Anti-KIR3DL3 Antibodies and Their Uses

Abstract

The present disclosure is based in part on the discovery of 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 prognostic, immunomodulatory, and therapeutic purposes. The present disclosure is based, at least in part, on the discovery that agents (e.g., antibodies) that target KIR3DL3 can specifically block HHLA2-KIR3DL3 interaction and can be used in methods for modulating immune responses.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 910,594, filed on October 04, 2019, the entire content of which is hereby incorporated herein by reference in its entirety.

[0002] Statement of Government Rights This invention was made with government support under grant number P50CA101942 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

Background Art

[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 the immune response based on complex combinatorial interactions among multiple inputs. Immune checkpoint inhibitors can modulate the immune response in some subjects, but immune checkpoint expression and interaction with native binding partners vary between subjects and within tissues of a subject. A significant proportion of patients do not respond to this treatment, and many patients who do respond ultimately develop resistance. Thus, there is an important unmet need to find additional immune pathways that are not redundant with the PD - 1 pathway.

[0004] HERV-H LTR-related 2 (HHLA2, also known as B7-H5 and B7-H7) is a B7 family member that regulates T cell function. HHLA2 is widely expressed in various tumors (such as solid tumors and hematological cancers including primary human renal cell carcinoma (RCC)) and antigen-presenting cells, and is involved as both an activating and an inhibitory ligand for T cells. HHLA2 has been identified as a specific ligand for TMIGD2 (CD28H, IGPR-1), and the HHLA2 / TMIGD2 interaction selectively co-stimulates human T cell proliferation and cytokine production via an AKT-dependent signaling cascade (Zhu et al. (2013) Nat. Comm. 4:2043, Janakiram et al. (2015) Clin. Cancer Res. 21:2359-2366). TMIGD2, which is expressed on naive T cells, is an activating receptor for HHLA2 and transmits co-stimulatory signals after T cell antigen receptor (TCR) ligation. TMIGD2 is downregulated after repeated TCR stimulation. A putative inhibitory receptor for HHLA2 is upregulated on activated T cells and may be capable of regulating T cell activation.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0006] Prior to the present disclosure, the existence of an uncharacterized receptor for HHLA2 on activated T cells that exerts a coinhibitory function has been suggested by several studies (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 has been discovered that HHLA2 binds to the receptor KIR3DL3 on T cells and NK cells, and that the consequence of the 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 the immune response.

[0007] The present disclosure is based at least in part on the discovery that an agent (e.g., an antibody) targeting KIR3DL3 can specifically block the HHLA2-KIR3DL3 interaction and can be used in methods for modulating the immune response. Importantly, it is presented herein that targeting KIR3DL3 does not disrupt the overall function of HHLA2, which includes activation of the immune response through interaction with TMIGD2. Accordingly, the present disclosure provides the important and surprising finding that targeting KIR3DL3 results in specificity to block only the immune inhibitory function of HHLA2, thereby inducing an effective immune response (e.g., against cancer cells) without downregulating the immune activating function of HHLA2. The development of agents that specifically block the immune inhibitory 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 the immune response and / or treat cancer. In some embodiments, the KIR3DL3×PD-1 bispecific antibodies described herein are useful as checkpoint immunotherapies, such as activating T cells and NK cells in tumors. In some embodiments, the KIR3DL3×PD-1 bispecific antibody is 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 KIR3DL3×PD-1 bispecific antibody checkpoint blockade.

[0009] An exemplary and representative panel of anti-KIR3DL3 human monoclonal antibodies (mAbs) is described herein as immunotherapy 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, there is provided a monoclonal 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, 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 an antigen-binding fragment thereof is provided, comprising: a) one, two, or three heavy chain CDR sequences each having at least about 95% identity with 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 with 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 an 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 an antigen-binding fragment thereof is provided, comprising: a) one, two, or three heavy chain CDR sequences each 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 selected from the group consisting of the sequences listed in Tables 2, 7, and 8.

[0014] Numerous embodiments are further provided that may 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, bispecific, murine, or human. In another embodiment, the monoclonal antibody or antigen-binding fragment thereof is (a) detectably labeled, (b) conjugated to a cytotoxic agent, optionally a chemotherapeutic agent, biological agent, toxin, and / or radioisotope, (c) comprises an effector domain, (d) comprises an Fc domain, and / or (e) is selected from the group consisting of Fv, Fab, 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 accession number ______. In still another embodiment, the monoclonal antibody or antigen-binding fragment thereof inhibits the binding of HHLA2 to KIR3DL3. A KIR3DL3 mAb that blocks the binding of HHLA2 to KIR3DL3 in a T cell activation assay has been shown to be a checkpoint blocker. In another embodiment, the monoclonal antibody or antigen-binding fragment thereof specifically binds to KIR3DL3.

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

[0016] In one aspect, a bispecific antibody or antigen-binding fragment thereof is provided herein, the 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 an antigen-binding fragment thereof is provided, which comprises: a) one, two, or three heavy-chain CDR sequences each having at least about 95% identity with 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 with 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, a bispecific antibody or an antigen-binding fragment thereof is provided, which comprises: 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, a bispecific antibody or an antigen-binding fragment thereof is provided, which comprises: a) one, two, or three heavy-chain CDR sequences each 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 selected from the group consisting of the sequences listed in Tables 2 and 7-9.

[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 is (a) detectably labeled, (b) conjugated to a cytotoxic agent, optionally a chemotherapeutic agent, biological agent, toxin, and / or radioisotope, (c) includes an effector domain, (d) includes 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 the accession number ______. In still another embodiment, the bispecific antibody or antigen-binding fragment thereof inhibits (a) the binding of HHLA2 to KIR3DL3 and (b) the 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 includes (a) the heavy chain sequence listed in Table 9 and / or (b) the light chain sequence listed in Table 9.

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

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

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

[0026] In yet another aspect, there is provided a method of producing at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or antigen-binding fragments thereof) described herein, the method comprising: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding at least one according to the present disclosure under conditions suitable to permit 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 of detecting the presence or level of a KIR3DL3 polypeptide, the method comprising detecting the polypeptide in a sample using at least one antibody or antigen-binding fragment thereof described herein (e.g., a monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof). 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 the form of an enzyme-linked immunosorbent assay (ELISA), in the form of a radioimmunoassay (RIA), immunochemically, in the form of a Western blot, or using an intracellular flow assay.

[0028] In yet another aspect, a method of predicting responsiveness to a therapy targeting KIR3DL3, 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, bispecific antibody, or antigen-binding fragment thereof); b) determining the level of KIR3DL3 and / or HHLA2 in a sample from at least one control subject having a good responsiveness 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 the sample from the control subject, wherein a same or higher level of KIR3DL3 and / or HHLA2 in the subject sample as 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 described herein.

[0029] In yet another aspect, provided is a method of predicting responsiveness to a therapy targeting KIR3DL3 using at least one antibody or antigen-binding fragment thereof (e.g., monoclonal antibody, bispecific antibody, or 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 having a good responsiveness to a 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 the sample from the control subject, wherein the same or higher level of KIR3DL3 and / or HHLA2 in the subject sample as compared to the level in the sample from at least one control subject indicates that the subject will respond to the therapy.

[0030] As described 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 a pooled sample 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 obtained from a subject, serum, peritumoral tissue, and / or intratumoral tissue).

[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., monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof) described herein.

[0032] As described 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., monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof) described herein (a) reduces the number of proliferating cancer cells in cancer, (b) reduces the tumor volume or size of cancer, and / or (c) activates T cells and / or NK cells. In another embodiment, at least one antibody or antigen-binding fragment thereof (e.g., monoclonal antibody, bispecific antibody, or 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 a subject a therapeutic agent or regimen for treating cancer. In still another embodiment, the method described herein further comprises administering to a subject an additional therapy selected from the group consisting of immunotherapy, checkpoint blockade, cancer vaccine, chimeric antigen receptor (e.g., CAR targeting CD19), chemotherapy, radiation, targeted therapy, and surgery. In another embodiment, cancer cells and / or tumor-infiltrating immune 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 cancer, thymoma, B-CLL, leukemia, B cell lymphoma, and cancer infiltrated by immune cells expressing a receptor for HHLA2. In still 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 still another embodiment, the subject is a mammal, such as a humanized mouse or a human.

[0033] In another aspect, provided is a method of modulating an immune response using at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof described herein. For example, in one embodiment, the at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof described herein inhibits or disrupts the interaction between HHLA2 and KIR3DL3, its inhibitory receptor. In another embodiment, the at least one anti-KIR3DL3 antibody or an 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 still 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 an antigen-binding fragment thereof is a checkpoint inhibitor of T cell activation for cancer immunotherapy. In yet another embodiment, modulating the immune response includes modulating T cell function or NK cell function (e.g., cytotoxicity against cancer cells such as cancer cells expressing HHLA2). In still 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 cancer, thymoma, B-CLL, leukemia, B cell lymphoma, and cancers infiltrated by immune cells expressing a receptor 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 includes administering to the subject an additional therapy selected from the group consisting of immunotherapy, checkpoint blockade, cancer vaccine, chimeric antigen receptor (e.g., 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 a bar histogram, curve, or other data related to the description, the bars, curves, or other data presented from left to right for each indicator correspond directly and in order from top to bottom or from left to right in the box of the description. In embodiments of the present invention, for example, the following items are provided. (Item 1) A monoclonal antibody or an antigen-binding fragment thereof, a) having a heavy chain sequence with 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 an 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 an 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 an 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 an antigen-binding fragment thereof, a) having 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 an 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 an antigen-binding fragment thereof, a) one, two, or three heavy chain CDR sequences each selected from the group consisting of the sequences listed in Tables 2, 7, and 8, and / or b) a monoclonal antibody or an antigen-binding fragment thereof comprising one, two, or three light chain CDR sequences each selected from the group consisting of the sequences listed in Tables 2, 7, and 8. (Item 5) The monoclonal antibody or an antigen-binding fragment thereof according to any one of Items 1 to 4, wherein the monoclonal antibody or an antigen-binding fragment thereof is chimeric, humanized, bispecific, murine, or human. (Item 6) The monoclonal antibody or antigen-binding fragment thereof is (a) detectably labeled, (b) conjugated to a cytotoxic agent, optionally a chemotherapeutic agent, a biological agent, a toxin, and / or a radioisotope, (c) contains an effector domain, (d) contains 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, the monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 5. (Item 7) The monoclonal antibody or antigen-binding fragment thereof can be obtained from hybridoma ______ deposited under the accession number ______, the monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 6. (Item 8) The monoclonal antibody or antigen-binding fragment thereof inhibits the binding of HHLA2 to KIR3DL3, the monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 7. (Item 9) The monoclonal antibody or antigen-binding fragment thereof specifically binds to KIR3DL3, the monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8. (Item 10) A bispecific antibody or antigen-binding fragment thereof, a) a heavy chain sequence having at least about 95% identity with a heavy chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7 to 9, and / or b) a bispecific antibody or antigen-binding fragment thereof comprising a light chain sequence having at least about 95% identity with a light chain sequence selected from the group consisting of the sequences listed in Tables 2 and 7 to 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 with a heavy chain CDR sequence selected from the group consisting of the sequences listed in Tables 2 and 7 to 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 with a light chain CDR sequence selected from the group consisting of the sequences listed in Tables 2 and 7 to 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 to 9, and / or b) A bispecific antibody or an 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 an antigen-binding fragment thereof, a) One, two, or three heavy chain CDR sequences respectively selected from the group consisting of the sequences listed in Tables 2 and 7-9, and / or b) A bispecific antibody or an antigen-binding fragment thereof comprising one, two, or three light chain CDR sequences respectively selected from the group consisting of the sequences listed in Tables 2 and 7-9. (Item 14) The bispecific antibody or an antigen-binding fragment thereof according to any one of Items 10-13, wherein the bispecific antibody or an antigen-binding fragment thereof is chimeric, humanized, composite, murine, or human. (Item 15) The bispecific antibody or an antigen-binding fragment thereof according to any one of Items 10-14, wherein the bispecific antibody or an antigen-binding fragment thereof is (a) detectably labeled, (b) 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) The bispecific antibody or an antigen-binding fragment thereof according to any one of Items 10-15, wherein the bispecific antibody or an antigen-binding fragment thereof can be obtained from hybridoma ______ deposited under deposit accession number ______. (Item 17) The bispecific antibody or an antigen-binding fragment thereof according to any one of Items 10-16, wherein the bispecific antibody or an antigen-binding fragment thereof inhibits (a) the binding of HHLA2 to KIR3DL3 and (b) the binding of PD-1 to PD-L1 and / or PD-L2. (Item 18) The bispecific antibody or an antigen-binding fragment thereof according to any one of Items 10-17, wherein the bispecific antibody or an antigen-binding fragment thereof specifically binds to KIR3DL3 and PD-1. (Item 19) The bispecific antibody or an antigen-binding fragment thereof, a) comprises the heavy chain sequence listed in Table 9, and / or b) comprises the light chain sequence listed in Table 9, according to any one of Items 10-18. (Item 20) An immunoglobulin heavy chain and / or light chain selected from the group consisting of the immunoglobulin heavy chain and light chain sequences listed in Tables 2 and 7-9. (Item 21) (a) A nucleic acid encoding an immunoglobulin heavy chain, an immunoglobulin light chain, and / or an antibody or antigen-binding fragment thereof as described in any one of Items 1-20, and / or (b) A 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 with a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 2 and 7-9, and hybridizing under stringent conditions, an isolated nucleic acid molecule. (Item 22) A vector comprising the isolated nucleic acid described in Item 21. (Item 23) A host cell comprising the isolated nucleic acid described in Item 21, or comprising the vector described in Item 22, or expressing an antibody or antigen-binding fragment thereof as described in any one of Items 1-19, or available under the deposit accession number ______. (Item 24) A device or kit comprising at least one antibody or antigen-binding fragment thereof as described in any one of Items 1-19, 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) A method for producing at least one antibody or antigen-binding fragment thereof as described in any one of Items 1-19, the method comprising: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding at least one antibody as described in any one of Items 1-19 under conditions suitable for enabling expression of the antibody or antigen-binding fragment thereof; and (ii) recovering the expressed antibody or antigen-binding fragment thereof. (Item 26) A method for detecting the presence or level of a KIR3DL3 polypeptide, comprising detecting the polypeptide in a sample by use of at least one antibody or antigen-binding fragment thereof as described in any one of Items 1-19. (Item 27) The method according to item 26, wherein 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), in the form of radioimmunoassay (RIA), immunochemically, in the form of Western blot, or using an intracellular flow assay. (Item 28) A method for predicting responsiveness to a therapy targeting KIR3DL3, the method comprising: a) determining the levels 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 levels of KIR3DL3 and / or HHLA2 in a sample from at least one control subject having a good responsiveness to a therapy targeting KIR3DL3 using the at least one antibody or antigen-binding fragment thereof; c) comparing the levels of KIR3DL3 and / or HHLA2 in the subject sample with the levels of KIR3DL3 and / or HHLA2 in the sample from the control subject, wherein the same or higher levels of KIR3DL3 and / or HHLA2 in the subject sample compared to the levels in the sample from the at least one control subject indicate that the subject responds to the therapy. (Item 29) The method according to item 28, wherein the therapy targets KIR3DL3 using at least one antibody or antigen-binding fragment thereof according to any one of items 1 to 19. (Item 30) 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 levels of KIR3DL3 and / or HHLA2 in a subject sample; b) determining the levels of KIR3DL3 and / or HHLA2 in a sample from at least one control subject having a good responsiveness to a therapy targeting KIR3DL3; c) comparing the levels of KIR3DL3 and / or HHLA2 in the subject sample with the levels of KIR3DL3 and / or HHLA2 in the sample from the control subject, A method wherein a same or higher level of KIR3DL3 and / or HHLA2 in the test sample as compared to the level in the sample from at least one control subject indicates that the subject responds to the therapy. (Item 31) The method according to 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 a pooled sample obtained from at least one subject. (Item 32) The method according to any one of Items 28 to 31, wherein the therapy blocks (a) the interaction and / or signal transduction between HHLA2 and KIR3DL3, and / or (b) the interaction and / or signal transduction between PD-1 and PD-L1 and / or PD-L2. (Item 33) The method according to 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) The method according to Item 33, wherein the cells are T cells or natural killer (NK) cells. (Item 35) A method for 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) The method according to Item 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 the volume or size of the tumor of the cancer, and / or (c) activates T cells and / or NK cells. (Item 37) The method according to Item 35 or 36, wherein the at least one antibody or antigen-binding fragment thereof is administered in a pharmaceutically acceptable formulation. (Item 38) The method according to any one of Items 35 to 37, further comprising administering to the subject a therapeutic agent or regimen for treating cancer. (Item 39) The method according to 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) The method according to Item 39, wherein the chimeric antigen receptor targets CD19. (Item 41) The method according to any one of items 35 to 40, wherein cancer cells and / or tumor-infiltrating immune cells in the subject express HHLA2. (Item 42) The method according to 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 cancer, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancer infiltrated by immune cells expressing a receptor for HHLA2. (Item 43) The method according to item 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) The method according to any one of items 35 to 43, wherein the subject is an animal model of cancer. (Item 45) The method according to item 44, wherein the animal model is a mouse model, and optionally, the mouse model is a humanized mouse model. (Item 46) The method according to any one of items 35 to 45, wherein the subject is a mammal. (Item 47) The method according to item 46, wherein the mammal is a humanized mouse or a human. (Item 48) The method according to item 47, wherein the mammal is a human. (Item 49) A method of modulating an immune response using at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof. (Item 50) The method according to item 49, wherein the at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof inhibits or disrupts the interaction between HHLA2 and KIR3DL3, which is its binding inhibitor receptor. (Item 51) The method according to item 49 or 50, wherein the at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof is conjugated to a cytotoxic agent. (Item 52) The method according to item 51, wherein the cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes. (Item 53) The method according to any one of items 49 to 52, wherein the immune response is downregulated or upregulated. (Item 54) The method according to any one of items 49 to 53, wherein the at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof is described in any one of items 1 to 19. (Item 55) The method according to any one of Items 49 to 54, wherein 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) The method according to any one of Items 49 to 55, wherein the anti-KIR3DL3 antibody or antigen-binding fragment thereof is a checkpoint inhibitor for T cell activation for cancer immunotherapy. (Item 57) The method according to any one of Items 49 to 56, wherein regulating the immune response includes regulating T cell function or NK cell function. (Item 58) The method according to Item 57, wherein the T cell function or NK cell function includes cytotoxic activity. (Item 59) The method according to Item 58, wherein the cytotoxic activity is against cancer cells. (Item 60) The method according to Item 59, wherein the cancer cells express HHLA2. (Item 61) The method according to 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 cancer, thymoma, B-CLL, leukemia, B cell lymphoma, and cancer infiltrated by immune cells expressing a receptor for HHLA2. (Item 62) The method according to 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) The method according to 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) The method according to Item 63, wherein the chimeric antigen receptor targets CD19. (Item 65) The method according to any one of Items 49 to 64, wherein the immune response is regulated in an animal model of cancer. (Item 66) The method according to Item 65, wherein the animal model is a mouse model, and optionally, the mouse model is a humanized mouse model. (Item 67) The method according to any one of Items 49 to 66, wherein the immune response is regulated in a mammal. (Item 68) The method according to item 67, wherein the mammal is a humanized mouse or a human. (Item 69) The method according to item 68, wherein the mammal is a human.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0036] HHLA2, a B7 gene family member, is widely expressed in various tumors and antigen-presenting cells and is involved as both an activating ligand and an inhibitory ligand for T cells. TMIGD2, expressed on naive T cells, is an activating receptor for HHLA2 and transmits a co-stimulatory signal after T cell antigen receptor (TCR) ligation. TMIGD2 is downregulated after repeated TCR stimulation. HHLA2 binds to KIR3DL3, another receptor expressed on T cells and NK cells. As described herein, the present disclosure, unlike the immunostimulatory function of the HHLA2-TMIGD2 interaction, includes the recognition that the HHLA2-KIR3DL3 interaction can inhibit the immune response and thus provides an attractive target for modulation in various diseases, disorders or conditions, including 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 the immune response. Importantly, targeting KIR3DL3 does not disrupt the overall function of HHLA2, which includes activation of the immune response via interaction with TMIGD2. Thus, precisely targeting KIR3DL3 provides specificity to block only the immunosuppressive function of HHLA2, thereby inducing an effective immune response against cancer cells, for example, without downregulating the immunostimulatory 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 the immune response and / or treat cancer. In some embodiments, the KIR3DL3×PD-1 bispecific antibodies described herein are checkpoint immunotherapies for activating T cells and NK cells in tumors. In some embodiments, the KIR3DL3×PD-1 bispecific antibody is 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 KIR3DL3×PD-1 bispecific antibody checkpoint blockade.

