[0244] Anti-KIR3DL3 agents can also be administered in combination with targeted therapies, such as immunotherapy. Immunotherapies designed to induce or amplify an immune response are referred to as "activating immunotherapy." Immunotherapies designed to reduce or suppress an immune response are referred to as "suppressive immunotherapy." Any agent believed to have an immune system effect on genetically modified transplanted cancer cells can be assayed to determine whether the agent is immunotherapeutic and to determine the effect a given genetic modification has on modulating the immune response. In some embodiments, the immunotherapy is cancer cell-specific. In some embodiments, the immunotherapy can be "non-targeted," which refers to the administration of an agent that does not selectively interact with immune system cells but modulates immune system function. Representative examples of non-targeted therapies include, but are not limited to, chemotherapy, gene therapy, and radiation therapy.

[0245] The term "targeted therapy" refers to the administration of an agent that selectively interacts with a selected biomolecule, e.g., thereby treating cancer. For example, targeted therapy involving the inhibition of immune checkpoint inhibitors is useful in combination with methods encompassed by the present disclosure. The term "immune checkpoint inhibitor" refers to a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by downregulating or inhibiting anti-tumor immune responses. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR (see, e.g., WO2012 / 177624). Inhibition of one or more immune checkpoint inhibitors blocks or otherwise neutralizes inhibitory signaling, thereby upregulating the immune response and allowing cancer to be treated more effectively.

[0246] Immunotherapy is a form of targeted therapy that may involve, for example, the use of one or more cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are viruses that can infect and lyse cancer cells while leaving normal cells unharmed, making them potentially useful in cancer therapy. Oncolytic virus replication promotes tumor cell destruction and also results in dose amplification at the tumor site. They can also act as vectors for anti-cancer genes, delivering them specifically to the tumor site. This immunotherapy may involve passive immunization to provide short-term host protection, achieved by administering preformed antibodies against cancer or disease antigens (e.g., monoclonal antibodies, optionally conjugated to chemotherapeutic agents or toxins, to tumor antigens). For example, anti-VEGF and mTOR inhibitors are known to be effective in treating renal cell carcinoma. This immunotherapy can also focus on the use of epitopes recognized by cytotoxic lymphocytes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc. can be used to selectively regulate biomolecules associated with the onset, progression, and / or pathology of tumors or cancer. This immunotherapy can also focus on the use of epitopes recognized by cytotoxic lymphocytes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc. can be used to selectively regulate biomolecules associated with the onset, progression, and / or pathology of tumors or cancer. As described above, immunotherapy against immune checkpoint targets, such as HHLA2 and KIR3DL3, is useful.

[0247] In some embodiments, immunotherapy may include one or more adoptive cell-based immunotherapies, including, but not limited to, irradiated autologous or allogeneic tumor cells, tumor lysates or apoptotic tumor cells, antigen-presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell transfer, adoptive CAR T cell therapy, autoimmune enhancement therapy (AIET), cancer vaccines, and / or antigen-presenting cells. Such cell-based immunotherapies may be further modified to express one or more gene products to further modulate the immune response, for example, to express cytokines such as GM-CSF and / or tumor-associated antigens (TAA) such as Mage-1, gp-100, or patient-specific neoantigen vaccines.

[0248] In some embodiments, immunotherapy may include one or more non-cell-based immunotherapies. In some embodiments, compositions containing antigens with or without vaccine-enhancing adjuvants are used. Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, and recombinant antigens, including fusion proteins. In yet another embodiment, immunomodulatory interleukins, such as IL-2, IL-6, IL-7, IL-12, IL-17, and IL-23, and modulators thereof (e.g., blocking antibodies or more potent or longer-lasting forms), are used. In yet another embodiment, immunomodulatory cytokines, such as interferons, G-CSF, imiquimod, and TNF-alpha, and modulators thereof (e.g., blocking antibodies or more potent or longer-lasting forms), are used. In another embodiment, immunomodulatory chemokines, such as CCL3, CCL26, and CXCL7, and modulators thereof (e.g., blocking antibodies or more potent or longer-lasting forms), are used. In another embodiment, immunomodulatory molecules that target immunosuppression are used, such as STAT3 signaling modulators, NF-kappa B signaling modulators, and immune checkpoint modulators. The terms "immune checkpoint" and "anti-immune checkpoint therapy" are explained above.

