Anti-kir3dl3 antibodies and uses thereof

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; and immunoglobulins, polypeptides, nucleic acids thereof, and methods of using such antibodies for prognostic purposes, immunomodulatory purposes, and therapeutic purposes.

Description

[0001] Cross-citation of related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 910,594, filed October 4, 2019; the entire contents of which are incorporated herein by reference.

[0003] Rights Statement

[0004] This invention was carried out with government support under license number P50CA101942 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention.

[0005] sequence list

[0006] This application contains an ASCII format sequence list submitted electronically, the entire contents of which are incorporated herein by reference. This ASCII copy was created on November 17, 2020, and is named DFS-277_25_SL.txt, with a size of 120,293 bytes. Background Technology

[0007] 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, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, prolactin, and A2aR, and many more, negatively regulate the immune response process based on complex and combinatorial interactions among numerous inputs. Immune checkpoint inhibitors can modulate the immune response in some subjects, but immune checkpoint expression and interactions with natural binding partners vary between subjects and within subject tissues. A significant percentage of patients do not respond to this treatment, and many responding patients eventually develop resistance. Therefore, there is an urgent need to find alternative immune pathways that are not redundant due to the PD-1 pathway.

[0008] HERV-H LTR-associated 2 (HHLA2, also known as B7-H5 or B7-H7) is a member of the B7 family that regulates T cell function. HHLA2 is widely expressed in a variety of tumors (e.g., solid tumors and hematologic malignancies, including primary human renal cell carcinoma (RCC)) and antigen-presenting cells, and has been identified as a ligand for both T cell activation and inhibition. HHLA2 has been identified as a specific ligand for TMIGD2 (CD28H, IGPR-1), and the HHLA2 / TMIGD2 interaction selectively co-stimulates human T cell growth and cytokine production through an AKT-dependent signaling cascade (Zhu et al. (2013) Nature Communications 4:2043; Janakiram et al. (2015) Clin. Cancer Res. 21:2359–2366). TMIGD2, expressed in naïve T cells, is an activating receptor for HHLA2 and transduces co-stimulatory signals in the presence of the T cell antigen receptor (TCR). TMIGD2 is downregulated after repeated TCR stimulation. The putative inhibitory receptor for HHLA2 may be upregulated on activated T cells to regulate T cell activation. Summary of the Invention

[0009] Prior to this disclosure, several studies have shown the presence of an uncharacterized HHLA2 receptor on activated T cells, which plays a co-inhibitory role (Zhao et al. (2013) Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 110:9879–9884; Xiao and Freeman et al. (2015) Clinical Cancer Research 21:2201–2203; Wang et al. (2014) Journal of Immunology 192:126.11). HHLA2 has been found to bind to KIR3DL3, a receptor on both T and NK cells, and the HHLA2-KIR3DL3 interaction results in the inhibition of T and NK cell activation (PCT / US2019 / 026034). Therefore, this disclosure covers the recognition that the KIR3DL3 receptor is a candidate for cancer immunotherapy, and this document provides compositions and methods for targeting KIR3DL3 to modulate immune responses.

[0010] This disclosure is based, at least in part, on the finding that agents (e.g., antibodies) targeting KIR3DL3 can specifically block the HHLA2-KIR3DL3 interaction and can be used in methods for modulating immune responses. Importantly, this document proposes that targeting KIR3DL3 does not disrupt the overall function of HHLA2, which also includes activating the immune response through its interaction with TMIGD2. Therefore, this disclosure provides an important and surprising finding that targeting KIR3DL3 provides the specificity to block only the immunosuppressive function of HHLA2, thereby evoking an effective immune response (e.g., against cancer cells) without downregulating the immune-activating function of HHLA2. Developing drugs that specifically block the immunosuppressive activity of the HHLA2 pathway while preserving its stimulatory function represents a novel approach to immune checkpoint blockade in patients with cancer (e.g., hematologic malignancies and solid tumors, including clear cell renal cell carcinoma (ccRCC)).

[0011] This disclosure is also based, at least in part, on the finding that agents targeting KIR3DL3 and PD-1 can be used to modulate immune responses and / or treat cancer. In some embodiments, the KIR3DL3 x PD-1 bispecific antibody described herein can be used as a checkpoint immunotherapy, such as activating T cells and NK cells in tumors. In some embodiments, the KIR3DL3 x 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 the tumor is a useful biomarker for determining responsiveness to checkpoint blockade of KIR3DL3 mAb and / or KIR3DL3 x PD-1 bispecific antibody.

[0012] A group of exemplary representative anti-KIR3DL3 human monoclonal antibodies (mAbs) are described herein as immune checkpoint inhibitors. Blocking and non-blocking anti-KIR3DL3 mAbs were identified, and anti-KIR3DL3 mAbs that block the binding of HHLA2 to KIR3DL3 were shown as checkpoint inhibitor antibodies in T-cell and NK-cell assays.

[0013] On the one hand, a monoclonal antibody or an antigen-binding fragment thereof is provided, comprising: a) a heavy chain sequence having at least about 95% identity with a heavy chain sequence selected from the group consisting of sequences listed in Tables 2, 7 and 8; and / or b) a light chain sequence having at least about 95% identity with a light chain sequence selected from the group consisting of sequences listed in Tables 2, 7 and 8.

[0014] On the other hand, a monoclonal antibody or its antigen-binding fragment is provided, comprising: a) one, two, or three heavy chain CDR sequences, each of which has at least about 95% identity with a heavy chain CDR sequence selected from the group consisting of sequences listed in Tables 2, 7, and 8; and / or b) one, two, or three light chain CDR sequences, each of which has at least about 95% identity with a light chain CDR sequence selected from the group consisting of sequences listed in Tables 2, 7, and 8.

[0015] On the other hand, a monoclonal antibody or an antigen-binding fragment thereof is provided, comprising: a) a heavy chain sequence selected from the group consisting of sequences listed in Tables 2, 7 and 8; and / or b) a light chain sequence selected from the group consisting of sequences listed in Tables 2, 7 and 8.

[0016] On the other hand, a monoclonal antibody or an antigen-binding fragment thereof is provided, comprising: a) one, two or three heavy chain CDR sequences, each of which is selected from the group consisting of sequences listed in Tables 2, 7 and 8; and / or b) one, two or three light chain CDR sequences, each of which is selected from the group consisting of sequences listed in Tables 2, 7 and 8.

[0017] Numerous embodiments are further provided, which can be applied to any aspect covered by this disclosure as described herein. For example, in one embodiment, the monoclonal antibody or its antigen-binding fragment is chimeric, humanized, complexed, mouse, or human. In another embodiment, the monoclonal antibody or its antigen-binding fragment: (a) is detectably labeled; (b) is conjugated to a cytotoxic agent, optionally a chemotherapeutic agent, a biological agent, a toxin, and / or a radioisotope; (c) 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 biantibody fragments. In yet another embodiment, the monoclonal antibody or its antigen-binding fragment can be obtained from a hybridoma deposited under accession number ______. In yet another embodiment, the monoclonal antibody or its antigen-binding fragment inhibits the binding of HHLA2 to KIR3DL3. In T-cell activation assays, KIR3DL3 mAb that blocks the binding of HHLA2 to KIR3DL3 has been shown to be a checkpoint inhibitor. In another embodiment, a monoclonal antibody or its antigen-binding fragment specifically binds to KIR3DL3.

[0018] A group of exemplary representative bispecific antibodies that bind to KIR3DL3 and PD-1 are described herein as immune checkpoint inhibitors.

[0019] On the one hand, this article provides a bispecific antibody or an antigen-binding fragment thereof, comprising: a) a heavy chain sequence having at least about 95% identity with a heavy chain sequence selected from the group consisting of sequences listed in Tables 2 and 7 to 9; and / or b) a light chain sequence having at least about 95% identity with a light chain sequence selected from the group consisting of sequences listed in Tables 2 and 7 to 9.

[0020] On the other hand, a bispecific antibody or antigen-binding fragment thereof is provided, comprising: a) one, two, or three heavy chain CDR sequences, each of which has at least about 95% identity with a heavy chain CDR sequence selected from the group consisting of sequences listed in Tables 2 and 7 to 9; and / or b) one, two, or three light chain CDR sequences, each of which has at least about 95% identity with a light chain CDR sequence selected from the group consisting of sequences listed in Tables 2 and 7 to 9.

[0021] In another aspect, a bispecific antibody or an antigen-binding fragment thereof is provided, comprising: a) a heavy chain sequence selected from the group consisting of sequences listed in Tables 2 and 7 to 9; and / or b) a light chain sequence selected from the group consisting of sequences listed in Tables 2 and 7 to 9.

[0022] In another aspect, a bispecific antibody or antigen-binding fragment thereof is provided, comprising: a) one, two, or three heavy chain CDR sequences, each of which is selected from the group consisting of sequences listed in Tables 2 and 7 to 9; and / or b) one, two, or three light chain CDR sequences, each of which is selected from the group consisting of sequences listed in Tables 2 and 7 to 9.

[0023] Numerous embodiments are provided, which can be applied to any aspect covered by this disclosure as described herein. For example, in one embodiment, the bispecific antibody or its antigen-binding fragment is chimeric, humanized, complexed, mouse, or human. In another embodiment, the bispecific antibody or its antigen-binding fragment: (a) is detectably labeled; (b) is conjugated to a cytotoxic agent, optionally a chemotherapeutic agent, a biological agent, a toxin, and / or a radioisotope; (c) 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 the bispecific antibody fragment. In yet another embodiment, the bispecific antibody or its antigen-binding fragment can be obtained from a hybridoma deposited under accession number ______. In yet another embodiment, the bispecific antibody or its antigen-binding fragment inhibits (a) the binding of HHLA2 to KIR3DL3 and (b) the binding of PD-1 to PD-L1 and / or PD-L2. Bispecific antibodies binding to both KIR3DL3 and PD-1 have been shown to be checkpoint blockers. In another embodiment, the bispecific antibody or its antigen-binding fragment specifically binds to KIR3DL3 and PD-1. In yet another embodiment, the bispecific antibody or its antigen-binding fragment comprises a) the heavy chain sequences listed in Table 9; and / or b) the light chain sequences listed in Table 9.

[0024] On the other hand, immunoglobulin heavy chains and / or immunoglobulin light chains are provided, selected from the group consisting of immunoglobulin heavy chain and immunoglobulin light chain sequences listed in Tables 2 and 7 to 9.

[0025] On the other hand, there is provided an isolated nucleic acid molecule that (a) encodes the immunoglobulin heavy chain, immunoglobulin light chain, and / or monoclonal antibody or antigen-binding fragment thereof as described herein and covered by this disclosure; and / or (b) hybridizes under stringent conditions to complement a nucleic acid of a polypeptide encoding the group consisting of polypeptide sequences selected from the group consisting of polypeptide sequences listed in Tables 2 and 7 to 9, or a sequence having at least about 95% homology with a nucleic acid of a polypeptide encoding the group consisting of polypeptide sequences listed in Tables 2 and 7 to 9.

[0026] On another front, a vector comprising the isolated nucleic acid described herein is provided.

[0027] On the other hand, a host cell is provided, which includes the isolated nucleic acids described herein, including the vectors described herein, expressing the antibodies described herein or their antigen-binding fragments, or which can be obtained with accession number ______.

[0028] In another aspect, an apparatus or kit is provided comprising at least one antibody or antigen-binding fragment thereof described herein (e.g., a monoclonal antibody, a bispecific antibody or antigen-binding fragment thereof); an apparatus or kit 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.

[0029] In another aspect, a method is provided for generating at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody or antigen-binding fragment thereof) described herein, the method comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid, the nucleic acid comprising encoding at least one sequence according to the present disclosure, under conditions suitable for allowing expression of the antibody or antigen-binding fragment thereof; and (ii) recovering the expressed antibody or antigen-binding fragment thereof.

[0030] On the other hand, a method for detecting the presence or level of the KIR3DL3 peptide includes detecting the peptide in a sample by using at least one antibody or antigen-binding fragment thereof described herein (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof). In one embodiment, at least one antibody or antigen-binding fragment thereof forms a complex with the KIR3DL3 peptide, and the complex is detected by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical methods, Western blotting, or intracellular flow assay.

[0031] In another aspect, a method for predicting responsiveness to a therapy targeting KIR3DL3 is provided, the method comprising: a) determining the levels of KIR3DL3 and / or HHLA2 in a subject sample using at least one antibody described herein or an antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof); b) determining the levels of KIR3DL3 and / or HHLA2 in a sample from at least one control subject, the at least one control subject having a good response to a therapy targeting KIR3DL3, using at least one antibody described herein or an antigen-binding fragment thereof; and 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 level of KIR3DL3 and / or HHLA2 in the subject sample being the same as or higher than the level of KIR3DL3 and / or HHLA2 in the sample from the at least one control subject indicates that the subject will respond to the therapy. In one embodiment, the therapy uses at least one antibody or antigen-binding fragment thereof described herein (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) to target KIR3DL3.

[0032] In another aspect, a method is provided for predicting responsiveness to a therapy targeting KIR3DL3 using at least one antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) described herein, the method comprising: a) determining the 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, the at least one control subject having a good response to a therapy targeting KIR3DL3; and 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 level of KIR3DL3 and / or HHLA2 in the subject sample being the same as or higher than the level of KIR3DL3 and / or HHLA2 in the sample from the at least one control subject indicates that the subject will respond to the therapy.

[0033] As described above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, the sample is a portion of a single sample obtained from at least one subject or a portion of a combined sample obtained from at least one subject. In another embodiment, the therapy blocks (a) HHLA2 with 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).

[0034] In another aspect, a method for treating a subject with cancer is provided, the method comprising administering to the subject at least one antibody or antigen-binding fragment thereof described herein (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof).

[0035] As described above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, at least one antibody described herein or an antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) (a) reduces the number of proliferating cancer cells in the cancer; (b) reduces the volume or size of the tumor in the cancer; and / or (c) activates T cells and / or NK cells. In another embodiment, at least one antibody described herein or an antigen-binding fragment thereof (e.g., a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof) is administered in a pharmaceutically acceptable formulation. In yet another embodiment, the method described herein further includes administering a therapeutic agent or regimen for treating cancer to a subject. In yet another embodiment, the method described herein further includes administering an additional therapy selected from the group consisting of: immunotherapy, checkpoint blockade, cancer vaccines, chimeric antigen receptors (e.g., CARs targeting CD19), chemotherapy, radiation, targeted therapy, and surgery to a subject. In another embodiment, the subject's cancer cells and / or tumor immune-infiltrating cells express HHLA2. In yet another embodiment, the cancer is selected from the group consisting of: adenocarcinoma, chronic myeloid leukemia (CML), lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-cell CLL, leukemia, B-cell lymphoma, and cancers infiltrated by immune cells expressing the HHLA2 receptor. In yet another embodiment, the cancer is selected from the group consisting of: lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), head and neck cancer, liver cancer, ovarian cancer, prostate cancer, and uterine cancer. In another embodiment, the subject is an animal model of cancer. In yet another embodiment, the animal model is a mouse model, optionally wherein the mouse model is a humanized mouse model. In yet another embodiment, the subject is a mammal, such as a humanized mouse or a human.

[0036] On the other hand, a method for modulating an immune response using at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein is provided. For example, in one embodiment, at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein inhibits or disrupts the interaction between HHLA2 and its binding inhibitor receptor KIR3DL3. In another embodiment, at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein is conjugated to a cytotoxic agent (e.g., a chemotherapeutic agent, a biological agent, a toxin, and / or a radioisotope). In yet another embodiment, the immune response is downregulated. In another embodiment, the immune response is upregulated. In yet another embodiment, the interaction between (a) HHLA2 and KIR3DL3; and / or (b) PD-1 and PD-L1 and / or PD-L2 is blocked. In another embodiment, the anti-KIR3DL3 antibody or antigen-binding fragment thereof is a T-cell activation checkpoint inhibitor for cancer immunotherapy. In yet another embodiment, modulating the immune response includes modulating T-cell function or NK-cell function (e.g., cytotoxicity, such as against cancer cells, such as cancer cells expressing HHLA2). In yet another embodiment, the cancer is selected from the group consisting of: adenocarcinoma, chronic myeloid leukemia (CML), lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-cell CLL, leukemia, B-cell lymphoma, and cancers infiltrated by immune cells expressing the HHLA2 receptor. 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 still another embodiment, a method further includes administering to a subject additional therapies selected from the group consisting of: immunotherapy, checkpoint blockade, cancer vaccines, chimeric antigen receptors (e.g., CARs targeting CD19), chemotherapy, radiation, targeted therapy, and surgery. In yet another embodiment, the immune response is modulated in a cancer animal model (e.g., a mouse model and / or a humanized animal model). In yet another embodiment, the immune response is modulated in a mammal, such as a humanized mouse or a human.

[0037] For any graph that shows bar charts, curves, or other data related to the legend, each bar, curve, or other data presented from left to right corresponds directly to a top-to-bottom or left-to-right box in the legend. Attached Figure Description

[0038] Figure 1A-Figure 1B The results of expression screening for identifying KIR3DL3 as the HHLA2 receptor are shown. Figure 1AThe results of a cell microarray analysis using soluble HHLA2-mIgG2a (HHLA2-Ig) to bind to the indicated cell surface receptor expressed alone in HEK293 cells are shown. HHLA2-Ig shows binding to TMIGD2, KIR3DL3, and the control (FCGR2A), but not to other members of the KIR family, PD-1, PD-L1, or HHLA2. Figure 1B Flow cytometry analysis is shown of HHLA2-Ig or control Ig combined with control 300.19 cells or 300.19 cells that stably express KIR3DL3, TMIGD2 or HHLA2 using specified concentrations of HHLA2-Ig or isotype control (0.1 μg / mL to 160 μg / mL).

[0039] Figures 2A-2D The identification and characterization of KIR3DL3 as the second receptor for HHLA2 are shown. Figure 2A A repeating microarray slide showing cells expressing 384 individual receptors and co-expressing GFP is presented, which identifies KIR3DL3 as the receptor for HHLA2-Ig (top panel) and shows GFP expression as a control for transfection and dot localization (bottom panel). Figure 2B This demonstrates the use of soluble HHLA2-mIgG2a (HHLA2-Ig) to bind to the indicated cell surface receptor expressed alone in HEK293 cells. Figure 1A The results of the cell microarray analysis shown are illustrated. HHLA2-Ig shows binding to TMIGD2, KIR3DL3 and control (FCGR2A), but not to other members of the KIR family, PD-1, PD-L1 or HHLA2. Figure 2C It shows Figure 2B (and Figure 1A The results of transfection control (GFP expression) of the receptor array are shown in the figure. Figure 2D The results of the positive control treatment of the receptor array are shown. Soluble PD-1-Ig and PD-L1-Ig, as well as those obtained with... Figure 2B and Figure 1A Microarray analysis of cells incubated with the same overexpressed receptor group showed binding to FCGR2A, PD-1, and PD-L1 but not to KIR. The PD-L1 site that does not bind to PD-1-Ig is an alternating splice isoform.

[0040] Figures 3A-3E Characterization of a set of KIR3DL3 and HHLA2 mAb is shown. Figure 3A Flow cytometry analysis of the binding of KIR3DL3 mAb to 300.19 cells expressing KIR3DL3. Figure 3BThe ability of KIR3DL3 mAb to block the binding of HHLA2-Ig to 300.19 cells expressing KIR3DL3 was demonstrated. Figure 3C The binding of HHLA2 mAb to 300.19 cells expressing HHLA2 was shown, with 2C4, 2G2 and 6F10 showing the strongest binding, while 6D10 showed less binding. Figure 3D The study demonstrated the ability of HHLA2mAb to block the binding of HHLA2-Ig to 300.19 cells expressing KIR3DL3, with 2C4, 2G2, and 6F10 cells exhibiting the strongest binding. Figure 3E The ability of HHLA2 mAb2G2 and 6F10 to block the binding of HHLA2-Ig to 300.19 cells expressing TMIGD2 was demonstrated.

[0041] Figures 4A-4C The binding of HHLA2-mIgG2a to KIR3DL3 and TMIGD2 was demonstrated. Figure 4A It shows Figure 1B Normalized binding data: binding of HHLA2-mIgG2a to 300.19 cells transfected with KIR3DL3 (blue), or binding of HHLA2-mIgG2a to 300.19 cells transfected with TMIGD2 (cyan), or binding of HHLA2-mIgG2a to 300.19 cells transfected with control HHLA2 (red), or binding of HHLA2-mIgG2a to parental 300.19 cells (green). Figure 4B and Figure 4C This shows 293T cells transfected with KIR3DL3 via flow cytometry. Figure 4B ) or TMIGD2 transfected 293T cells ( Figure 4C HHLA2-mIgG2a or isotype control (10 μg / ml) bound to HHLA2-mIgG2a.

[0042] Figure 5 The binding data of anti-KIR3DL3 mAb on the KIR3DL3-transfected 300.19 mouse pre-B-cell leukemia cell line are shown by flow cytometry.

[0043] Figure 6 Data on the binding of anti-KIR3DL3 mAb to KIR3DL3 by Western blot are presented. Specifically, the results of Western blot analysis of KIR3DL3 mAb using Jurkat cells transfected with KIR3DL3 are shown.

[0044] Figure 7KIR3DL3 expression in Jurkat parental cells, Jurkat cells transfected with KIR3DL3, NK-92 cells, and NK-92-MI cells is shown. The lysate was obtained using 5 μg / ml of anti-KIR3DL3 mAb 574.1F12 blot lysate.

[0045] Figure 8 Single-cell RNA sequencing analysis of KIR3DL3 expression evaluated in publicly available databases is shown (see the EMBL-EBI database available at ebi.ac.uk / gxa / sc / home and the corresponding publication entitled “Reconstructing the human first trimester fetal-maternal interface using single cell transcriptomics”, available at biorxiv.org / content / 10.1101 / 429589v1). KIR3DL3 expression is indicated by blue dots in the right-hand figure. Black boxes highlight decidual NK cells with the highest KIR3DL3 expression.

[0046] Figure 9 This demonstrates the anti-KIR3DL3 mAb blockade caused by HHLA2 binding to KIR3DL3.

[0047] Figures 10A-10D KIR3DL3 expression was shown on activated human T cells and NK92-MI cells. Figure 10A The results are shown for T cells purified from whole blood of four normal donors, activated with CD3 / CD28 antibody tetramer, and subjected to two FACS analyses on specified days to evaluate KIR3DL3 expression in gated CD3+CD4+ and CD3+CD8+ T cells. The results are shown on day 0 (unactivated)... Figure 10B ) and 21 days after activation ( Figure 10C A representative FACS plot of KIR3DL3 expression. Figure 10D The expression of KIR3DL3 on NK92-MI is shown (left panel), but its expression is lowest on NK-92 cells (right panel).

[0048] Figures 11A-11C It was shown that KIR3DL3 is an inhibitory receptor in T cells, and that HHLA2 / KIR3DL3 blockade enhances T cell activation. Figure 11AResults are shown for Jurkat IL-2 reporter T cells expressing KIR3DL3, co-cultured with CHO cells expressing anti-CD3scFV, CHO cells co-expressing anti-CD3scFV and HHLA2, or untransfected CHO cells, with or without the indicated CD28 mAb. Luciferase activity is expressed as relative light units (RLU). Figure 11B and Figure 11C This shows the presence of CD28 mAb and HHLA2 mAb ( Figure 11B ) or KIR3DL3 mAb ( Figure 11C Results of Jurkat IL-2 reporter T cells expressing KIR3DL3, co-cultured with CHO cells co-expressing anti-CD3 scFV and HHLA2. Activation fold of IL-2 reporter luciferase activity is expressed as mean ± SD (n ≥ 3; **** P ≤ 0.0001).

[0049] Figure 12 The HHLA2 / TMIGD2 interaction was shown to enhance T cell activation. Jurkat T cells expressing TMIGD2 and carrying an NFAT promoter linked to luciferase were co-cultured with anti-CD3 scFV CHO cells or HHLA2-anti-CD3 scFVCHO cells, and luciferase activity (RLU) was measured. Quantifications are presented as mean ± SD (n ≥ 3; *** P ≤ 0.001).

[0050] Figure 13 This study demonstrates enhanced expression of anti-KIR3DL3 mAb of IL-2 promoter-driven luciferase in Jurkat-KIR3DL3 T cells in response to anti-CD3-scFV and HHLA2-mediated signaling.