[0039] A panel of exemplary, representative anti-KIR3DL3 human monoclonal antibodies (mAbs) is described herein as immunotherapy 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. 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 immunotherapy checkpoint inhibitor agents. Targeting two immune checkpoints with non-overlapping expression provides a combination therapy with additive or synergistic anti-tumor activity.

[0041] Accordingly, the present disclosure provides 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, and immunoglobulins, polypeptides, nucleic acids, and methods of using such antibodies for immunomodulatory and therapeutic purposes and the like.

[0042] I. Definitions The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, “an 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 as 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 as 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 increases or decreases, for example, in a certain pathological condition in a biological sample as compared to the activity of the marker in a normal control sample. The altered activity of a marker can be, for example, the result of altered expression of the marker, altered protein level of the marker, altered structure of the marker, or altered interaction of the marker with other proteins involved in the same or different pathways, for example, or altered interaction with a transcriptional activator or inhibitor.

[0045] The term "altered structure" of a marker refers to the presence of a mutation or allelic variant in a marker gene or marker protein, such as 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 be present in the coding or non-coding regions of the marker.

[0046] The term "activating receptor" includes immune cell receptors that bind to an antigen, a complex antigen (e.g., in the context of an MHC polypeptide), or an antibody. 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 (and by polyclonal T cell activation reagents) in the context of MHC polypeptides. Activation of T cells via the TCR results in a number of changes, such as 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 an engineered T cell receptor (TCR) with a 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 specialized antigen-presenting cells (APCs) that provide additional co-stimulatory signals. TCR activation in the absence of co-stimulation can lead to anergy and clonal anergy. To bypass immunization, different approaches have been developed to induce cytotoxic effector cells grafted with recognition specificity. CARs have been constructed consisting of a binding domain derived from a natural ligand or antibody specific for a cell surface component of the TCR-associated CD3 complex. Upon antigen binding, such chimeric antigen receptors link to the endogenous signaling pathways in effector cells and generate activation signals similar to those initiated by the TCR complex. For example, a CAR targeting CD19, a protein highly expressed on blood cancer cells, has shown good clinical efficacy. Since the first report on chimeric antigen receptors, this concept has been steadily improved, the molecular design of chimeric receptors has been optimized, and several well-known binding domains, such as scFV, Fab, and other protein binding fragments described herein, are commonly used.

[0049] Generally, CAR is a type of "cell therapy" (e.g., T cell therapy) contemplated for use according to the present disclosure. By modulating the KIR3DL3 pathway, for example, by modulating the interaction between KIR3DL3 and its natural binding partners such as HHLA2, numerous representative embodiments of agents and methods for modulating immune cell activity are encompassed, but immune cell-based therapies and methods are also encompassed. For example, T cells engineered to have a knockout, knockdown, or increased expression of KIR3DL3 are contemplated. Similarly, immune cells or other cells engineered to have a knockout, knockdown, or increased expression of KIR3DL3, ligands for HHLA2 are 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 induced by cross-linking of the receptor polypeptide by oligomeric or multimeric antigens. B cells may also be activated by anti-immunoglobulin antibodies. Upon BCR activation, numerous changes occur in B cells, including tyrosine phosphorylation.

[0051] Fc receptors are found on many cells involved in the immune response. Fc receptors (FcR) are cell surface receptors for the Fc portion of immunoglobulin polypeptides (Ig). Among the human FcRs that have been identified to date, there are those that recognize IgG (designated FcγR), IgE (FcεR1), IgA (Fcα), and polymeric IgM / A (FcμαR). FcRs are found on the following cell types: FcεRI (mast cells), FcεR.II (many leukocytes), FcαR (neutrophils), and FcμαR (glandular epithelium, hepatocytes) (Hogg, N. (1988) Immunol. Today 9:185-86). The widely studied FcγR is central to cellular immune defense and is involved in stimulating the release of inflammatory mediators and hydrolases that are involved in the development of autoimmune diseases (Unkeless, J.C. et al. (1988) Annu. Rev. Immunol. 6:251-81). Since macrophage / monocyte, polymorphonuclear leukocyte, and natural killer (NK) cell FcγRs confer a specific recognition element mediated by IgG, FcγRs provide an important link between effector cells and lymphocytes that secrete Ig. Human leukocytes have at least three different receptors for IgG: hFcγRI (found on monocytes / macrophages), hFcγRII (found on monocytes, neutrophils, eosinophils, platelets, probably B cells, and the K562 cell line), and FcγIII (found on NK cells, neutrophils, eosinophils, and macrophages).

[0052] Regarding T cells, the transmission of co-stimulatory signals to T cells involves a signaling pathway that is not inhibited by cyclosporin A. In addition, co-stimulatory signals can induce cytokine secretion (e.g., IL-2 and / or IL-10) in T cells and / or 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, such as KIR3DL3 and / or a KIR3DL3 natural binding partner, such as HHLA2, includes the activity inherent in 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 modulating inhibitory signals in immune cells (e.g., by engaging a natural receptor on the immune cell). One of ordinary skill 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.) on an immune cell to a polypeptide. Such a signal antagonizes signals through activating receptors (e.g., through TCR, CD3, BCR, TMIGD2, or an Fc polypeptide) and can result in, for example, inhibition of second messenger production, inhibition of proliferation, inhibition of effector functions in immune cells, such as reduced phagocytosis, reduced antibody production, reduced cytotoxicity, the inability of immune cells to produce mediators (such as cytokines (e.g., IL-2) and / or allergy response mediators, etc.), or the induction of anergy.

[0055] The amount of biomarker in the subject is "significantly" higher or lower than the normal amount of the biomarker if the amount of the biomarker is, respectively, higher or lower than the normal level or control level by an amount exceeding the standard error of the assay used to assess the amount, preferably by 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 biomarker in the subject can be considered "significantly" higher or lower than the normal amount and / or control amount of the biomarker if the amount is, respectively, higher or lower than the normal amount and / or control amount of the biomarker by 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%, 195%, 2-fold, 3-fold, 4-fold, 5-fold, or more, or any range between them such as 5%-100%. Such significant regulatory values can be applied to any of the measurement criteria described herein, such as changed expression levels, changed activities, changes in cancer cell overproliferative growth, changes in cancer cell death, changes in biomarker inhibition, changes in test agent binding, etc.

[0056] The term "altered expression level" of a marker refers to the expression level or copy number of a marker in a test sample, e.g., a sample derived from a subject suffering from cancer, that is higher or lower than the standard error of the assay used to evaluate expression or copy number, 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 the related disease), preferably the average expression level or copy number of the marker or chromosomal region in several control samples. The altered expression level is higher or lower than the standard error of the assay used to evaluate 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 the related disease).

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

[0058] As used herein, the term "antibody" includes the "antigen-binding portion" (or simply "antibody portion") of the 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 included within the scope of the term "antigen-binding portion" of an antibody include (i) monovalent fragments consisting of a Fab fragment, VL domain, VH domain, CL domain, and CH1 domain, (ii) F(ab')2 fragments, divalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region, (iii) Fd fragments consisting of a VH domain and a CH1 domain, (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546), and (vi) isolated complementarity determining regions (CDRs). Further, although the two domains VL and VH of an Fv fragment are encoded by separate genes, they can be linked by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide (known as a single-chain Fv (scFv), see, for example, 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 included within the scope of the term "antigen-binding portion" of an antibody. Any VH and VL sequences of a specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences to generate an expression vector encoding a full IgG polypeptide or other isotype. VH and VL can also be used in the production of Fab fragments, Fv fragments, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology.Single-chain antibodies and other forms such as diabodies are also included. A diabody is expressed with a VH domain and a VL domain on a single polypeptide chain, using a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing these domains with the complementary domains of another chain to create two antigen-binding sites, a bivalent bispecific antibody (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, Poljak, R.J., et al. (1994) Structure 2:1121-1123).

[0059] Furthermore, the antibody or its antigen-binding portion can be part of a larger immunoadhesin polypeptide formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesin polypeptides include the use of the streptavidin core region to create a tetrameric scFv polypeptide (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101), as well as the use of cysteine residues, a marker peptide, and a C-terminal polyhistidine tag to create a bivalent biotinylated scFv polypeptide (Kipriyanov, S.M., 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 digestion or pepsin digestion of the whole antibody, respectively. Additionally, antibodies, antibody portions, and immunoadhesin polypeptides can be obtained using the standard recombinant DNA techniques described herein.

[0060] The antibodies can be polyclonal or monoclonal, heterologous, homologous, or syngeneic, or modified forms thereof (e.g., humanized, chimeric, etc.). The antibodies may be fully human. In one embodiment, the antibodies encompassed by the present disclosure specifically or substantially specifically bind to the KIR3DL3 polypeptide or fragments thereof. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one antigen-binding site capable of immunoreacting with a specific epitope of an antigen, and 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 specific antigen. Monoclonal antibody compositions typically exhibit a single binding affinity for the specific antigen to which they immunoreact.

[0061] The term "body fluid" refers to fluids excreted or secreted from the body, as well as fluids that are not normally excreted or secreted from the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, cerumen and earwax, Cowper's fluid or pre-ejaculatory semen, 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 lubricant, vitreous humor, vomit).

[0062] The terms "cancer", "tumor", or "hyperproliferative disorder" refer to the presence of cells having characteristics specific to cancer-causing cells, such as uncontrolled growth, immortality, metastatic ability, rapid growth and proliferation rates, and certain specific morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone in an animal or can be non-tumor-forming cancer cells such as leukemia cells. Cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenström macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, as well as immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach 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, etc.Other non-limiting examples of types of cancer applicable to the methods encompassed by this disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias, e.g., acute lymphocytic leukemia and acute myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is inherently an epithelial carcinoma, and the cancer includes, but is 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 still other embodiments, the epithelial carcinoma is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. The epithelial carcinoma can be characterized in various other ways including, but not limited to, serous, endometroid, mucinous, clear cell, Brenner, or undifferentiated.