[0249] In some embodiments, immunomodulatory agents include, for example, immune cell antiproliferative agents, glucocorticoids, cytostatics, immunophilins, and modulators thereof (e.g., rapamycin, calcineurin inhibitors, tacrolimus, cyclosporin, pimecrolimus, avetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone (doca) acetate, aldosterone, non-glucocorticoid steroids, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid analogs, methotrexate, antithymocyte growth factor receptor 1 (ATR), thrombus growth factor receptor 2 (THR), thrombus growth factor receptor 3 (THR), thrombus growth factor receptor 4 (THR), thrombus growth factor receptor 5 (THR), thrombus growth factor receptor 6 (THR), thrombus growth factor receptor 7 (THR), thrombus growth factor receptor 8 (THR), thrombus growth factor receptor 9 (THR), thrombus growth factor receptor 10 (THR), thrombus growth factor receptor 11 (THR), thrombus growth factor receptor 12 (THR), thrombus growth factor receptor 13 (THR), thrombus growth factor receptor 14 (THR), thrombus growth factor receptor 15 (THR), thrombus growth factor receptor 16 (THR), thrombus growth factor receptor 17 (THR), thrombus growth factor receptor 18 (THR), thrombus growth factor receptor 19 (THR), thrombus growth factor globulin, antilymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, opioids, IMPDH inhibitors, mycophenolate, myriocin, fingolimod, NF-kB inhibitors, raloxifene, drotrecogin alfa, denosumab, NF-kB signaling cascade inhibitors, disulfiram, olmesartan, dithiocarbamate, proteasome inhibitors In yet another embodiment, immunomodulatory agents such as NFKBr1, NFKBr2, NFKBr2, NFKBr3, NFKBr4, NFKBr5, NFKBr6, NFKBr7, NFKBr8, NFKBr9, NFKBr10, NFKBr21, NFKBr22, NFKBr13, NFKBr24, NFKBr15, NFKBr25, NFKBr16, NFKBr26, NFKBr17, NFKBr18, NFKBr19, NFKBr27, NFKBr28, NFKBr29 ...For example, CD40, Toll-like receptor (TLR), OX40, GITR, CD27, or 4-1BB, T cell bispecific antibodies, anti-IL-2 receptor antibodies, anti-CD3 antibodies, OKT3 (muromonab), otelixizumab, teplizumab, visilizumab, anti-CD4 antibodies, clenoliximab, keliximab, zanolimumab, anti-CD11a antibodies, efalizumab, anti-CD18 antibodies, erulizumab, rovelizumab, anti-CD20 antibodies, afutuzumab, and ocrelizumab , ofatumumab, pascolizumab, rituximab, anti-CD23 antibody, lumiliximab, anti-CD40 antibody, teneliximab, toralizumab, anti-CD40L antibody, ruplizumab, anti-CD62L antibody, aselizumab, anti-CD80 antibody, galiximab, anti-CD147 antibody, gavilimomab, B-lymphocyte stimulator (BLyS) inhibitor antibody, belimumab, CTLA4-Ig fusion protein, abatacept, belatacept, anti-CTLA4 antibody, ipilimumab, tremelimumab Mumab, anti-eotaxin 1 antibody, bertilimumab, anti-a4-integrin antibody, natalizumab, anti-IL-6R antibody, tocilizumab, anti-LFA-1 antibody, odulimomab, anti-CD25 antibody, basiliximab, daclizumab, inolimomab, anti-CD5 antibody, zolimomab, anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atolimumab, cedelizumab, dorlimomab-allitoxin, dorlixizumab, fontolizumab, cancer Antibodies that bind to tenerumab, gomiliximab, levrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, terimomab-alitonx, bapaliximab, beparimomab, aflibercept, alefacept, rilonacept, IL-1 receptor antagonists, anakinra, anti-IL-5 antibodies, mepolizumab, IgE inhibitors, omalizumab, talizumab, IL12 inhibitors, IL23 inhibitors, ustekinumab, etc.