[0051] Figure 14 This study demonstrates enhanced anti-KIR3DL3 mAb expression of IL-2 promoter-driven luciferase in Jurkat-HHLA2 T cells in response to anti-CD3-scFV and HHLA2-mediated signaling.

[0052] Figures 15A-15D The study demonstrated the cytotoxicity of KIR3DL3-CD19-CAR-T cells against HeLa tumors expressing CD19 and HHLA2. Figure 15A The KIR3DL3 / CAR-19 expression plasmid and lentivirus production are shown. Specifically, Figure 15A A schematic diagram of the PMC456-Ef1a expression plasmid is shown. Figure 15B The generation and expansion of KIR3DL3 / CD19-CAR-T cells were demonstrated. Specifically, Figure 15BThe FACS image of KIR3DL3 / CAR-19T cells (PMC456 cells) is shown. Figure 15C The generation of stable HeLa-CD19 and HeLa-CD19+KIR3DL3-expressing cells was demonstrated. Specifically, Figure 15C FACS images of HeLa-CD19 and HeLa-CD19-KIR3DL3 tumor cells are shown. Figure 15D This study demonstrated that HHLA2 mAb enhances the cytotoxicity of KIR3DL3 CD19-CAR-T cells against HHLA2+CD19-transfected HeLa tumor cells.

[0053] Figures 16A-16C The cytotoxicity assay of NK92 cells expressing KIR3DL3 against HeLa tumor target cells expressing or not expressing HHLA2 is shown. Figure 16A The KIR3DL3 expression plasmid and lentivirus generation are shown. Specifically, Figure 16A A schematic diagram of the PMC579 KIR3DL3 expression plasmid is shown. Figure 16B This illustrates the derivation of NK92 cells transduced with KIR3DL3. Specifically, Figure 16B The KIR3DL3 / NK92 FACS plot is shown. Figure 16C The images show the derivation of K562 and HeLa cells transfected or transduced with HHLA2, respectively. Specifically, Figure 16C The FACS diagrams of HHLA2-transfected K562 cells and HHLA2-transduced HeLa tumor cells are shown.

[0054] Figures 17A-17C The cytotoxicity of NK92 on HeLa alone and HeLa-transduced tumor target cells expressing HHLA2 was demonstrated. Figure 17A The KIR3DL3-HHLA2 interaction / pathway was shown to inhibit NK92 cytotoxicity. Figure 17B The enhanced cytotoxicity of HHLA2 mAb and KIR3DL3 mAb to NK92-KIR3DL3 cells was demonstrated. Figure 17C A schematic diagram of some cytotoxicity assays is shown.

[0055] Figure 18 The expression of β2-microglobulin and HHLA2 in Raji-B2M KO transfected with Raji-B2M KO and HHLA2 is shown by flow cytometry.

[0056] Figures 19A-19E It was shown that KIR3DL3 is an inhibitory receptor in NK cells, and that HHLA2 / KIR3DL3 blockade enhances NK cell cytotoxicity. Figure 19AThe cytotoxicity of NK92-MI against Raji cells with B2M deletion (Raji-B2M KO cells) and Raji-B2M KO cells expressing HHLA2 was demonstrated. Figure 19B and Figure 19C This shows that in the presence of 10ug / ml KIR3DL3 antibody ( Figure 19B ) or HHLA2 antibody ( Figure 19C NK92-MI showed cytotoxicity against Raji-B2M KO cells expressing HHLA2 at an indicated E / T ratio in the presence of both NK92-MI and isotype controls. Figure 19D Results of NK92-MI cells incubated with Raji B2M KO cells or with Raji B2M KO cells overexpressing HHLA2 are shown. Degranulation was measured as the percentage of CD107a-positive cells in the CD56+ population. Controls were effector cells alone or effector cells with PMA / ION, which resulted in complete degranulation. Figure 19E This study demonstrates enhanced degranulation of NK92-MI cells targeting HHLA2-overexpressing RajiB2M KO cells in the presence of KIR3DL3 mAb (1G7) compared to isotype controls. Quantification is presented as mean ± SD (N≥3; P≥0.05; *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001).

[0057] Figure 20 This shows that HHLA2 expression is different from PD-L1 expression. Figure 20 The expression levels of B7 gene family members in RCC are shown compared to those in normal kidneys from Cancer Genome Atlas (TCGA) samples.

[0058] Figures 21A-21B A model of the HHLA2 pathway is shown. HHLA2 delivers immune stimulation signals via TMIGD2 in primary T cells or NK cells. Figure 21A The study illustrates T cell activation leading to loss of TMIGD2 expression and gain of KIR3DL3. HHLA2 delivers immunosuppressive signals via KIR3DL3 in activated T cells. Figure 21BThis study illustrates NK cell lysis activity regulated by inhibitory and activating receptors. Inhibitory receptors include most KIRs, CD94 / NKG2A, and LILRBI, which recognize MHC classes C1, E, and G, respectively. Activating receptors include NKG2D, NKp30, NKp44, NKp46, CD94 / NKG2C, and TMIGD2, which recognize ULBP-1, MICA, MICB, B7-H6, HLA-E, HHLA2, and others. If tumors lose MHC expression (self-loss), inhibitory signaling decreases, and activating signaling becomes dominant, leading to NK cell lysis of the tumor. HHLA2 on tumors is an MHC-independent inhibitory signal that inhibits the lysis of KIR3DL3-positive NK cells.

[0059] Figure 22 A schematic diagram of the construction of the KIR3DL3 x PD-1 bispecific antibody is shown.

[0060] Figure 23 The binding sensing diagram of KIR3DL3 and PD-1 human IgG4 and scFV antibodies in the Octet assay is shown. Detailed Implementation

[0061] HHLA2, a member of the B7 gene family, is widely expressed in various tumor and antigen-presenting cells and is considered a ligand for both T cell activation and inhibition. TMIGD2, expressed in primary T cells, is the activating receptor for HHLA2 and transduces co-stimulatory signals upon involvement of the T cell antigen receptor (TCR). TMIGD2 is downregulated upon repeated TCR stimulation. HHLA2 binds to another receptor, KIR3DL3, which is expressed on both T and NK cells. As described herein, this disclosure covers the understanding that, unlike the immune-activating function of the HHLA2-TMIGD2 interaction, the HHLA2-KIR3DL3 interaction can suppress immune responses and provides an attractive target for regulating a variety of diseases, conditions, or symptoms, including, for example, cancer.

[0062] This disclosure is based, at least in part, on the finding that targeting KIR3DL3 can specifically block the HHLA2-KIR3DL3 interaction that suppresses the immune response. Importantly, targeting KIR3DL3 does not disrupt the overall function of HHLA2, which includes activating the immune response through its interaction with TMIGD2. Therefore, precise targeting of KIR3DL3 provides the specificity to block only the immunosuppressive function of HHLA2, thereby evoking an effective immune response, for example, against cancer cells, without downregulating the immune-activating function of HHLA2.

[0063] This disclosure is also based, at least in part, on the finding that agents targeting KIR3DL3 and PD-1 can be used to modulate immune responses and / or treat cancer. In some embodiments, the KIR3DL3 x PD-1 bispecific antibody described herein is a checkpoint immunotherapy for activating T cells and NK cells in tumors. In some embodiments, the KIR3DL3 x 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 checkpoint blockade of KIR3DL3 mAb and / or KIR3DL3 x PD-1 bispecific antibody.

[0064] A group of exemplary representative anti-KIR3DL3 human monoclonal antibodies (mAbs) are described herein as immune checkpoint inhibitors. Blocking and non-blocking anti-KIR3DL3 mAbs were identified, and anti-KIR3DL3 mAbs that block HHLA2 binding to KIR3DL3 showed up as checkpoint inhibitor antibodies in T-cell and NK-cell assays. The binding characteristics of these candidate therapeutic anti-KIR3DL3 antibodies and their variable region heavy and light chain gene sequences are described herein.

[0065] A group of exemplary representative bispecific antibodies or their antigen-binding fragments that bind to both KIR3DL3 and PD-1 are also described herein as immune checkpoint inhibitors. Targeting two immune checkpoints with non-overlapping expression provides combination therapy with additive or synergistic antitumor activity.

[0066] Therefore, this disclosure provides monoclonal antibodies that specifically bind to KIR3DL3 and their antigen-binding fragments, bispecific antibodies that bind to KIR3DL3 and PD-1 and their antigen-binding fragments, as well as their immunoglobulins, peptides, nucleic acids, and methods of using such antibodies, such as for immunomodulatory and therapeutic purposes.

[0067] I. Definition

[0068] The articles “a” and “an” used in this article refer to one or more of the grammatical objects of the article (i.e., at least one). For example, “an element” refers to one or more elements.

[0069] The term "altered amount" of a biomarker refers to an increase or decrease in the copy number of the biomarker and / or an increase or decrease in the nucleic acid level of one or more specific biomarker genes in the sample compared to the amount of the biomarker in the control sample. The term "altered amount" of a biomarker also includes an increase or decrease in the protein level of the biomarker in the sample compared to the protein level of the biomarker in a normal control sample.

[0070] The term "altered activity" of a biomarker refers to an increase or decrease in the activity of a biomarker in a disease state compared to its activity in a normal control sample, such as in a biological sample. Altered activity of a biomarker can be, for example, altered expression of the biomarker, altered protein levels of the biomarker, altered structure of the biomarker, or altered interactions with other proteins involved in the same or different pathways as the biomarker, or altered interactions with transcriptional activators or inhibitors.

[0071] The term "altered structure" of a biomarker refers to the presence of mutations or allele variants within the biomarker gene or protein compared to the normal or wild-type gene or protein. These mutations can affect the expression or activity of the biomarker. For example, mutations include, but are not limited to, substitution, deletion, or addition mutations. Mutations may be present in either the coding or non-coding regions of the biomarker.

[0072] The term "activating receptor" includes immune cell receptors that bind to antigens, complex antigens (e.g., in the case of MHC peptides), or antibodies. Such activating receptors include T-cell receptors (TCRs), B-cell receptors (BCRs), cytokine receptors, LPS receptors, complement receptors, and Fc receptors.

[0073] T cell receptors are present on T cells and are associated with the CD3 peptide. In the presence of MHC peptides, T cell receptors are stimulated by antigens (and polyclonal T cell activating agents). T cell activation via the TCR leads to numerous changes, such as protein phosphorylation, membrane lipid changes, ion flux alterations, cyclic nucleotide changes, RNA transcriptional changes, protein synthesis changes, and cell volume changes.

[0074] The terms "chimeric antigen receptor," "CAR," or "CAR-T" refer to engineered T-cell receptors (TCRs) with desired antigen specificity. T lymphocytes recognize specific antigens by interacting with short peptides presented by T-cell receptors (TCRs) with major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, primordial 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 unresponsiveness and clonal non-reactivity. To bypass immunity, various methods have been developed to derive cytotoxic effector cells with transplantation recognition specificity. CARs have been constructed consisting of binding domains derived from natural ligands or antibodies specific to cell surface components of the TCR-associated CD3 complex. Upon antigen binding, these chimeric antigen receptors connect to endogenous signaling pathways in effector cells and generate activation signals similar to those initiated by the TCR complex. For example, CARs targeting CD19 (a protein highly expressed on hematologic malignancies) have shown promising clinical efficacy. Since the first report on chimeric antigen receptors, the concept has been steadily improved, and the molecular design of chimeric receptors has been optimized, with the routine use of any number of well-known binding domains, such as scFV, Fav, and another protein-binding fragment described herein.

[0075] Generally, CAR is a type of "cell therapy" (e.g., T-cell therapy) contemplated for use according to this disclosure. While many representative embodiments of agents and methods for modulating immune cell activity by regulating the KIR3DL3 pathway, such as regulating the interaction between KIR3DL3 and its natural binding partner such as HHLA2, are also included, immune cell-based therapies and methods are also included. For example, T cells engineered to have KIR3DL3 knockout, knockdown, or increased expression are contemplated. Similarly, immune cells or other cells engineered to have KIR3DL3, HHLA2 ligand knockout, knockdown, or increased expression are also contemplated.

[0076] B cell receptors (BCRs) are located on B cells. B cell antigen receptors are complexes between membrane Ig (mIg) and other transmembrane polypeptides (such as Igα and Igβ). The signal transduction function of mIg is triggered by oligomeric or multimeric antigen-crosslinked receptor polypeptides. B cells can also be activated by anti-immunoglobulin antibodies. Upon BCR activation, B cells undergo numerous changes, including tyrosine phosphorylation.

[0077] Fc receptors are found on many cells involved in the immune response. Fc receptors (FcRs) are cell surface receptors for the Fc portion of immunoglobulin polypeptides (Ig). Human FcRs identified to date are those that recognize IgG (called FcγR), IgE (FcεR1), IgA (Fcα), and polymeric IgM / A (FcμαR). FcRs are found in the following cell types: FcεR I (mast cells), FcεR II (many leukocytes), FcαR (neutrophils), and FcμαR (glandular epithelial cells, hepatocytes) (Hogg, N. (1988) *Immunol. Today* 9:185-86). Extensively studied FcγRs are located at the heart of cellular immune defense and are responsible for stimulating the release of inflammatory mediators and hydrolases involved in the pathogenesis of autoimmune diseases (Unkeless, JC et al. (1988), *Annu. Rev. Immunol.* 6:251-81)). FcγRs provide a crucial link between effector cells and Ig-secreting lymphocytes, as macrophage / monocyte, polymorphonuclear leukocyte, and natural killer (NK) cell FcγRs confer specific recognition elements mediated by IgG. Human leukocytes possess at least three distinct IgG receptors: hFcγRI (found on monocytes / macrophages), hFcγRII (on monocytes, neutrophils, eosinophils, platelets, possibly B cells, and the K562 cell line), and FcγIII (on NK cells, neutrophils, eosinophils, and macrophages).

[0078] For T cells, the transmission of co-stimulatory signals to T cells involves signal transduction pathways that are not inhibited by cyclosporine A. Additionally, co-stimulatory signals can induce cytokine secretion (e.g., IL-2 and / or IL-10) in T cells and / or prevent the induction of antigen nonresponsiveness, induce unresponsiveness, or induce cell death (loss) in T cells.

[0079] The term "activity," when used with respect to peptides such as KIR3DL3 and / or KIR3DL3's natural binding mate such as HHLA2, encompasses the activity inherent in the protein structure. For example, with respect to HHLA2 ligands, the term "activity" includes the ability to modulate immunosuppression by modulating inhibitory signals in immune cells (e.g., by binding to natural receptors on immune cells). Those skilled in the art will recognize that when the activated form of the HHLA2 ligand peptide binds to an inhibitory receptor such as KIR3DL3, an inhibitory signal is generated in the immune cell.

[0080] The term "inhibitory signaling" refers to signals transmitted via inhibitory receptors (e.g., KLRB1, CTLA4, PD-1, etc.) on peptides on immune cells. Such signals antagonize signals by activating receptors (e.g., via TCR, CD3, BCR, TMIGD2, or Fc peptides) and may result in, for example, inhibition of second messenger production; inhibition of proliferation; inhibition of effector functions in immune cells, such as reduced phagocytosis, decreased antibody production, reduced cytotoxicity, inability of immune cells to produce mediators (e.g., cytokines (e.g., IL-2) and / or mediators of allergic responses); or the development of unresponsiveness.

[0081] If the amount of a biomarker is greater than or less than the normal or control level by a factor greater than the standard error of the determination used to assess the amount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% of the amount, then the amount of the biomarker in the subject is “significantly” higher than or lower than the normal amount of the biomarker. Alternatively, if the stated amount is 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%, two times, three times, four times, five times or more, or any range between the two such as 5%-100%, whichever is greater or less than the normal and / or control amount of the biomarker, then the amount of the biomarker in the subject can be considered “significantly” higher or lower than the normal and / or control amount. Such significant modulatory values ​​can be applied to any of the metrics described in this article, such as altered expression levels, altered activity, changes in cancer cell hyperproliferative growth, changes in cancer cell death, changes in biomarker inhibition, changes in assay binding, etc.

[0082] The term "altered expression level" for a biomarker refers to the expression level or copy number of the biomarker in a test sample, such as a sample from a subject with cancer, that is greater than or less than the standard error of the determination used to assess expression or copy number, and preferably is at least two times, more preferably three times, four times, five times, or ten times or more, the expression level or copy number of the biomarker or chromosomal region in control samples (e.g., samples from healthy subjects without the relevant disease) and preferably the average expression level or copy number of the biomarker or chromosomal region in several control samples. The altered expression level is greater than or less than the standard error of the determination used to assess expression or copy number, and preferably is at least two times, more preferably three times, four times, five times, or ten times or more, the expression level or copy number of the biomarker in control samples (e.g., samples from healthy subjects without the relevant disease) and preferably the average expression level or copy number of the biomarker in several control samples.

[0083] Unless otherwise stated herein, the terms “antibody” and “antibodies” broadly encompass naturally occurring antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric antibodies, humanized antibodies, and multispecific antibodies, as well as all of the above fragments and derivatives having at least an antigen-binding site. Antibody derivatives may include protein or chemical moieties conjugated to antibodies. An “antibody” is a glycoprotein or its antigen-binding moiety comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as V). H Each light chain contains a heavy chain constant region and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as V in this paper). L ) and a light chain constant region. The light chain constant region contains a structural domain, CL. V H District and V L The region can be further subdivided into hypervariable regions called complementary determinant regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The term "inactivated antibody" refers to an antibody that does not induce the complement system.

[0084] As used herein, the term “antibody” also includes the “antigen-binding portion” (or simply “antibody portion”) of an antibody. As used herein, the term “antigen-binding portion” refers to one or more fragments of an antibody (e.g., the KIR3DL3 polypeptide or a fragment thereof) that retain the ability to bind specifically to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments covered in the term “antigen-binding portion” of an antibody include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′)2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be linked using recombination methods by enabling them to act as synthetic linkers for a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide (referred to as a single-chain Fv (scFv)); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proceedings of the National Academy of Sciences 85:5879-5883; and Osbourn et al. (1998) Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of antibody. Any VH and VL sequence of a particular scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used to generate Fab, Fv, or other immunoglobulin fragments using protein chemistry or recombinant DNA techniques. Other forms of single-chain antibodies, such as biantibodies, are also covered. Biantibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow the two domains on the same chain to pair, thus forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, for example, Holliger, P. et al. (1993), Proceedings of the National Academy of Sciences 90:6444-6448; Poljak, RJ et al. (1994), Structure 2:1121-1123).

[0085] Furthermore, the antibody or its antigen-binding moiety can be part of a larger immunoadhesion polypeptide formed by the covalent or non-covalent binding of the antibody or antibody moiety to one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include the preparation of tetrameric scFv polypeptides using the streptavidin core region (Kipriyanov, SM et al. (1995), Human Antibodies and Hybridomas, 6:93-101) and the preparation of divalent and biotinylated scFv polypeptides using cysteine ​​residues, marker peptides, and C-terminal multihistidine tags (Kipriyanov, SM et al. (1994), Molecular Immunology, 31:1047-1058). Antibody moieties such as Fab and F(ab′)2 fragments can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of the whole antibody. Additionally, as described herein, antibodies, antibody moieties, and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques.

[0086] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or homologous; or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies can also be fully human. In one embodiment, the antibody covered by this disclosure binds specifically or substantially specifically to a KIR3DL3 polypeptide or a fragment thereof. As used herein, the terms “monoclonal antibody” and “monoclonal antibody composition” refer to a group of antibody polypeptides containing only one species of antigen-binding sites capable of immune responses to specific epitopes of an antigen, while the terms “polyclonal antibody” and “polyclonal antibody composition” refer to a group of antibody polypeptides containing multiple species of antigen-binding sites capable of interacting with specific antigens. Monoclonal antibody compositions typically exhibit a single binding affinity to the specific antigen with which they immune responses.

[0087] The term "body fluid" refers to fluids that are excreted or secreted from the body, as well as fluids that are not normally excreted or secreted (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, cerumen and earwax, Cowper's fluid or pre-ejaculate fluid, chyle, chyme, feces, female ejaculation, interstitial fluid, intracellular fluid, lymph, menstruation, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubricant, vitreous humor, vomitus).

[0088] The terms "cancer," "tumor," or "hyperplastic disorder" refer to cells possessing the typical characteristics of cancerous cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain distinctive morphological features. Cancer cells typically take the form of tumors, but these cells can exist alone in an animal or as non-tumorigenic cancer cells, such as leukemia cells. Cancer includes, but is not limited to, B-cell carcinomas such as multiple myeloma and Waldenström macroglobulinemia. Macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease and μ chain disease, benign monoclonal gammopathy and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematologic malignancies, etc. Other non-limiting examples of cancer types suitable for the methods covered by this disclosure include human sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor. Tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hidradenoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, 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 Cytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal tumors, angioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, retinoblastomas; leukemias, such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); as well as 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 epithelial in nature and 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 cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancer can be characterized in various other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0089] For example, the term "CDR" and its plural "CDR" refer to complementarity-determining regions (CDRs), where three constitute the binding features of the light chain variable regions (CDR-L1, CDR-L2, and CDR-L3), and three constitute the binding features of the heavy chain variable regions (CDR-H1, CDR-H2, and CDR-H3) on the antibody. CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences comprising scaffold or framework regions. The precisely defined boundaries and lengths of CDRs depend on different classification and numbering systems. Therefore, Kabat, Chothia, contact, or any other boundary definition may refer to CDRs. Despite the different boundaries, each of these systems has a degree of overlap in the content constituting the so-called "hypervariate region" within the variable sequence. Therefore, CDR definitions can differ relative to adjacent framework regions in terms of length and boundary regions according to these systems. See, for example, Kabat, Chothia and / or MacCallum et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, 1992; Chothia et al. (1987), Journal of Molecular Biology, 196, 901; and MacCallum et al., Journal of Molecular Biology (1996), 262, 732, each of which is incorporated herein by reference in its entirety).

[0090] As used herein, the term "classification" encompasses "associating" or "categorizing" a sample with a disease state. In some cases, "classification" is based on statistical evidence, empirical evidence, or both. In some embodiments, classification methods and systems use a so-called training sample set with known disease states. Once established, the training dataset serves as a basis, model, or template for comparing features of unknown samples in order to classify the unknown disease state of the samples. In some cases, classifying a sample is analogous to diagnosing the disease state of the sample. In other cases, classifying a sample is analogous to distinguishing the disease state of the sample from another disease state.

[0091] As used herein, the term “coding region” refers to a region of a nucleotide sequence that includes codons that translate into amino acid residues, while the term “non-coding region” refers to a region of a nucleotide sequence that does not translate into amino acids (e.g., the 5′ and 3′ untranslated regions).

[0092] "Complementary" or "complementary" is a broad concept referring to sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that if the residue is thymine or uracil, 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. Similarly, it is known that if the residue is guanine, 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. If, when the two regions are arranged in an antiparallel manner, at least one nucleotide residue in the first region can base pair with a residue in the second region, then the first region of the nucleic acid is complementary to the second region of the same or different nucleic acid. 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%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues in the first portion can base pair with nucleotide residues in the second portion. In another embodiment, all nucleotide residues in the first part are capable of base pairing with nucleotide residues in the second part.

[0093] As used herein, the term "complex antibody" refers to an antibody having a variable region comprising germline or non-germline immunoglobulin sequences derived from two or more unrelated variable regions. Additionally, the term "complex human antibody" refers to an antibody having a constant region derived from a human germline or non-germline immunoglobulin sequence and a variable region comprising human germline or non-germline sequences derived from two or more unrelated human variable regions. Complex human antibodies can be used as an effective component in therapeutic agents according to this disclosure because they exhibit reduced antigenicity in the human body.