[0063] The term "CDR" and its plural form "CDRs" refer to Complementary Determining Regions (CDRs) which consist of three that constitute the binding characteristics of the light chain variable regions (CDR-L1, CDR-L2, and CDR-L3) and three that, for example, constitute the binding characteristics of the heavy chain variable regions (CDR-H1, CDR-H2, and CDR-H3) on an antibody. CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences that include the scaffold or framework regions. Precise definitions of CDR boundaries and lengths are assigned to different classification and numbering systems. Thus, CDRs can be referred to by Kabat, Chothia, contact, or any other boundary definition. Despite 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 can differ in terms of length and boundary regions with respect to the adjacent framework regions. For example, see Kabat, Chothia, and / or MacCallum et al. (Kabat et al., "Sequences of Proteins of Immunological Interest," 5 th th Edition, U.S. 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), each incorporated by reference in its entirety).

[0064] As used herein, the term "classifying" includes "associating" a sample with a medical condition or "categorizing" a sample by a medical condition. In certain instances, "classifying" is based on statistical evidence, experimental evidence, or both. In certain embodiments, the methods and systems for classifying use a training set of samples having so-called known medical conditions. Once established, the training data set functions as a basis, model, or template against which the characteristics of an unknown sample are compared to classify the unknown medical condition of the sample. In certain instances, classifying a sample is similar to diagnosing the medical condition of the sample. In certain other instances, classifying a sample is similar to differentiating the medical condition of the sample from another medical condition.

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

[0066] The term "complement to" 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 a specific hydrogen bond ("base pairing") with a residue in a second nucleic acid region that is antiparallel to the first region when 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 that is antiparallel to the first strand when the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if at least one nucleotide residue of the first region can base pair with a residue of the second region when the two regions are arranged in an antiparallel manner. 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 of the first portion can base pair with the nucleotide residues of the second portion. In another embodiment, all of the nucleotide residues of the first portion can base pair with the nucleotide residues of the second portion.

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

[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, the control includes obtaining a "control sample" in which the expression product level is detected and compared with the expression product level from the test sample. Such control samples include samples from control cancer patients with known outcomes (which can be stored samples or prior sample measurements), normal tissues or cells isolated from subjects such as healthy patients or cancer patients, cultured primary cells / tissues isolated from subjects such as healthy subjects or cancer patients adjacent to normal cells / tissues obtained from the same organ or body location of a cancer patient, tissue or cell samples isolated from healthy subjects, or primary cells / tissues obtained from a depository, and can include any suitable sample, not limited to these. In another preferred embodiment, the control includes reference standard expression product levels from any suitable source, including but not limited to housekeeping genes, the range of expression product levels from normal tissue (or other previously analyzed control samples), the range of expression product levels previously determined in test samples from a group of patients having a particular outcome (e.g., survival period of 1, 2, 3, 4 years, etc.) or receiving a particular treatment (e.g., standard cancer therapy). One of ordinary skill in the art will understand that such control samples and reference standard expression product levels can be used in combination with the methods encompassed by the present disclosure as controls. In one embodiment, the control can include normal or non-cancerous cell / tissue samples. In another preferred embodiment, the control can include the expression levels of a group of patients, e.g., a group of cancer patients, or a group of cancer patients receiving a particular treatment, or the expression levels of a group of patients having one outcome versus another outcome. In the former case, the specific expression product level of each patient can be assigned to a percentile level of expression or represented as either higher or lower than the average value or average of the reference standard expression level. In another preferred embodiment, the control can include normal cells, cells from patients treated with combination chemotherapy, and cells from patients with benign cancer. In another embodiment, the control can also include a measured value, e.g., the average expression level of a particular gene in the same population compared to the expression level of a housekeeping gene in the population.Such a group may include healthy subjects, cancer patients who have not received any treatment (i.e., untreated), cancer patients who have received standard treatment therapies, or patients with benign cancer. In another preferred embodiment, the control determines the ratio of the expression product levels of two genes in a test sample and compares it to any suitable ratio of the same two genes in a reference standard, determines the expression product levels of two or more genes in a test sample and determines the difference in the expression product levels in any suitable control, and determines the expression product levels of two or more genes in a test sample, normalizes their expression to the expression of a housekeeping gene in the test sample, and compares it to any suitable control, including, but not limited to, ratio conversion of expression product levels. In a particularly preferred embodiment, the control includes a control sample that is of the same strain and / or type as the test sample. In another embodiment, the control may include expression product levels that are grouped as or based on percentiles in a set of patient samples such as all patients with cancer. In one embodiment, control expression product levels are established and, for example, higher or lower expression product levels compared to a particular percentile are used as a basis for predicting outcome. In another preferred embodiment, control expression product levels are established using expression product levels from cancer control patients with known outcomes, and expression product levels from test samples are compared to the control expression product levels as a basis for predicting outcome. As demonstrated by the following data, the methods encompassed by the present disclosure are not limited to the use of a particular cut point when comparing expression product levels in a test sample to a control.

[0069] The term "co-stimulate", when used with respect to activated immune cells, includes the ability of a co-stimulatory polypeptide to provide a second non-activating receptor-mediated signal ("co-stimulatory signal") that induces proliferation or effector function. For example, the co-stimulatory signal can result in cytokine secretion, for example, in T cells that have 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, such as CD28. As used herein, the term "inhibitory receptor" includes receptors that transmit negative signals to immune cells (e.g., CTLA4, KIR3DL3, or PD-1). Inhibitory signals transmitted by inhibitory receptors can occur even when costimulatory receptors (such as CD28) are not present on the immune cell and thus are not simply a function of competition between the inhibitory receptor and the costimulatory receptor for binding of the 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 that can be characterized using techniques known in the art. Apoptotic cell death can be characterized, for example, by cell shrinkage, membrane blebbing, and chromatin condensation that ultimately lead to cell fragmentation. Cells undergoing apoptosis also exhibit a characteristic pattern of internucleosomal DNA fragmentation. Depending on the form of the polypeptide that binds to the receptor, a signal can be transmitted (e.g., by a multivalent form of the HHLA2 and / or KIR3DL3 polypeptide) or inhibited (e.g., by a soluble monovalent form of HHLA2 and / or KIR3DL3) by competing, for example, for binding to one or more natural binding partners. However, there are also examples where the soluble polypeptide can be stimulatory. The effect of the modulator can be readily demonstrated using the screening assays described herein as a general rule.

[0071] The term "determining a treatment regimen suitable for a subject" is considered to mean the determination of a subject's treatment regimen (i.e., a single therapy or a combination of different therapies used for the prevention and / or treatment of cancer in a subject) that is initiated, changed, and / or terminated based on or essentially based on or at least in part based on the results of the analysis according to the present disclosure. An example is determining whether to provide a targeted therapy for cancer to provide an 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 is initiating an adjuvant therapy after surgery aimed at reducing the risk of recurrence, and another example would be changing the dosage of a particular chemotherapy. The determination can be based on the individual characteristics of the subject being treated in addition to the results of the analysis according to the present disclosure. In many cases, the actual determination of a treatment regimen suitable for a subject will be made by an attending physician or a doctor.

[0072] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes the native sequence Fc region and variant Fc regions. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. Native sequence Fc regions suitable for use in the 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. Preferred FcRs are native sequence human FcRs. Further, preferred FcRs are those that bind to 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), and the FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that mainly differ in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory 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 to be identified in the future, are also encompassed by the term "FcR" as used herein.

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

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

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

[0077] As used herein, the term "heterologous antibody" is defined in the context of a transgenic non - human organism that produces such an antibody. This term does not refer to a transgenic non - human animal per se, but rather to an antibody having an amino acid sequence or a coding nucleic acid sequence corresponding to one found in an organism generally from a species other than the transgenic non - human animal species.

[0078] The terms "high," "low," "intermediate," and "negative" as related to cell biomarker expression refer to the amount of biomarker expressed as compared to the cell expression of the biomarker by one or more reference cells. Biomarker expression can be determined according to any method described herein, including, but not limited to, analysis of the cell level, activity, structure, etc. of one or more biomarker genomic nucleic acids, ribonucleic acids, and / or polypeptides. In one embodiment, these terms refer to defined percentages of cell populations that express the biomarker at the highest level, intermediate level, or lowest level, respectively. Such percentages can 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, or any range (including boundary values) therebetween of cell populations that express the biomarker either highly or weakly. The term "low" excludes cells that do not detectably express the biomarker, as such cells are "negative" for biomarker expression. The term "intermediate" includes cells that express the biomarker but at a level lower than the population that expresses it at the "high" level. In another embodiment, these terms can also refer to, or alternatively refer to, cell populations of biomarker expression identified by qualitative or statistical plot regions. For example, cell populations sorted using flow cytometry can be distinguished based on biomarker expression levels by identifying separate plots based on analysis of detectable moieties, such as, for example, mean fluorescence intensity, according to methods well known in the art. Such plot regions can be refined according to number, shape, overlap, etc. based on methods well known in the art for the biomarker of interest. In yet another embodiment, these terms may be determined according to the presence or absence of the expression of additional biomarkers.

[0079] As used herein, "homologous" refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When the nucleotide residue positions in both regions are occupied by the same nucleotide residue, those regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position in each of the regions 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, a first region includes a first portion and a second region includes 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 of each of those portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of those 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 to not only a particular target cell but also the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not actually be identical to the parental cell, but are still included within the scope of the present term as used herein.

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

[0082] As used herein, a humanized mouse is a mouse that has 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 mice, NOG mice, and NSG mice can be used to more efficiently engraft human cells and tissues than other models. Such humanized mouse models can be used to model the human immune system in healthy and pathological scenarios and can enable the evaluation of therapeutic candidates in in vivo situations relevant to human physiology.

[0083] As used herein, the terms "hypervariable region", "HVR", or "HV" refer to regions of the 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 cells that play a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes such as B cells and T cells, natural killer cells, and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

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

[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 such as cytokine production and cytotoxicity. In addition, the term "immune response" includes immune responses indirectly brought about by T cell activation such as antibody production (humoral response) and activation of cytokine-responsive cells such as macrophages.

[0087] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate the host immune system to bring about an immune response against a tumor or cancer in a subject. Various 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 finely tune the immune response by downregulating or inhibiting the anti-tumor immune response. 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 meets the homology description 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 having PD-L1 and PD-L2 as known ligands. PD-1 was previously identified using a cloning-based subtraction approach to select genes that were 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 PD-L1. Similar to CTLA-4, PD-1 is rapidly induced on the surface of T cells in response to anti-CD3 (Agata et al. 25 (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 subsets of thymocytes and myeloid cells (Agata et al. (1996) (supra), Nishimura et al. (1996) Int. Immunol. 8:773).

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

[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., the binding of HHLA2 to TMIGD2 or the binding of HHLA2 to KIR3DL3). Generally, such an interaction results in the activation of one or both of the molecules (thereby resulting in a biological effect). The activation can be the direct activation (e.g., signal transduction) of one or both of those molecules. Alternatively, one or both of the molecules in the interaction may be prevented from binding to their ligands and are thus kept inactive with respect to ligand binding activity (e.g., binding to its ligand and inducing or inhibiting an immune response). By inhibiting such an interaction, disruption of the activity of one or more of the molecules involved in the interaction is brought about. Enhancing such an interaction is extending or increasing the likelihood of such physical contact and extending or increasing the likelihood of such activity.

[0092] The term "neoadjuvant therapy" refers to a treatment administered prior to primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiotherapy, 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 having 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, such as other animals like humans and non-rodent animals, or other mammalian or non-mammalian species. However, in some embodiments, the antibody maintains a higher or actually specific affinity and selectivity for human KIR3DL3. In addition, an isolated antibody typically is substantially free of other cellular materials and / or chemical substances. In one embodiment encompassed by the present disclosure, combinations of "isolated" monoclonal antibodies having different specificities for human KIR3DL3 are combined in a defined composition.

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

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

[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 present invention disclosed can be measured or determined by standard antibody-antigen assays, such as competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA.

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

[0098] A "marker" or "biomarker" is a gene or protein whose change in expression level in a given tissue or cell from its expression level in normal or healthy tissue or cells is associated with a medical condition such as cancer. A "marker nucleic acid" is a nucleic acid (e.g., mRNA, cDNA) that is encoded by or corresponds to a marker encompassed by the present disclosure. Such marker nucleic acids include DNA (e.g., cDNA) that includes the entire sequence or a partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing, or a complement of such a sequence. Marker nucleic acids also include RNA that includes the entire sequence or a partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing, or a complement of such a sequence, with all thymidine residues replaced by uridine residues. A "marker protein" is a protein that is encoded by or corresponds to a marker encompassed by the present disclosure. Marker proteins include the entire sequence or a partial sequence of any of the sequences set forth in the Sequence Listing. In some embodiments, the entirety of 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 synonymously.

[0099] As used herein, the term "modulate" includes up-regulation and down-regulation, e.g., enhancing or inhibiting a response.

[0100] The term "predetermined" biomarker amount and / or activity measurement can be, by way of example only, for evaluating a subject who may be selected for a particular treatment, one or more modulators of the KIR3DL3 pathway such as a KIR3DL3 modulator, and / or one or more natural binding partners such as HHLA2, for evaluating the response to treatment either alone or in combination with one or more immunotherapies, and / or for evaluating a disease state, and can be the biomarker amount and / or activity measurement used. The predetermined biomarker amount and / or activity measurement can be determined in a patient population with cancer or a patient population without cancer. The predetermined biomarker amount and / or activity measurement can be a single value that is equally applicable to all patients, or the predetermined biomarker amount and / or activity measurement can vary by a particular patient subset. The age, weight, height, and other factors of a subject can affect the predetermined biomarker amount and / or activity measurement of that individual. Further, the predetermined biomarker amount and / or activity can be determined individually for each subject. In one embodiment, the amount determined and / or compared by the methods described herein is based on an absolute measurement. In another embodiment, the amount determined and / or compared by the methods described herein is based on a relative measurement such as a ratio (e.g., a cell ratio, or a serum biomarker normalized to the expression of a housekeeping or otherwise generally constant biomarker). The predetermined biomarker amount and / or activity measurement can be any suitable criterion. For example, the predetermined biomarker amount and / or activity measurement can be obtained from the same or different humans from whom patient selection is being evaluated. In one embodiment, the predetermined biomarker amount and / or activity measurement can be obtained from a previous evaluation of the same patient. In such a manner, the course of a patient's selection can be monitored over time. Additionally, the control can be obtained from the evaluation of another human or multiple humans, e.g., if the subject is human, from the evaluation of a selected group of humans. In such a manner, the degree of selection of the humans for whom selection is being evaluated can be compared to other humans in a similar situation to the intended humans, e.g., humans suffering from a similar or the same condition and / or humans of the same ethnic group.

[0101] The term "predictive" refers to the use of pre-, during-, or post-treatment biomarker nucleic acid and / or protein states, such as over- or under-activity, appearance, expression, growth, remission, recurrence, or tumor resistance, to determine the likelihood of cancer response to immunomodulatory therapies such as KIR3DL3 pathway modulator therapies (e.g., either alone or in combination with one or more additional therapies such as immunotherapy, e.g., immune checkpoint inhibitor therapy, etc., a modulator of the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2). Such predictive use of biomarkers can be determined, for example, by (1) increased or decreased copy number (e.g., by FISH, FISH+SKY, single molecule sequencing, e.g., as described at least in J. Biotechnol., 86:289-301 in the art, or by qPCR), overexpression or underexpression of biomarker nucleic acid (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., about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, over 100%, or more of an assayed human cancer type or cancer sample), (2) the absolute or relatively regulated presence or absence thereof in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal swab, saliva, cerebrospinal fluid, urine, feces, or bone marrow from a subject having cancer, e.g., a human, and (3) the absolute or relatively regulated presence or absence thereof in a clinical subset of patients having cancer (e.g., patients responsive to a particular immunomodulatory therapy (e.g., a KIR3DL3 pathway modulator therapy (e.g., either alone or in combination with immunotherapy, a modulator of the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2)), or patients developing resistance thereto).