[0250] In some embodiments, nutritional supplements that enhance immune responses, such as vitamin A, vitamin E, vitamin C, and the like, are known in the art (see, e.g., U.S. Pat. Nos. 4,981,844 and 5,230,902 and PCT Publication No. WO2004 / 004483), and may be used in the methods described herein.

[0251] Similarly, drugs and non-immunotherapeutic therapies can be used in combination with anti-KIR3DL3 antibodies to stimulate an immune response and thereby treat conditions that would benefit therefrom, such as chemotherapy, radiation, epigenetic modifiers (e.g., histone deacetylase (HDAC) modifiers, methylation modifiers, phosphorylation modifiers, etc.), targeted therapies, etc., which are well known in the art.

[0252] The term "non-targeted therapy" refers to the administration of a drug that does not selectively interact with a selected biomolecule but treats cancer. Representative examples of non-targeted therapy include, but are not limited to, chemotherapy, gene therapy, and radiation therapy.

[0253] In one embodiment, chemotherapy is used. Chemotherapy involves the administration of a chemotherapeutic agent. Such chemotherapeutic agents may be selected from, but are not limited to, the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, antimitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogs, plant alkaloids, and toxins, and their synthetic derivatives. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, and docetaxel; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; antifolates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and mitotic inhibitors: halichondrin, colchicine, and rhizoxin. Compositions containing one or more chemotherapeutic agents (e.g., FLAG, CHOP) can also be used. FLAG includes fludarabine, cytosine arabinoside (Ara-C), and G-CSF. CHOP includes cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiment, a PARP (e.g., PARP-1 and / or PARP-2) inhibitor is used, and such inhibitors are well known in the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.), INO-1001 (Inotek Pharmaceuticals Inc.), PJ34 (Soriano et al., 2001; Pacher et al., 2002b), 3-aminobenzamide (Trevigen), 4-amino-1,8-naphthalimide (Trevigen), 6(5H)-phenanthridinone (Trevigen), benzamide (U.S. Patent Re. 36,397), and NU1025 (Bowman et al.).The mechanism of action is generally related to the ability of PARP inhibitors to bind to and reduce the activity of PARP. PARP catalyzes the conversion of beta-nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly-ADP-ribose (PAR). Both poly(ADP-ribose) and PARP are associated with transcriptional regulation, cell proliferation, genome stability, and carcinogenesis (Bouchard VJ et. al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 June 2001, pp. 97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule in the repair of DNA single-strand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame JC, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang ZQ, et al. (1997) Genes Dev 11:2347-2358). Knockout of SSB repair by inhibiting PARP1 function induces DNA double-strand breaks (DSBs), which can induce synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant HE, et al. (2005) Nature 434:913-917, Farmer H, et al. (2005) Nature 434:917-921).The above examples of chemotherapeutic agents are illustrative and are not intended to be limiting.

[0254] In another embodiment, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external beam radiation therapy, interstitial implants of radioisotopes such as I-125, palladium, iridium, strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or whole abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J.B. Lippencott Company, Philadelphia. Radiation therapy can be administered as external beam radiation or teletherapy, in which radiation is directed from a remote source. Radiation therapy can also be administered as internal therapy or brachytherapy, in which a radioactive source is placed inside the body in close proximity to cancer cells or tumor masses. Also encompassed is the use of photodynamic therapy, which involves the administration of photosensitizing agents such as hematoporphyrin and its derivatives, vertoporphin (BPD-MA), phthalocyanines, the photosensitizer Pc4, demethoxy-hypocrelin A, and 2BA-2-DMHA.

[0255] In another embodiment, hormone therapy is used.Hormonal therapeutic treatment can include, for example, hormone agonist, hormone antagonist (for example, flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonist), hormone biosynthesis and processing inhibitor, and steroid (for example, dexamethasone, retinoid, deltoid, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen, testosterone, progestin), vitamin A derivative (for example, all-trans retinoic acid (ATRA)), vitamin D3 analog, antigestagen (for example, mifepristone, onapristone) or antiandrogen (for example, cyproterone acetate).The duration and / or dosage of treatment using therapy can vary depending on specific therapeutic agent or their combination. Those skilled in the art will understand the appropriate treatment duration for a particular cancer therapeutic agent. The present disclosure contemplates the ongoing evaluation of the optimal treatment schedule for each cancer therapeutic agent, where the subject's cancer phenotype, as determined by the methods encompassed by the present disclosure, is a factor in determining the optimal treatment dose and schedule.