[0094] The term "control" refers to any reference standard suitable for providing comparison with the expressed product in the test sample. In one embodiment, control includes obtaining a "control sample," detecting the level of the expressed product from the control sample, and comparing it with the level of the expressed product from the test sample. Such a control sample may include any suitable sample, including but not limited to samples from control cancer patients with known outcomes (which may be stored samples or measurements from previous samples); normal tissues or cells isolated from subjects such as normal patients or cancer patients; cultured primary cells / tissues isolated from subjects such as normal subjects or cancer patients; adjacent normal cells / tissues obtained from the same organ or body site of a cancer patient; tissue or cell samples isolated from normal subjects; or primary cells / tissues obtained from a collection. In another preferred embodiment, control may include reference standard expressed product levels from any suitable source, including but not limited to housekeeping genes, ranges of expressed product levels from normal tissues (or other previously analyzed control samples), and ranges of previously determined expressed product levels from test samples from a group of patients or a group of patients with specific outcomes (e.g., survival for one, two, three, four years, etc.) or receiving a certain treatment (e.g., standards of care for cancer therapy). Those skilled in the art will understand that such control samples and reference standard expression product levels can be combined as controls in the methods covered by this disclosure. In one embodiment, a control may include normal or non-cancer cell / tissue samples. In another preferred embodiment, a control may include expression levels of a group of patients, such as a group of cancer patients or a group of cancer patients receiving a certain treatment or a group of patients with one outcome versus another. In the former case, the specific expression product level for each patient may be specified as a percentile expression level or expressed as an average or mean above or below a reference standard expression level. In another preferred embodiment, a control may include normal cells, cells from patients treated with combination chemotherapy, and cells from patients with benign cancer. In another embodiment, a control may also include a measurement, such as the average expression level of a particular gene in a population compared to the expression levels of housekeeping genes in the same population. Such a population may include normal subjects, cancer patients who have not undergone any treatment (i.e., have not received treatment), cancer patients receiving standard care, or patients with benign cancer. In another preferred embodiment, the control includes a ratio conversion of expression product levels, including, but not limited to, determining a ratio of the expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard; determining the expression product levels of two or more genes in the test sample and determining the difference in expression product levels in any suitable control; and determining the expression product levels of two or more genes in the test sample, normalizing their expression relative to the expression of housekeeping genes in the test sample, and comparing them to any suitable control.In a particularly preferred embodiment, the control includes a control sample having the same lineage and / or type as the test sample. In another embodiment, the control may include expression product levels grouped into percentiles within or based on a set of patient samples, such as all patients with cancer. In one embodiment, control expression product levels are established where expression product levels higher or lower than, for example, a specific percentile are used as the basis for predictive outcomes. 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 the test sample are compared to control expression product levels as the basis for predictive outcomes. As shown in the data below, the methods covered by this disclosure are not limited to using specific cutoff points when comparing expression product levels in the test sample with controls.

[0095] The term "co-stimulation," when used in relation to activated immune cells, refers to 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, a co-stimulatory signal may lead to cytokine secretion, for instance, in T cells that have received a signal mediated by a T cell receptor. Immune cells that have received a cell receptor-mediated signal, for example, through an activating receptor, are referred to herein as "activated immune cells."

[0096] The term “co-stimulatory receptor” includes receptors that transmit co-stimulatory 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 transduced by inhibitory receptors may occur even in the absence of co-stimulatory receptors (such as CD28) on immune cells, and therefore it is not merely a competition between inhibitory receptors and co-stimulatory receptors for the function of binding co-stimulatory peptides (Fallarino et al. (1998) J. Exp. Med. 188:205). The transmission of inhibitory signals to immune cells may result in unresponsive or non-reactive or programmed cell death in the immune cells. Preferably, the transmission of inhibitory signals is carried out through mechanisms that do not involve apoptosis. As used herein, the term “apoptosis” includes programmed cell death, which can be characterized using techniques known in the art. Apoptotic cell death can be characterized, for example, by cell contraction, membrane bubbling, and chromatin condensation that ultimately leads to cell fragmentation. Cells undergoing apoptosis also exhibit characteristic patterns of internuclear DNA cleavage. Depending on the form of the peptide binding to the receptor, signals can be transmitted (e.g., via multivalent forms of HHLA2 and / or KIR3DL3 peptides) or the signal can be inhibited (e.g., via soluble monovalent forms of HHLA2 and / or KIR3DL3), for example by competing with the activated forms of HHLA2 and / or KIR3DL3 for binding to one or more natural binding partners. However, the presence of soluble peptides can be stimulating. The role of modulators can be readily demonstrated using routine screening assays as described herein.

[0097] The term "determining an appropriate treatment regimen for a subject" means determining a treatment regimen (i.e., a single therapy or combination of different therapies for the prevention and / or treatment of the subject's cancer) based on, substantially based on, or at least partially based on the analytical results of this disclosure. One example is determining whether to provide a targeted therapy against the cancer to provide an immunomodulatory 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 KIR3DL3)). Another example is initiating adjuvant therapy after surgery to reduce the risk of recurrence, and yet another is modifying the dosage of a specific chemotherapy therapy. In addition to the analytical results of this disclosure, determination can also be based on the individual characteristics of the subject to be treated. In most cases, the actual determination of an appropriate treatment regimen for the subject will be made by the attending physician or doctor.

[0098] As used herein, the term "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, comprising the native sequence Fc region and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for antibodies covered by this disclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0099] As used herein, “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are receptors that bind to IgG antibodies (γ receptors) and comprise subclasses of FcγRI, FcγRII, and FcγRIII, including allelic variants and alternative splice forms of these receptors. FcγRII receptors comprise FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”) having similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activation motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain (see M). Annual Review of Immunology 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annual Review of Immunology 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). The term “FcR” in this article encompasses other FcRs, including those to be identified in the future.

[0100] If the molecule is covalently or non-covalently associated with the substrate, the molecule is "fixed" or "attached" to the substrate, so that the substrate can be washed with a fluid (e.g., standard citrate, pH 7.4) without most of the molecules dissociating from the substrate.

[0101] As used herein, and as defined herein, “frame region” or “FR” residues are those variable domain residues other than CDR residues.

[0102] "Functionally conserved variants" are variants in which a given amino acid residue in a protein or enzyme has been altered without changing the overall conformation and function of the polypeptide. This includes, but is not limited to, amino acid substitutions with amino acids having similar properties (e.g., polarity, hydrogen bond potential, acidity, basicity, hydrophobicity, aromaticity, etc.). The amino acids, other than those indicated as conserved, may differ in the protein, such that the percentage of protein or amino acid sequence similarity between any two proteins with similar functions can vary and may be, for example, 70% to 99% as determined by an alignment scheme, such as by a clustering method, where the similarity is based on the MEGALIGN algorithm. "Functionally conserved variants" also include polypeptides having at least 60% amino acid identity, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, as determined by BLAST or FASTA algorithms, and having the same or substantially similar properties or functions as the native or parent protein to which they are compared.

[0103] As used herein, the term "heterologous antibody" is defined in relation to a transgenic nonhuman organism that produces such antibodies. This term refers to antibodies that have an amino acid sequence or a nucleic acid-coding sequence found in an organism not composed of transgenic nonhuman animals, and are generally derived from a species other than the transgenic nonhuman animal species.

[0104] The terms “high,” “low,” “intermediate,” and “negative” related to cellular biomarker expression refer to the amount of a biomarker expressed relative to the cellular expression of a biomarker in one or more reference cells. Biomarker expression can be determined according to any of the methods described herein, including, but not limited to, analyzing the cellular level, activity, structure, etc., of one or more biomarker genomic nucleic acids, ribonucleic acids, and / or peptides. In one embodiment, the terms refer to a defined percentage of a cell population expressing a biomarker at the highest, intermediate, 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 between both, including extreme values, of the cell population expressing either high or weak biomarkers. The term "low" does not include cells that cannot detectably express the biomarker, as such cells are "negative" for biomarker expression. The term "moderate" includes cells that express the biomarker, but at a level lower than that of the population expressing the biomarker at a "high" level. In another embodiment, the term may also refer to, or alternatively to, a cell population expressing the biomarker as identified by qualitative or statistical plots. For example, according to methods well-known in the art, cell populations sorted using flow cytometry can be differentiated based on biomarker expression levels by identifying different plots based on detectable portion analysis, such as based on average fluorescence intensity. Such plot areas can be refined based on the number, shape, overlap, etc., of the biomarker of interest using methods well-known in the art. In yet another embodiment, terms can also be determined based on the presence or absence of expression of other biomarkers.

[0105] As used herein, “homology” refers to the nucleotide sequence similarity between two regions of the same nucleic acid chain or between regions of two different nucleic acid chains. Regions are homologous at said positions when nucleotide residue positions in two regions are occupied by the same nucleotide residue. A first region and a second region are homologous if at least one nucleotide residue position in each region is occupied by the same residue. Homology between two regions is expressed as the proportion of nucleotide residue positions in the two regions occupied by the same nucleotide residue. For example, a region having the nucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotide sequence 5′-TATGGC-3′ have 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95%, of the nucleotide residue positions in each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions in each of the portions are occupied by the same nucleotide residue.

[0106] As used herein, the term "host cell" is intended to refer to a cell in which a nucleic acid covered by this disclosure, such as a recombinant expression vector covered by this disclosure, has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to specific subject cells but also to the progeny or potential progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell but are still included within the scope of the terminology used herein.

[0107] As used herein, the term "humanized antibody" is intended to encompass antibodies produced by non-human cells having variable and constant regions that have been modified to more closely resemble antibodies produced by human cells. This can be achieved, for example, by altering the amino acid sequence of the non-human antibody to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies may contain, for example, amino acid residues in the CDR that are not encoded by human germline immunoglobulin sequences (e.g., through in vitro random mutagenesis or site-specific mutagenesis, or through mutations introduced via in vivo somatic mutations). The term "humanized antibody," as used herein, also encompasses antibodies in which a CDR sequence derived from another mammalian species, such as a mouse, has been grafted onto a human scaffold sequence.

[0108] Humanized mice, as used in this article, are mice carrying 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 severely combined immunodeficient (SCID) mice can be used for this purpose. NCG mice, NOG mice, and NSG mice can be used to transplant human cells and tissues more effectively than other models. Such humanized mouse models can be used to model the human immune system under both healthy and pathological conditions and can enable the evaluation of therapeutic candidates in an in vivo environment relevant to human physiology.

[0109] As used herein, the terms “hypervariant region,” “HVR,” or “HV” refer to regions where the sequence of the antibody variable domain is hypervariable and / or forms a structure-defined loop, and which contain a CDR.

[0110] 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.

[0111] As used herein, the term "immunocondition" includes immune diseases, symptoms, and susceptibility to, but is not limited to, cancer, chronic inflammatory diseases and conditions (including, for example, Crohn's disease, inflammatory bowel disease, reactive arthritis, and Lyme disease), insulin-dependent diabetes mellitus, and organ-specific autoimmunity (including, for example, multiple sclerosis, Hashimoto's thyroiditis, autoimmune uveitis, and Grave's disease). Diseases such as contact dermatitis, psoriasis, graft rejection, graft-versus-host disease, sarcoidosis, atopic conditions (including, for example, asthma and allergies, including but not limited to allergic rhinitis and gastrointestinal allergies such as food allergies), eosinophilia, conjunctivitis, glomerulonephritis, systemic lupus erythematosus, scleroderma, susceptibility to certain pathogens such as helminthiasis (including, for example, leishmaniasis), and certain viral infections (including, for example, HIV and bacterial infections such as tuberculosis and leprosy) and malaria.

[0112] 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. Additionally, the term immune response includes immune responses indirectly influenced by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells such as macrophages.

[0113] The term "immunotherapy agent" can include any molecule, peptide, antibody, or other agent that can stimulate the host's immune system to generate an immune response against a tumor or cancer in a subject. A variety of immunotherapy agents can be used in the compositions and methods described herein.

[0114] The term "immune checkpoint" refers to a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune immune responses by downregulating or inhibiting antitumor immune responses. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, lactolipoprotein, and A2aR (see, for example, WO2012 / 177624). The terminology further encompasses bioactive protein fragments, as well as nucleic acids encoding full-length immune checkpoint proteins and their bioactive protein fragments. In one embodiment, the terminology further encompasses any fragments described in accordance with the homology description provided herein.

[0115] Immune checkpoints and their sequences are well known in the art, and representative examples are described below. For example, the term "PD-1" refers to a member of the immunoglobulin gene superfamily that functions as a co-inhibitory receptor with PD-L1 and PD-L2 as known ligands. PD-1 was previously identified using a subtractive cloning-based approach to select genes upregulated during TCR-induced activated T cell death. Based on its ability to bind to PD-L1, PD-1 is a member of the CD28 / CTLA-4 molecular family. Like CTLA-4, PD-1 is rapidly induced on the surface of T cells in response to anti-CD3 (Agata et al. 25 (1996) International Journal of Immunology 8:765). However, unlike CTLA-4, PD-1 is also induced on the surface of B cells (in response to anti-IgM). PD-1 is also expressed in subsets of thymocytes and bone marrow cells (Agata et al. (1996) (ibid.); Nishimura et al. (1996) International Journal of Immunology 8:773).

[0116] As used herein, the term “inhibition” and its grammatical equivalents refer to the reduction, restriction, and / or prevention of a particular action, function, or interaction. In one embodiment, the term refers to reducing the level of a given output or parameter to a quantity (e.g., background staining, KIR3DL3 signaling, KIR3DL3 immunosuppressive function, etc.) that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or less than the quantity in a corresponding control. A reduction in the level of a given output or parameter does not necessarily (though may) mean the absolute absence of the output or parameter. The present invention does not require, and is not limited to, methods for the complete elimination of an output or parameter. A given output or parameter can be determined using methods well known in the art, including, but not limited to, immunohistochemical, molecular biological, cell biological, clinical, and biochemical assays as discussed herein and in examples. The opposite terms “promote,” “increase,” and their grammatical equivalents refer to an increase in a given level of output or parameter, as described in relation to suppression or reduction.

[0117] As used herein, when referring to the interaction between two molecules, the term "interaction" means the physical contact (e.g., binding) between the molecules (e.g., the binding of HHLA2 to TMIGD2 or HHLA2 to KIR3DL3). Typically, such interactions result in the activity (biological effect) of one or both of the molecules. This activity can be the direct activity of one or both molecules (e.g., signal transduction). Alternatively, one or both interacting molecules can be prevented from binding to their ligands, thus remaining inactive in terms of ligand-binding activity (e.g., binding to their ligands and triggering or inhibiting an immune response). Inhibiting such interactions would disrupt the activity of one or more of the interacting molecules. Enhancing such interactions is intended to prolong or increase the likelihood of said physical contact and the likelihood of said activity.

[0118] The term "neoadjuvant therapy" refers to treatment given prior to primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy.

[0119] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., isolated antibodies that specifically bind to KIR3DL3 and are substantially free of antibodies that do not bind to KIR3DL3). However, isolated antibodies that specifically bind to KIR3DL3 may have cross-reactivity with other KIR family proteins from different species. For example, in some embodiments, the antibody maintains specific binding affinity to at least two species, such as humans and other animals, such as non-rodents or other mammalian or non-mammal species. However, in some embodiments, the antibody maintains a higher or more specific affinity and selectivity for human KIR3DL3. Additionally, isolated antibodies are generally substantially free of other cellular material and / or chemicals. In one embodiment covered by this disclosure, "isolated" monoclonal antibodies with different specificities for human KIR3DL3 are combined in a well-defined composition.

[0120] As used herein, "isolated protein" means 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 technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. "Isolated" or "purified" proteins, or their biologically active portions, are substantially free of cellular material or other contaminating proteins from cell or tissue sources, or are substantially free of chemical precursors or other chemicals when chemically synthesized, from which antibodies, polypeptides, peptides, or fusion proteins are derived. The term "substantially free of cellular material" includes formulations containing target polypeptides (e.g., immunoglobulins) or fragments thereof, wherein the protein is isolated from the cellular components of the cells from which the protein was isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes formulations containing target proteins or fragments thereof, the formulation having less than about 30% (on a dry weight basis) of non-target proteins (also referred to herein as "contaminating proteins"), more preferably less than about 20% of non-target proteins, still more preferably less than about 10% of non-target proteins, and most preferably less than about 5% of non-target proteins. When recombinant protein produces antibodies, polypeptides, peptides or fusion proteins or fragments thereof, such as their bioactive fragments, it is also preferably substantially free of culture medium, i.e., the culture medium accounts for less than about 20% of the volume of the protein preparation, more preferably less than about 10% and most preferably less than about 5%.

[0121] As used herein, the term “isotype” refers to an antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene.

[0122] As used in this article, the term "K" D"This refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. The binding affinity of the antibodies of the present invention can be measured or determined by standard antibody-antigen assays, such as competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA."

[0123] As used herein, a "kit" is any article (e.g., packaging or container) comprising at least one reagent, such as a probe, for the specific detection or modulation of the expression of a biomarker covered by this disclosure. Kits may be promoted, distributed, or sold as units for performing the methods covered by this disclosure.

[0124] A “marker” or “biomarker” is a gene or protein whose expression level in a tissue or cell is associated with a disease state such as cancer, based on its altered expression level in normal or healthy tissues or cells. A “marker nucleic acid” is a nucleic acid (e.g., mRNA, cDNA) encoded by or corresponding to a marker covered by this disclosure. Such marker nucleic acids comprise DNA (e.g., cDNA) comprising all or part of any nucleic acid sequence shown in the sequence listing or complement of such sequences. Marker nucleic acids also comprise RNA comprising all or part of any nucleic acid sequence shown in the sequence listing or complement of such sequences, wherein all thymidine residues are replaced by uridine residues. A “marker protein” is a protein encoded by or corresponding to a marker covered by this disclosure. Marker proteins comprise all or part of any sequence shown in the sequence listing. In some embodiments, whole KIR3DL3 or HHLA2 is used as a marker. In other embodiments, fragments of KIR3DL3 or HHLA2 are used as markers. The terms “protein” and “peptide” are used interchangeably.

[0125] As used herein, the term “regulation” includes both upregulation and downregulation, such as enhancing or suppressing a response.

[0126] The term "predetermined" biomarker quantity and / or activity measurement can be used, by way of example only, to assess the following: a subject who may be selected for a specific treatment; an assessment of the response to treatment (such as one or more modulators of the KIR3DL3 pathway, such as KIR3DL3 and one or more natural binding couplers such as HHLA2), alone or in combination with one or more immunotherapies; and / or an assessment of disease status. Predetermined biomarker quantity and / or activity measurements can be determined in patient populations with or without cancer. Predetermined biomarker quantity and / or activity measurements can be a single number equally suitable for each patient, or they can vary depending on a specific subgroup of patients. A subject's age, weight, height, and other factors may affect the individual's predetermined biomarker quantity and / or activity measurement. Furthermore, predetermined biomarker quantity and / or activity can be determined individually for each subject. In one embodiment, the quantities determined and / or compared in the methods described herein are based on absolute measurements. In another embodiment, the quantities determined and / or compared in the methods described herein are based on relative measurements, such as ratios (e.g., cell ratios normalized to the expression of a biomarker or serum biomarker relative to a housekeeper or otherwise generally constant biomarker). The predetermined biomarker quantity and / or activity measurement can be any suitable criterion. For example, the predetermined biomarker quantity and / or activity measurement can be obtained from the same or different individuals selected for the patient being evaluated. In one embodiment, the predetermined biomarker quantity and / or activity measurement can be obtained from a previous evaluation of the same patient. In this way, the progress of patient selection can be monitored over time. Additionally, if the subject is human, controls can be obtained from the evaluation of another person or groups of people, such as a selected cohort. In this way, the degree of selection of the person being evaluated can be compared with suitable other individuals, such as others in similar circumstances to the person of interest, such as those with similar or identical symptoms and / or of the same ethnicity.

[0127] The term “predictive” includes the use of biomarkers of nucleic acid and / or protein states, such as the overactivity or underactivity of tumors, their appearance, expression, growth, remission, relapse, or resistance before, during, or after therapy, to determine the likelihood of a cancer response to immunomodulatory therapies such as KIR3DL3 pathway modulators (e.g., modulators of the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2, alone or in combination with one or more other therapies such as immunotherapy, such as immune checkpoint inhibitor therapy). Such predictive use of biomarkers can be confirmed by, for example, (1) increased or decreased copy number (e.g., by FISH, FISH plus SKY, single-molecule sequencing, e.g., as described in the art at least in the Journal of Biotechnology, 86:289-301 or qPCR), overexpression or underexpression of biomarker nucleic acid (e.g., by ISH, Northern blotting or qPCR), increased or decreased biomarker protein (e.g., by IHC) and / or increased or decreased activity of biomarker target or biomarker, e.g., at more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%). (1) 60%, 70%, 80%, 90%, 95%, 100% or more of the determined human cancer type or cancer sample; (2) the absolute or relative regulation of the biomarker in a biological sample, such as a sample containing tissue, whole blood, serum, plasma, oral scraping, saliva, cerebrospinal fluid, urine, feces or bone marrow from a subject with cancer; (3) the absolute or relative regulation of the biomarker in a clinical subgroup of patients with cancer (e.g., those who respond to or are resistant to a specific immunomodulatory therapy (e.g., KIR3DL3 pathway modulator therapy (e.g., modulators of the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2, alone or in combination with immunotherapy)).

[0128] The terms “prevent,” “preventing,” “prevention,” and “prophylactic treatment” refer to reducing the risk of developing a disease, condition, or symptom in subjects who do not have such a disease, condition, or symptom, or who are susceptible to such a disease, condition, or symptom.

[0129] The term "prognosis" includes a prediction of the likely course and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis for an individual's cancer. For example, a prognosis may be the outcome of surgery, the development of a clinical subtype of cancer (e.g., solid tumors such as lung cancer, melanoma, and renal cell carcinoma), the development of one or more clinical factors, the development of colorectal cancer, or recovery from the disease.

[0130] When used with respect to a reference polypeptide, the term "peptide fragment" or "fragment" refers to a polypeptide that, compared to the reference amino acid itself, lacks amino acid residues, but whose remaining amino acid sequence is generally identical to the corresponding position in the reference polypeptide. Such deletions can occur at the N-terminus, internally, or C-terminus of the reference polypeptide, or alternatively, both. Fragments are typically at least 5, 6, 8, or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40, or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500, or more amino acids long. The fragment may be, for example, at least and / or contain 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 longer, as long as the fragment is shorter than the length of the full-length polypeptide. Alternatively, the fragment may not exceed and / or may not contain such ranges, as long as the fragment is shorter than the length of the full-length polypeptide.

[0131] The term "probe" refers to any molecule capable of selectively binding to a specific intended target molecule, such as a nucleotide transcript or protein encoded by a marker or corresponding to it. Probes can be synthesized by those skilled in the art or derived from suitable biological agents. For the purpose of detecting the target molecule, probes can be specifically designed to be labeled, as described herein. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0132] As used herein, the term "rearrangement" refers to the locus configuration of heavy or light chain immunoglobulins, where the V segment encodes essentially complete V...H and V L The domain is located immediately adjacent to the DJ or J segment in its conformation. Rearranged immunoglobulin loci can be identified by comparison with germline DNA; rearranged loci will have at least one recombinant heptamer / nonamer homologous element.

[0133] As used herein, the term “recombinant host cell” (or simply “host cell”) is intended to refer to the cell that has been introduced into the recombinant expression vector. It should be understood that such terms are intended to refer not only to the specific subject cell but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0134] The term "resistance" refers to acquired or natural resistance to immunomodulatory therapy in a cancer sample or mammal (i.e., no response or reduced or limited response to therapeutic treatment), such as a reduction in response to therapeutic treatment by 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. The reduction in response can be measured by comparison with the same cancer sample or mammal before resistance is acquired, or by comparison with different cancer samples or mammals known to be resistant to therapeutic treatment. Typical acquired resistance to chemotherapy is called "multidrug resistance." Multidrug resistance can be mediated by P-glycoproteins or by other mechanisms, or it can occur when a mammal is infected with multidrug-resistant microorganisms or combinations of microorganisms. Determining resistance to therapeutic treatments is routine in the art and within the skill level of a typical clinician; for example, it can be measured by cell proliferation and cell death assays, as described herein as “sensitization.” In some embodiments, the term “reverse resistance” means that the combination of a second agent with a primary cancer therapy (e.g., chemotherapy or radiotherapy) results in a statistically significant reduction in tumor volume (e.g., p < 0.05) compared to the untreated tumor volume, where the primary cancer therapy alone (e.g., chemotherapy or radiotherapy) does not result in a statistically significant reduction in tumor volume compared to the untreated tumor volume. This generally applies to tumor volume measurements performed when an untreated tumor is growing rhythmically logarithmically.