[0102] The terms "prevent", "preventing", "prevention", "preventive treatment", etc. 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 is at risk of developing it or is susceptible to it.

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

[0104] The term "polypeptide fragment" or "fragment", when used with respect 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 usually identical to the corresponding position in the reference polypeptide. Such deletions can occur internally at the amino terminus of the reference polypeptide, at its carboxyl terminus, or alternatively at 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 can 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, 620, 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 in length, and / or can include it, provided they are less than the length of the full-length polypeptide. Alternatively, they can be outside of and / or exclude such ranges, provided 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 specifically intended target molecule, e.g., a nucleotide transcript or protein encoded by or corresponding to a marker. A probe can be either synthesized by one of ordinary skill in the art or derived from a suitable biological preparation. For the detection of a target molecule, a probe can be specifically designed to be labeled as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0106] As used herein, the term "rearranged" refers to the configuration of a heavy or light chain immunoglobulin locus in which the V segments are positioned directly adjacent to a D-J or J segment in a conformation that essentially encodes the V H and V L domains. A rearranged immunoglobulin locus can be identified by comparison to germline DNA, and the rearranged locus has 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 also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, 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 an immunomodulatory therapy (i.e., being non-responsive to a therapeutic treatment or having a reduced or limited response to a therapeutic treatment), for example, having a response that is 5% or more, for example, 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 compared to the same cancer sample or mammal before resistance is acquired, or compared to a different cancer sample or mammal known not to have resistance to the therapeutic treatment. A typical acquired resistance to chemotherapy is called "multi-drug resistance". Multi-drug resistance can be mediated by the P-glycoprotein or by other mechanisms, or can occur when a mammal is infected with a multi-drug resistant microorganism or combination of microorganisms. Determination of resistance to a therapeutic treatment is routine in the art and within the skill of the artisan, and can be measured, for example, by the cell proliferation assays and cell death assays described herein as "sensitization". In some embodiments, the term "reversing resistance" means that the use of a second agent in combination with a primary cancer therapy (e.g., chemotherapy or radiation therapy) results in a statistically significant decrease in tumor volume at a statistically significant level (e.g., p<0.05) compared to the tumor volume of tumors not being treated, in a situation where the primary cancer therapy (e.g., chemotherapy or radiation therapy) alone cannot result in a statistically significant decrease in tumor volume compared to the tumor volume of tumors not being treated. This generally applies to tumor volume measurements taken when untreated tumors are growing logarithmically.

[0109] As described above, the term "response" is generally related to, for example, determining the course, effectiveness, or outcome of a clinical intervention. For example, a response to a therapy (e.g., a KIR3DL3 pathway modulator therapy (e.g., either alone or in combination with an immunotherapy such as immune checkpoint inhibitor therapy, a modulator of the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2)) is, with respect to any response to a therapy (e.g., a KIR3DL3 pathway modulator therapy (e.g., either alone or in combination with an immunotherapy such as immune checkpoint inhibitor therapy, a modulator of the interaction between KIR3DL3 and one or more natural binding partners such as KIR3DL3)), in the case of cancer, preferably related to the change in the number of cancer cells, tumor mass, and / or volume after the initiation of neoadjuvant or adjuvant chemotherapy. The hyperproliferative disorder response can be evaluated, for example, with respect to effectiveness or in a neoadjuvant or adjuvant setting, and the tumor size after a systemic intervention can be compared to the initial size and dimensions measured by CT, PET, mammogram, ultrasound, or palpation. The response can also be evaluated by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. The response can be recorded in a quantitative manner such as the percentage change in tumor volume or in a qualitative manner such as "pathological complete remission" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. The evaluation of the hyperproliferative disorder response can be performed early after the initiation of neoadjuvant or adjuvant therapy, for example, hours, days, weeks, or preferably months later. A typical endpoint for response evaluation is at the end of neoadjuvant chemotherapy or at the time of surgical removal of residual tumor cells and / or the tumor bed. This is typically 3 months after the start of neoadjuvant therapy. In some embodiments, the clinical effectiveness of the therapeutic treatment described herein can be determined by measuring the clinical benefit rate (CBR).The clinical utility rate is measured by determining the percentage of patients in complete remission (CR) at least 6 months after the end of therapy, the number of patients in partial remission (PR), and the total number of patients with stable disease (SD). An abbreviated expression of 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 evaluating the response to cancer therapy are related to "survival period", which includes the survival period until death, also known as the overall survival period (the death can be due to any cause or be tumor-related), "recurrence-free survival period" (the term recurrence is to include both local and distant recurrences), metastasis-free survival period, and disease-free survival period (the term disease is to include cancer and related diseases). The length of the survival period can be calculated by referring to a defined starting point (e.g., at diagnosis or at the start of treatment) and an end point (e.g., death, recurrence, or metastasis). Additionally, the criteria for treatment effectiveness can be expanded to include the response to chemotherapy, the probability of survival, the probability of metastasis within a given period, and the probability of tumor recurrence. For example, to determine appropriate thresholds, a particular cancer treatment regimen can be administered to a subject population, and the outcome can be correlated with biomarker measurements determined prior to the administration of any immunomodulatory therapy. The outcome measurement can be the pathological response to therapy administered in a neoadjuvant setting. Alternatively, for subjects after an immunomodulatory therapy where the biomarker measurements are known, outcome measurements such as overall survival and disease-free survival can be monitored over a period of time. In certain embodiments, the dose administered is the standard dose of a cancer therapeutic agent known in the art. The period for which the subject is monitored can vary. For example, the subject can 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 given antigen. Typically, an antibody binds with an affinity (K -7 ) of less than about 10 -8 M, e.g., less than about 10 -9 M, 10 -10 M, or less than 10 D M, or with an even lower affinity, as determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using human KIR3DL3 as the analyte and the antibody as the ligand, and binds to the given 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, or more, higher than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the given antigen or an antigen closely related thereto. 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 targeted condition (e.g., cancer). The term "subject" is synonymous with "patient". In some embodiments, the term is intended to include living organisms in which an immune response can be induced. Representative non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

[0112] As used herein, the term "survival period" includes all of the survival period until death, also known as the overall survival period (said death may be due to any cause or may be tumor-related), "recurrence-free survival period" (the term recurrence shall include both local recurrence and distant recurrence), metastasis-free survival period, and disease-free survival period (the term disease shall include cancer and related diseases). The length of the survival period can be calculated by referring to a defined starting point (e.g., at the time of diagnosis or the start of treatment) and an end point (e.g., death, recurrence, or metastasis). Additionally, the criteria for treatment effectiveness can be expanded to include response to chemotherapy, survival probability, metastasis probability within a given period, and tumor recurrence probability.

[0113] The terms "tolerance" or "unresponsiveness" include the refractivity of cells such as immune cells to stimuli, e.g., stimuli by activating receptors or cytokines. Unresponsiveness can occur, for example, by exposure to immunosuppressive agents or exposure to high doses of antigens. Several independent methods can cause tolerance. One mechanism is termed "anergy" and is defined as a state in which cells survive in vivo as non-responsive cells rather than differentiating into cells with effector functions. Such refractivity is generally antigen-specific and persists after the exposure to the tolerizing antigen has ended. For example, anergy in T cells is characterized by the lack of cytokine production such as IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive the first signal (T cell receptor or CD-3 mediated signal) in the absence of the second signal (co-stimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if the re-exposure occurs in the presence of a co-stimulatory polypeptide) fails to produce cytokines and, therefore, fails 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 measured by ELISA or a proliferation assay using an indicator cell line. Alternatively, a reporter gene construct 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 multimerization of AP1 sequences 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 many chronic infections and cancer development. This is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from functional effector or memory T cells.

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

[0115] As used herein, the term "T cell" includes CD4+ T cells and CD8+ T cells. The term T cell also includes both type 1 T helper cells and type 2 T helper cells. The term "antigen-presenting cell" 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.) for enhancing the immune response by expression of one or more T cell receptors. Tcon or Teff is generally defined as any T cell population that is not a Treg, and includes, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated into, for example, the Th1 or Th2 lineage. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is CD4+ Teff or CD8+ Teff, 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 that has differentiated in the bone marrow and successfully undergone positive and negative central selection processes in the thymus but has not been activated by exposure to an antigen. +They are T cells. Naive Tcon are generally characterized by surface expression of L-selectin (CD62L), absence of activation markers such as CD25, CD44, or CD69, and absence of memory markers such as CD45RO. Thus, naive Tcon are in a resting state, are non-dividing, and are thought to 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 are undesirable in the context of suppression of the immune response. Unlike Treg, Tcon are not anergic and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. B Biol. Sci. 356:625-637). In tumors, exhausted cells may exhibit characteristics of anergy.

[0116] As used herein, the terms "not rearranged" or "germline configuration" with respect to a V segment refer to a configuration in which the V segment is not rearranged to be directly adjacent to a D segment or a 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) produced against KIR3DL3. Such molecules are characterized, in part, by their ability to recognize the KIR3DL3 protein in diagnostic assays, such as immunohistochemical (IHC), Western blot, flow cytometry, ELISA, etc. Such molecules are characterized, in part, by their ability to inhibit the binding of KIR3DL3 to binding partners such as HHLA2.

[0118] The term "HHLA2", its aliases, human endogenous retrovirus H long terminal repeat-associated protein 2, HERV-H LTR-associated 2, B7y, B7H7, B7-H5, B7-H7 refers to a member of the B7 family. The HHLA2 protein has limited expression in normal human tissues but is widely expressed in human cancers. While the 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). The HHLA2 protein in normal human tissues is expressed in the epithelium of the kidney, intestine, gallbladder, and breast, as well as in placental trophoblast cells. In the immune system, the 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 production and cytokine production. HHLA2 is expressed at higher levels in a wide range of human cancers derived from the colon, rectum, kidney, lung, pancreas, ovary, and prostate. 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 HHLA2 are well known in the art (see, for example, 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. U.S.A. 110:9879-9884, and Zhu 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 are 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 the use of an alternative promoter and having a different 5'UTR compared to Variant 1), Variant 3 (NM_001282557.1 and NP_001269486.1, representing the use of an alternative promoter and having a different 5'UTR compared to Variant 1), Variant 4 (NM_001282558.1 and NP_001269487.1, encoding isoform b, representing the use of an alternative promoter, having a different 5'UTR compared to Variant 1, lacking an alternative in-frame exon within the 3' coding region, and resulting in an isoform shorter than isoform a), and Variant 5 (NM_001282559.1 and NP_001269488.1, encoding isoform c, representing the use of an alternative promoter, having multiple differences compared to Variant 2, resulting in a distinct 5'UTR, starting translation with an alternative start codon compared to Variant 1, resulting in a distinct N-terminus and an isoform shorter than isoform a). Nucleic acid and polypeptide sequences of HHLA2 orthologs in organisms other than human are well known, and for example, Xenopus HHLA2 (NM_001128644.1 and NP_001122116.1) is included. Representative sequences of HHLA2 orthologs are presented in Table 1 below.

[0121] Anti-HHLA2 antibodies suitable for the detection of HHLA2 protein 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), etc.Furthermore, a plurality of siRNAs, shRNAs, CRISPR constructs for reducing HHLA2 expression, 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, iAAV00961608, 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), etc. can be found in the commercial product lists of the above companies. It should be noted that this term can be further used to refer to any combination of the features described herein with respect to the HHLA2 molecule. For example, any combination of sequence composition, percentage 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 the interaction of HHLA2 and one or more of its natural binding partners such as TMIGD2 and KIR3DL3.

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

[0124] The term "TMIGD2" refers to a transmembrane and immunoglobulin domain containing 2, CD28H, IGPR1, and IGPR-1, which is a membrane protein having about 10% amino acid identity with 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. The TMIGD2 protein is constitutively expressed on most naive T cells and the majority of natural killer (NK) cells, but not constitutively expressed on T regulatory cells or B cells. TMIGD2 expression is slowly lost upon repeated stimulation of T cells. Consistent with this, TMIGD2 is expressed in only about half of memory T cells, and TMIGD2-negative T cells have a terminally differentiated senescent phenotype. It has also been shown that TMIGD2 is expressed in endothelial and epithelial cells and functions to reduce cell migration and promote capillary formation during angiogenesis.

[0125] The structure and function of certain TMIGD2 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 its fragments, variants (e.g., allelic variants), and derivatives. Representative human TMIGD2 cDNA and human TMIGD2 protein sequences are well known in the art and are 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 uses an alternative in-frame splice site within the 3' coding region compared to variant 1, resulting in an isoform shorter than isoform 1), and isoform 3 (NM_001308232.1 and NP_001295161.1, which lacks an alternative in-frame exon within the 5' coding region compared to variant 1, resulting in an isoform shorter than isoform 1). Nucleic acid and polypeptide sequences of TMIGD2 orthologs in organisms other than human are well known, and for example, include chimpanzee TMIGD2 (XM_009434393.2 and XP_009432668.2, as well as XM_001138228.4 and XP_001138228.3), and bovine TMIGD2 (XM_005208980.3 and XP_005209037.1, XM_005208979.3 and XP_005209036.1, as well as XM_002688933.5 and XP_002688979.1). Representative sequences of TMIGD2 orthologs are presented in Table 1 below.

[0127] Anti-TMIGD2 antibodies suitable for the detection of 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), etc.Furthermore, multiple siRNAs, shRNAs, CRISPR constructs for reducing TMIGD2 expression, 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, iAfAV02491407, iAAV02491408, and iAAV02491409, and 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, and 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), etc. can be found in the commercial product lists of the above companies.Furthermore, multiple CRISPR constructs for increasing TMIGD2 expression, 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), etc., can be found in the commercial product lists of the above companies. Note that this term can be further used to refer to any combination of the features described herein with respect to the TMIGD2 molecule. For example, any combination of sequence composition, percentage identity, sequence length, domain structure, functional activity, etc. can be used to describe the TMIGD2 molecules encompassed by the present disclosure.

[0128] The interaction between TMIGD2 and HHLA2 described above, 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", alias killer cell immunoglobulin-like receptor 3DL3, CD158Z, KIR3DL7, KIR44, KIRC1, KIR2DS2, killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 3, refers to a member of the transmembrane glycoprotein family expressed by subsets of natural killer cells and T cells. Killer cell immunoglobulin-like receptor (KIR) genes are polymorphic and highly homologous, and they are found in clusters 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, KIR3DL2). KIR proteins are classified by the number of extracellular immunoglobulin domains (2D or 3D) and whether they have a long (L) cytoplasmic domain or a short (S) cytoplasmic domain. KIR proteins with long cytoplasmic domains transmit inhibitory signals upon ligand binding via immunoreceptor tyrosine-based inhibitory motifs (ITIMs), while KIR proteins with short cytoplasmic domains lack the ITIM motif and instead bind to TYRO protein tyrosine kinase-binding proteins to transmit activating signals. The ligands for some KIR proteins are subsets of HLA class I molecules, and thus KIR proteins are thought to play an important role in the regulation of the immune response. 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 KIR3DL3 are well known in the art (see, for example, 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 are 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). Nucleic acid and polypeptide sequences of KIR3DL3 orthologs in organisms other than human are well known, and include, 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 orthologs are presented in Table 1 below.