[0256] Any means for introducing polynucleotides into mammals, humans, or non-humans, or their cells, can be adapted to the practice of the present invention for delivering the various constructs encompassed by this disclosure to the intended recipient. In one embodiment encompassed by this disclosure, DNA constructs are delivered to cells by transfection, i.e., by delivery of "naked" DNA, or in a complex with a colloidal dispersion system. Colloidal systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the present invention is lipid-complexed DNA or liposome-formulated DNA. In the former approach, for example, a plasmid containing a transgene bearing the desired DNA construct is subjected to expression (e.g., inclusion of introns in the 5' untranslated region and elimination of unwanted sequences (Felgner, et al., Ann NY Acad Sci The DNA can be first experimentally optimized for the purpose (126-139, 1995). Then, for example, the formulation of DNA with various lipid or liposome materials can be achieved using known methods and materials, and delivered to the recipient mammal. For example, see Canonico et al., Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al., Am J Physiol 268; Alton et al., Nat Genet. 5:135-142, 1993, and U.S. Patent No. 5,679,647 (Carson et al.).

[0257] Liposome targeting can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, such as organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based on whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) within organs, including the sinusoidal capillaries. On the other hand, active targeting involves modifying liposomes by conjugating them to specific ligands, such as monoclonal antibodies, sugars, glycolipids, or proteins, or by changing their composition or size, to achieve targeting to organs and cell types other than their naturally occurring localization sites.

[0258] The surface of targeted delivery system can be modified in various ways.In the case of liposome targeted delivery system, lipid groups can be incorporated into the lipid bilayer of liposome to maintain targeting ligand in stable association with the liposome bilayer.Various linking groups can be used to link lipid chains to targeting ligand.Delivery vehicle, for example, naked DNA or DNA associated with liposome, can be administered to several sites in a subject (see below).

[0259] Nucleic acids can be delivered by any desired vector. These include viral or non-viral vectors, including adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and plasmid vectors. Exemplary viral types include HSV (herpes simplex virus), AAV (adeno-associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired form that provides a sufficiently efficient delivery level, for example, in viral particles, liposomes, nanoparticles, and complexed with polymers.

[0260] The nucleic acid encoding the protein or nucleic acid of interest can be present in a plasmid or viral vector, or other vectors known in the art. Such vectors are well known, and any vector can be selected for a particular application. In one embodiment encompassed by the present disclosure, the gene delivery vehicle comprises a promoter and a demethylase coding sequence. Preferred promoters are tissue-specific promoters and promoters activated by cell proliferation, such as thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters that can be activated by viral infection, such as α- and β-interferon promoters, and promoters that can be activated by hormones such as estrogen. Other promoters that can be used include Moloney virus LTR, CMV promoter, and mouse albumin promoter. The promoter can be constitutive or inducible.

[0261] In another embodiment, naked polynucleotide molecules can be used as gene delivery vehicles, as described in WO90 / 11092 and U.S. Patent No. 5,580,859. Such gene delivery vehicles can be either growth factor DNA or RNA, and in certain embodiments, are linked to killed adenovirus. Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other vehicles that may optionally be used include DNA-ligands (Wu et al., J. Biol. Chem. 264:16985-16987, 1989), lipid-DNA combinations (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et al., Proc. Natl. Acad. Sci. 84:7851-7855, 1987), and microprojectiles (Williams et al., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).

[0262] The gene delivery vehicle may optionally include one or more viral sequences, such as a viral origin of replication or packaging signal. These viral sequences may be selected from viruses such as astroviruses, coronaviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, retroviruses, togaviruses, or adenoviruses. In a preferred embodiment, the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and their various uses are described in numerous references, e.g., Mann et al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al., Human Gene Therapy 1:5-14, 1990, U.S. Pat. Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos. 89 / 02,468, 89 / 05,349, and 90 / 02,806. For example, EP 0,415,731, WO90 / 07936, WO94 / 03622, WO93 / 25698, WO93 / 25234, U.S. Pat. No. 5,219,740, WO9311230, WO9310218, Vile and Hart, Cancer Res. 53:3860-3864, 1993, Vile and Hart, Cancer Res. 53:962-967, 1993, Ram et al., Cancer Res. 53:83-88, 1993, Takamiya et al., J. Neurosci. Res. 33:493-503, 1992, Baba et al. A number of retroviral gene delivery vehicles can be utilized in the present disclosure, including those described in [End Page 110] al., J. Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242, and WO 91 / 02805).