[0135] As described above, the term "response" is generally associated with, for example, determining the role of a clinical intervention in its progress, efficacy, or outcome. For example, a response to 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, used alone or in combination with immunotherapies such as immune checkpoint inhibitors)) refers to any response to 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 KIR3DL3, used alone or in combination with immunotherapies such as immune checkpoint inhibitors)), and for cancer, preferably involves changes in the number of cancer cells, tumor quality, and / or volume after the initiation of neoadjuvant or adjuvant chemotherapy. Responses to hyperproliferative conditions can be assessed, for example, for efficacy or in neoadjuvant or adjuvant settings, where the size of the tumor after systemic intervention can be compared to the initial size and dimensions measured by CT, PET, mammography, ultrasound, or palpation. Responses can also be assessed by caliper measurements or tumor pathology following biopsy or surgical resection. Responses can be recorded quantitatively, such as by change in tumor volume percentage, or qualitatively, such as “pathological complete response” (pCR), “clinical complete response” (cCR), “clinical partial response” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD), or other qualitative criteria. Assessment of response in hyperproliferative disorders can be performed early after the initiation of neoadjuvant or adjuvant therapy, such as hours, days, weeks, or preferably months later. The typical endpoint for response assessment is at the termination of neoadjuvant chemotherapy or at surgical resection of residual tumor cells and / or the tumor bed. This is typically three months after the initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatments described herein can be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by summing the percentage of patients in complete response (CR), the number of patients in partial response (PR), and the number of patients with stable disease (SD) at least 6 months after the end of therapy. The abbreviated form of this formula is CBR = CR + PR + SD over 6 months. In some embodiments, the CBR for a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for assessing response to cancer therapy relate to “survival,” which includes all of the following: survival to death, also known as overall survival (where death may be unrelated to the cause or related to the tumor); “recurrence-free survival” (where the term recurrence should include both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term disease should include both cancer and related diseases).The length of survival can be calculated by referencing a defined start point (e.g., time of diagnosis or treatment initiation) and end point (e.g., death, recurrence, or metastasis). Additionally, the criteria for treatment efficacy can be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence. For example, to determine appropriate thresholds, specific cancer treatment regimens can be administered to a subject population, and the results can be correlated with biomarker measurements determined prior to the administration of any immunomodulatory therapy. Outcome measurements can be pathological responses to therapy administered in a neoadjuvant setting. Alternatively, outcome measurements such as overall survival and disease-free survival can be monitored over a period of time following immunomodulatory therapy with known biomarker measurements. In some embodiments, the administered dose is a standard dose of a cancer therapeutic agent known in the art. The time period for monitoring subjects can vary. For example, subjects 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.

[0136] As used herein, the term "specific binding" refers to the binding of an antibody to a predetermined antigen. Typically, when human KIR3DL3 is used as the analyte and the antibody as the ligand, in... When measured using surface plasmon resonance (SPR) technology in the instrument, the antibody reacts at a rate of approximately less than 10. -7 M is approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower affinity (K) D The antibody binds to a 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. The phrases “antibody that recognizes an antigen” and “antibody specific to an antigen” are used interchangeably herein with the term “antibody that specifically binds to an antigen.”

[0137] The term "subject" means any healthy animal, mammal, or human, or any animal, mammal, or human suffering from a condition of interest (e.g., cancer). The term "subject" may be used interchangeably with "patient." In some embodiments, the term is intended to include a living organism in which an immune response can be elicited. Representative, non-limiting examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species.

[0138] As used herein, the term “survival” encompasses all of the following: survival to death, also known as overall survival (where death may be unrelated to the cause of the disease or related to the tumor); “recurrence-free survival” (where the term “recurrence” should include both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term “disease” should include both cancer and related diseases). The length of said survival can be calculated by referring to a defined start point (e.g., time of diagnosis or treatment initiation) and an end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for treatment efficacy can be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0139] The terms “tolerance” or “unresponsiveness” encompass the refraction of cells, such as immune cells, to stimuli, such as those that activate receptors or cytokines. Unresponsiveness can occur, for example, due to exposure to immunosuppressants or high doses of antigens. Several independent mechanisms can induce tolerance. One mechanism, known as “non-responsiveness,” is defined as the state in which cells persist in vivo as non-responsive cells instead of differentiating into cells with effector functions. This refraction is typically antigen-specific and persists after cessation of exposure to the tolerant antigen. For example, T cell non-responsiveness is characterized by the lack of cytokine production, such as IL-2. T cell non-responsiveness occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, re-exposure to the same antigen (even in the presence of co-stimulatory peptides) results in the inability to produce cytokines and therefore cannot proliferate. However, if cultured with cytokines (e.g., IL-2), non-responsive T cells can proliferate. For example, T cell non-responsiveness can also be observed when T lymphocytes do not produce IL-2, as measured by ELISA or by using a proliferation assay of an indicator cell line. Alternatively, reporter gene constructs can be used. For example, non-responsive T cells cannot initiate IL-2 gene transcription induced by a heterologous promoter or AP1 sequence multimer visible within the enhancer, controlled by the 5′ IL-2 gene enhancer (Kang et al. (1992) Science 257:1134). Another mechanism is called “exhaustion.” T cell exhaustion is a state of T cell dysfunction that occurs during many chronic infections and cancers. T cell exhaustion is defined by poor effector organelle function, persistent expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells.

[0140] “Transcribed polynucleotides” or “nucleotide transcripts” are polynucleotides (e.g., mRNA, hnRNA, cDNA, or analogs of such RNA or cDNA) that are complementary to or homologous to all or part of mature mRNA, which is prepared by transcription of markers covered in this disclosure and normal post-transcriptional processing (e.g., splicing) of the RNA transcript (if any) and reverse transcription of the RNA transcript.

[0141] As used herein, the term "T cell" includes CD4+ T cells and CD8+ T cells. The term T cell also includes T helper type 1 T cells and T helper type 2 T cells. The term "antigen-presenting cell" includes specialized antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) and 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 immune responses by expressing one or more T cell receptors. Tcon or Teff is generally defined as any population of T cells that is not Treg and includes, for example, primordial T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated into, for example, Th1 or Th2 lineages. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is a CD4+ Teff or a CD8+ Teff, such as 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. “Primary Tcon” refers to CD4+ T cells differentiated in the bone marrow. +T cells, having successfully undergone positive and negative central selection processes in the thymus, are not yet activated by exposure to antigens. Primitive Tcon are typically characterized by surface expression of L-selectin (CD62L), the absence of activation markers such as CD25, CD44, or CD69, and the absence of memory markers such as CD45RO. Therefore, primitive Tcon are considered to be quiescent and non-dividing, requiring interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostasis (see at least WO 2010 / 101870). The presence and activity of such cells are undesirable in the context of suppressed immune responses. Unlike Tregs, Tcon are not unresponsive and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001), Proceedings of the Royal Society B: Biosciences, 356:625-637). In tumors, exhausted cells can exhibit unresponsive characteristics.

[0142] As used herein, the term “unrearranged” or “phylogenetic configuration” for the V segment refers to a configuration in which the V segment is not rearranged to be adjacent to the D or J segment.

[0143] II. Monoclonal antibodies, immunoglobulins, and peptides

[0144] This disclosure relates in part to isolated monoclonal antibodies or fragments thereof targeting KIR3DL3 (as listed herein as monoclonal and polyclonal antibodies). A characteristic of such molecules is that they exhibit the ability to recognize the KIR3DL3 protein in diagnostic assays such as immunohistochemistry (IHC), Western blotting, intercellular flow assays, and ELISA. A characteristic of such molecules is that they exhibit the ability to inhibit the binding of KIR3DL3 to binding partners such as HHLA2.

[0145] Also known as human endogenous retrovirus-H long terminal repeat-associated protein 2, HERV-H LTR-associated 2, B7y, B7H7, B7-H5, and B7-H7, the term "HHLA2" refers to a member of the B7 family. HHLA2 protein expression is limited in normal human tissues but is widely expressed in human cancers. HHLA2 is a membrane protein with three Ig-like domains (IgV-IgC-IgV), while other members of the B7 family typically have only two Ig domains (IgV-IgC). In normal human tissues, HHLA2 is expressed in the epithelium of the kidney, intestine, gallbladder, and breast, as well as in placental trophoblast cells. In the immune system, HHLA2 is constitutively expressed on human monocytes / macrophages. HHLA2 regulates human T cell function, including, for example, inhibiting T cell proliferation and cytokine production, and increasing T cell production and cytokine production. HHLA2 is expressed at high levels in a variety of human cancers, including colorectal cancer, kidney cancer, lung cancer, pancreatic cancer, ovarian cancer, and prostate cancer. HHLA2 is also expressed in human thyroid cancer, melanoma, liver cancer, bladder cancer, colon cancer, kidney cancer, breast cancer, and esophageal cancer.

[0146] As described above, certain HHLA2 structures and functions are well known in the field (see, for example, Xiao et al. (2015) Clinical Cancer Research 21:2201-2203, Janakiram et al. (2015) Clinical Cancer Research 21:2359-2366, Mager et al. (1999) Genomics 21:2359-2366, Flajnik et al. (2012) Immunogenetics 64:571-590, Zhao et al. (2013) Proceedings of the National Academy of Sciences 110:9879-9884, and Zhu et al. (2013) Nature Communications 4:2043).

[0147] The term "HHLA2" is intended to encompass its fragments, variants (e.g., allelic variants), and derivatives. 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 isotype a), variant 2 (NM_001282556.1 and NP_001269485.1, representing the use of an alternative promoter and differing in the 5' UTR compared to variant 1), variant 3 (NM_001282557.1 and NP_001269486.1, representing the use of an alternative promoter and differing in the 5' UTR compared to variant 1), and variant 4 (NM_001282558...). Variant 1 and NP_001269487.1, which encode isotype b, indicate the use of a substitute promoter, differ from variant 1 in the 5' UTR and lack substitute in- and out-of-frame units in the 3' coding region, resulting in a shorter isotype than isotype a), and variant 5 (NM_001282559.1 and NP_001269488.1, which encode isotype c, indicate the use of a substitute promoter and have several differences compared to variant 2, producing a unique 5' UTR compared to variant 1 and causing translation to begin at the substitute start codon, producing a unique N-terminus and a shorter isotype than isotype a). The nucleic acid and polypeptide sequences of HHLA2 orthologs in organisms other than humans are well known, including, for example, frog HHLA2 (NM_001128644.1 and NP_001122116.1). Representative sequences of HHLA2 orthologs are presented in Table 1 below.

[0148] Anti-HHLA2 antibodies suitable for detecting 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 (Thermo Fisher Scientific), and antibodies MAB80841, AF8084, FAB80841R, FAB80841T, and MAB8084 (R&D Systems). 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 Biosciences), etc.In addition, several siRNA, shRNA, and CRISPR constructs for reducing HHLA2 expression are available in the commercial product lists of the aforementioned companies, such as shRNA product numbers TL312462, TF312462, TR312462, TG312462, and TL312462V; siRNA product number SR323358 from Aorui Gene Technology Co., Ltd.; and siRNA product numbers i009616, i009616a, i009616b, i009616c, i009616d, iV009616, iV009616a, iV009616b, iV009616c, iV009616d, iAAV00961600, iAAV00961601, iAAV00961602, and iAAV00961. 603, iAAV00961604, iAAV00961605, iAAV00961606, iAAV00961607, iAAV00961608 and iAAV00961609; CRISPR product numbers K0950321, K0950301, K0950302, K0950303, K0950304, K09503 05, 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 sand c-411576-NIC-2 (Santa Cruz Biotechnology), etc. It should be noted that the terminology can be further used to refer to any combination of 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 molecule covered by this disclosure.

[0149] The term "HHLA2 pathway" includes HHLA2 and its interaction with one or more of its natural binding partners such as TMIGD2 and KIR3DL3.

[0150] The term "KIR3DL3 pathway" encompasses KIR3DL3 and its interactions with one or more of its natural binding partners, such as HHLA2.

[0151] The term "TMIGD2" refers to a transmembrane and immunoglobulin domain containing CD28H, IGPR1, and IGPR-1. It is a membrane protein with approximately 10% amino acid identity to CD28, CTLA-4, ICOS, and PD-1. TMIGD2 possesses an extracellular IgV-like domain, a transmembrane region, and a proline-rich cytoplasmic domain with two tyrosine signaling motifs. TMIGD2 protein is constitutively expressed on all primary T cells and most natural killer (NK) cells, but not on regulatory T cells or B cells. TMIGD2 expression is slowly lost with repeated stimulation by T cells. Consistent with this, TMIGD2 is expressed only on about half of memory T cells, and TMIGD2-negative T cells exhibit a terminally differentiated senescent phenotype. TMIGD2 has also been shown to be expressed in endothelial and epithelial cells and to reduce cell migration and promote capillary formation during angiogenesis.

[0152] As described above, certain TMIDD2 structures and functions are well known in the art (see, for example, Xiao et al. (2015) Clinical Cancer Research 21:2201-2203, Janakiram et al. (2015) Clinical Cancer Research 21:2359-2366, Zhu et al. (2013) Nature Communications 4:2043 and Rahimi (2012) Cell 23:1646-1656).

[0153] The term “TMIGD2” is intended to encompass its fragments, variants (e.g., allele 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 isotypes include isotype 1 (NM_144615.2 and NP_653216.2), isotype 2 (NM_001169126.1 and NP_001162597.1, which, compared to variant 1, use alternative in-frame splicing sites in the 3′ coding region, resulting in shorter isotypes compared to isotype 1), and isotype 3 (NM_001308232.1 and NP_001295161.1, which, compared to variant 1, lack alternative in-frame and out-of-frame splicing sites in the 5′ coding region, resulting in shorter isotypes compared to isotype 1). The nucleic acid and polypeptide sequences of TMIGD2 orthologs in organisms other than humans are well known, including, for example, chimpanzee TMIGD2 (XM_009434393.2 and XP_009432668.2 and XM_001138228.4 and XP_001138228.3) and bovine TMIGD2 (XM_005208980.3 and XP_005209037.1, XM_005208979.3 and XP_005209036.1 and XM_002688933.5 and XP_002688979.1). Representative sequences of TMIGD2 orthologs are presented in Table 1 below.

[0154] Anti-TMIGD2 antibodies suitable for detecting TMIGD2 protein are well known in the art and include, for example, antibody catalog numbers MAB8316, MAB83162, FAB8316R, FAB83162R, FAB83162G, FAB83162N, FAB83162S, FAB83162T, FAB83162U and FAB83162V (R&D Systems), antibody TA326695 (Aurora Gene), antibodies PA5-52787 and PA5-38055 (Thermo Fisher Scientific), antibodies MAB83161 and NBP1-81164 (Novex Biopharmaceuticals), etc. In addition, several siRNA, shRNA, and CRISPR constructs for reducing TMIGD2 expression are available in the commercial product lists of the aforementioned companies, such as shRNA product numbers TF317829, TG317829, TL317829, TR317829, and TL317829V; siRNA product number SR314913; and CRISPR product numbers KN204938, KN204938LP, and KN2049 from Aorui Gene Technology Co., Ltd. 38RB and KN204938BN; siRNA product numbers i024914, i024914a, i024914b, i024914c, i024914d, iV024914, iV024914a, iV024914b, iV024914c, iV024914d, iAAV02491400, iAAV02491401, iAAV02491402, iAAV02491403, iAAV0 2491404, iAAV02491405, iAAV02491406, iAAV02491407, 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.In addition, several CRISPR constructs for increasing TMIDD2 expression are available in the commercial product lists of the aforementioned companies, such as CRISPR product numbers K2409378, K2409377, K2409376, K2409375, K2409374, K2409373, K2409372, and K2409371 (Abm); and CRISPR product numbers sc-414261-ACT, sc-414261-ACT-2, sc-414261-LAC, and sc-414261-LAC-2 (Santa Cruz Biotechnology). It should be noted that the terminology can be further used to refer to any combination of features described herein with respect to the TMIDD2 molecule. For example, any combination of sequence composition, percentage identity, sequence length, domain structure, functional activity, etc., can be used to describe the TMIDD2 molecule covered by this disclosure.

[0155] The interaction between TMIGD2 and HHLA2 and their functions are well known in the field (see, for example, Xiao et al. (2015) Clinical Cancer Research 21:2201-2203 and Janakiram et al. (2015) Clinical Cancer Research 21:2359-2366).

[0156] The term "KIR3DL3," also known as killer cell immunoglobulin-like receptor 3DL3, CD158Z, KIR3DL7, KIR44, KIRC1, KIR2DS2, killer cell immunoglobulin-like receptor, with three Ig domains and a long cytoplasmic tail, refers to a member of a transmembrane glycoprotein family expressed by natural killer cells and T cell subsets. Killer cell immunoglobulin-like receptor (KIR) genes are polymorphic and highly homologous, and they are located in a cluster on chromosome 19q13.4 within the 1 Mb leukocyte receptor complex (LRC). Although several "framework" genes (KIR3DL3, KIR3DP1, KIR3DL4, KIR3DL2) have been found in all haplotypes, the gene content of the KIR gene cluster varies from haplotype to haplotype. KIR proteins are classified according to the number of extracellular immunoglobulin domains (2D or 3D) and whether they have long (L) or short (S) cytoplasmic domains. KIR proteins with long cytoplasmic domains transduce inhibitory signals upon ligand binding via immunotyrosine-based inhibitory motifs (ITIMs), while KIR proteins with short cytoplasmic domains lack ITIM motifs and instead transduce activation signals by binding to TYRO protein tyrosine kinase-binding proteins. The ligands of several KIR proteins are subsets of HLAI molecules; therefore, KIR proteins are considered to play an important role in regulating immune responses. This gene is one of the "framework" loci present in all haplotypes. The KIR3DL3 protein possesses an N-terminal signaling 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 stem region found in other KIRs.

[0157] As described above, certain KIR3DL3 structures and functions are well known in the art (see, for example, Hsu et al. (2002) Immunol Rev. 190:40-52, Trompeter et al. (2005) Journal of Immunology 174:4135-4143, Trundley et al. (2006) Immunogenetics 57:904-916 and Jones et al. (2006) Immunogenetics 58:614-627).

[0158] The term "KIR3DL3" is intended to encompass its fragments, variants (e.g., allelic variants), and derivatives. 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 isotype is known: human KIR3DL3 (NM_153443.4) is encoded by the transcript (NP_703144.3). The nucleic acid and polypeptide sequences of KIR3DL3 orthologs in organisms other than humans are well known, including, for example, chimpanzee KIR3DL3 (XM_003316679.3 and XP_003316727.3), rhesus monkey 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.

[0159] Anti-KIR3DL3 antibodies suitable for detecting KIR3DL3 protein are well known in the art and include, for example, antibody catalog numbers: FAB8919R, MAB8919, FAB8919G, FAB8919N, FAB8919S, FAB8919T, FAB8919U and FAB8919V (R&D Systems), antibody AP52374PU-N (Aurora Gene), antibody PA5-26178 (Thermo Fisher Scientific), antibodies OAAB05761, OAAF08125, OAAN04122, OACA09134, OACA09135, OACD04988 and OASG01190 (Aviva Systems Biology), etc. In addition, several siRNA, shRNA, and CRISPR constructs for reducing KIR3DL3 expression are available in the commercial product lists of the aforementioned companies, such as shRNA product numbers TF303684, TR303684, TG303684, TL303684, and TL303684V; siRNA product number SR314516; and CRISPR product numbers KN224383 and KN224383BN from Aorui Gene Technology Co., Ltd. KN224383RB and KN224383LP; siRNA product numbers i011627, i011627a, i011627b, i011627c, i011627d, iV011627, iV011627a, iV011627b, iV011627c, iV011627d, iAAV01162700, iAAV01162701, iAAV01162702, iAAV 01162703, iAAV01162704, iAAV01162705, iAAV01162706, iAAV01162707, iAAV01162708 and iAAV01162709, and CRISPR product numbers K1151421, K1151401, K1151402, K1151403, K1151404, K1151405, K1151406, K115140 7. 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. It should be noted that the terminology can be further used to refer to any combination of features described herein regarding 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 molecule covered by this disclosure.

[0160] The term "peripheral blood cell subtype" refers to the cell types that are normally present in peripheral blood, including but not limited to eosinophils, neutrophils, T cells, monocytes, NK cells, granulocytes, and B cells.

[0161] The term "recombinant human antibody" encompasses all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomally modified animals (e.g., mice) or hybridomas prepared therefrom (further described below) that are transgenic with respect to the human immunoglobulin gene; (b) antibodies isolated from host cells transformed to express antibodies, such as from transfected tumors; (c) antibodies isolated from recombinant combined human antibody libraries; and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may undergo in vitro mutagenesis (or, when using animals with transgenic human Ig sequences, in vivo somatic cell mutagenesis), and therefore the V of the recombinant antibody... H District and V L The amino acid sequence of the region is as follows: Although it is derived from human lineage V H Sequence and V L The sequence is associated with it, but it may not be naturally present in the human antibody germline library in vivo.

[0162] The term "sample" used to detect or determine the presence or level of at least one biomarker is generally whole blood, plasma, serum, saliva, urine, feces (e.g., excrement), tears, and any other bodily fluids (e.g., as described above in the definition of "bodily fluids") or tissue samples (e.g., biopsies) such as small intestine, colon samples, or surgically removed tissue. In some cases, the methods covered by this disclosure further include obtaining the sample from the individual prior to detecting or determining the presence or level of at least one biomarker in the sample.

[0163] As used herein, “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 containing RNA molecules or fragments thereof homologous to the target biomarker genes covered by this disclosure, short interfering RNAs (siRNAs), and small molecules that interfere with or inhibit the expression of target biomarker nucleic acids by RNA interference (RNAi).

[0164] RNA interference (RNAi) is an evolutionarily conserved process in which the expression or introduction of RNA with a sequence identical or highly similar to that of a target biomarker nucleic acid leads to sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of the messenger RNA (mRNA) transcribed from the target gene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting the expression of the target biomarker nucleic acid. In one embodiment, the RNA is double-stranded RNA (dsRNA). This process has been described in plant, invertebrate, and mammalian cells. Essentially, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes the progressive cleavage of long dsRNA into double-stranded fragments called siRNA. The siRNA is incorporated into a protein complex that recognizes and cleaves the target mRNA. RNAi can also be initiated by introducing nucleic acid molecules, such as synthetic siRNA, shRNA, 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 reduction in the expression, activity, or level of the target biomarker nucleic acid or the protein encoded by the target biomarker nucleic acid. The reduction may be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to the expression of the target biomarker nucleic acid or the activity or level of the protein encoded by a target biomarker nucleic acid not targeted by the RNA interference agent.

[0165] Besides RNAi, genome editing can also be used to regulate the copy number or gene sequence of a biomarker of interest, such as constitutive or inducible knockout or mutation of the biomarker of interest, such as KIR3DL3 pathway components, such as HHLA2, TMIGD2, and / or KIR3DL3. For example, the CRISPR-Cas system can be used to precisely edit genomic nucleic acids (e.g., to create nonfunctional or null mutations). In such embodiments, CRISPR guide RNA and / or Cas enzymes can be expressed. For example, a vector containing only the guide RNA can be administered to animals or cells transgenic with the Cas9 enzyme. Similar strategies can be used (e.g., designer zinc fingers, transcription activator-like effectors (TALEs), or homing broad-spectrum nucleases). Such systems are well known in the art (see, for example, U.S. Patent No. 8,697,359; Sander and Joung (2014) Nature Biotechnology 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Molecular Cell 37:7; U.S. Patent Publications 2014 / 0087426 and 2012 / 0178169; Boch et al. (2011) Nature Biotechnology 29:135-136; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al. (2011) PLOS ONE One) 6:e19722; Li et al. (2011) Nucleic Acids Res. 39:6315-6325; Zhang et al. (2011) Nature Biotechnology 29:149-153; Miller et al. (2011) Nature Biotechnology 29:143-148; Lin et al. (2014) Nucleic Acids Res. 42:e47). According to the methods in this art, such genetic strategies can be used with constitutive expression systems or inducible expression systems.

[0166] Piwi-interacting RNAs (piRNAs) are the largest class of non-coding small RNA molecules. piRNAs form RNA-protein complexes by interacting with piwi proteins. These piRNA complexes are involved in epigenetic and posttranscriptional gene silencing of retrotransposons and other genetic elements in germline cells, particularly those involved in spermatogenesis. They differ from microRNAs (miRNAs) in size (26–31 nt instead of 21–24 nt), 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 targets of piRNAs. In mammals, piRNA activity in transposon silencing appears to be most important during embryonic development, and in *C. elegans* and humans, piRNAs are essential for spermatogenesis. piRNAs function in RNA silencing by forming RNA-induced silencing complexes (RISCs).