[0132] Anti-KIR3DL3 antibodies suitable for the detection of KIR3DL3 protein are well-known in the art, 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), antibody OAAB05761, OAAF08125, OAAN04122, OACA09134, OACA09135, OACD04988, and OASG01190 (Aviva Systems Biology), etc. are included.Furthermore, a plurality of siRNAs, shRNAs, CRISPR constructs for reducing KIR3DL3 expression, such as shRNA product numbers TF303684, TR303684, TG303684, TL303684, 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 commercial product lists of the above companies. Note that this term may be further used to refer to any combination of the features described herein with respect to the KIR3DL3 molecule. For example, any combination of sequence composition, percentage identity, sequence length, domain structure, functional activity, etc. can be used to describe the KIR3DL3 molecules encompassed by the present disclosure.

[0133] The term "peripheral blood cell subtype" refers to cell types commonly 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 transgenic or transchromosomal animals (e.g., mice) to human immunoglobulin genes or hybridomas prepared therefrom (further described below), (b) antibodies isolated from host cells transformed to express an 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, if transgenic animals to human Ig sequences are used, in vivo somatic mutagenesis), and thus, the amino acid sequences of the V H region and V L region are sequences that are derived from and related to human germline V H sequences and V L sequences, while being sequences that may not naturally occur in the human antibody germline repertoire in vivo.

[0135] The term "sample" as used herein to detect or determine the presence or level of at least one biomarker typically includes whole blood, plasma, serum, saliva, urine, feces (e.g., stool), tears, and any other body fluid (e.g., those described under the definition of "body fluid" above), or a tissue sample (e.g., a biopsy), such as a small intestine, colon sample, or surgically excised tissue. In certain instances, the 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 that contain 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 the 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 having the same or highly similar sequences to a target biomarker nucleic acid results in sequence-specific degradation of messenger RNA (mRNA) transcribed from the target gene or specific post-transcriptional gene silencing (PTGS) (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 progressive cleavage of long dsRNA into double-stranded fragments called small interfering RNAs (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 siRNAs, shRNAs, or other RNA interference agents, to inhibit or silence the expression of the target biomarker nucleic acid. As used herein, "inhibition of target biomarker nucleic acid expression" or "inhibition of marker gene expression" includes any decrease in the expression or protein activity or level of the target biomarker nucleic acid or the protein encoded by the target biomarker nucleic acid. This decrease is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or more, as compared to the expression of the target biomarker nucleic acid not targeted by the RNA interference agent or the activity or level of the protein encoded by the target biomarker nucleic acid.

[0138] In addition to RNAi, genome editing can be used to modulate constitutive or inducible knockouts or mutations of the copy number or gene sequence of a biomarker of interest, such as components of the KIR3DL3 pathway, such as HHLA2, TMIGD2, and / or KIR3DL3. For example, the CRISPR-Cas system can be used for precise editing of genomic nucleic acids (e.g., to create non-functional or null mutations). In such embodiments, CRISPR guide RNAs and / or Cas enzymes can be expressed. For example, a vector containing only the guide RNA can be administered to a transgenic animal or cell that is 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 (see, e.g., U.S. Patent 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 gene strategies can use constitutive or inducible expression systems according to methods well known in the art.

[0139] "Piwi-interacting RNA (piRNA)" is the largest class of small non-coding RNAs. piRNAs form RNA-protein complexes through interaction with Piwi proteins. These piRNA complexes are involved in both epigenetic gene silencing and post-transcriptional gene silencing of retrotransposons and other genetic elements in germline cells, particularly in germline cells during spermatogenesis. They differ from microRNAs (miRNAs) in terms of 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. Most piRNAs are antisense to transposon sequences, suggesting that transposons are piRNA targets. In mammals, the activity of piRNAs 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 by repeated in vitro selection rounds or equivalently by 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 sequences presented by the protein backbone. They are typically isolated from combinatorial libraries and then often improved by site-directed mutagenesis or variable region mutagenesis and selection rounds. An "affimer protein", which is the evolution of peptide aptamers, is a highly stable small protein engineered to present a peptide loop that provides a high-affinity binding surface to a specific target protein. It is a low molecular weight (12 - 14 kDa) protein derived from the cystatin cysteine protease inhibitor family. Aptamers are useful for biotechnological and therapeutic applications because they provide molecular recognition properties that compete with the commonly used biomolecule antibodies. In addition to their discriminatory 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] As used herein, "short interfering RNA" (siRNA), also referred to as "small interfering RNA", is defined, for example, as an agent that functions to inhibit the expression of a target biomarker nucleic acid by RNA interference (RNAi). siRNA can be chemically synthesized, produced by in vitro transcription, or produced in a host cell. In one embodiment, the siRNA is a double-stranded RNA (dsRNA) molecule that is 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 each strand may have a 3' and / or 5' overhang having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhangs is independent between these two strands, i.e., the length of the overhang on one strand does not depend on the length of the overhang on the second strand. Preferably, the siRNA can promote RNA interference by degradation of the 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 loop of 5-9 nucleotides, and a sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow. These shRNAs can be included in plasmids, retroviruses, and lentiviruses and can be expressed, for example, from a polymerase III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA Apr;9(4):493-501, which is incorporated herein by reference).

[0143] RNA interference agents, such as siRNA molecules, can inhibit the expression of biomarker genes that are overexpressed in cancer and can thereby be administered to patients having cancer or at risk of having cancer to treat, prevent, or suppress cancer in the subject.

[0144] The term "small molecule" is a term in the art and includes molecules having a molecular weight of less than about 1000 or less than about 500. In one embodiment, the small molecule exclusively comprises no peptide bonds. In another embodiment, the small molecule is not an oligomer. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, organic small molecules (e.g., polyketides) (Cane et al. 1998. Science 282:63), and natural product extract libraries. In another embodiment, these compounds are non-peptidic organic small molecule compounds. In a further embodiment, the small molecule is not biosynthetic.

[0145] The terms "selective modulator" or "selectively modulates" as applied to a biologically active agent refers to the ability of the agent to modulate a target of a cell population, signaling activity, etc. as compared to off-target cell populations, signaling activity, etc. by direct or interacting interactions with the target. For example, an agent that selectively inhibits the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2 more than another interaction between KIR3DL3 and another binding partner, and / or such an interaction in a target cell population, is 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 of the activity of the agent against at least one other binding partner (e.g., at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 105-fold, 110-fold, 120-fold, 125-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-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 (including boundary values) therebetween). Such a measure is 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 in a particular target as compared to other targets. For example, a measured variable (e.g., regulation of Treg / Breg relative to other cells (such as other immune cells like Tcon)) can be 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, 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 the boundary values) (e.g., 50% to 16-fold), and can be different for the target of interest compared to unintended or undesirable targets. The same fold analysis can be used to confirm 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 growth rate or number, etc.

[0147] In contrast, the term "specific" refers to an exclusive action or function. For example, specific regulation of the HHLA2-KIR3DL3 interaction refers to exclusive regulation of the HHLA2-KIR3DL3 interaction and not regulation of the interaction between KIR3DL3 and another ligand. In another example, specific binding of an antibody to a given antigen refers to the ability of the antibody to bind to the antigen of interest without binding to other antigens. Typically, an antibody has an affinity of approximately less than 1×10 -7 M, e.g., approximately 10 -8 M, 10 -9 M, 10 -10 M, or less than 10 -11 M, or even lower affinity (KD ) is conjugated, 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, or more, higher than its affinity for binding to non-specific antigens other than the predetermined antigen (e.g., BSA, casein) or closely related antigens. In addition, K D is A the reciprocal of. The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" may be used synonymously with the term "antibody that specifically binds to an antigen" herein.

[0148] The term "sensitize" means to modify cells such as cancer cells or tumor cells in a manner that enables more effective treatment using a therapy (e.g., a 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 an immune checkpoint inhibitor therapy). In some embodiments, normal cells are not affected to the extent of being overly damaged by a therapy (e.g., a 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 an immune checkpoint inhibitor therapy). An increase or decrease in sensitivity to a therapeutic treatment is measured according to methods known in the art for the particular treatments and methods described herein below, including, but not limited to, cell proliferation assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1982;42:2159-2164), cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984;94:161-173, Weisenthal L M, Lippman M E, Cancer Treat Rep 1985;69:615-632, Weisenthal L M, In:Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A:Harwood Academic Publishers, 1993:415-432, Weisenthal L M, 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 primes a response to a therapeutic treatment if the increased sensitivity or decreased resistance is 5% or more, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or more compared to the sensitivity or resistance in the absence of such composition or method. Determination of sensitivity or resistance to a therapeutic treatment is conventional in the art and within the skill of the artisan. It should be understood that any method described herein for enhancing the effectiveness of immunomodulation is equally applicable to methods of priming hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to therapy.

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

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

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

[0152] As used herein, the terms "therapeutically effective amount" and "effective amount" mean an amount of a compound, material, or composition comprising a compound encompassed by the present disclosure that is effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. The toxicity and therapeutic efficacy of the subject compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD 50 and ED 50 . Compositions that exhibit a high therapeutic index are preferred. In some embodiments, LD 50 (lethal dose) can be measured, and the agent 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 compared to administration of the agent without the agent. Similarly, ED 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be measured, and the agent can be increased, 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 compared to administration of the agent without the agent. Also, similarly, IC 50(i.e., the concentration that achieves half-maximal cytotoxicity or cell growth inhibitory effect on cancer cells) can be measured, and for the agent, compared to without administration of the agent, can increase, 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. 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 can 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 decrease of 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 the number of cancer cells and / or solid malignant tumors can be achieved.

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

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

[0155] The term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid" which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector to which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of self-replicating in the host cells 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) integrate into the genome of the host cell upon introduction into the host cell and are thereby replicated with the host genome. Further, 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 since the plasmid is the most commonly used form of a vector. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

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

Table 1A

[0157] An important 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 (illustrated above). Thus, several different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered to be functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although a particular organism may translate some sequences more efficiently than others). Additionally, occasionally, methylated variants of purines or pyrimidines can 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 for translating 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 (due to its redundancy, multiple nucleic acid sequences are yielded for any given amino acid sequence). Thus, the description and / or disclosure herein of a nucleotide sequence encoding a polypeptide should be considered to also include the description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, the description and / or disclosure herein of a polypeptide amino acid sequence should be considered to also include the description and / or disclosure of all possible nucleotide sequences capable of encoding the amino acid sequence.

[0159] Finally, nucleic acid and amino acid sequence information for nucleic acids 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 derived 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, for example, the ability of a KIR3DL3 polypeptide to regulate inhibitory signals in activated immune cells by binding to the natural HHLA2 ligand on cancer cells. Regulation of inhibitory signals in immune cells results in regulation of immune cell proliferation and / or cytokine secretion by immune cells. Accordingly, the term "KIR3DL3 activity" includes the ability of a KIR3DL3 polypeptide to bind to its natural ligand, the ability to regulate immune cell inhibitory signals, and the ability to regulate an immune response.

[0161] In some embodiments, a condition such as cancer is responsive to KIR3DL3 blockade alone. In other embodiments, a condition such as cancer is responsive to KIR3DL3 blockade alone, but is significantly or synergistically more responsive when treated with a combination of KIR3DL3 blockade and at least one other therapy. Many conditions responsive to KIR3DL3 blockade alone or in combination include 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 lymphoma, as well as bladder cancer, gastric cancer, head and neck cancer, kidney cancer, prostate cancer, gynecological cancer, colorectal cancer, ovarian cancer, adenocarcinoma, adenocarcinoma, chronic myelogenous leukemia (CML), and blood cancers, but are not limited thereto.

[0162] Preferred B7 polypeptides can provide co-stimulatory or inhibitory signals to immune cells, thereby promoting or inhibiting immune cell responses. For example, B7 family members that bind to co-stimulatory receptors increase T cell activation and proliferation, while B7 family members that bind to inhibitory receptors decrease co-stimulation. Furthermore, the same B7 family member can either increase or decrease T cell co-stimulation. For example, when binding to co-stimulatory receptors, HHLA2 can induce co-stimulation of immune cells, and when binding to inhibitory receptors, HHLA2 can inhibit immune cells. When binding to inhibitory receptors, 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, a B7 family member that binds to one or more receptors on immune cells, such as TMIGD2, KIR3DL3, CTLA4, CD28, ICOS, PD-1, and / or other receptors, has the ability to transmit inhibitory or co-stimulatory signals to immune cells, preferably T cells, depending on the receptor.

[0163] Regulation of co-stimulatory signals results in regulation of effector functions of immune cells. Thus, the term "KIR3DL3 activity" includes the ability to bind to its natural receptor (e.g., HHLA2) of a KIR3DL3 ligand polypeptide, the ability to regulate co-stimulatory or inhibitory signals in immune cells, and the ability to regulate 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, the agents encompassed by the present disclosure described herein that modulate the interaction between KIR3DL3 and one or more natural binding partners can upregulate or downregulate the immune system, directly or indirectly, thereby upregulating or downregulating the immune response. Agents that modulate such interaction can do so either directly or indirectly.

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

[0166] In other exemplary embodiments, an agent that promotes the binding of an HHLA2 polypeptide to one or more natural binding partners, such as a KIR3DL3 polypeptide, promotes an inhibitory signal to immune cells. An agent that modulates such interaction can do so either directly or indirectly. Thus, in one embodiment, an agent that directly enhances the interaction between HHLA2 and KIR3DL3 (an HHLA2 agonist and / or a KIR3DL3 agonist) can promote inhibitory signaling and downregulate the immune response. Alternatively, an agent that blocks the binding of KIR3DL3 to other targets increases the effective concentration of KIR3DL3 available for binding to HHLA2. Exemplary agents for downregulating the immune response 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 bind to and / or activate or inhibit a protein biomarker encompassed by the present disclosure or a fragment thereof that includes the biomarker listed in Table 1; RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and / or activity of a biomarker encompassed by the present disclosure or fragments thereof that include the biomarkers listed in Table 1.

[0168] Isolated monoclonal antibodies or fragments thereof produced against KIR3DL3 are provided. In some embodiments, the mAbs produced by the hybridomas are deposited with the American Type Culture Collection (ATCC) under accession number ______ at ______ in accordance with the terms of the Budapest Treaty.

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

[0170] The CDR1, 2, and / or 3 regions of the engineered antibodies described above may include the exact amino acid sequences as those of the variable regions encompassed by the present disclosure (e.g., including the sequences of Table 2 or a portion thereof). However, one of ordinary 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 bind effectively to KIR3DL3 (e.g., conservative sequence modifications). Thus, in another embodiment, the engineered antibody may consist of, for example, one or more CDRs (e.g., including the sequences of Table 2 or a portion thereof) that are 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.

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

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

[0173] Similarly, a monoclonal antibody that can bind to human KIR3DL3 is provided, wherein the variable domain comprises a light chain that comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the light chain CDR group presented in Table 2.

[0174] Also provided is a monoclonal antibody that can bind to human KIR3DL3, wherein the variable domain comprises a heavy chain that comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the heavy chain CDR group presented in Table 2, and the variable domain comprises a light chain that comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the light chain CDR group presented in Table 2.