[0263] Other viral vector systems that can be used to deliver polynucleotides encompassed by the present disclosure include herpes viruses, such as herpes simplex virus (U.S. Pat. No. 5,631,236 (Woo et al.), issued May 20, 1997) and WO 00 / 08191 (Neurovex)), vaccinia virus (Ridgeway (1988) Ridgeway, "Mammalian expression vectors," In: Rodriguez RL, Denhardt DT, eds. Vectors: A survey of molecular cloning vectors and their uses. Stoneham: Butterworth; Baichwal and Sugden (1986) "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press; Coupar et al. (1988) Gene, 68:1-10), and several RNA viruses. Preferred viruses include alphaviruses, poxyviruses, arenaviruses, vaccinia viruses, polioviruses, etc. They offer several attractive features to various mammalian cells (Friedmann (1989) Science, 244:1275-1281; Ridgeway, 1988 (see above); Baichwal and Sugden, 1986 (see above); Coupar et al. 1988; Horwich et al. (1990) J. Virol., 64:642-650).

[0264] In other embodiments, the target DNA in the genome can be manipulated using methods well known in the art.For example, the target DNA in the genome can be manipulated by deletion, insertion, and / or mutation, which are retroviral insertion, artificial chromosome technique, gene insertion, random insertion by tissue-specific promoter, gene target, transposable element, and / or any other method for introducing foreign DNA or producing modified DNA / modified nuclear DNA.Other modification techniques include deleting DNA sequence from genome and / or modifying nuclear DNA sequence.For example, nuclear DNA sequence can be modified by site-directed mutagenesis.

[0265] In other embodiments, recombinant biomarker polypeptides and fragments thereof may be administered to a subject. In some embodiments, fusion proteins with enhanced biological properties may be constructed and administered. In addition, biomarker polypeptides and fragments thereof may be modified according to pharmacological methods well known in the art (e.g., pegylation, glycosylation, oligomerization, etc.) to further enhance desirable biological activities, such as increased bioavailability and reduced proteolysis.

[0266] 2. Assays and Screening Methods Another aspect encompassed by the present disclosure relates to screening assays, including non-cell-based assays and xenograft animal model assays. In one embodiment, these assays provide methods for identifying agents that modulate KIR3DL3 signaling, such as in human or animal model assays, to identify agents that decrease KIR3DL3 signaling, thereby increasing immune responses, and / or to identify agents that increase KIR3DL3 signaling, thereby decreasing immune responses.

[0267] In one embodiment, the present disclosure relates to assays for screening test agents that bind to or modulate the biological activity of at least one biomarker described herein (e.g., in a table, figure, example, or otherwise herein), such as HHLA2, TMIGD2, and KIR3DL3. In one embodiment, a method for identifying such agents involves determining the ability of the agent to modulate, e.g., inhibit, at least one biomarker described herein.

[0268] In one embodiment, the assay is a cell-free or cell-based assay that comprises contacting at least one biomarker described herein with a test agent and determining the ability of the test agent to modulate (e.g., inhibit) the enzymatic activity of the biomarker, such as by measuring direct binding of a substrate or by measuring an indirect parameter as described below.

[0269] For example, in direct binding assays, the biomarker proteins (or their respective target polypeptides or molecules) can be coupled to radioisotopes or enzyme labels, such that binding can be determined by detecting the labeled protein or molecule in the complex. 125 I, 35 S, 14 C, or 3 The target may be labeled with H, and the radioisotope may be detected by direct counting of radioactive emissions or by scintillation counting. Alternatively, the target may be enzymatically labeled, for example, with horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label may be detected by determining the conversion of an appropriate substrate to a product. Determination of the interaction between the biomarker and the substrate may also be achieved using standard binding or enzyme analytical assays. In one or more embodiments of the above-described assay methods, it may be desirable to immobilize the polypeptide or molecule to facilitate separation of the complexed form from the uncomplexed form of one or both of the proteins or molecules and to accommodate automation of the assay.