[0167] "Aptamers" are oligonucleotides or peptide molecules that bind to specific target molecules. "Nucleic acid aptamers" are nucleic acid species engineered through repeated rounds of in vitro selection or equivalently, using SELEX (ligand index-enhanced systemic evolution technology), to bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. "Peptide aptamers" are artificial proteins selected or engineered to bind to specific target molecules. These proteins consist of one or more peptide loops with variable sequences displayed on a protein scaffold. These proteins are typically isolated from combinatorial libraries and are usually subsequently modified through directed mutagenesis or multiple rounds of variable region mutagenesis and selection. "Affimer proteins," the evolution of peptide aptamers, are small, highly stable proteins engineered to display peptide loops that provide a high-affinity binding surface for specific target proteins. Affimer proteins are low-molecular-weight proteins, 12-14 kDa, derived from the cysteine ​​protease inhibitor family of cystatin. Aptamers can be used in biotechnology and therapeutic applications because they provide molecular recognition properties comparable to those of commonly used biomolecular antibodies. In addition to their discriminative recognition, aptamers offer advantages over antibodies because they can be fully engineered in vitro, are easily produced through chemical synthesis, possess desired storage properties, and induce little or no immunogenicity in therapeutic applications.

[0168] "Short interfering RNA" (siRNA), also referred to herein as "small interfering RNA," is defined as an agent used, for example, to inhibit the expression of target biomarker nucleic acids via RNAi. siRNA can be chemically synthesized, produced by in vitro transcription, or generated within host cells. In one embodiment, siRNA is a double-stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides, and even more preferably about 19, 20, 21, or 22 nucleotides, and may contain 3' and / or 5' overhangs on each strand of about 0, 1, 2, 3, 4, or 5 nucleotides in length. The length of the overhangs is independent between the two strands; that is, the length of the overhang on one strand does not depend on the length of the overhang on the second strand. Preferably, siRNA is capable of promoting RNA interference through the degradation of target messenger RNA (mRNA) or specific posttranscriptional gene silencing (PTGS).

[0169] In another embodiment, the siRNA is a small hairpin (also known as stem-loop) RNA (shRNA). In one embodiment, these shRNAs consist of a short (e.g., 19 to 25 nucleotides) antisense strand, followed by a 5 to 9 nucleotide loop and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow it. These shRNAs may be contained in plasmids, retroviruses, and lentiviruses, and expressed from, for example, the pol III U6 promoter or other promoters (see, for example, Stewart et al. (2003) RNA April; 9(4):493-501, which is incorporated herein by reference).

[0170] RNA interference agents, such as siRNA molecules, can be administered to patients with cancer or at risk of developing cancer to suppress the expression of biomarker genes overexpressed in cancer, thereby treating, preventing, or inhibiting cancer in the subject.

[0171] The term "small molecule" is a term used in the art and includes molecules with a molecular weight of less than about 1000 or less than about 500. In one embodiment, a small molecule nonexclusively includes peptide bonds. In another embodiment, the small molecule is not oligomeric. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, peptide mimics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketide compounds) (Cane et al. 1998. Science 282:63), and libraries of natural product extracts. In another embodiment, the compound is a small organic non-peptide compound. In yet another embodiment, the small molecule is not biosynthetic.

[0172] The term "selective modulator" or "selective modulation" applied to bioactive agents refers to the ability of an agent to modulate a target, such as a cell population, or signal transduction activity, by direct or indirect interaction with the target, compared to off-target cell populations or signal transduction activities. For example, agents that selectively inhibit the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2 (rather than another interaction between KIR3DL3 and another binding partner) and / or such interactions on the cell population of interest may be active against KIR3DL3 pathway modulator therapies (e.g., modulators of the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2), wherein the activity is 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%, 2x (times) or more of the activity of the agent against at least one other binding partner. Many (e.g., at least approximately 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 105x, 110x, 120x, 125x, 150x, 200x, 250x, 300x, 350x, 400x, 450x), 500x, 600x, 700x, 800x, 900x, 1000x, 1500x, 2000x, 2500x, 3000x, 3500x, 4000x, 4500x, 5000x, 5500x, 6000x, 6500x, 7000x, 7500x, 8000x, 8500x, 9000x, 9500x, 10000x or more, or any range between the two, including the extreme values. Such measures are typically expressed as the relative amount required to reduce the interaction / activity by half.

[0173] More generally, the term "selectivity" refers to preferential action or function. The term "selectivity" can be quantified based on the preferential effect of a particular target of interest relative to other targets. For example, the measured variable (e.g., the regulation of Treg / Breg with other cells, such as other immune cells, such as Tcon) could 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, or 7-fold. 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 70x, 80x, 90x, 100x or more, or any range between these (e.g., 50% to 16x), differing in terms of the target of interest from an unexpected or undesirable target. The same fold-up analysis can be used to confirm the magnitude of a role in a given tissue, cell population, measurement variable, measurement effect, such as the Treg:Tcon ratio, Breg:Tcon ratio, growth rate or volume of overproliferating cells, Treg / Breg proliferation rate or number, etc.

[0174] In contrast, the term "specificity" refers to an exclusionary behavior or function. For example, the specific regulation of HHLA2-KIR3DL3 interaction refers to the exclusive regulation of the HHLA2-KIR3DL3 interaction, rather than the regulation of the interaction between KIR3DL3 and another ligand. In another instance, the specific binding of an antibody to a predetermined antigen refers to the ability of an antibody to bind to the antigen of interest without binding to other antigens. Typically, when using the antigen of interest as the analyte and the antibody as the ligand, in When measured using surface plasmon resonance (SPR) technology in the instrument, the antibody reacts at a rate of approximately less than 1 x 10⁻⁶. -7 M is approximately less than 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or even lower affinity (K) DThe antigen binds to a 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. Additionally, K... D It is K A The reciprocal of the term. The phrases “antibody that recognizes an antigen” and “antibody specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen” in this document.

[0175] The term "sensitization" refers to altering cells, such as cancer cells or tumor cells, in a manner that allows for more effective treatment with 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), alone or in combination with immunotherapies such as immune checkpoint inhibition therapy). In some embodiments, normal cells are not affected to the extent that they would be excessively harmed 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), alone or in combination with immunotherapies such as immune checkpoint inhibition therapy). The measurement of increased or decreased sensitivity to therapeutic treatments according to methods known in the art, used for the specific treatments and methods described below, includes, but is not limited to, cell proliferation assays (Tanigawa N, Kern DH, Kikasa Y, Morton DL, *Cancer Research* 1982; 42:2159-2164), cell death assays (Weisenthal LM, Shoemaker RH, Marsden JA, Dill PL, Baker JA, Moran EM, *Cancer Research* 1984; 94:161-173; Weisenthal LM, Lippman ME, *Cancer Treat Rep* 1985; 69:615-632; Weisenthal LM, Yu: Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, ed. *Drug Resistance in Leukemia*). (and Lymphoma) Harwood Academic Publishers, Langhorne, Pennsylvania (1993: 415-432; Weisenthal LM, *Contrib Gynecol Obstet*, 1994; 19: 82-90). Animal sensitivity or resistance can also be measured by measuring tumor size reduction over a period of time, such as 6 months in humans and 4 to 6 weeks in mice.If, compared to the treatment sensitivity or resistance in the absence of such a composition or method, the increase in treatment sensitivity or the decrease in resistance 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 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times or more, then the composition or method sensitizes the response to therapeutic treatment. The determination of sensitivity or resistance to therapeutic treatment is routine in the art and within the skill of an ordinary clinician. It should be understood that any method described herein for enhancing immunomodulatory efficacy can be equally applied to methods for sensitizing overproliferating or other cancer cells (e.g., resistant cells) to therapy.

[0176] The term "synergistic effect" refers to the combined effect of two or more therapeutic agents, such as two or more KIR3DL3 pathway modulators, or KIR3DL3 pathway modulators and immunotherapy, which can be greater than the sum of the effects of individual anticancer agents alone, or KIR3DL3 pathway modulators alone or in combination with immunotherapies such as immune checkpoint inhibitory therapy.

[0177] The term "survival" encompasses all of the following: survival to death, also known as overall survival (where death may be unrelated to the cause of the disease or related to the tumor); "recurrence-free survival" (where the term recurrence should include both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term disease should include both cancer and related diseases). The length of survival can be calculated using a reference-defined start point (e.g., time of diagnosis or treatment initiation) and end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for treatment efficacy can be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0178] The term "therapeutic effect" refers to the local or systemic effect caused by a pharmacologically active substance in animals, particularly mammals, and more particularly humans. Therefore, the term means any substance intended for the diagnosis, cure, relief, treatment, or prevention of disease or for enhancing the desired physical or mental development and condition of an animal or human.

[0179] As used herein, the terms "therapeutic effective amount" and "effective amount" mean the amount of a compound, material, or composition comprising compounds covered by this disclosure that effectively produces some desired therapeutic effect in at least a subpopulation of cells in animals at a reasonable benefit / risk ratio suitable for any medical treatment. The toxicity and therapeutic efficacy of the subject matter compounds can be determined by standard pharmaceutical procedures in cell cultures or laboratory animals, such as those used to determine LD50. 50 and ED 50Compounds exhibiting a large therapeutic index are preferred. In some embodiments, the LD50 can be measured. 50 (Lethal dose) and relative to no administration, the dose can be reduced by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more. Similarly, ED can be measured. 50 (i.e., the concentration at which the half-maximal inhibitory concentration (MCC) of symptoms is achieved) and relative to no medication, the effect of medication 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. Similarly, IC50 can be measured. 50 (i.e., the concentration at which the half-maximal cytotoxicity or cell inhibition is achieved against cancer cells) and the effect of the agent can be increased by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to no agent application. In some embodiments, the growth of cancer cells being measured 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%. It can promote cancer cell death 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, it can achieve a reduction in the number of cancer cells and / or solid malignancies 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%.

[0180] The term "substantially free of chemical precursors or other chemicals" includes formulations containing antibodies, peptides, peptides, or fusion proteins, wherein the protein is separated from the chemical precursors or other chemicals involved in protein synthesis. In one embodiment, the term "substantially free of chemical precursors or other chemicals" includes formulations containing antibodies, peptides, peptides, or fusion proteins having less than about 30% (on a dry weight basis) of chemical precursors or non-antibody, peptide, peptide, or fusion protein chemicals, more preferably less than about 20% of chemical precursors or non-antibody, peptide, peptide, or fusion protein chemicals, still more preferably less than about 10% of chemical precursors or non-antibody, peptide, peptide, or fusion protein chemicals, and most preferably less than about 5% of chemical precursors or non-antibody, peptide, peptide, or fusion protein chemicals.

[0181] "Transcribed polynucleotides" or "nucleotide transcripts" are polynucleotides (e.g., mRNA, hnRNA, cDNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or miRNA binding sites or variants thereof, or analogs of such RNA or cDNA) that are complementary to or homologous to all or part of mature mRNA, said mature mRNA being prepared by transcription of markers covered in this disclosure and normal post-transcriptional processing (e.g., splicing, if any) of the RNA transcript and reverse transcription of the RNA transcript.

[0182] The term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) are integrated into the genome of the host cell upon introduction and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Generally, expression vectors used in recombinant DNA technologies are often in plasmid form. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, the present invention is intended to include other forms of such expression vectors that provide equivalent functionality, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0183] There is a known and well-defined correspondence between the amino acid sequence of a specific protein and the nucleotide sequence that can encode the protein, as defined by the genetic code (shown below). Similarly, there is a known and well-defined correspondence between the nucleotide sequence of a specific nucleic acid and the amino acid sequence encoded by said nucleic acid, as defined by the genetic code.

[0184] Genetic code

[0185] Alanine (Ala, A) GCA, GCC, GCG, GCT

[0186] Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT

[0187] Asparagine (Asn, N) AAC, AAT

[0188] Aspartic acid (Asp, D) GAC, GAT

[0189] Cysteine ​​(Cys, C) TGC, TGT

[0190] Glutamic acid (Glu, E) GAA, GAG

[0191] Glutamine (Gln, Q) CAA, CAG

[0192] Glycine (Gly, G) GGA, GGC, GGG, GGT

[0193] Histidine (His, H) CAC, CAT

[0194] Isoleucine (Ile, I) ATA, ATC, ATT

[0195] Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG

[0196] Lysine (Lys, K) AAA, AAG

[0197] Methionine (Met, M) ATG

[0198] Phenylalanine (Phe, F) TTC, TTT

[0199] Proline (Pro, P) CCA, CCC, CCG, CCT

[0200] Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT

[0201] Threonine (Thr, T) ACA, ACC, ACG, ACT

[0202] Tryptophan (Trp, W) TGG

[0203] Tyrosine (Tyr, Y) TAC, TAT

[0204] Valine (Val, V) GTA, GTC, GTG, GTT

[0205] Termination signals (end): TAA, TAG, TGA

[0206] A key and well-known characteristic of the genetic code is its redundancy; therefore, for most amino acids used to make proteins, more than one coding nucleotide triplet can be used (as shown above). Thus, many different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the same amino acid sequence in all organisms (although some organisms may translate some sequences more efficiently than others). Furthermore, methylated variants of purines or pyrimidines may sometimes be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0207] In light of the foregoing, the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any part thereof) can be used to derive a polypeptide amino acid sequence, using the genetic code to translate DNA or RNA into amino acids. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence encoding the polypeptide can be inferred from the genetic code (due to the redundancy of the genetic code, it will generate multiple nucleic acid sequences for any given amino acid sequence). Therefore, the description and / or disclosure herein of nucleotide sequences encoding polypeptides should be considered to also include the description and / or disclosure of amino acid sequences encoded by nucleotide sequences. Likewise, the description and / or disclosure herein of polypeptide amino acid sequences should be considered to also include the description and / or disclosure of all possible nucleotide sequences that can encode amino acid sequences.

[0208] Finally, the nucleic acid and amino acid sequence information of the nucleic acid and polypeptide molecules useful in this 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, Table 1 below provides exemplary nucleic acid and amino acid sequences from publicly available sequence databases.

[0209] Table 1

[0210] SEQ ID NO:1 Human HHLA2 variant 1 cDNA sequence (NM_007072.3, CDS region at positions 415-1659)

[0211]

[0212]

[0213] SEQ ID NO:2 Human HHLA2 variant 1 amino acid sequence (NP_009003.1)

[0214]

[0215] SEQ ID NO:3 Human HHLA2 variant 2 cDNA sequence (NM_001282556.1, CDS region at positions 224-1468)

[0216]

[0217]

[0218] SEQ ID NO:4 Human HHLA2 variant 2 amino acid sequence (NP_001269485.1)

[0219]

[0220] SEQ ID NO:5 Human HHLA2 variant 3cDNA sequence (NM_001282557.1, CDS region at positions 155-1399)

[0221]

[0222]

[0223] SEQ ID NO:6 Human HHLA2 variant 3 amino acid sequence (NP_001269486.1)

[0224]

[0225] SEQ ID NO:7 Human HHLA2 variant 4 cDNA sequence (NM_001282558.1, CDS region at positions 302-1495)

[0226]

[0227]

[0228] SEQ ID NO:8 Human HHLA2 variant 4 amino acid sequence (NP_001269487.1)

[0229]

[0230] SEQ ID NO:9 Human HHLA2 variant 5 cDNA sequence (NM_001282559.1, CDS region at positions 232-1284)

[0231]

[0232]

[0233] SEQ ID NO:10 Human HHLA2 variant 5-amino acid sequence (NP_001269488.1)

[0234]

[0235] SEQ ID NO:11 Human KIR3DL3 cDNA sequence (NM_153443.4, CDS region at positions 51-1283)

[0236]

[0237] SEQ ID NO:12 Human KIR3DL3 amino acid sequence (NP_703144.3)

[0238]

[0239]

[0240] *Table 1 includes RNA nucleic acid molecules (e.g., thymine replaced by uridine), nucleic acid molecules encoding orthologs of encoded proteins, and DNA, cDNA, or RNA nucleic acid sequences comprising nucleic acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity in their full length with any of the nucleic acid sequences of SEQ ID NO listed in Table 1 or a portion thereof. Such nucleic acid molecules may have the functions of full-length nucleic acids as further described herein.

[0241] *Table 1 contains orthologs of proteins and polypeptide molecules comprising amino acid sequences, wherein any amino acid sequence of SEQ ID NO listed in Table 1, or a portion thereof, has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity over its full length. Such polypeptides may have the functions of full-length polypeptides as further described herein.

[0242] *Table 1 contains other known HHLA2 and KIR3DL3 nucleic acid and amino acid sequences.

[0243] The term "KIR3DL3 activity" encompasses the ability of the KIR3DL3 peptide to modulate inhibitory signaling in activated immune cells, for example, by binding to the native HHLA2 ligand on cancer cells. Modulation of inhibitory signaling in immune cells leads to regulation of immune cell proliferation and / or cytokine secretion. Therefore, the term "KIR3DL3 activity" includes the ability of the KIR3DL3 peptide to bind to its native ligand, its ability to modulate inhibitory signaling in immune cells, and its ability to regulate immune responses.

[0244] In some embodiments, a condition such as cancer responds only to KIR3DL3 blockade. In other embodiments, a condition such as cancer responds to KIR3DL3 blockade alone, but responds significantly or synergistically when treated with KIR3DL3 blockade in combination with at least one other therapy. Many conditions that respond to KIR3DL3 blockade, alone or in combination, include, but are not limited to, melanoma (e.g., advanced or metastatic melanoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), breast cancer (e.g., HER-2 negative breast cancer, estrogen receptor+ / HER-2- 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, chronic myeloid leukemia (CML), and blood cancers.

[0245] Preferred B7 peptides can deliver 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 reduce co-stimulation. Furthermore, the same B7 family members can increase or decrease T cell co-stimulation. For example, HHLA2 can induce immune cell co-stimulation when bound to a co-stimulatory receptor, or it can inhibit immune cells when bound to an inhibitory receptor. When bound to an inhibitory receptor, HHLA2 can transmit inhibitory signals to immune cells. Preferred B7 family members include HHLA2, B7-1, B7-2, B7h, PD-L1, or PD-L2 and their soluble fragments or derivatives. In one embodiment, a member of the B7 family binds to one or more receptors on an immune cell, such as TMIGD2, KIR3DL3, CTLA4, CD28, ICOS, PD-1 and / or other receptors, and, depending on the receptor, has the ability to transmit inhibitory or co-stimulatory signals to the immune cell, preferably a T cell.

[0246] Regulation of co-stimulatory signals leads to the regulation of effector functions of immune cells. Therefore, the term "KIR3DL3 activity" encompasses the ability of KIR3DL3 ligand peptides to bind to their natural receptors (e.g., HHLA2), the ability to regulate co-stimulatory or inhibitory signals of immune cells, and the ability to modulate immune responses.

[0247] The KIR3DL3 pathway is a negative regulator of immune function, meaning that modulating the interaction between KIR3DL3 and one or more natural binding partners, such as HHLA2, can modulate immune function. Therefore, agents that directly or indirectly modulate the interaction between KIR3DL3 and one or more natural binding partners, as described herein, can upregulate or downregulate the immune system, thereby upregulating or downregulating the immune response. Agents that modulate such interactions can do so directly or indirectly.

[0248] Exemplary agents for upregulating immune responses include antibodies against HHLA2 or KIR3DL3 that block the interaction between HHLA2 and KIR3DL3; inactive forms of HHLA2 or KIR3DL3 (e.g., dominant inactive peptides), small molecules or peptides that block the interaction between HHLA2 and KIR3DL3; fusion proteins that bind to HHLA2 or KIR3DL3 and inhibit the interaction between HHLA2 and KIR3DL3 (e.g., extracellular portions of HHLA2 or KIR3DL3 fused to the Fc portion of an antibody or immunoglobulin); nucleic acid molecules and / or genetic modifications that block the transcription or translation of HHLA2 and / or KIR3DL3; inactive forms of natural HHLA2 ligands and soluble forms of natural KIR3DL3 ligands.

[0249] In other exemplary embodiments, agents that promote the binding of the HHLA2 peptide to one or more natural binding partners, such as the KIR3DL3 peptide, promote inhibitory signaling against immune cells. Agents that modulate such interactions can do so directly or indirectly. Thus, in one embodiment, agents that directly enhance the interaction between HHLA2 and KIR3DL3 (HHLA2 agonists and / or KIR3DL3 agonists) can promote inhibitory signaling and downregulate the immune response. Alternatively, agents that block the binding of KIR3DL3 to other targets increase the effective concentration of KIR3DL3 available for binding to HHLA2. Exemplary agents for downregulating the immune response comprise an antibody against HHLA2 or KIR3DL3 that activates or promotes the interaction between HHLA2 and KIR3DL3; a small molecule or peptide that activates or promotes the interaction between HHLA2 and KIR3DL3; and a blocking antibody that binds to natural binding partners of HHLA2 and KIR3DL3, respectively, rather than HHLA2 and KIR3DL3.

[0250] Other agents that can be used in the methods covered by this disclosure include antibodies, small molecules, peptides, peptide mimics, natural ligands, and derivatives of natural ligands, which can bind to and / or activate or inhibit the protein biomarkers covered by this disclosure, said protein biomarkers including the biomarkers listed in Table 1 or fragments thereof; RNA interference, antisense, nucleic acid aptamers, etc., which can downregulate the expression and / or activity of the biomarkers covered by this disclosure, including the biomarkers listed in Table 1 or fragments thereof.

[0251] An isolated monoclonal antibody or fragment thereof targeting KIR3DL3 is provided. In some embodiments, the mAb produced by the hybridoma has been deposited in accordance with the terms of the Budapest Treaty at the American Type Culture Collection (ATCC) ______, accession number ______.

[0252] Since it is well known in the art that the CDR3 domains of the antibody heavy and light chains play a particularly important role in the antibody's binding specificity / affinity to the antigen, the recombinant monoclonal antibody covered by this disclosure prepared as described above preferably includes the heavy and light chain CDR3 domains of the variable region covered by this disclosure (e.g., comprising the sequences or portions thereof in Table 2). The antibody may further include the CDR2 domain of the variable region covered by this disclosure (e.g., comprising the sequences or portions thereof in Table 2). The antibody may further include the CDR1 domain of the variable region covered by this disclosure (e.g., comprising the sequences or portions thereof in Table 2). In other embodiments, the antibody may include any combination of CDRs.

[0253] The CDR1, 2, and / or 3 regions of the engineered antibody described above may include identical amino acid sequences to those of the variable regions covered by this disclosure (e.g., sequences comprising the sequences in Table 2 or portions thereof). However, those skilled in the art will understand that some deviations from the exact CDR sequence are possible while still retaining the antibody's ability to bind effectively to KIR3DL3 (e.g., conserved sequence modifications). Therefore, in another embodiment, the engineered antibody may consist of one or more CDRs, such as one or more CDRs having, for example, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with one or more CDRs covered by this disclosure (e.g., sequences comprising the sequences in Table 2 or portions thereof).

[0254] Structural features of known non-human or human antibodies (e.g., mouse or non-rodent anti-human KIR3DL3 antibodies) can be used to generate structure-related human anti-human KIR3DL3 antibodies that retain at least one functional property of the antibodies covered by this disclosure, such as binding to KIR3DL3. Another functional property includes inhibiting the binding of the original known non-human or human antibody in a competitive ELISA assay.

[0255] In some embodiments, a monoclonal antibody capable of binding to human KIR3DL3 is provided, the monoclonal antibody comprising a heavy chain, wherein the variable domain comprises at least one CDR, the sequence of which has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with a set of heavy chain variable domain CDRs presented in Table 2.

[0256] Similarly, monoclonal antibodies capable of binding to human KIR3DL3 are also provided, the monoclonal antibodies comprising a light chain wherein a variable domain comprises at least one CDR, the sequence of which has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with a set of light chain variable domain CDRs presented in Table 2.

[0257] Also provided are monoclonal antibodies capable of binding to human KIR3DL3, wherein the monoclonal antibody comprises a heavy chain, wherein the variable domain comprises at least one CDR, the sequence of which has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with a set of heavy chain variable domain CDRs presented in Table 2; and the monoclonal antibody comprises a light chain, wherein the variable domain comprises at least one CDR, the sequence of which has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with a set of light chain variable domain CDRs presented in Table 2.

[0258] Skilled technicians will notice that such percentage homology is equivalent to and can be achieved by introducing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conserved amino acid substitutions within a given CDR.