[0175] One of ordinary skill in the art will note that such percentage identities are equivalent to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more conservative amino acid substitutions within a given CDR, and can be achieved by introducing such conservative amino acid substitutions.

[0176] The monoclonal antibodies encompassed by the present disclosure may include a heavy chain comprising 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 comprising 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 include a light chain comprising 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 comprising 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, a monoclonal antibody capable of binding to human KIR3DL3 comprises or consists of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 described herein.

[0178] The heavy chain variable domain of the monoclonal antibodies encompassed by the present disclosure may comprise or consist of the vH amino acid sequence described in Table 2, and / or the light chain variable domain of the monoclonal antibodies encompassed by the present disclosure may comprise or consist of the vL amino acid sequence described in Table 2.

[0179] The monoclonal antibodies encompassed by the present disclosure can be produced and modified by any technique well known in the art. For example, such monoclonal antibodies can be obtained from mouse or non-rodent antibodies, such as those available from hybridomas deposited with the ATCC under the deposit number ______ at ______. Similarly, such monoclonal antibodies can be chimeric antibodies, preferably chimeric mouse / human antibodies. In some embodiments, the monoclonal antibody is a humanized antibody such that the variable domain comprises a human acceptor framework region and optionally a human constant domain (if present), as well as non-human donor CDRs such as the mouse or non-rodent CDRs 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 antibody fragments. For example, some immune inhibitory molecules such as HHLA2, PD-L2, PD-L1, CTLA-4, KIR3DL3, etc. 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, and their variable domains include at least one CDR presented in Table 2. In one embodiment, the immunoglobulin heavy chain includes at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the group of heavy or light chain variable domain CDRs presented in Table 2. In another embodiment, the immunoglobulin light chain includes at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the group 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 chain includes a variable domain that includes at least one of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3 described herein. Such an immunoglobulin heavy chain can include or consist of at least one of CDR-H1, CDR-H2, and CDR-H3. Such an immunoglobulin light chain can include 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 each comprise, or consist of, a vH variable domain sequence or a vL variable domain sequence provided in Table 2.

[0184] The present disclosure further provides a polypeptide having a sequence selected from the group consisting of the vH variable domain, 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 the present disclosure can be used in isolated (e.g., purified) form or can be included in a vector such as a membrane or lipid vesicle (e.g., liposome).

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

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

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

[0188] The vector may include control elements, such as promoters, enhancers, terminators, etc., for causing or directing the 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 early promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney murine leukemia virus (Kuwana Y et al. 1987), the promoter of immunoglobulin H chain (Mason J O et al. 1985) and enhancer (Gillies S D et al. 1983), etc.

[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), pSG1 beta d2-4 (Miyaji H et al. 1990), etc. Other typical examples of plasmids include replicating plasmids containing an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, etc. Typical examples of viral vectors include adenoviral vectors, retroviral vectors, herpesviral 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 a helper plasmid or virus. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and WO94 / 19478.

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

[0191] The recombinant polypeptides encompassed by the present disclosure can be produced using the nucleic acids encompassed by the present disclosure in a suitable expression system. The term "expression system" means, for example, a host cell and a compatible vector under conditions suitable for the expression of a protein encoded by foreign DNA carried by a 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 (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (such as those derived from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also 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 the "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"), etc. 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 a method for producing a recombinant host cell that expresses an antibody or polypeptide encompassed by the present disclosure according to the present disclosure, comprising the steps of: (i) introducing the above-described recombinant nucleic acid or vector into competent host cells in vitro or ex vivo; (ii) culturing the resulting recombinant host cells 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 for the production of the antibodies and polypeptides described herein.

[0194] In another aspect, the present disclosure provides an isolated nucleic acid that hybridizes to a polynucleotide disclosed herein under selective hybridization conditions. Thus, the polynucleotides of this embodiment can be used for the isolation, detection, and / or quantification of 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 polynucleotide is isolated from a human or mammalian nucleic acid library or is otherwise a genomic or cDNA sequence complementary to a cDNA 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, more preferably at least 95% full-length sequences. The cDNA library can be normalized to increase the presentation of rare sequences. Low or medium stringency hybridization conditions are typically, but not exclusively, used for sequences with reduced sequence identity compared to complementary sequences. Medium and high stringency conditions can optionally be used for sequences with higher identity. Low stringency conditions allow for the selective hybridization of sequences having about 70% sequence identity and can be used to identify orthologous or paralogous sequences. Optionally, the polynucleotides of the invention encode at least a portion of an antibody encoded by a polynucleotide described herein. The polynucleotides of the invention include nucleic acid sequences that can be used for the selective hybridization to polynucleotides encoding antibodies encompassed by the present disclosure. See, for example, Ausubel (supra), Colligan (supra), each of which is hereby incorporated by reference in its entirety.

[0195] IV. Methods for Producing Antibodies The antibodies and their fragments, immunoglobulins, and polypeptides encompassed by the present disclosure can be produced by any chemical, biological, genetic, or enzymatic technique, among others, known in the art, including but not limited to, any technique known in the art and can be produced by any technique known in the art either alone or in combination.

[0196] By knowing the amino acid sequence of the desired sequence, one of ordinary skill in the art can readily produce the antibody or polypeptide by standard techniques for producing polypeptides. For example, they can be synthesized using well-known solid-phase methods, preferably using a commercially available peptide synthesizer (e.g., one manufactured by Applied Biosystems (Foster City, Calif.)) according to the manufacturer's instructions. Alternatively, the 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 the DNA sequence encoding the desired (poly)peptide into an expression vector and introduction of such vector into a suitable eukaryotic or prokaryotic host that expresses the desired polypeptide, and the desired polypeptide can be subsequently isolated using well-known techniques.

[0197] Specifically, the present disclosure further relates to a method for 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, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), which are hereby incorporated by reference in their entirety, for example, see Chapters 1, 4, 6, 8, 9, 10.

[0199] Chimeric antibodies encompassed by the present disclosure (e.g., mouse-human chimeric or non-rodent-human chimeric) can be produced by obtaining nucleic acid sequences encoding the VL and VH domains as described above and inserting them into an expression vector for animal cells having genes encoding human antibody CH and human antibody CL to construct a human chimeric antibody expression vector, and introducing the expression vector into animal cells to express the coding sequences. The CH domain of a human chimeric antibody can be any region belonging to human immunoglobulins of the IgG class or its subclasses, such as IgG1, IgG2, IgG3, and IgG4, etc. Similarly, the CL of a human chimeric antibody can be any region belonging to Ig such as the kappa class or the lambda class. Chimeric and humanized monoclonal antibodies containing both human and non-human portions that 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. Patent 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. (1985) Nature 314:446-449, Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559), Morrison, S.L. (1985) Science 229:1202-1207, Oi et al. (1986) Biotechniques 4:214, U.S. Patent 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] In addition, the humanized antibody can be produced according to a standard protocol such as the protocol disclosed in U.S. Patent No. 5,565,332. In some embodiments, the antibody chain or specific binding pair member is a nucleic acid molecule encoding a fusion of the polypeptide chain of the specific binding pair member and a component of a replicable generic display package using techniques known in the art, such as those described in U.S. Patent No. 5,565,332, No. 5,871,907, or No. 5,733,743, and a vector containing a nucleic acid molecule encoding the second polypeptide chain of a single binding pair member. The humanized antibody encompassed by the present disclosure can be produced by obtaining nucleic acid sequences encoding the CDR domains as described above and inserting them into an expression vector for animal cells having 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 to construct a humanized antibody expression vector, and expressing the genes by introducing the expression vector into animal cells. The humanized antibody expression vector can be 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 (tandem type) in which both of these genes are present on the same vector.

[0201] Methods for producing humanized antibodies based on conventional recombinant DNA techniques 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 humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP239,400, PCT Publication No. 91 / 09967, U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP592,106, EP519,596, Padlan EA (1991), Studnicka G M et al. (1994), Roguska M A. et al. (1994)), and chain shuffling (U.S. Patent No. 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application No. EP125023 and International Patent Application No. WO96 / 02576).

[0202] Similarly, the bispecific or multispecific antibodies described herein can be made according to standard procedures. For example, triomas and hybrid hybridomas are two examples of cell lines that can secrete 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 by 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 also be generated by creating heterohybridomas by fusing hybridomas or other cells that make different antibodies, and then identifying clones that produce and co-aggregate the desired antibodies. They can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or parts thereof such as Fab and Fv sequences. The antibody component can bind to a polypeptide or a fragment thereof of one or more biomarkers encompassed by the present disclosure, including one or more immunosuppressive 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. Also, Fab can be produced by inserting DNA encoding the Fab of the antibody into a vector of a prokaryotic or eukaryotic expression system, and introducing the vector into a prokaryote or eukaryote (where appropriate) to express the Fab.

[0204] Similarly, the F(ab’)2 fragments encompassed by the present disclosure can be obtained by treating an antibody that specifically reacts with KIR3DL3 with a protease, pepsin. Also, the F(ab’)2 fragments can be produced by binding to the Fab’ described below by a thioether bond or a disulfide bond.

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

[0206] In addition, the scFvs encompassed by the present disclosure can be produced by obtaining cDNA encoding the VH and VL domains as described above, constructing DNA encoding the scFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryote or a eukaryote (where appropriate) to express the scFv. To generate a humanized scFv fragment, a well-known technique called CDR grafting can be used, which includes selecting complementarity-determining regions (CDRs) from a donor scFv fragment and grafting them onto a human scFv fragment framework having a known three-dimensional structure (see, for example, WO98 / 45322, WO87 / 02671, U.S. Patent No. 5,859,205, U.S. Patent No. 5,585,089, U.S. Patent No. 4,816,567, EP0173494).

[0207] V. Modification 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 within 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 decreases as compared to the original antibody derived from the non-human animal. Some amino acid residues in the VH and VL of the non-human antibody, not only within the CDRs but also within the FRs, are considered to be directly or indirectly related to the antigen-binding activity. Thus, substitution of these amino acid residues with different amino acid residues derived from the FRs of the VH and VL of a human antibody results in a decrease in binding activity, which can be corrected by replacing the amino acids with the amino acid residues of the original antibody derived from the non-human animal.

[0208] Modifications and changes may be made in the structures of the antibodies encompassed by the present disclosure and in the DNA sequences encoding them, and functional molecules encoding antibodies and polypeptides having desirable characteristics can still be obtained. For example, certain amino acids can be substituted by other amino acids in the protein structure without a recognizable loss of activity. Since the interaction ability and properties of a protein define the biological functional activity of the protein, certain amino acid substitutions can be made in the protein sequence and thus, a fortiori, in its DNA coding sequence, and yet a protein having similar characteristics can also be obtained. Thus, it is contemplated that various changes may be made in the antibody sequences encompassed by the present disclosure or in the corresponding DNA sequences encoding said polypeptides without a recognizable loss of their biological activity.

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

[0210] When changing the amino acid sequence of a polypeptide, the hydrophobicity-hydrophilicity index of the amino acids can be considered. The importance of the hydrophobicity-hydrophilicity amino acid index in conferring an interactive biological function to a protein is generally understood in the art. It is recognized that the relative hydrophobicity-hydrophilicity characteristics 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 is assigned a hydrophobicity-hydrophilicity index based on their hydrophobic 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), glutamate (-3.5), glutamine (-3.5), aspartic acid (<RTI 3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0211] It is known in the art that a particular amino acid may be substituted by other amino acids having a similar hydrophobicity-hydrophilicity index or score, and a protein having a similar biological activity may still be brought about, i.e., a functionally equivalent biological protein can still be obtained.

[0212] Thus, as outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side chains of the substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions taking into account the various foregoing characteristics 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 the antibodies encompassed by the present disclosure can be useful, for example, in modifying the original glycosylation pattern of the antibodies to increase stability. "Modifying" means deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites not present in the antibody. Glycosylation of the antibody can 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 the 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 a glycosylation site to an antibody is conveniently achieved by modifying the amino acid sequence to include one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Another type of covalent modification involves chemically or enzymatically coupling a glycoside to the antibody. These procedures are advantageous in that they do not require the production of the antibody in a host cell having glycosylation capacity for N-linked or O-linked glycosylation. Depending on the coupling mode used, the sugar may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as the free sulfhydryl group of cysteine, (d) free hydroxyl groups, such as the free hydroxyl group of serine, threonine, or hydroxyproline, (e) aromatic residues, such as the aromatic residue of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. Such methods are described, for example, in WO87 / 05330.

[0214] Similarly, removal of any carbohydrate moieties present on the antibody can be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in cleavage of most or all of the sugars, except the bound sugars (N-acetylglucosamine or N-acetylgalactosamine), while keeping 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 the antibody can be achieved by use of various endoglycosidases and exoglycosidases as described by Thotakura, N R. et al. (1987).

[0215] Other modifications can include the formation of immunoconjugates. For example, in one type of covalent modification, an antibody or protein is covalently linked to one of various non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, 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] The conjugation of an antibody or other protein encompassed by the present disclosure with a heterologous agent can 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-azide 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-isothiocyanatobenzylmethyl diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radio nucleotides to antibodies (WO94 / 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.” Cytotoxins or cytotoxic agents include any agent that is harmful to cells (e.g., kills cells). Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Therapeutic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiammine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine), but are not limited thereto. The antibodies of the present disclosure can be conjugated to a radioisotope, e.g., radioactive iodine, to generate a cytotoxic radiopharmaceutical for treating related disorders such as cancer.

[0218] Using the conjugated anti-KIR3DL3 antibody, among other things, as part of clinical trial procedures, the polypeptide levels in tissues can be diagnostically or prognostically monitored to, for example, determine the effectiveness of a given treatment regimen or select patients most likely to respond to immunotherapy. For example, cells can be permeabilized in a flow cytometry assay to target the antibody that binds to KIR3DL3 and the recognized intracellular epitope, and the detection of binding can be enabled by 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, avidin-biotin complexes, 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 avidin-biotin complexes include streptavidin / biotin and avidin / biotin, examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin (PE), an example of a luminescent substance includes luminol, examples of bioluminescent substances include luciferase, luciferin, and aequorin, and examples of suitable radioactive substances 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 both direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance, such as a radiopharmaceutical or fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)) to the antibody, and indirect labeling of the antibody by reaction with a detectable substance.

[0219] Using the antibody conjugates encompassed by the present disclosure, a given biological response can be modulated. The chemical moiety should not be construed as being limited to classical chemical agents. For example, the drug moiety can be a protein or polypeptide having the 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, see, for example, 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 Prospective Of The Therapeutic Use Of Radiolabeled Antibody 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 promotes the release of a cytotoxic agent or growth inhibitor in a cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photosensitive linker, a dimethyl linker, or a disulfide-containing linker (see, e.g., U.S. Patent No. 5,208,020) can be used. Alternatively, a fusion protein comprising an antibody and a growth inhibitor can be produced by recombinant techniques or peptide synthesis. The lengths of the DNA can include regions encoding each of 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 disclosure described herein can 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. Further, the anti-KIR3DL3 antibodies, immunoglobulins, polypeptides, and nucleic acids encompassed by the disclosure described herein can be used in a number of predictive pharmaceutical assays based on the detection of KIR3DL3 levels. For example, the disclosure provides a prognostic (or predictive) assay for determining whether an individual will respond to a particular therapy (e.g., a therapy targeting KIR3DL3). As described herein, the KIR3DL3 polypeptide or a fragment thereof encompassed by the disclosure has one or more of the following activities: 1) binding to its natural binding partner such as HHLA2 and / or modulating its activity, 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 a mammal such as a mouse, non-rodent animal, or human, and 5) modulating immune cell anergy.