[0270] The binding of test agent to target can be achieved in any suitable container for containing reactants.Non-limiting examples of such container include microtiter plate, test tube and microcentrifuge tube.The immobilized form of antibody described herein can also include the antibody bound to solid phase, such as porous, microporous (having an average pore size of less than about 1 micron) or macroporous (having an average pore size of more than about 10 microns) material, for example, membrane, cellulose, nitrocellulose or glass fiber; beads, for example, made of agarose or polyacrylamide or latex; or the surface of dish, plate or well, for example, made of polystyrene.

[0271] In alternative embodiments, determining the ability of an agent to modulate the interaction between a biomarker and a substrate or a biomarker and its natural binding partner can be accomplished by determining the ability of the test agent to modulate the activity of a polypeptide or other product that functions downstream or upstream from its position in a signal transduction pathway (e.g., a feedback loop). Such feedback loops are well known in the art (see, e.g., Chen and Guillemin (2009) Int. J. Tryptophan Res. 2:1-19).

[0272] The status of KIR3DL3 can be measured using the anti-KIR3DL3 antibody described herein. A decrease in KIR3DL3 binding to HHLA2 indicates that the agent inhibits KIR3DL3 activity / signal transduction, identifying the agent as useful for inhibiting KIR3DL3 activity / signal transduction and increasing immune responses. In contrast, an increase in KIR3DL3 binding to HHLA2 indicates that the agent promotes KIR3DL3 activity / signal transduction, identifying the agent as useful for promoting KIR3DL3 activity / signal transduction and decreasing immune responses.

[0273] The present disclosure further relates to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of the present invention to further use agents identified as described herein in appropriate animal models, etc. For example, agents identified as described herein can be used in animal models to determine the efficacy, toxicity, or side effects of treatment with such agents. Alternatively, antibodies identified as described herein can be used in animal models to determine the mechanism of action of such agents.

[0274] One aspect encompassed by the present disclosure relates to screening assays, including non-cell-based assays and xenograft animal model assays. In one embodiment, these assays provide a method for identifying whether cancers in humans, etc., are likely to respond to anti-KIR3DL3 antibody therapy and / or for identifying whether an agent can inhibit the growth of or kill cancer cells unlikely to respond to anti-KIR3DL3 antibody therapy by using xenograft animal model assays.

[0275] 3. Preventive methods In one aspect, the present disclosure provides a method for preventing a disease or condition associated with an undesired or less-than-desired immune response in a subject. Subjects at risk of developing a disease that would benefit from treatment with the claimed agents or methods can be identified, for example, by any or a combination of diagnostic or prognostic assays known in the art. Administration of a prophylactic agent can occur before the onset of symptoms associated with an undesired or less-than-desired immune response. Appropriate agents (e.g., antibodies, peptides, fusion proteins, or small molecules) used for treatment can be determined based on clinical indications and can be identified, for example, using the screening assays described herein.

[0276] 4. Prognostic Assay Furthermore, the detection methods described herein can be used to identify subjects who will respond to a particular therapy, such as a therapy that targets KIR3DL3 to modulate its activity and / or its interaction with a binding partner such as HHLA2. Similarly, the prognostic assays described herein can be used to determine whether a drug (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) can be administered to a subject to treat such a disorder associated with excessive or insufficient KIR3DL3 activity. For example, such methods can be used to determine whether a subject can be effectively treated with one or a combination of drugs. Thus, the present disclosure provides a method for determining whether a subject can be effectively treated with one or more drugs for treating a disorder associated with excessive or insufficient KIR3DL3 activity, wherein a test sample is obtained and KIR3DL3 is detected. The test sample can be a biological sample obtained from a subject of interest. The test sample may be obtained from a subject of interest. For example, the sample can be a biological fluid (e.g., cerebrospinal fluid or serum), a cell sample, or a tissue, e.g....

Claims

[Claim 1] The invention described in the specification.

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