[0259] The monoclonal antibodies covered by this disclosure may include a heavy chain, wherein the variable domain includes at least one CDR having a sequence selected from the group consisting of heavy chain variable domain CDRs and light chains presented in Table 2, wherein the variable domain includes at least one CDR having a sequence selected from the group consisting of light chain variable domain CDRs presented in Table 2.

[0260] Such monoclonal antibodies may include a light chain, wherein a variable domain includes at least one CDR having a sequence selected from the group consisting of CDR-L1, CDR-L2, and CDR-L3, as described herein; and / or include a heavy chain, wherein a variable domain includes at least one CDR having a sequence selected from the group consisting of CDR-H1, CDR-H2, and CDR-H3, as described herein. In some embodiments, monoclonal antibodies capable of binding to human KIR3DL3 include or consist of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, as described herein.

[0261] The heavy chain variable domain of the monoclonal antibodies covered by this disclosure may include or consist of the vH amino acid sequence shown in Table 2 and / or the light chain variable domain of the monoclonal antibodies covered by this disclosure may include or consist of the vL amino acid sequence shown in Table 2.

[0262] The monoclonal antibodies covered by this disclosure can be produced and modified using any techniques well known in the art. For example, such monoclonal antibodies can be mouse or non-rodent antibodies, such as those obtainable from hybridomas deposited at ______, ATCC as the storage site ______. Similarly, such monoclonal antibodies can be chimeric, preferably chimeric mouse / human antibodies. In some embodiments, the monoclonal antibody is a humanized antibody, such that the variable domain includes a human receptor framework region and optionally present human constant domains, as well as a non-human donor CDR as defined above, such as a mouse or non-rodent CDR.

[0263] This disclosure further provides fragments of the monoclonal antibodies, which include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and biantibodies; as well as multispecific antibodies formed from antibody fragments. For example, many immunosuppressive molecules such as HHLA2, PD-L2, PD-L1, CTLA-4, KIR3DL3, etc., can be detected in a bispecific or multispecific manner to effectively characterize the expression of such molecules.

[0264] Other fragments of monoclonal antibodies covered by this disclosure are also considered. For example, separate immunoglobulin heavy chains and / or light chains are provided, wherein their variable domains include at least one CDR presented in Table 2. In one embodiment, the immunoglobulin heavy chain includes at least one CDR whose sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with a set of heavy chain or light chain variable domain CDRs presented in Table 2. In another embodiment, the immunoglobulin light chain includes at least one CDR whose sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with (e.g., presented in Table 2) a set of light or heavy chain variable domain CDRs described herein.

[0265] In some embodiments, the immunoglobulin heavy and / or light chains include variable domains, said variable domains including at least one of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3 as described herein. Such immunoglobulin heavy chains may include or consist of at least one of CDR-H1, CDR-H2, and CDR-H3. Such immunoglobulin light chains may include or consist of at least one of CDR-L1, CDR-L2, and CDR-L3.

[0266] In other embodiments, the immunoglobulin heavy and / or light chains according to this disclosure respectively include or consist of the vH or vL variable domain sequences provided in Table 2.

[0267] This disclosure further provides polypeptides having sequences 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.

[0268] The antibodies, immunoglobulins, and peptides covered by this disclosure may be used in isolated (e.g., purified) form or contained in a carrier, such as a membrane or lipid vesicle (e.g., liposome).

[0269] Table 2: Anti-KIR3DL3 monoclonal antibodies containing mAbs 1C7, 1D12, 1G7, 2A3, 2D8, 2F11, 2H1, 8C2, and 8F7 Characteristics and sequence of representative variable regions of antibodies

[0270]

[0271] 1C7 heavy chain variable (vH) and light chain variable (vL) DNA and amino acid sequences*

[0272] AVS-3698HC[574.2.1C7.D2.6.5 Heavy Chain]

[0273]

[0274] AVS-3698LC[574.2.1C7.D2.6.5 Light Chain]

[0275]

[0276]

[0277]

[0278]

[0279] 2A3 Heavy chain variable (vH) and light chain variable (vL) DNA and amino acid sequences*

[0280] AVS-3699HC [574.2.2A3.5.9 Heavy Chain]

[0281]

[0282] AVS-3699LC[574.2.2A3.5.9 Light Chain]

[0283]

[0284]

[0285]

[0286]

[0287] 8F7 heavy chain variable (vH) and light chain variable (vL) DNA and amino acid sequences*

[0288] AVS-3700HC [574.2.8F7.1.7.4 Heavy Chain]

[0289]

[0290] AVS-3700LC [574.2.8F7.1.7.4 Light Chain]

[0291]

[0292]

[0293]

[0294]

[0295] 1G7 heavy chain variable (vH) and light chain variable (vL) DNA and amino acid sequences*

[0296] AVS-3701HC [574.2.1G7.9.9 Heavy Chain]

[0297]

[0298]

[0299] AVS-3701LC [574.2.1G7.9.9 Light Chain]

[0300]

[0301]

[0302]

[0303]

[0304] 2D8 heavy chain variable (vH) and light chain variable (vL) DNA and amino acid sequences*

[0305] AVS-3702HC [574.2.2D8.6.9 Heavy Chain]

[0306]

[0307] AVS-3702LC [574.2.2D8.6.9 Light Chain]

[0308]

[0309]

[0310]

[0311]

[0312] 2F11 heavy chain variable (vH) and light chain variable (vL) DNA and amino acid sequences*

[0313] AVS-3703HC [574.2.2F11.2.7.4 Heavy Chain]

[0314]

[0315] AVS-3703LC[574.2.2F11.2.7.4 Light Chain]

[0316]

[0317]

[0318]

[0319]

[0320] 2H1 heavy chain variable (vH) and light chain variable (vL) DNA and amino acid sequences*

[0321] AVS-3704HC[574.2.2H1.11.6 Heavy Chain]

[0322]

[0323]

[0324] AVS-3704LC[574.2.2H1.11.6 Light Chain]

[0325]

[0326]

[0327]

[0328]

[0329] 1D12 heavy chain variable (vH) and light chain variable (vL) DNA and amino acid sequence*

[0330] AVS-3705HC [574.2.1D12.1.6 Heavy Chain]

[0331]

[0332] AVS-3705LC [574.2.1D12.1.6 Light Chain]

[0333]

[0334]

[0335]

[0336]

[0337] 8C2 heavy chain variable (vH) and light chain variable (vL) DNA and amino acid sequences*

[0338] AVS-3706HC [574.2.8C2.12.3.10 Heavy Chain]

[0339]

[0340] AVS-3706LC [574.2.8C2.12.3.10 Light Chain]

[0341]

[0342]

[0343]

[0344]

[0345] *Based on Kabat's CDR definition and protein sequence number.

[0346] *Table 2 includes RNA nucleic acid molecules (e.g., thymine replaced by uridine), nucleic acid molecules encoding orthologs of encoded proteins, and DNA, cDNA, or RNA nucleic acid sequences comprising nucleic acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity in their full length with the nucleic acid sequence of any of the SEQ ID NOs listed in Table 2 or a portion thereof. Such nucleic acid molecules may have the function of a full-length nucleic acid as further described herein.

[0347] III. Nucleic Acids, Vectors, and Recombinant Host Cells

[0348] Other aspects covered by this disclosure relate to nucleic acid sequences encoding the monoclonal antibodies and fragments thereof, immunoglobulins, and polypeptides covered by this disclosure.

[0349] Typically, nucleic acids are DNA or RNA molecules that can be contained in any suitable vector such as plasmids, granules, episomes, artificial chromosomes, bacteriophages, or viral vectors.

[0350] Vectors may include regulatory elements such as promoters, enhancers, terminators, etc., to induce or guide the expression of the polypeptide upon administration to a subject. Examples of promoters and enhancers for animal cell expression vectors include the early promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), and the promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of the immunoglobulin H chain.

[0351] Expression vectors from any animal cell can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al., 1990), pAGE103 (Mizukami T et al., 1987), pHSG274 (Brady G et al., 1984), pKCR (O'Hare K et al., 1981), and pSG1βd2-4- (Miyaji H et al., 1990). Other representative examples of plasmids include replication plasmids or integration plasmids that include the origin of replication, such as pUC, pcDNA, and pBR. Representative examples of viral vectors include adenoviruses, retroviruses, herpesviruses, and AAV vectors. Such recombinant viruses can be generated using techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, and 293 cells. Detailed protocols for generating such replication-defective recombinant viruses can be found, for example, WO 95 / 14785, WO 96 / 22378, U.S. Patent Nos. 5,882,877, 6,013,516, 4,861,719, 5,278,056, and WO 94 / 19478.

[0352] Further aspects covered by this disclosure relate to cells that have been transfected, infected, or transformed according to the nucleic acids and / or vectors disclosed herein. The term “transformation” means the introduction of a “foreign” (i.e., extrinsic or extracellular) gene, DNA, or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. Host cells that accept and express the introduced DNA or RNA have been “transformed.”

[0353] The nucleic acids covered by this disclosure can be used to generate the recombinant polypeptides covered by this disclosure in a suitable expression system. The term "expression system" means a host cell and a compatible vector under suitable conditions, for example, for expressing proteins encoded by exogenous DNA carried by a vector and introduced into the host cell.

[0354] 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 yeast, or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., those produced by 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 with a dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") defect (Urlaub G et al.; 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"). YB2 / 0 cells are preferred because the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in these cells.

[0355] This disclosure also relates to a method for generating recombinant host cells expressing antibodies or peptides covered by this disclosure, the method comprising the steps of: (i) introducing a recombinant nucleic acid or vector as described above into competent host cells in vitro or in vitro; (ii) culturing the obtained recombinant host cells in vitro or in vitro; and (iii) optionally, selecting cells that express and / or secrete said antibodies or peptides. Such recombinant host cells can be used to generate antibodies and peptides as described herein.

[0356] On the other hand, this disclosure provides isolated nucleic acids that hybridize with the polynucleotides disclosed herein under selective hybridization conditions. Therefore, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids comprising such polynucleotides. For example, the polynucleotides covered by this disclosure can be used to identify, isolate, or amplify partial or full-length clones in a library. In some embodiments, the polynucleotide is a genomic or cDNA sequence isolated from or complementary to cDNA from a human or mammalian nucleic acid library. Preferably, the cDNA library comprises at least 80% of the full-length sequence, preferably at least 85% or 90% of the full-length sequence, and more preferably at least 95% of the full-length sequence. The cDNA library can be normalized to increase the representativeness of rare sequences. Low or moderately stringent hybridization conditions are generally, but not exclusively, used with sequences having reduced sequence identity relative to the complementary sequence. For sequences with higher identity, moderate and high stringent conditions can optionally be used. Low stringent conditions allow selective hybridization of sequences with about 70% sequence identity and can be used to identify orthologous or paralogous sequences. Optionally, the polynucleotides of the present invention will encode at least a portion of an antibody encoded by the polynucleotides described herein. The polynucleotides of the present invention encompass nucleic acid sequences that can be used for selective hybridization with polynucleotides encoding antibodies covered by this disclosure. See, for example, Ausubel (ibid.); Colligan (ibid.), each incorporated herein by reference in its entirety.

[0357] IV. Methods of Antibody Production

[0358] The antibodies and their fragments, immunoglobulins and peptides covered by this disclosure can be produced by any technology known in the art, such as, but not limited to, any chemical, biological, genetic or enzymatic technology, alone or in combination.

[0359] Knowing the amino acid sequence of the desired sequence, those skilled in the art can readily produce the antibody or peptide using standard techniques for generating peptides. For example, the antibody or peptide can be synthesized using well-known solid-phase methods, preferably using commercially available peptide synthesis equipment (such as equipment manufactured by Applied Biosystems, Foster City, Calif.) and following the manufacturer's instructions. Alternatively, the antibodies and other peptides covered by this disclosure can be synthesized using recombinant DNA techniques well-known in the art. For example, after incorporating a DNA sequence encoding the desired (multi)peptide into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host from which the desired peptide will be expressed, these fragments can be obtained as DNA expression products, from which they can then be isolated using well-known techniques.

[0360] Specifically, this disclosure further relates to a method for generating antibodies or peptides covered by this disclosure, the method comprising the steps of: (i) culturing host cells transformed according to this disclosure under conditions suitable for allowing the expression of the antibody or peptide; and (ii) recovering the expressed antibody or peptide.

[0361] The antibodies and other peptides covered by this disclosure can be appropriately separated from the culture medium using conventional immunoglobulin purification procedures, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, hydroxyapatite 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, New York, NY (1997–2001), e.g., sections 1, 4, 6, 8, 9, 10, each incorporated herein by reference in its entirety.

[0362] Chimeric antibodies (e.g., mouse-human chimeras or non-rodent-human chimeras) covered by this disclosure can be generated by: obtaining nucleic acid sequences encoding VL and VH domains as previously described; constructing a human chimeric antibody expression vector by inserting them into an animal cell expression vector having genes encoding human antibody CH and human antibody CL; and expressing the encoding sequences by introducing the expression vector into animal cells. The CH domain of a human chimeric antibody can be any region belonging to human immunoglobulins, such as IgG class or its subclasses, such as IgG1, IgG2, IgG3, and IgG4. Similarly, the CL domain of a human chimeric antibody can be any region belonging to Ig, such as κ or λ class. Chimeric and humanized monoclonal antibodies (including human and non-human portions) prepared using standard recombinant DNA techniques are within the scope of this disclosure. Such chimeric and humanized monoclonal antibodies can be produced using recombinant DNA techniques known in the art, for example, using methods described in: Robinson et al., International Patent Publication PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86 / 01533; US 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) Proceedings of the National Academy of Sciences 84:3439-3443; Liu et al. (1987) Journal of Immunology 139:3521-3526; Sun et al. (1987) Proceedings of the National Academy of Sciences 84:214-218; Nishimura et al. (1987) Cancer Research 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) Journal of the National Cancer Institute (J. Natl. Cancer) Inst.) 80:1553-1559); Morrison, SL (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter US Patent 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) Journal of Immunology 141:4053-4060.

[0363] Additionally, humanized antibodies can be prepared according to standard protocols, such as those disclosed in U.S. Patent 5,565,332. In some embodiments, antibody chains or specific binding pair members can be generated by recombination between a vector comprising a fused nucleic acid molecule of a polypeptide chain encoding a specific binding pair member and a component of a reproducible universal display package and a vector containing a nucleic acid molecule encoding a second polypeptide chain encoding a single binding pair member, using techniques known in the art, such as those described in U.S. Patents 5,565,332, 5,871,907, or 5,733,743. Humanized antibodies covered by this disclosure can be generated by obtaining nucleic acid sequences encoding CDR domains as previously described; constructing humanized antibody expression vectors by inserting them into expression vectors in animal cells, wherein the gene encodes (i) a heavy chain constant region identical to the heavy chain constant region of a human antibody and (ii) a light chain constant region identical to the light chain constant region of a human antibody; and expressing the gene by introducing the expression vector into animal cells. Humanized antibody expression vectors can be of the type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain exist on separate vectors, or in the type in which both genes exist on the same vector (tandem type).

[0364] Methods for generating humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger M S. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-transplantation (EP 239,400; PCT Publication WO91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), faceting or surface repair (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska M A. et al. (1994)) and chain tampering (U.S. Patent No. 5,565,332). Common recombinant DNA techniques for preparing such antibodies are also known (see European patent application EP 125023 and international patent application WO 96 / 02576).

[0365] Similarly, the bispecific or multispecific antibodies described herein can be prepared according to standard procedures. For example, trigeneous hybridomas and hybridomas are two examples of cell lines capable of secreting bispecific or multispecific antibodies. Examples of bispecific and multispecific antibodies produced by hybridomas or trigeneous hybridomas are disclosed in U.S. Patent 4,474,893. Such antibodies can also be constructed by chemical methods (Staerz et al. (1985) Nature 314:628 and Perez et al. (1985) Nature 316:354) and hybridoma techniques (Staerz and Bevan (1986) Proceedings of the National Academy of Sciences 83:1453 and Staerz and Bevan (1986) Immunology Today 7:241). Alternatively, such antibodies can also be produced by generating xenogeneic hybridomas by fusing hybridomas or other cells that produce different antibodies, and then identifying clones that produce and co-assemble the desired antibodies. The antibody can also be produced by chemical or genetic conjugation of the complete immunoglobulin chain or portions thereof, such as Fab and Fv sequences. The antibody component can bind to a polypeptide or fragment thereof of one or more biomarkers covered in this disclosure, including one or more immunosuppressive biomarkers described herein.

[0366] Furthermore, methods for generating antibody fragments are well known. For example, the Fab fragments covered in this disclosure can be obtained by treating an antibody that specifically reacts with human KIR3DL3 with a protease such as papain. Alternatively, Fab can be generated by inserting DNA encoding the Fab of an antibody into a vector in a prokaryotic or eukaryotic expression system and introducing the vector into a prokaryote or eukaryote (as applicable) to express the Fab.

[0367] Similarly, the F(ab′)2 fragment covered by this disclosure can be obtained by treating an antibody that specifically reacts with KIR3DL3 with a protease, namely papain. Furthermore, the F(ab′)2 fragment can be generated by binding Fab′ as described below via a thioether bond or a disulfide bond.

[0368] The Fab' fragment covered by this disclosure can be obtained by treating F(ab')2, which specifically reacts with human KIR3DL3, with a reducing agent, namely dithiothreitol. Furthermore, the Fab' fragment can be generated by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector, and then introducing the vector into a prokaryote or eukaryote (as applicable) for expression.

[0369] Additionally, the scFv covered by this disclosure can be generated by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote (as the case may be) to express scFv. To generate humanized scFv fragments, a well-known technique known as CDR transplantation can be used. This technique involves selecting a complementarity-determining region (CDR) from a donor scFv fragment and transplanting the CDR onto a human scFv fragment framework with a known three-dimensional structure (see, for example, WO 98 / 45322; WO 87 / 02671; U.S. Patent No. 5,859,205; U.S. Patent No. 5,585,089; U.S. Patent No. 4,816,567; EP0173494).

[0370] V. Modification of antibodies, immunoglobulins, and peptides

[0371] Amino acid sequence modifications of the antibodies described herein are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. It is known that when humanized antibodies are produced by simply transplanting only the CDR of the VH and VL of an antibody derived from a non-human animal into the FR of the VH and VL of a human antibody, the antigen-binding activity is reduced compared to that of the original antibody derived from the non-human animal. It is believed that several amino acid residues of the VH and VL of the non-human antibody are directly or indirectly related to antigen-binding activity, not only in the CDR but also in the FR. Therefore, replacing these amino acid residues with different amino acid residues of the FR of the VH and VL of the human antibody reduces binding activity and can be corrected by replacing the amino acids with amino acid residues of the original antibody derived from the non-human animal.

[0372] The structure of the antibodies covered by this disclosure and the DNA sequence encoding the antibodies can be modified and altered, and these modifications and alterations can still yield functional molecules encoding antibodies and peptides with desired properties. For example, certain amino acids can be substituted for other amino acids in the protein structure without significant loss of activity. Since the interaction capabilities and properties of proteins define their biological functional activity, certain amino acid sequences can be substituted in the protein sequence, and of course, in its DNA-coding sequence, while simultaneously obtaining proteins with similar properties. Therefore, it is contemplated that various alterations can be made to the antibody sequences covered by this disclosure or the corresponding DNA sequences encoding the peptides without significant loss of their biological activity.

[0373] In some embodiments, amino acid changes can be achieved by altering codons in the DNA sequence to encode conservative substitutions based on the conservation of the genetic code. Specifically, there is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequence that can encode the protein, as defined by the genetic code (shown below). Similarly, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by the nucleic acid, as defined by the genetic code (see the genetic code chart above).

[0374] When making changes to the amino acid sequence of a polypeptide, the hydrophilicity index of the amino acids can be considered. The importance of the hydrophilicity index of amino acids in conferring interactive biological functions to proteins is generally understood in the art. It is accepted that the relative hydrophilic nature of amino acids contributes 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 hydrophilicity index based on its hydrophobic and charge characteristics, which 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); aspartate (<RTI3.5); asparagine (–3.5); lysine (–3.9); and arginine (–4.5).

[0375] It is known in the art that certain amino acids can be replaced by other amino acids having similar hydrophilicity indices or scores and still result in a protein having similar biological activity, i.e., still obtaining a biologically functional equivalent protein.

[0376] As outlined above, amino acid substitutions are therefore generally based on the relative similarity of the amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various前述特性的示例性取代是本领域技术人员众所周知的并且包含:精氨酸和赖氨酸;谷氨酸盐和天冬氨酸盐;丝氨酸和苏氨酸;谷氨酰胺和天冬酰胺;以及缬氨酸、亮氨酸和异亮氨酸。

[0377] It seems there is an error in the description in item , where "各种前述特性的示例性取代是本领域技术人员众所周知的并且包含:精氨酸和赖氨酸;谷氨酸盐和天冬氨酸盐;丝氨酸和苏氨酸;谷氨酰胺和天冬酰胺;以及缬氨酸、亮氨酸和异亮氨酸。" has some unclear表述. I translated it as best as possible based on the context. You may want to check and correct it if needed.Another type of amino acid modification of antibodies covered by this disclosure can be used to alter the original glycosylation pattern of the antibody to, for example, increase stability. "Alteration" means the deletion of one or more carbohydrate moieties found in the antibody and / or the addition of one or more glycosylation sites not present in the antibody. Antibody glycosylation is typically N-linked. N-linking refers to the attachment of a carbohydrate moieties to a side chain of asparagine residues. Where X is a tripeptide sequence of any amino acid other than proline, asparagine-X-serine and asparagine-X-threonine are recognition sequences for the enzymatic attachment of the carbohydrate moieties to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in the polypeptide creates a potential glycosylation site. Adding glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Another type of covalent modification involves chemically or enzymatically coupling a glycoside to the antibody. These procedures are advantageous because they do not require antibody generation in host cells with glycosylation capacity for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar can be linked to: (a) arginine and histidine, (b) a free carboxyl group, (c) a free thiol group, such as those of cysteine, (d) a free hydroxyl group, such as those of serine, threonine, or hydroxyproline, (e) an aromatic residue, such as those of phenylalanine, tyrosine, or tryptophan, or (f) an amide group of glutamine. Such methods are described, for example, in WO87 / 05330.

[0378] Similarly, chemical deglycosylation requires exposing the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylglucosamine), while maintaining the integrity of the antibody. Chemical deglycosylation is described by Sojahr H. et al. (1987) and Edge, AS. et al. (1981). Enzymatic cleavage of the carbohydrate moiety on the antibody can be achieved using various endo- and exo-glycosidases, as described by Thotakura, N R. et al. (1987).

[0379] Other modifications may involve the formation of immunoconjugates. For example, in one type of covalent modification, an antibody or protein is covalently linked to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyethylene, in the manner shown in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0380] The conjugation of antibodies or other proteins covered by this disclosure with heterologous agents can be performed using a variety of bifunctional protein conjugates, including, but not limited to, N-(2-pyridyldithio)propionate succinimide ester (SPDP), (N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester, iminothiones (IT), bifunctional derivatives of imine esters (such as dimethyl hexamethylenediimide hydrochloride), active esters (such as disuccinimide ester), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazide derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bifunctional fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, carbon-labeled 1-isothiocyanobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies (WO 94 / 11026).

[0381] On the other hand, this disclosure is characterized by antibodies that specifically bind to KIR3DL3, which is conjugated to therapeutic components such as cytotoxins, drugs, and / or radioisotopes. When conjugated to cytotoxins, these antibody conjugates are referred to as “immunotoxins.” A cytotoxin or cytotoxic agent contains any agent that is harmful to cells (e.g., killing). Examples include paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, styracin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologs. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatin(II)(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., actinomycin (formerly known as actinomycin), bleomycin, styraxycin and anthraxycin (AMC), and antimitotic agents (e.g., vincristine and vinblastine). The antibodies disclosed herein can be conjugated with radioisotopes such as radioactive iodine to generate cytotoxic radiopharmaceuticals for the treatment of related conditions such as cancer.

[0382] Conjugated anti-KIR3DL3 antibodies can be used, among other things, to diagnostically or prognostively monitor peptide levels in tissues as part of clinical testing procedures, such as to determine the efficacy of a given treatment regimen or to select patients most likely to respond to a particular immunotherapy. For example, cells can be permeabilized in flow cytometry assays to allow antibodies binding to KIR3DL3 to target the intracellular epitopes they recognize, and binding can be detected by analyzing signals emitted from the conjugated molecule. Detection can be facilitated by conjugating (i.e., physically linking) antibodies to detectable substances. Examples of detectable substances include a variety of enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, luciferin, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin (PE); examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and jellyfish luminescent protein; and examples of suitable radioactive materials include... 125 I, 131 I, 35 S or 3 H. As used herein, the term “labeled” for antibodies is intended to cover both direct labeling of antibodies by conjugation (i.e., physical linking) to detectable substances such as radiopharmaceuticals or fluorophores (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)) and indirect labeling of antibodies by reactivity with detectable substances.