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

[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 interventions and / or the individualized high-resolution diagnosis of disorder subtypes. In some embodiments, KIR3DL3 is detected combinatorially with another marker.

[0225] 1. Therapeutic Methods and Uses In some embodiments, the antibodies, fragments, or immunoconjugates (e.g., anti-KIR3DL3 antibodies) encompassed by the present disclosure are useful for the treatment of any disorder associated with abnormal or undesirable KIR3DL3 activation (e.g., cancer). In certain embodiments, the treatment is for mammals such as humans. 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, it is contemplated that a therapeutic synergistic effect will occur when cells are treated with an anti-KIR3DL3 mAb and another immune checkpoint inhibitor or cell therapy, such as a therapy comprising a CAR.

[0226] The antibodies or fragments thereof encompassed by the present disclosure described herein are useful for modulating an immune response by blocking or disrupting the interaction of KIR3DL3 with 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. Accordingly, an object encompassed by the present disclosure is a method for modulating an immune response associated with abnormal KIR3DL3 activation and / or for treating a disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or fragment thereof encompassed by the present disclosure.

[0227] Upregulation of an immune response can be in the form of enhancing an existing immune response or inducing a primary immune response. For example, enhancement of an immune response using the subject compositions and methods is useful in improving immunological defenses against cancer and microbial (e.g., bacterial, viral, or parasitic) infections. For example, upregulation or enhancement of the immune response functions described herein is useful in inducing tumor immunity.

[0228] In another embodiment, the immune response can be stimulated by the methods described herein such that 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 initiate a significant immune response, such as a self-antigen such as a tumor-specific antigen, can be induced by administering an appropriate agent described herein that upregulates the immune response. In one embodiment, a self-antigen such as a tumor-specific antigen can be co-administered. In another embodiment, the immune response can be stimulated against an antigen (e.g., a self-antigen) for treating a neurological disorder. In another embodiment, the subject agent can be used as an adjuvant for enhancing the response to a foreign antigen during the process of active immunization.

[0229] In certain instances, it may be desirable to further administer other agents that upregulate the immune response, such as forms of other B7 family members that signal through co-stimulatory receptors, to further enhance the immune response. Also, agents that upregulate the immune response can be used prophylactically in vaccines against various polypeptides (e.g., polypeptides derived from pathogens). Immunity to a pathogen (e.g., a virus) can be induced by vaccinating with viral proteins together with an agent that upregulates the immune response in a suitable adjuvant.

[0230] Alternatively or additionally, in some embodiments, the antibodies and antigen-binding fragments encompassed by the present disclosure are useful for therapeutic applications (such as treating the disease and delaying its onset or progression, etc.) for suppressing immune responses in diseases, such as asthma, autoimmune diseases (glomerulonephritis, arthritis, dilated cardiomyopathy-like diseases, ulcerative colitis, Sjogren's syndrome, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, scleroderma, periarteritis nodosa, rheumatic fever, vitiligo, insulin-dependent diabetes, Behcet'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, celiac disease, Cushing's syndrome, dermatomyositis, discoid lupus erythematosus, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes, Lambert-Eaton syndrome, lupoid hepatitis, some cases of lymphopenia, mixed connective tissue disease, pemphigoid, pemphigus vulgaris, pernicious anemia, lens-induced uveitis, polyarteritis nodosa, polyglandular autoimmune syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt's syndrome, localized scleroderma (or CREST syndrome), sympathetic ophthalmia, systemic lupus erythematosus, Takayasu 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 therapeutic uses (such as treating the disease and delaying its onset or progression) for infectious diseases that persist, such as viral infectious diseases including human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), retroviruses such as human immunodeficiency virus (HIV-1 and HIV-2), herpesviruses such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, and influenza virus. Other antigens related to pathogens that can be used as described herein are antigens of various parasites, including malaria, preferably malaria peptides based on the repeat of NANP. In addition, bacterial, fungal, and other pathogenic diseases are included, such as those caused by Aspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma, and Vibrio cholerae.Exemplary species include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group 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, or fungi such as Paracoccidioides brasiliensis, etc., or other pathogens such as Plasmodium falciparum. Also included are the National Institute of Allergy and Infectious Diseases (NIAID) priority pathogens.These include Category A agents, such as smallpox (variola), 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 burnetii (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), alphaviruses (Venezuelan equine encephalitis, eastern and western equine encephalitis), ricin toxin from Ricinus communis (castor bean), 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, the antibodies or antigen-binding fragments encompassed by the present disclosure are useful for therapeutic applications with respect to induction of diseases caused by attenuation of immunological tolerance, organ transplant rejection, graft-versus-host disease (GVHD), allergic diseases, and immune responses mediated by KIR3DL3, in addition to prognostic and prophylactic uses.

[0233] In the context of the present invention, the terms "treating" or "treatment" as used herein mean reversing, reducing, or suppressing the progression of a disorder or condition to which such terms apply, or one or more symptoms of such disorder or condition. The term "treating cancer" as used herein means suppressing 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] A therapeutic formulation comprising one or more of the antibodies encompassed by the present disclosure is prepared for storage by mixing an antibody having the desired purity, in the form of a lyophilized formulation or an aqueous solution, with any physiologically acceptable carrier, excipient, or stabilizer (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). The antibody composition can be formulated, dosed, and administered in any manner consistent with good medical practice. Factors to be considered in this context 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 for drug delivery, the method of administration, the dosing schedule, and other factors known to the physician.

[0235] A therapeutic dose can 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. One of ordinary skill in the art will understand that such guidelines are adjusted according to, for example, the molecular weight of the active agent when using antibody fragments or antibody conjugates. The dosage can also vary depending on whether the administration is local, such as nasal, inhalation, etc., or systemic, such as intramuscular, intraperitoneal, intravenous, etc.

[0236] The composition does not require one or more agents to enhance activity or otherwise improve therapeutic effect, but may optionally be formulated therewith.

[0237] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; 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 nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Formulations for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0238] These active ingredients may be encapsulated, for example, in microcapsules prepared by droplet formation techniques or interfacial polymerization, such as hydroxy methyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0239] The compositions described herein can be administered by any suitable means, including parenterally, subcutaneously, intraperitoneally, intraluminally, and intranasally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the compositions can be preferably administered by pulse infusion, particularly with a decrease in the dose of the present antibody.

[0240] For the prevention or treatment of a disease, the appropriate dosage of the antibody will depend on the type of disease to be treated as defined above, the severity and course of the disease, whether the antibody is administered for prevention, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is preferably administered to the patient over one or 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 block KIR3DL3 signaling and its downstream immune responses. Alternatively, agents that indirectly block the interaction between KIR3DL3 and HHLA2 can block KIR3DL3 signaling and its downstream immune responses. For example, in some embodiments, soluble forms 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 the ligand, inactivated 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 that bind to KIR3DL3 and / or HHLA2 and inhibit the interaction between the receptor and the ligand (e.g., the extracellular portion of HHLA2 and / or KIR3DL3 fused to the Fc portion of an antibody or immunoglobulin), inactivated forms of native KIR3DL3 and / or HHLA2, and soluble forms of native KIR3DL3 and / or HHLA2.

[0242] In some embodiments, anti-KIR3DL3 antibody therapy or a 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) can be administered. The combination therapy can 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, and each of these combinations can be in combination with anti-immune checkpoint therapy. In addition, any representative embodiments of agents for modulating a particular target can be adapted by those skilled in the art to any other target described herein and below (e.g., the direct and indirect KIR3DL3 inhibitors described herein can be applied to other immune checkpoint inhibitors and / or monospecific antibodies, bispecific antibodies, inactivated forms, small molecules, peptides, interfering nucleic acids, etc.).

[0243] Accordingly, the therapeutic agents encompassed by the present disclosure can be used alone or administered, for example, in combination therapy with chemotherapeutic agents, hormones, anti-angiogenic agents, CARs, radiolabeled compounds, or surgery, cryotherapy, and / or radiation therapy. The aforementioned therapies can be administered in conjunction with, before, or after conventional therapies, and in combination with other forms of conventional therapies (e.g., standard-of-care cancer treatments well known to those skilled in the art). For example, the agents encompassed by the present disclosure can be administered with a therapeutically effective dose of a chemotherapeutic agent. In another embodiment, the agents encompassed by the present disclosure are administered in combination with chemotherapy to enhance the activity and effectiveness of the chemotherapeutic agent. The Physician's Desk Reference (PDR) discloses the dosages of chemotherapeutic agents used in the treatment of various cancers. The dosing regimens and dosages of these aforementioned chemotherapeutic agents that are therapeutically effective depend on the particular cancer being treated, the extent of the disease, and other factors with which physicians in the art are familiar and can be determined by a physician.

[0244] An anti-KIR3DL3 agent can also be administered in combination with a targeted therapy, such as an immunotherapy. An immunotherapy designed to induce or amplify an immune response is referred to as an "activating immunotherapy". An immunotherapy designed to reduce or suppress an immune response is referred to as a "suppressive immunotherapy". Any agent that is thought to have an effect on the immune system in genetically modified transplanted cancer cells can be assayed to determine whether the agent is an immunotherapy and to determine the effect that a given genetic modification has on the regulation of 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 and thereby treats cancer. For example, a targeted therapy related to the inhibition of immune checkpoint inhibitors in combination with the methods encompassed by the present disclosure is useful. 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 the anti-tumor immune response. 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, TMIGD2, KIR3DL3, and A2aR (see, for example, 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 can include, 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 intact, making them potentially useful for cancer therapy. The replication of oncolytic viruses 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 can involve passive immunity to provide short-term protection to the host achieved by the administration of pre-formed antibodies produced against cancer antigens or disease antigens (e.g., the administration of 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 the treatment of 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 modulate biomolecules associated with the development, 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 modulate biomolecules associated with the development, progression, and / or pathology of tumors or cancer. As described above, immunotherapy against immune checkpoint targets, such as HHLA2, KIR3DL3, etc., is useful.

[0247] In some embodiments, immunotherapy may include one or more adoptive cell-based immunotherapies. Well-known adoptive cell-based immunotherapy modalities include, but are 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, autologous immune 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 regulate the immune response, for example, to express cytokines such as GM-CSF and / or to express tumor-associated antigen (TAA) antigens such as Mage-1, gp-100, patient-specific neoantigen vaccines, etc.

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

[0249] In some embodiments, immunomodulatory agents such as immunosuppressive agents, glucocorticoids, cytostatic agents, immunophilins, and their modulators (e.g., rapamycin, calcineurin inhibitors, tacrolimus, ciclosporin), pimecrolimus, abetimus, gusperimus, ridafolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, non-glucocorticoid steroids, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid analogs, methotrexate, antithymocyte globulin, antilymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechins, opioids, IMPDH inhibitors, mycophenolic acid, miltefosine, fingolimod, NF-κB inhibitors, raloxifene, drotrecogin alpha, denosumab, NF-κB signaling cascade inhibitors, disulfiram, olmesartan, dithiocarbamate, proteasome inhibitors, bortezomib, MG132, Prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, non-steroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethyl epoxyquinomycin (DHMEQ), I3C (indole-3-carbinol) / DIM (diindolylmethane) (13C / DIM), Bay 11-7082, luteolin, cell-permeable peptide SN-50, IκBα-superrepressor overexpression, NFκB decoy oligodeoxynucleotide (ODN), or any derivatives or analogs thereof are used. In yet another embodiment, immunomodulatory antibodies or proteins are used.For example, antibodies that bind to 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), otrexup, teprotumumab, visilizumab, anti-CD4 antibodies, clenoliximab, keliximab, zanilimumab, anti-CD11a antibodies, efalizumab, anti-CD18 antibodies, erlizumab, loflizumab, anti-CD20 antibodies, ofatumumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, anti-CD23 antibodies, lumiliximab, anti-CD40 antibodies, teneliximab, toralizumab, anti-CD40L antibodies, rupalizumab, anti-CD62L antibodies, aselizumab, anti-CD80 antibodies, galiximab, anti-CD147 antibodies, gavrilimomab, B lymphocyte stimulator (BLyS) inhibitory antibodies, belimumab, CTLA4-Ig fusion proteins, abatacept, belatacept, anti-CTLA4 antibodies, ipilimumab, tremelimumab, anti-eotaxin 1 antibodies, belinostat, anti-a4-integrin antibodies, natalizumab, anti-IL-6R antibodies, tocilizumab, anti-LFA-1 antibodies, odevixibat, anti-CD25 antibodies, basiliximab, daclizumab, inolimomab, anti-CD5 antibodies, zolimomab, anti-CD2 antibodies, siprilimumab, nerelimomab, faralimumab, atorolimumab, atorixibat, dolutegravir, dolutegravir, fontolizumab, gantenerumab, gomiliximab, lebrikizumab, maslimomab, mololizumab, paxalisib, reslizumab, loflizumab, talizumab, terixibat, bapalixibat, bepalizumab, 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 the immune response, such as vitamin A, vitamin E, vitamin C, etc., are well known in the art (see, for example, U.S. Patent Nos. 4,981,844 and 5,230,902 and PCT Publication No. WO2004 / 004483), and can be used in the methods described herein.

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

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

[0253] In one embodiment, chemotherapy is used. Chemotherapy includes the administration of chemotherapeutic agents. Such chemotherapeutic agents can be selected from the group of the following compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, antimitotics, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogs, plant alkaloids, and toxins, as well as synthetic derivatives thereof, but are not limited thereto. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxel; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; antifolates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytarabine; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and mitotic inhibitors: halichondrin, colchicine, and lysocine. Compositions containing one or more chemotherapeutic agents (e.g., FLAG, CHOP) can also be used. FLAG includes fludarabine, cytarabine (Ara-C), and G-CSF. CHOP includes cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiment, PARP (e.g., PARP-1 and / or PARP-2) inhibitors are 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 No. 36,397), and NU1025 (Bowman et al.).The mechanism of action is generally related to the ability of PARP inhibitors to bind to PARP and reduce its activity. PARP catalyzes the conversion of beta-nicotinamide adenine dinucleotide (NAD+) to nicotinamide and poly-ADP-ribose (PAR). Both poly(ADP-ribose) and PARP have been associated with transcriptional control, cell proliferation, genomic stability, and carcinogenesis (Bouchard V.J.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 Jun.2001,pp.97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is an important 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 J C,de Murcia G(2006)Nat Rev Mol Cell Biol 7:517-528,Wang Z Q,et al.(1997)Genes Dev 11:2347-2358). Knockout of SSB repair by inhibition of PARP1 function induces DNA double-strand breaks (DSBs) that can induce synthetic lethality in cancer cells defective in homologous recombination-directed DSB repair (Bryant H E,et al.(2005)Nature 434:913-917,Farmer H,et al.(2005)Nature 434:917-921). The foregoing examples of chemotherapeutic agents are illustrative and 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, radioisotopes (I-125, palladium, iridium), interstitial implantation of radioisotopes such as strontium-89, chest radiation therapy, intraperitoneal P-32 radiation therapy, and / or whole abdomen 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 where the radiation is directed from a remote source. Radiation treatment can also be administered as internal therapy or brachytherapy where the radioactive source is placed within the body in close proximity to the cancer cells or tumor mass. The use of photodynamic therapy including the administration of hematoporphyrin and its derivatives, verteporfin (BPD-MA), phthalocyanines, photosensitizer Pc4, demethoxy-hypocrellin A, and photosensitizers such as 2BA-2-DMHA is also encompassed.