[0383] The antibody conjugates covered by this disclosure can be used to modify a given biological response. The chemical portion should not be construed as limited to classical chemical agents. For example, the pharmaceutical portion can be a protein or peptide with the desired biological activity. Such proteins may contain, for example, tumor necrosis factor or interferon-γ; or biological response modulators such as 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.

[0384] The technique of combining such therapeutic components with antibodies is 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 in Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Immunol. Rev.)》, 62:119 58(1982). .

[0385] In some embodiments, conjugation may be performed using a “cleavable adapter” that promotes the release of cytotoxic agents or growth inhibitors into cells. For example, acid-labile adapters, peptidase-sensitive adapters, light-labile adapters, dimethyl adapters, or disulfide-bonded adapters may be used (see, for example, U.S. Patent No. 5,208,020). Alternatively, fusion proteins comprising antibodies and growth inhibitors may be prepared via recombinant techniques or peptide synthesis. The length of the DNA may include corresponding regions encoding two parts of the conjugate, said two parts being adjacent to each other or separated by regions encoding adapter peptides that do not disrupt the desired properties of the conjugate.

[0386] VI. Uses and Methods

[0387] The anti-KIR3DL3 antibodies, immunoglobulins, peptides, and nucleic acids covered by this disclosure can be used alone or in combination with other therapies for a variety of purposes, such as KIR3DL3 detection methods, therapeutic purposes (e.g., therapeutic, prophylactic, and immunomodulatory). Furthermore, the anti-KIR3DL3 antibodies, immunoglobulins, peptides, and nucleic acids covered by this disclosure can be used in many predictive pharmacological assays based on the detection of KIR3DL3 levels. For example, this 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 peptides or fragments thereof covered by this disclosure have one or more of the following activities: 1) binding to and / or modulating the activity of their natural binding partners, such as HHLA2; 2) modulating intracellular or intercellular signaling, such as co-immunosuppressive signaling; 3) modulating the activation of T cells or NK cells; 4) modulating the immune response of an organism, such as a mammalian organism like a mouse, non-rodent, or human; and 5) modulating immune cell unresponsiveness.

[0388] This disclosure also provides a means of detecting KIR3DL3 as a means of identifying agents that transduce KIR3DL3 signaling. Agents that transduce KIR3DL3 signaling may weaken the immune response and may be useful for autoimmune diseases, asthma, and for building tolerance.

[0389] 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 co-stimulatory molecules. Combined detection of several molecules (e.g., sequentially or simultaneously) can provide useful information about the synergistic effects of therapeutic interventions and / or disease subtype personalization and higher resolution diagnosis. In some embodiments, KIR3DL3 is detected in combination with one or more biomarkers.

[0390] 1. Treatment methods and uses

[0391] In some embodiments, the antibodies, fragments, or immunoconjugates covered by this disclosure (e.g., anti-KIR3DL3 antibodies) can be used to treat any condition (e.g., cancer) associated with abnormal or undesirable activation of KIR3DL3. In some embodiments, the treatment is directed at mammals such as humans. Such antibodies covered by this disclosure can be used alone or in combination with any suitable agent or appropriate therapy to treat the condition of interest. For example, therapeutic synergy is believed to emerge when cells are treated with a therapy comprising an anti-KIR3DL3 mAb and another immune checkpoint inhibitor or a cell therapy such as CAR.

[0392] The antibodies or fragments thereof covered by this disclosure can be used to modulate immune responses by preventing or disrupting the interaction between KIR3DL3 and its natural ligand HHLA2. Similarly, the antibodies or fragments thereof described herein can be used to treat diseases, such as cancer, by increasing immune responses and the activity of T cells and / or NK cells against cancer cells. Therefore, the purposes covered by this disclosure relate to methods for modulating immune responses and / or treating conditions associated with aberrant KIR3DL3 activation, said methods comprising administering a therapeutically effective amount of the antibodies and fragments thereof covered by this disclosure to a subject in need.

[0393] Upregulation of the immune response can take the form of enhancing an existing immune response or inducing an initial immune response. For example, enhancing the immune response using the subject composition and methods is useful in improving immune defense against cancer and microbial infections (e.g., bacteria, viruses, or parasites). For example, upregulation or enhancement of the immune response function as described herein can be used to induce tumor immunity.

[0394] In another embodiment, an immune response can be stimulated by the methods described herein to overcome pre-existing tolerance, clonal loss, and / or exhaustion (e.g., T cell exhaustion). For example, an immune response to an antigen to which a subject cannot elicit a significant immune response, such as an autoantigen like a tumor-specific antigen, can be induced by administration of an appropriate agent that upregulates the immune response as described herein. In one embodiment, an autoantigen such as a tumor-specific antigen can be co-administered. In another embodiment, an immune response can be stimulated against an antigen (e.g., an autoantigen) to treat a neurological disorder. In another embodiment, the subject agent can be used as an adjuvant to enhance the response to foreign antigens during active immunization.

[0395] In some cases, it may be desirable to further enhance the immune response by administering other agents that upregulate the immune response, such as other B7 family members that transduce signals via co-stimulatory receptors. Furthermore, agents that upregulate the immune response can be used prophylactically in vaccines against various peptides (e.g., peptides derived from pathogens). Immunity against pathogens (e.g., viruses) can be induced by inoculating viral proteins with an agent that upregulates the immune response in an appropriate adjuvant.

[0396] Alternatively, in some embodiments, the antibody and antigen-binding fragments covered by this disclosure, in addition to diagnostic, prognostic, and preventative applications (such as treating and delaying the onset or progression of disease), may also be used for therapeutic applications to suppress diseases that upregulate immune responses, such as asthma, autoimmune diseases (glomerulonephritis, arthritis, dilated cardiomyopathy-like disease, ulcerative colitis, Sjogren's syndrome, Crohn's disease, erythema generalis, chronic rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, hypertrophic skin, periarteritis nodosa, rheumatic fever, leukoderma vulgaris, insulin-dependent diabetes mellitus, Behcet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, pernicious anemia, Goodpasture syndrome). syndrome), infertility, chronic active hepatitis, pemphigus, autoimmune thrombocytopenic purpura and autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, antiphospholipid syndrome, atopic hypersensitivity, autoimmune atrophic gastritis, gastric acid deficiency autoimmune disease, celiac disease, Cushing's syndrome, dermatomyositis, discoid lupus, erythema, Goodpasture's syndrome, Hashimoto's thyroiditis, primary adrenal atrophy, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus, Lambert-Eaton syndrome. Syndrome), lupus-like hepatitis, some lymphopenias, mixed connective tissue diseases, bullous pemphigoid, pemphigus vulgaris, pernicious anemia, lens-derived uveitis, polyarteritis nodosa, polyglandular syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, recurrent osteitis, Schmidt's syndrome, localized scleroderma (or Crest syndrome), sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, insulin resistance type B, ulcerative colitis, and Wegener's granulomatosis.

[0397] Similarly, in addition to diagnostic, prognostic, and preventative applications (such as treatment and delaying the onset or progression of disease) of persistent infectious diseases (e.g., viral infectious diseases including HPV, 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), the antibodies and antigen-binding fragments covered in this disclosure can be used for therapeutic applications. Other pathogen-related antigens that can be used as described herein are antigens of various parasites, including malaria, preferably based on NANP repeats of the malaria peptide. In addition, it includes bacteria, fungi, and other pathogenic organisms, such as *Aspergillus*, *Brugia*, *Candida*, *Chlamydia*, *Coccidia*, *Cryptococcus*, *Dirofilaria*, *Gonococcus*, *Histoplasma*, *Leishmania*, *Mycobacterium*, and *Mycoplasma*. The genera include *Plasmodium*, *Paramecium*, *Pertussis*, *Plasmodium*, *Pneumococcus*, *Pneumocystis*, *Rickettsia*, *Salmonella*, *Shigella*, *Staphylococcus*, *Streptococcus*, *Toxoplasma*, and *Vibrio cholerae*.Exemplary species include Neisseriagonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Haemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, and Brucella abortus.Brucella melitensis (sheep), Brucella suis (swine), Brucella canis (canine), Campylobacter fetus (fetal), Campylobacter fetus intestinalis (fetal), Leptospira pomona, Listeria monocytogenes, Brucella ovis (sheep), Chlamydia psittaci, Trichomonas foetus, Toxoplasma gondii, Escherichia coli, Actinobacillus equuli (foal), Salmonella abortus ovis (sheep abortion), Salmonella abortus equi (equine abortion), Pseudomonas aeruginosa Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa, Trypanosoma equiperdum, Babesiacaballi, Clostridium tetani, Clostridium botulinum; or fungi such as Paracoccidioides brasiliensis; or other pathogens such as Plasmodium falciparum. It also includes priority pathogens of the National Institute of Allergy and Infectious Diseases (NIAID).These include Category A drugs, such as smallpox, Bacillus anthracis, Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularensis), filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagic fever), arenaviruses (Lassa fever, Junin hemorrhagic fever, and related viruses); and Category B drugs, such as Coxiella burnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei (horse glanders), and alphaviruses (Venezuelan encephalomyelitis). Encephalomyelitis, equine encephalomyelitis, ricin (from castor beans), ε-toxin from Clostridium perfringens; Staphylococcus enterotoxin B, Salmonella species, Shigella dysenteriae, Escherichia coli strain O157:H7, Vibrio cholerae, Cryptosporidium parvum; Category C drugs, such as Nipah virus, Hantaviruses, tick-borne hemorrhagic fever virus, tick-borne encephalitis virus, yellow fever, and multidrug-resistant tuberculosis; worms such as schistosomes and tapeworms; and protozoa such as Leishmania (e.g., Leishmania mexicana) and Plasmodium.

[0398] In some embodiments, in addition to prognostic and preventative applications, the antibodies or antigen-binding fragments covered by this disclosure may be used for therapeutic applications relating to induction of immune tolerance, organ transplant rejection, graft-versus-host disease (GVHD), allergic diseases, and diseases caused by KIR3DL3-mediated attenuated immune responses.

[0399] In the context of this invention, as used herein, the term "treating" means reversing, alleviating, or inhibiting the progression of a condition or symptom to which such terms apply, or one or more symptoms of such a condition or symptom. The term "treating cancer" as used herein means inhibiting the growth and / or proliferation of cancer cells. Preferably, such treatment also results in the regression of tumor growth (i.e., a measurable reduction in tumor size). Most preferably, such treatment results in complete tumor regression.

[0400] Therapeutic formulations comprising one or more antibodies covered herein are prepared for storage by mixing antibodies of desired purity with optional physiologically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Antibody compositions can be formulated, administered, and applied in any manner consistent with good medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the drug, the method of administration, the schedule of administration, and other factors known to the practicing physician.

[0401] The therapeutic dose may be at least about 0.001 μg / kg body weight, 0.005 μg / kg body weight, 0.01 μg / kg body weight, at least about 0.05 μg / kg body weight; at least about 0.1 μg / kg body weight, at least about 0.5 μg / kg body weight, at least about 1 μg / kg body weight, at least about 2.5 μg / kg body weight, at least about 5 μg / kg body weight, at least about 50 μg / kg body weight, or at least about 100 μg / kg body weight. Those skilled in the art will understand that such guidelines will be adjusted for the molecular weight of the active agent, for example, in the use of antibody fragments or antibody conjugates. The dose may also vary for local administration, such as intranasal, inhalation, etc., or for systemic administration, such as intramuscular, intraperitoneal, intravenous, etc.

[0402] The composition does not need to be formulated with, but may optionally be formulated with, one or more agents that enhance activity or otherwise increase therapeutic effect.

[0403] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used and contain: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenolic alcohols, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). Polypeptides (containing 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG). Formulations intended for in vivo administration must be sterile. This is easily accomplished through filtration using a sterile filter membrane.

[0404] The active ingredient can also be encapsulated in prepared microcapsules, for example, by coagulation techniques or by interfacial polymerization reactions. These microcapsules include, for example, hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed., 1980).

[0405] The compositions described herein can be administered by any suitable method, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Additionally, the compositions can be appropriately administered via pulsatile infusion, particularly in cases of antibody dose reduction.

[0406] For the prevention or treatment of disease, the appropriate dosage of an antibody will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is being administered for prophylactic purposes, previous treatments, the patient's clinical history and response to the antibody, and the attending physician's considerations, as defined above. Antibodies may be administered to the patient in a single dose or as part of a series of treatments.

[0407] Agents that directly block the interaction between KIR3DL3 and HHLA2, such as anti-HHLA2 antibodies, anti-KIR3DL3 antibodies, and anti-KIR3DL3 / anti-immune checkpoint bispecific antibodies (e.g., anti-KIR3DL3 / PD-1 bispecific antibodies), can inhibit KIR3DL3 signaling and its downstream immune responses. Alternatively, agents that indirectly block the interaction between KIR3DL3 and HHLA2 can inhibit KIR3DL3 signaling and its downstream immune responses. For example, in some embodiments, 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 comprise monospecific or bispecific blocking antibodies against KIR3DL3 and / or HHLA2 that block the interaction between the receptor and ligand; inactive forms of HHLA2 and / or KIR3DL3 (e.g., dominantly inactivated or soluble peptides); 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 ligand (e.g., extracellular portions of HHLA2 and / or KIR3DL3 fused to the Fc portion of an antibody or immunoglobulin); inactive forms of native KIR3DL3 and / or HHLA2; and soluble forms of native KIR3DL3 and / or HHLA2.

[0408] In some embodiments, anti-KIR3DL3 antibody therapy or combinations of therapies may be administered (e.g., one or more anti-KIR3DL3 antibody therapies combined with one or more additional anticancer therapies such as another immune checkpoint inhibitor). Combination therapies may include, for example, one or more chemotherapeutic agents and radiation, one or more chemotherapeutic agents and immunotherapy, or one or more chemotherapeutic agents, radiation, and chemotherapy, each of which may be used in conjunction with an anti-immune checkpoint therapy. Furthermore, those skilled in the art can apply any representative embodiment of an agent modulating a specific target to any other target described herein and below (e.g., the direct and indirect KIR3DL3 inhibitors described herein may be applied to other immune checkpoint inhibitors and / or monospecific antibodies, bispecific antibodies, inactive forms, small molecules, peptides, interfering nucleic acids, etc.).

[0409] Therefore, the therapeutic agents covered by this disclosure can be used alone or in combination with, for example, chemotherapy agents, hormones, anti-angiogenic agents, CARs, radiolabeled compounds, or surgical procedures, cryotherapy, and / or radiation therapy. The aforementioned treatments can be administered in combination with other forms of conventional therapy (e.g., standard cancer care treatments well known to those skilled in the art), administered sequentially with, before, or after conventional therapy. For example, the agents covered by this disclosure can be administered together with a therapeutically effective dose of a chemotherapy agent. In another embodiment, the agents covered by this disclosure are administered in combination with chemotherapy to enhance the activity and efficacy of the chemotherapy agent. The Physicians' Desk Reference (PDR) discloses dosages of chemotherapy agents used to treat various cancers. The dosing regimen and dosage of these effective chemotherapy agents will depend on the specific cancer being treated, the severity of the disease, and other factors familiar to those skilled in the art, and can be determined by a physician.

[0410] Anti-KIR3DL3 agents can also be administered in combination with targeted therapies, such as immunotherapy. Immunotherapy designed to elicit or amplify an immune response is called "activating immunotherapy." Immunotherapy designed to reduce or suppress an immune response is called "suppressive immunotherapy." Any agent believed to have an immune system effect on genetically modified transplanted cancer cells can be tested to determine whether the agent is an immunotherapy and the effect of a given genetic modification on the regulation of the immune response. In some embodiments, immunotherapy is cancer cell-specific. In some embodiments, immunotherapy can be "non-targeted," which refers to the administration of agents that do not selectively interact with immune system cells but modulate immune system function. Representative examples of non-targeted therapies include, but are not limited to, chemotherapy, gene therapy, and radiation therapy.

[0411] The term "targeted therapy" refers to the administration of an agent that selectively interacts with chosen biomolecules, for example, to treat cancer. For instance, targeted therapy with immune checkpoint inhibitors can be useful in combination with the methods covered in this disclosure. The term "immune checkpoint inhibitor" refers to a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by downregulating or inhibiting 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, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR (see, for example, WO2012 / 177624). Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling, thereby upregulating the immune response for more effective cancer treatment.

[0412] Immunotherapy is a form of targeted therapy that may include, for example, the use of one or more cancer vaccines and / or sensitized antigen-presenting cells. Oncolytic viruses, for example, are viruses capable of infecting and lysing cancer cells while leaving normal cells unharmed, making them potentially useful in cancer therapy. Oncolytic virus replication both promotes tumor cell destruction and generates dose amplification at the tumor site. Oncolytic viruses can also serve as vectors for anticancer genes, enabling them to be specifically delivered to tumor sites. Immunotherapy can involve passive immunization for short-term host protection, achieved by administering pre-formed antibodies against cancer antigens or disease antigens (e.g., administering monoclonal antibodies optionally linked to chemotherapeutic agents or toxins to the tumor antigen). For example, anti-VEGF and mTOR inhibitors are known to be effective in treating renal cell carcinoma. Immunotherapy can also focus on using cytotoxic lymphocyte recognition epitopes from cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple-helical polynucleotides, etc., can be used to selectively modulate biomolecules associated with the initiation, progression, and / or pathology of tumors or cancers. Immunotherapy can also focus on using cytotoxic lymphocyte recognition epitopes from cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple-helix polynucleotides, etc., can be used to selectively modulate biomolecules associated with tumor or cancer initiation, progression, and / or pathology. As described above, immunotherapy targeting immune checkpoints such as HHLA2 and KIR3DL3 is useful.

[0413] In some embodiments, immunotherapy may include one or more adoptive cell-based immunotherapies. Well-known forms of adoptive cell-based immunotherapy 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 modulate the immune response, such as expressing cytokines like GM-CSF and / or expressing tumor-associated antigens (TAA) such as Mage-1, gp-100, patient-specific neoantigen vaccines, etc.

[0414] In some embodiments, immunotherapy may include one or more cell-based immunotherapies. In some embodiments, compositions comprising antigens with or without vaccine adjuvants are used. Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, recombinant antigens comprising fusion proteins, etc. 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 durable forms) are used. In yet another embodiment, immunomodulatory cytokines such as interferon, G-CSF, imiquimod, TNFα, etc., and their modulators (e.g., blocking antibodies or more potent or durable forms) are used. In yet another embodiment, immunomodulatory chemokines such as CCL3, CCL26, and CXCL7, etc., and their modulators (e.g., blocking antibodies or more potent or durable forms) are used. In yet another embodiment, immunomodulatory molecules targeting immunosuppression, such as STAT3 signaling modulators, NFκB signaling modulators, and immune checkpoint modulators, are used. The above text describes the terms "immune checkpoint" and "anti-immune checkpoint therapy".

[0415] In some embodiments, immunomodulatory drugs are used, such as immunosuppressive drugs, glucocorticoids, cell inhibitors, immunosuppressants and their modulators (e.g., rapamycin, calmodulin inhibitors, tacrolimus, cyclosporin, pimecrolimus, abetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), and cortisone acetate. Acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone Acetate, doca), aldosterone, non-glucocorticoids, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid, methotrexate, anti-thymocyte globulin, anti-lymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, opioid, IMPDH inhibitors, mycophenolic acid, myriocin, fingolimod, NF-xB inhibitors, raloxifene, drotrecogin Alfa, denosumab, NF-κB signaling cascade inhibitors, disulfiram, olmesartan, dithiocarbamate, proteasome inhibitors, bortezomib, MG132, Prol,NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, nonsteroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethylepoxyquinoline (DHMEQ), I3C (indole-3-carbinol) / DIM (disindolemethane) (I3C / DIM), Bay 11-7082, luteolin, cell-penetrating peptide SN-50, IKBa.-superrepressor overexpression, NFKB-induced oligodeoxynucleotides (ODNs) or any derivatives or analogs thereof. In yet another embodiment, an immunomodulatory antibody or protein is 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); otelixizumab; teplizumab; visilizumab; anti-CD4 antibodies; clenoliximab; keliximab; zanolimumab; anti-CD11 antibodies; efalizumab; anti-CD18 antibodies; erlizumab; rovelizumab; anti-CD20 antibodies; afutuzumab; ocrelizumab; ofatumumab; and pascolizumab. (mab), rituximab, anti-CD23 antibody, lumiliximab, anti-CD40 antibody, teneliximab, totalizumab, anti-CD40L antibody, ruplizumab, anti-CD62L antibody, acelizumab, anti-CD80 antibody, galiximab, anti-CD147 antibody, gavilimomab, B-lymphocyte stimulating factor (BLyS) inhibitory antibody, belimumab, CTLA4-Ig fusion protein, abatacept, belacept, anti-CTLA4 antibody, ipilimumab, tremelimumab, anti-eosinophil activating chemokine 1 antibody, bertilimumab.Anti-a4-integrin antibody, natalizumab, anti-IL-6R antibody, tocilizumab, anti-LFA-1 antibody, odulimomab, anti-CD25 antibody, basiliximab, daclizumab, inoximomab, anti-CD5 antibody, zolimomab, anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atorolimumab, cedelizumab, dorlimomab aritox, dolixizumab, fontolizumab, gantenerumab, gomiliximab, lebrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, telimomab Arritox, valliximab, vepalimomab, aflibercept, alefacept, rilonacept, IL-1 receptor antagonists, anakinra, anti-IL-5 antibodies, mepolizumab, IgE inhibitors, omalizumab, talizumab, IL-12 inhibitors, IL-23 inhibitors, ustekinumab, etc.

[0416] In some embodiments, nutritional supplements that enhance immune responses, 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.

[0417] Similarly, agents and therapies other than immunotherapy can be combined with anti-KIR3DL3 antibodies to stimulate an immune response, thereby treating the condition from which the patient will benefit. Examples of such therapies include chemotherapy, radiation, epigenetic modifiers (e.g., histone deacetylase (HDAC) modifiers, methylation modifiers, phosphorylation modifiers, etc.), and targeted therapies, which are well known in the art.

[0418] The term "non-targeted therapy" refers to the administration of drugs that do not selectively interact with chosen biomolecules but still treat cancer. Representative examples of non-targeted therapies include, but are not limited to, chemotherapy, gene therapy, and radiation therapy.

[0419] In one embodiment, chemotherapy is used. Chemotherapy involves the administration of a chemotherapeutic agent. Such chemotherapeutic agents may be, but are not limited to, those selected from the group consisting of platinum compounds, cytotoxic antibiotics, antimetabolites, antimitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogs, plant alkaloids and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, and docetaxel; DNA topoisomerase inhibitors: teniposide, crisantol, and mitomycin; antifolate agents: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytarabine; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouracil, glycine afedipine, and pyrazolimidazole; and antimitotic agents: leucospirin, colchicine, and rhizomycin. Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may 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-naphthalenedimide (Trevigen); 6(5H)-phenanthridine (Trevigen); benzamide (US Patent Re. 36,397); and NU1025 (Bowman et al.). The mechanism of action is generally related to the ability of PARP inhibitors to bind to PARP and reduce its activity. PARP catalyzes the conversion of β-nicotinamide adenine dinucleotide (NAD+) to nicotinamide and poly-ADP-ribose (PAR).Both poly(ADP-ribose) and PARP are associated with transcriptional regulation, cell proliferation, genome stability and carcinogenicity (Bouchard VJ et al., Experimental Hematology, Vol. 31, No. 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q., Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 477, No. 1, June 2, 2001, pp. 97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule for repairing DNA single-strand breaks (SSBs) (de Murcia J. et al. 1997. Proceedings of the National Academy of Sciences 94:7303-7307; Schreiber V, Dantzer F, Ame JC, de Murcia G (2006. Nature Review of Molecular Cell Biology 7:517-528; Wang ZQ et al. (1997. Genes Dev 11:2347-2358). Knocking out SSB repair-induced DNA double-strand breaks (DSBs) by inhibiting PARP1 function can trigger synthetic killing in cancer cells with defective homologous DSB repair (Bryant HE et al. (2005. Nature 434:913-917; Farmer H et al. (2005. Nature 434:917-921). The examples of the aforementioned chemotherapeutic agents are illustrative and not intended to be limiting.