[0255] In another embodiment, hormone therapy is used. Hormonal therapeutic treatments include, for example, hormone agonists, hormone antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), hormone biosynthesis and processing inhibitors, and steroids (e.g., dexamethasone, retinoids, deltoid, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestin), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)), vitamin D3 analogs, antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate). The duration and / or dosage of treatment with the therapy can vary depending on the particular therapeutic agent or combination thereof. One of ordinary skill in the art will understand the appropriate treatment times for particular cancer therapeutic agents. The present disclosure contemplates the continued evaluation of the optimal treatment schedule for each cancer therapeutic agent, where the phenotype of the subject's cancer 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 for the practice of the invention for delivering the various constructs encompassed by this disclosure to the recipient subject. In one embodiment encompassed by this disclosure, the DNA construct is delivered to cells by transfection, i.e., by delivery of "naked" DNA, or as a complex with a colloidal dispersion system. Colloidal systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. Preferred colloidal systems of the invention are lipid-complexed DNA or liposome-formulated DNA. In the former approach, for example, a plasmid containing a transgene with the desired DNA construct can first be experimentally optimized for expression (e.g., inclusion of the 5' untranslated region of an intron and elimination of unwanted sequences (Felgner, et al., Ann NY Acad Sci 126-139, 1995)) prior to formulation of the DNA with lipids. Thereafter, formulation of the DNA with various lipid or liposome materials can be achieved using known methods and materials and delivered to the recipient mammal. See, for example, 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] The targets of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, e.g., organ-specific, cell-specific, and organelle-specific. Mechanistic targets can be distinguished based on whether they are passive or active. Passive targets utilize the innate tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) within organs that contain fenestrated capillaries. On the other hand, active targets involve modification of liposomes by binding the liposomes to specific ligands such as monoclonal antibodies, sugars, glycolipids, or proteins, or by changing the composition or size of the liposomes, in order to achieve targeting to organs and cell types other than naturally occurring localization sites.

[0258] The surface of the targeted delivery system can be modified in various ways. In the case of liposomal target delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain the target ligand in stable association with the liposome bilayer. Various linking groups can be used to link the lipid chain to the target ligand. Delivery vehicles, e.g., naked DNA or DNA associated with liposomes, can be administered to several sites in the 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 virus 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 format that provides a sufficiently efficient delivery level, e.g., as viral particles, liposomes, nanoparticles, and can be complexed to 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 the thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters activatable by viral infection, such as the α- and β-interferon promoters, and promoters activatable by hormones such as estrogen. Other promoters that can be used include the Moloney virus LTR, the CMV promoter, and the 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 inactivated adenoviruses. Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other vehicles that can 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 contain one or more viral sequences such as a viral replication origin or a packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus, or adenovirus. 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, such as 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. Patent Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Applications Nos. 89 / 02,468, 89 / 05,349, and 90 / 02,806. For example, a number of retroviral gene delivery vehicles can be utilized in the present disclosure, including those described in EP0,415,731, WO90 / 07936, WO94 / 03622, WO93 / 25698, WO93 / 25234, U.S. Patent 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., J. Neurosurg. 79:729-735, 1993 (U.S. Patent No. 4,777,127, GB2,200,651, EP0,345,242, and WO91 / 02805).

[0263] Other viral vector systems that can be used to deliver the polynucleotides encompassed by the present disclosure are derived from herpes viruses, such as herpes simplex virus (U.S. Patent No. 5,631,236 (Woo et al., issued May 20, 1997) and WO00 / 08191 (Neurovex)), vaccinia virus (Ridgeway (1988) Ridgeway, “Mammalian expression vectors,” In: Rodriguez R L, Denhardt D T, ed. 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, poxviruses, arenaviruses, vaccinia virus, poliovirus, etc. They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244:1275-1281, Ridgeway, 1988 (supra), Baichwal and Sugden, 1986 (supra), 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 can be retroviral insertion, artificial chromosome techniques, gene insertion, random insertion by tissue-specific promoters, gene targeting, transposable elements, and / or any other method for introducing foreign DNA or producing modified DNA / modified nuclear DNA. Other modification techniques include deleting a DNA sequence from the genome and / or modifying the nuclear DNA sequence. For example, the nuclear DNA sequence can be modified by site-directed mutagenesis.

[0265] In other embodiments, recombinant biomarker polypeptides and fragments thereof can be administered to a subject. In some embodiments, fusion proteins having enhanced biological properties can be constructed and administered. Additionally, biomarker polypeptides and fragments thereof can 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 decreased proteolysis.

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

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

[0268] In one embodiment, the assay 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 measuring indirect parameters as described below, in a cell-free assay or a cell-based assay.

[0269] For example, in a direct binding assay, the biomarker protein (or their respective target polypeptides or molecules) can be coupled to a radioisotope or enzyme label such that binding can be determined by detecting the labeled protein or molecule in the complex. For example, the target can be labeled either directly or indirectly with 125 I, 35 S, 14 C, or 3 H, and the radioisotope can be detected by direct counting of radiation emission or scintillation counting. Alternatively, the target can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzyme label can be detected by determination of 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 assay assays. In one or more embodiments of the assay methods described above, 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 protein or molecule and to accommodate automation of the assay.

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

[0271] In an alternative embodiment, determination of the ability of an agent to modulate the interaction between a biomarker and a substrate or between a biomarker and its natural binding partner can be achieved by determining the ability of a test agent to modulate the activity of a polypeptide or other product that functions downstream or upstream of its position within a signaling 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 antibodies 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 the immune response. 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 the immune response.

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

[0274] One aspect encompassed by the present disclosure relates to screening assays including cell-free based assays and xenograft animal model assays. In one embodiment, these assays are used to identify whether cancer in a human or the like is likely to respond to anti-KIR3DL3 antibody therapy by using xenograft animal model assays, and / or to identify whether an agent can inhibit or kill the growth of cancer cells that are unlikely to respond to anti-KIR3DL3 antibody therapy.

[0275] 3. Preventive methods In one aspect, the present disclosure provides a method for preventing a disease or condition associated with an unwanted or never-desirable immune response in a subject. A subject at risk of developing a disease that would benefit from treatment with the claimed agent or method can be identified, for example, by any one or a combination of diagnostic assays or prognostic assays known in the art. Administration of a prophylactic agent can occur before the onset of symptoms associated with an unwanted or never-desirable immune response. Suitable agents for treatment (e.g., antibodies, peptides, fusion proteins, or small molecules) can be determined based on clinical indications and can be identified, for example, using the screening assays described herein.

[0276] 4. Prognostic assays Furthermore, using the detection methods described herein, it is possible to identify a subject responsive to a particular therapy, such as a therapy targeting KIR3DL3 for modulating activity and / or an interaction with a binding partner such as HHLA2. Similarly, using the prognostic assays described herein, it is possible to determine whether a drug (e.g., an agonist, ant...

Claims

1. A monoclonal anti-KIR3DL3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) containing the VH CDR1 amino acid sequence of SEQ ID NO: 67, the VH CDR2 amino acid sequence of SEQ ID NO: 69, and the VH CDR3 amino acid sequence of SEQ ID NO: 71, and a light chain variable region (VL) containing the VL CDR1 amino acid sequence of SEQ ID NO: 73, the VL CDR2 amino acid sequence of SEQ ID NO: 75, and the VL CDR3 amino acid sequence of SEQ ID NO:

77.

2. The monoclonal antibody or an antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or an antigen-binding fragment thereof comprises a VH sequence having at least 90% identity with SEQ ID NO: 79 and a VL sequence having at least 90% identity with SEQ ID NO:

81.

3. The monoclonal antibody or an antigen-binding fragment thereof according to claim 2, wherein the monoclonal antibody or an antigen-binding fragment thereof comprises the VH sequence of SEQ ID NO: 79 and the VL sequence of SEQ ID NO:

81.

4. The monoclonal antibody or an antigen-binding fragment thereof, a) inhibits the binding of HHLA2 to KIR3DL3, and / or b) specifically binds to KIR3DL3, The monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A bispecific anti-KIR3DL3 antibody or an antigen-binding fragment thereof, comprising a VH containing the VH CDR1 amino acid sequence of SEQ ID NO: 67, the VH CDR2 amino acid sequence of SEQ ID NO: 69, and the VH CDR3 amino acid sequence of SEQ ID NO: 71, and a VL containing the VL CDR1 amino acid sequence of SEQ ID NO: 73, the VL CDR2 amino acid sequence of SEQ ID NO: 75, and the VL CDR3 amino acid sequence of SEQ ID NO:

77.

6. The bispecific antibody or an antigen-binding fragment thereof according to claim 5, comprising a VH sequence having at least 90% identity with SEQ ID NO: 79 and a VL sequence having at least 90% identity with SEQ ID NO:

81.

7. The bispecific antibody or an antigen-binding fragment thereof according to claim 5, comprising the VH sequence of SEQ ID NO: 79 and the VL sequence of SEQ ID NO:

81.

8. The monoclonal antibody, bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the monoclonal antibody, bispecific antibody or an antigen-binding fragment thereof is chimeric, humanized, composite, or murine.

9. The monoclonal antibody, bispecific antibody or antigen-binding fragment thereof is (a) detectably labeled, (b) conjugated to a cytotoxic agent, and optionally a chemotherapeutic agent, biological agent, toxin, and / or radioisotope, (c) contains an effector domain, (d) contains an Fc domain, and / or (e) is selected from the group consisting of Fv, F av, F(ab’)2, Fab’, dsFv, scFv, sc(Fv)2, and diabody fragments, the monoclonal antibody, bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

10. The bispecific antibody or antigen-binding fragment thereof is a) inhibiting (i) the binding of HHLA2 to KIR3DL3 and (ii) the binding of PD-1 to PD-L1 and / or PD-L2, and / or b) specifically binding to KIR3DL3 and PD-1, the bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 7.

11. An isolated nucleic acid molecule encoding the monoclonal antibody, bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. A vector comprising the isolated nucleic acid according to claim 11.

13. A host cell comprising the isolated nucleic acid according to claim 11, or comprising the vector according to claim 12, or expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

14. A device or kit comprising at least one monoclonal antibody, bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the device or kit optionally comprises a label for detecting the at least one monoclonal antibody, bispecific antibody or antigen-binding fragment thereof, or a complex comprising the monoclonal antibody, bispecific antibody or antigen-binding fragment thereof.

15. A method for producing at least one antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the method comprising (i) culturing the transformed host cell according to claim 13 under conditions suitable for enabling the expression of the antibody or antigen-binding fragment thereof, and (ii) recovering the expressed antibody or antigen-binding fragment thereof. 【Claim sixteen】 A composition for detecting the presence or level of a KIR3DL3 polypeptide, comprising at least one antibody, bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, characterized in that the polypeptide is detected in a sample.

17. The composition according to claim 16, wherein the at least one antibody or antigen-binding fragment thereof forms a complex with the KIR3DL3 polypeptide, and the complex is detected in the form of an enzyme-linked immunosorbent assay (ELISA), in the form of a radioimmunoassay (RIA), immunochemically, in the form of a Western blot, or using an intracellular flow assay.

18. A composition for use in a method of predicting responsiveness to a therapy targeting KIR3DL3, comprising at least one antibody, bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the method comprises a) determining the level of KIR3DL3 and / or HHLA2 in a test sample using the at least one antibody or antigen-binding fragment thereof; b) determining the level of KIR3DL3 and / or HHLA2 in a sample from at least one control subject having a good responsiveness 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 test sample with the level of KIR3DL3 and / or HHLA2 in the sample from the at least one control subject, wherein the same or higher level of KIR3DL3 and / or HHLA2 in the test sample compared to the level in the sample from the at least one control subject indicates that the subject responds to the therapy. Composition.

19. The composition according to claim 18, wherein the therapy targets KIR3DL3 using at least one antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

20. a) the sample is part of a single sample obtained from at least one subject or part of a pooled sample obtained from at least one subject b) the therapy blocks (i) the interaction and / or signal transduction between HHLA2 and KIR3DL3, and / or (ii) the interaction and / or signal transduction between PD-1 and PD-L1 and / or PD-L2, and / or c) the sample comprises cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject d) the cells are T cells or natural killer (NK) cells The composition according to claim 18 or claim 19

21. A composition for treating a subject suffering from cancer, comprising at least one antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10

22. a) the at least one antibody or an antigen-binding fragment thereof reduces (i) the number of proliferating cancer cells in the cancer, (ii) the volume or size of the tumor of the cancer, and / or (iii) activates T cells and / or NK cells, and / or b) the at least one antibody or an antigen-binding fragment thereof is administered in a pharmaceutically acceptable formulation The composition according to claim 21

23. a) further comprising administering to the subject a therapeutic agent or regimen for treating cancer, and / or b) 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, wherein, optionally, the chimeric antigen receptor targets CD19 The composition according to claim 21 or claim 22

24. a) the cancer cells and / or tumor immune infiltrating cells in the subject express HHLA2 b) 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 cancer, thymoma, B-CLL, leukemia, B cell lymphoma, and cancer infiltrated by immune cells expressing a receptor for HHLA2, and / or c) 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 The composition according to any one of claims 21 to 23.

25. a) the subject is an animal model of cancer, optionally the animal model is a mouse model, and optionally the mouse model is a humanized mouse model, and / or b) the subject is a mammal, and optionally the mammal is a humanized mouse or a human. The composition according to any one of claims 21 to 24.

26. A composition for modulating an immune response, comprising at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof according to claim 1.

27. The composition according to claim 26, wherein the at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof is as described in any one of claims 1 to 10.

28. a) the at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof inhibits or disrupts the interaction between HHLA2 and KIR3DL3, which is its binding inhibitor receptor; b) the at least one anti-KIR3DL3 antibody or an antigen-binding fragment thereof is conjugated to a cytotoxic agent, and optionally the cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes; c) the immune response is downregulated or upregulated; d) (a) the interaction between HHLA2 and KIR3DL3, and / or (b) the interaction between PD-1 and PD-L1 and / or PD-L2 is blocked; and / or e) the anti-KIR3DL3 antibody or an antigen-binding fragment thereof is a checkpoint inhibitor for T cell activation for cancer immunotherapy. The composition according to claim 26 or 27.

29. Modulating the immune response includes modulating T cell function or NK cell function, and optionally the T cell function or NK cell function includes cytotoxic activity, and optionally the cytotoxic activity is against cancer cells expressing HHLA2. The composition according to any one of claims 26 to 28.

30. a) 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 cancer, thymoma, B-CLL, leukemia, B cell lymphoma, and cancer infiltrated by immune cells expressing a receptor for HHLA2, and / or b) 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, The composition according to claim 29.

31. The composition is administered in combination with an additional therapy selected from the group consisting of immunotherapy, checkpoint blockade, cancer vaccine, chimeric antigen receptor, chemotherapy, radiation, targeted therapy, and surgery, and optionally, the chimeric antigen receptor targets CD19. The composition according to any one of claims 26 to 30.

32. a) the immune response is regulated in an animal model of cancer, and optionally, the animal model is a mouse model, and optionally, the mouse model is a humanized mouse model, and / or b) the immune response is regulated in a mammal, and optionally, the mammal is a humanized mouse or a human, The composition according to any one of claims 26 to 31.

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