[0420] In another embodiment, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external beam radiation therapy, interstitial implantation of radioactive isotopes (I-125, palladium, iridium), radioactive isotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or whole abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th ed., 2001, edited by DeVita et al., JBLippencott Company, Philadelphia, Pennsylvania. Radiation therapy can be administered as external beam radiation or teletherapy, where the radiation is directed from a remote source. Radiation therapy can also be administered as internal therapy or brachytherapy, where the radiation source is placed inside the body close to cancer cells or tumor masses. It also covers the use of photodynamic therapy, which includes the application of photosensitizers such as hematoporphyrin and its derivatives, verteporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypochlorous A; and 2BA-2-DMHA.

[0421] In another embodiment, hormone therapy is used. Hormone therapeutic treatment may 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, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogens, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; anti-estrogens (e.g., mifepristone, onapristone) or anti-androgens (e.g., cyproterone acetate)).

[0422] The duration and / or dosage of treatment with a therapy can vary depending on the specific therapeutic agent or combination thereof. A skilled technician will understand the appropriate duration of treatment for a particular cancer therapeutic agent. This disclosure considers the ongoing evaluation of the optimal treatment regimen for each cancer therapeutic agent, wherein the phenotype of the subject's cancer, determined by the methods covered in this disclosure, is a factor in determining the optimal treatment dosage and regimen.

[0423] Any method used to introduce polynucleotides into mammals, humans, or non-humans or their cells can be adapted for the practice of this invention to deliver the various constructs covered by this disclosure to the intended recipient. In one embodiment covered by this disclosure, the DNA construct is delivered to cells by transfection, i.e., by delivering “naked” DNA or in a complex having a colloidal dispersion system. Colloidal systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Preferred colloidal systems of this invention are lipid-complexed or liposome-formulated DNA. In the former approach, before, for example, preparing the DNA with lipids, the plasmid containing the transgene carrying the desired DNA construct can first be experimentally optimized for expression (e.g., including introns in the 5′ untranslated region and eliminating unwanted sequences (Felgner et al., Ann NY Acad Sci. 126-139, 1995)). DNA prepared, for example, with various lipid or liposome materials can then be realized and delivered to recipient mammals using known methods and materials. See, for example, Canonico et al., Am J Respir Cell & Molecular Biology 10:24-29, 1994; Tsan et al., Am J Physiology 268; Alton et al., Nature Genetics 5:135-142, 1993; and U.S. Patent No. 5,679,647 to Carson et al.

[0424] Liposome targeting can be categorized based on anatomical and mechanistic factors. Anatomical classification is based on levels of selectivity, such as organ specificity, cell specificity, and organelle specificity. Mechanotargeting can be distinguished based on whether the mechanotargeting is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs containing sinusoidal capillaries. On the other hand, active targeting involves altering liposomes by conjugating them to specific ligands such as monoclonal antibodies, sugars, glycolipids, or proteins, or by changing the composition or size of the liposomes, to achieve targeting to organ and cell types, rather than naturally occurring localization sites.

[0425] The surface of a targeted delivery system can be modified in a variety of ways. In the case of a liposome-based targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain stable binding of the targeting ligand to the liposome bilayer. Various linker groups can be used to link the lipid chain to the targeting ligand. Naked DNA or DNA associated with the delivery medium, such as liposomes, can be applied to several sites on the subject (see below).

[0426] Nucleic acids can be delivered in any desired vector. These include viral or non-viral vectors, including adenovirus vectors, adeno-associated virus 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 (micetic leukemia virus). Nucleic acids can be administered in any desired form that provides a sufficiently efficient delivery level, including in the form of viral particles, liposomes, nanoparticles, and polymer complexes.

[0427] The nucleic acid encoding the protein or nucleic acid of interest can be in a plasmid, viral vector, or other vector known in the art. Such vectors are well known and can be selected for specific applications. In one embodiment covered by this disclosure, the gene delivery medium includes a promoter and a demethylase-coding sequence. Preferred promoters are tissue-specific promoters and promoters activated by cell proliferation, such as thymidine kinase and thymidine synthase promoters. Other preferred promoters include promoters that can be activated by viral infection, such as α- and β-interferon promoters, and promoters that can be activated by hormones such as estrogen. Other promoters that can be used include Moloni virus LTR, CMV promoters, and mouse albumin promoters. The promoter can be constitutive or inducible.

[0428] In another embodiment, naked polynucleotide molecules can be used as gene delivery mediators, as described in WO 90 / 11092 and U.S. Patent 5,580,859. Such gene delivery mediators can be growth factor DNA or RNA, and in some embodiments, are associated with cytotoxic adenoviruses. (Curiel et al., Human Gene Therapy 3:147-154, 1992.) Other mediators that may be used include DNA-ligands (Wu et al., Journal of Biochemistry 264:16985-16987, 1989), lipid-DNA combinations (Felgner et al., Proceedings of the National Academy of Sciences 84:74137417, 1989), liposomes (Wang et al., Proceedings of the National Academy of Sciences 84:7851-7855, 1987) and microparticles (Williams et al., Proceedings of the National Academy of Sciences 88:2726-2730, 1991).

[0429] The gene delivery medium may optionally include one or more viral sequences, such as viral origin of replication or packaging signals. These viral sequences may be selected from viruses such as astroviruses, coronaviruses, orthomyxoviruses, papillomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, retroviruses, cloacal viruses, or adenoviruses. In a preferred embodiment, the growth factor gene delivery medium is a recombinant retroviral vector. Recombinant retroviruses and their various uses are described in numerous references, including, for example, Mann et al., Cell 33:153, 1983; Cane and Mulligan, Proceedings of the National Academy of Sciences 81:6349, 1984; Miller et al., Human Gene Therapy 1:5-14, 1990; U.S. Patents 4,405,712, 4,861,719, and 4,980,289; and PCT applications WO 89 / 02,468, WO 89 / 05,349, and WO 90 / 02,806. Various retroviral gene delivery agents may be used in this disclosure, including, for example, those described in: EP 0,415,731; WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Research 53:3860-3864, 1993; Vile and Hart, Cancer Research 53:962-967, 1993; Ram et al., Cancer Research 53:83-88, 1993; Takamiya et al., Journal of Neuroscience Research 33:493-503, 1992; Baba et al., Journal of Neurosurgery 79:729-735, 1993 (US Patent No. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91 / 02805).

[0430] Other viral vector systems that can be used to deliver the polynucleotides covered in this disclosure have been derived from herpesviruses, such as herpes simplex virus (US Patent No. 5,631,236 to Woo et al., issued May 20, 1997, and Neurovex WO 00 / 08191), vaccinia virus (Ridgeway (1988), “Mammalian expression vectors” in: Rodriguez RL, Denhardt DT, edited “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, edited “Gene Transfer (Gene Transfer (Gene Transfer)” (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, polioviruses, etc. The viruses provide several attractive features for various mammalian cells (Friedmann (1989) Science, 244:1275-1281; Ridgeway, 1988 (ibid.); Baichwal and Sugden, 1986 (ibid.); Coupar et al., 1988; Horwich et al. (1990) Journal of Virology, 64:642-650).

[0431] In other embodiments, the target DNA in the genome can be manipulated using methods well known in the art. For example, manipulation of the target DNA in the genome by deletion, insertion, and / or mutation includes retroviral insertion, artificial chromosome techniques, gene insertion, random insertion using tissue-specific promoters, gene targeting, transposons, and / or any other methods for introducing exogenous DNA or generating modified DNA / modified nuclear DNA. Other modification techniques include deleting DNA sequences from the genome and / or altering nuclear DNA sequences. For example, nuclear DNA sequences can be altered by site-directed mutagenesis.

[0432] In other embodiments, recombinant biomarker peptides and fragments thereof may be administered to a subject. In some embodiments, fusion proteins with enhanced biological properties may be constructed and administered. Additionally, biomarker peptides and fragments thereof may be modified according to pharmacological methods well known in the art (e.g., PEGylation, glycosylation, oligomerization, etc.) to further enhance desired biological activities, such as increased bioavailability and reduced proteolytic degradation.

[0433] 2. Measurement and screening methods

[0434] Another aspect covered by this disclosure relates to screening assays, including cell-based assays and xenograft animal model assays. In one embodiment, the assay provides a method for identifying agents that modulate KIR3DL3 signaling, such as in human or animal model assays, to identify agents that reduce KIR3DL3 signaling thereby increasing an immune response and / or identify agents that increase KIR3DL3 signaling thereby reducing an immune response.

[0435] In one embodiment, this disclosure relates to the determination of a test agent for screening at least one biomarker, such as HHLA2, TMIGD2, and KIR3DL3, described herein (e.g., in tables, figures, examples, or otherwise in the specification). In one embodiment, a method for identifying such a agent requires determining the agent's ability to regulate, for example, inhibit at least one of the biomarkers described herein.

[0436] In one embodiment, the assay is a cell-free or cell-based assay that includes 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 enzyme activity of the biomarker, such as by measuring direct binding to the substrate or by measuring indirect parameters as described below.

[0437] For example, in direct binding assays, biomarker proteins (or their corresponding target peptides or molecules) can be conjugated to radioactive isotopes or enzyme labels, allowing binding to be determined by detecting the labeled protein or molecule in the complex. For example, binding can be determined directly or indirectly using… 125 I, 35 S, 14 C or 3 H-labeled targets are used, and radioisotopes are detected by direct counting or scintillation counting of radiation emissions. Alternatively, targets can be enzyme-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzyme labeling is detected by determining the conversion of a suitable substrate to a product. Standard binding or enzyme assays can also be used to determine the interaction between the biomarker and the substrate. In one or more embodiments of the assay methods described above, it may be desirable to immobilize peptides or molecules to facilitate the separation of one or both of the complexed and uncomplexed forms of proteins or molecules, and to facilitate the automation of the assay.

[0438] The binding of the test reagent to the target can be completed in any suitable container for containing the reactants. Non-limiting examples of such containers include microtiter plates, test tubes, and microcentrifuge tubes. The immobilized form of antibodies described herein may also contain antibodies bound to the immobilized material, such as porous, microporous (average pore size less than about 1 micrometer) or macroporous (average pore size greater than about 10 micrometer) materials, such as membranes, cellulose, nitrocellulose, or glass fibers; beads, such as beads made of agarose, polyacrylamide, or latex; or the surface of a plate, plate, or well, such as a surface made of polystyrene.

[0439] In alternative embodiments, the ability to determine the interaction between a drug-regulated biomarker and its substrate or its natural binding partner can be accomplished by determining the activity of the drug-regulated peptide or other products that act downstream or upstream of its signaling pathway (e.g., a feedback loop). Such feedback loops are well known in the art (see, for example, Chen and Guillemin (2009), International Journal of Tryptophan Research, 2:1-19).

[0440] KIR3DL3 status can be measured using the anti-KIR3DL3 antibody described herein. Reduced binding of KIR3DL3 to HHLA2 indicates that the agent inhibits KIR3DL3 activity / signaling, and the agent is identified as potentially effective in inhibiting KIR3DL3 activity / signaling and increasing the immune response. Conversely, increased binding of KIR3DL3 to HHLA2 indicates that the agent promotes KIR3DL3 activity / signaling, and the agent is identified as potentially effective in promoting KIR3DL3 activity / signaling and reducing the immune response.

[0441] This disclosure further relates to novel pharmaceutical agents identified by the screening assays described above. Therefore, further use of pharmaceutical agents identified as described herein is within the scope of the invention, such as in suitable animal models. For example, pharmaceutical agents identified as described herein can be used in animal models to determine the efficacy, toxicity, or side effects of treatment with such pharmaceutical agents. Alternatively, antibodies identified as described herein can be used in animal models to determine the mechanism of action of such pharmaceutical agents.

[0442] One aspect covered by this disclosure relates to screening assays, which include cell-based assays and xenograft animal model assays. In one embodiment, the assay provides a method for identifying, as in humans, whether cancer is likely to respond to anti-KIR3DL3 antibody therapy and / or whether an agent can inhibit the growth of or kill cancer cells that are unlikely to respond to anti-KIR3DL3 antibody therapy.

[0443] 3. Prevention methods

[0444] On the one hand, this disclosure provides methods for preventing disease or condition associated with an unwanted or less desirable immune response in subjects. For example, subjects at risk of disease who will benefit from treatment using the claimed agent or method can be identified by any diagnostic or prognostic assay known in the art, or a combination thereof. Administration of the prophylactic agent can occur before the onset of symptoms associated with the unwanted or less 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.

[0445] 4. Prognostic determination

[0446] Furthermore, the detection methods described herein can be used to identify subjects who will respond to a therapy, such as a therapy targeting KIR3DL3, for modulating the activity and / or interaction with binding mates such as HHLA2. Similarly, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., agonist, antagonist, peptide mimic, polypeptide, peptide, nucleic acid, small molecule, or other candidate drug) for treating such conditions associated with excessive or insufficient KIR3DL3 activity. For example, such methods can be used to determine whether a subject can be effectively treated with a single agent or combination of agents. Therefore, this disclosure provides a method for determining whether a subject can be effectively treated with one or more agents for treating conditions associated with excessive or insufficient KIR3DL3 activity, wherein a test sample is obtained and KIR3DL3 is detected. The test sample can be a biological sample obtained from the subject of interest. The test sample can be obtained from the subject of interest. For example, the sample can be a biological fluid (e.g., cerebrospinal fluid or serum), a cell sample, or a tissue, such as a histopathological slide of the tumor microenvironment, peritumoral region, and / or intratumoral region. In some embodiments, the test sample may include cells expressing mature membrane-bound KIR3DL3 and / or KIR3DL3 fragments.

[0447] The methods described herein can be performed, for example, by using a pre-packaged diagnostic kit comprising at least one of the antibody reagents described herein, which can be conveniently used, for example, in a clinical setting to predict patients with diseases presenting with symptoms or a family history of disease or diseases involving KIR3DL3.

[0448] Furthermore, any cell type or tissue expressing KIR3DL3 can be used for the prognostic assays described herein.

[0449] Another aspect of this disclosure includes the use of the compositions and methods described herein for association and / or stratification analysis, wherein KIR3DL3 in biological samples from individuals suffering from a condition associated with excessive or insufficient KIR3DL3 activity is analyzed, and said information is compared with information from controls (e.g., individuals without said condition; controls may also be referred to as “healthy” or “normal” individuals or at an earlier time point in a given time-lapse study), said controls preferably having similar age and ethnicity. Alternatively, controls may be individuals suffering from a condition with excessive or insufficient KIR3DL3 activity who respond well to therapies targeting KIR3DL3, such as therapies modulating KIR3DL3 activity or the interaction of KIR3DL3 with one or more of its binding mates. Since appropriate selection of patients and controls is useful for association and / or stratification studies in some embodiments, it may be desirable to have a group of individuals with well-characterized phenotypes. Different study designs can be used for stratified studies (Modern Epidemiology, Lippincott Williams & Wilkins (1998), 609-622).

[0450] VII. Pharmaceutical Composition

[0451] Agents containing, for example, blocking antibodies, peptides, fusion proteins, or small molecules that regulate (e.g., inhibit or promote) the interaction between KIR3DL3 and one or more natural binding partners such as HHLA2, can be incorporated into pharmaceutical compositions suitable for administration to a subject. Such pharmaceutical compositions may further comprise additional components and / or therapeutic agents, as described herein. Pharmaceutical compositions typically comprise one or more pharmaceutical agents and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to encompass any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and adsorption delay agents, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Their use in the composition should be considered unless any conventional media or reagent is incompatible with the active compound. Additional active compounds may also be incorporated into these compositions.

[0452] The pharmaceutical compositions covered by this disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or glucose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multiple dosing vials made of glass or plastic.

[0453] Suitable injectable pharmaceutical compositions comprise a sterile aqueous solution (when water-soluble) or dispersion and a sterile powder for the ad hoc preparation of a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition should be sterile and should have a flowability sufficient for easy injection. It must be stable under the conditions of manufacture and storage and should be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the dispersed state, and by using surfactants. Antimicrobial action can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include an isotonic agent in the composition, such as sugar, polyol (e.g., mannitol, sorbitol), or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0454] As needed, sterile injectable solutions can be prepared by incorporating the active compound in the desired amount into a suitable solvent having one or a combination of the components listed above, followed by sterile filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which produce powders of the active ingredient and any other desired components from their previously sterile filtered solutions.

[0455] Oral compositions typically contain an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with excipients and administered in tablet, lozenge, or capsule form. Oral compositions can also be prepared using a fluid carrier as used in mouthwashes, wherein the compound in the fluid carrier is administered orally and swished and spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or hydrogenated vegetable oil (Sterotes); flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavorings.

[0456] For administration by inhalation, the compound is delivered from a pressurized container or dispenser containing a suitable propellant, such as a gas (e.g., carbon dioxide), or from a nebulizer as an aerosol spray.

[0457] Systemic application can also be performed via mucosal or transdermal routes. For mucosal or transdermal application, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and contain, for example, detergents, bile salts, and clostridial acid derivatives for mucosal application. Mucosal application can be achieved by using nasal sprays or suppositories. For transdermal application, the active compound is formulated as an ointment, cream, gel, or lotion, as commonly known in the art.

[0458] The composition can also be prepared as a suppository (e.g., using a conventional suppository base, such as cocoa butter and other glycerides) or as a retention enema for rectal delivery.

[0459] In one embodiment, the modifier is prepared together with a carrier that protects these compounds from rapid elimination from the body, such as a controlled-release formulation, comprising an implant and a microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. Methods for preparing such formulations will be readily apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeting infected cells with monoclonal antibodies against antiviral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0460] Of particular advantage is the formulation of oral or parenteral compositions in unit dosage form for ease of administration and dosage uniformity. As used herein, unit dosage form refers to a physically discrete unit suitable as a single dose for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired drug carrier. The specifications of the unit dosage forms covered by this disclosure are determined and directly depended upon by and the unique characteristics of the active compound, the specific therapeutic effect to be achieved, and the inherent limitations in the art of compounding such active compounds for use in treating individuals.

[0461] The toxicity and therapeutic efficacy of such compounds can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals, for example, by determining the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and the therapeutic index can be expressed as the ratio LD50 / ED50. Compounds exhibiting a large therapeutic index are preferred. Although compounds exhibiting toxic side effects can be used, care should be taken to design delivery systems that target such compounds to the site of the affected tissue, thereby minimizing potential damage to uninfected cells and thus reducing side effects.

[0462] Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for human use. The doses of such compounds are preferably within a range of circulating concentrations having little or no toxicity, including the ED50. The dose can vary within this range depending on the dosage form used and the route of administration. For any compound used in the methods covered by this disclosure, a therapeutically effective dose can be initially estimated based on cell culture assays. In animal models, the dose can be formulated to achieve a range of circulating plasma concentrations including the IC50 (i.e., the concentration at which the test compound achieves half-maximal inhibition of symptoms) as determined in cell culture. This information can be used to more precisely determine the dose that can be used in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0463] The modulators described above can be administered in the form of an expressible nucleic acid encoding the drug. Such nucleic acids and compositions containing said nucleic acids are also covered in this disclosure. For example, nucleic acid molecules covered in this disclosure can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to subjects by, for example, intravenous injection, topical administration (see U.S. Patent 5,328,470), or stereotactic injection (see, for example, Chen et al. (1994), Proceedings of the National Academy of Sciences 91:3054-3057). Pharmaceutical formulations of gene therapy vectors can be contained in gene therapy vectors in an acceptable diluent, or can include a sustained-release matrix incorporating a gene delivery medium. Alternatively, where a complete gene delivery vector can be generated intact from recombinant cells, such as a retroviral vector, the pharmaceutical article can contain one or more cells that generate the gene delivery system.

[0464] The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use.

[0465] VIII. Drug Administration

[0466] The immunomodulators covered in this disclosure are administered to subjects in a biocompatible form suitable for in vivo drug administration to enhance or inhibit immune cell-mediated immune responses. "Biocompatible form suitable for in vivo administration" means the form of the protein to be administered, wherein any toxic effects outweigh the therapeutic effects of the protein. Administration of the drug as described herein can be in any pharmacological form, comprising a therapeutically effective amount of the drug, alone or in combination with a pharmaceutically acceptable carrier.

[0467] The application of a therapeutically active amount of the therapeutic composition covered by this disclosure is defined as the amount that effectively achieves the desired result at the necessary dose and over the required time period. For example, the therapeutically active amount of the agent can vary depending on factors such as an individual's disease state, age, sex, and weight, as well as the peptide's ability to elicit the desired response in the individual. Dosing regimens can be adjusted to provide an optimal therapeutic response. For example, several separate doses can be administered daily, or the dose can be reduced proportionally, as indicated by the urgency of the treatment situation.

[0468] The pharmaceutical agents or inventions described herein can be conveniently administered, such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound may be encapsulated in a material to protect it from enzymes, acids, and other natural conditions that could inactivate it. For example, for the administration of pharmaceutical agents, in addition to parenteral administration, it may be desirable to encapsulate the agent in a material or administer it co-with the agent to prevent its inactivation.

[0469] The drug can be administered to an individual in a suitable carrier, diluent, or adjuvant, either co-administered with an enzyme inhibitor or in a suitable carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvants are used in their broadest sense and include any immunostimulatory compound such as interferon. Adjuvants considered in this article include resorcinol, nonionic surfactants such as polyoxyethylene oleyl ether and hexadecyl polyethylene ether. Enzyme inhibitors include trypsin inhibitors, diisopropyl fluorophosphate (DEEP), and aprotinin. Liposomes include water-in-oil-in-water emulsions and conventional liposomes (Sterna et al. (1984), *J. Neuroimmunol.* 7:27).

[0470] Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, as well as in oils. Under normal storage and use conditions, these products may contain preservatives to prevent microbial growth.

[0471] Pharmaceutical compositions suitable for injectable applications comprise sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the composition will preferably be sterile and must have a flowability sufficient for easy injection. The composition will preferably be stable under manufacturing and storage conditions and be able to withstand contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the dispersed state, and by using surfactants. Antimicrobial action can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0472] As needed, sterile injectable solutions can be prepared by incorporating the pharmaceutical agents covered by this disclosure (e.g., antibodies, peptides, fusion proteins, or small molecules) in desired amounts into a suitable solvent having one or a combination of the components listed above, followed by sterile filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which produce powders containing the pharmaceutical agent and any other desired components from their previously sterile filtered solutions.

[0473] When the pharmaceutical preparation is properly protected, as described above, the protein can be administered orally, for example, with an inert diluent or an assimilateable, edible carrier. As used herein, a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. The use of such media and preparations for pharmaceutically active substances is well known in the art. Unless any conventional medium or preparation is incompatible with the active compound, its use in therapeutic compositions should be considered. Additional active compounds may also be incorporated into these compositions.

[0474] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. As used herein, “unit dosage form” means a physically discrete unit suitable as a single dose for use in a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired drug carrier. Specifications for unit dosage forms covered by this disclosure are subject to and directly depend on: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art imposed by individual sensitivities on the combination of such active compounds for treatment.

[0475] In one embodiment, the agent covered by this disclosure is an antibody. As defined herein, the therapeutically effective amount (i.e., effective dose) of the antibody ranges from about 0.001 to 100 mg / kg body weight, preferably from about 0.01 to 25 mg / kg body weight, more preferably from about 0.1 to 20 mg / kg body weight, and even more preferably from about 1 to 10 mg / kg, 2 to 9 mg / kg, 3 to 8 mg / kg, 4 to 7 mg / kg, or 5 to 6 mg / kg body weight. Those skilled in the art will understand that certain factors may affect the dose required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatment, the subject's overall health and / or age, and any other pre-existing conditions. Furthermore, treating a subject with a therapeutically effective amount of antibody may comprise a single treatment, or preferably, a series of treatments. In a preferred embodiment, the subject is treated with an antibody at a dose ranging from about 0.1 to 20 mg / kg body weight, once weekly for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably about 4, 5, or 6 weeks. It should also be understood that the effective dose of antibodies used for treatment can be increased or decreased during a particular treatment course. Dosage variations may arise from the results of diagnostic assays.

[0476] As described above, in some embodiments, the agent used for administration is cell-based. Cell-based agents have an immunocompatibility relationship with the subject host, and any such relationship is contemplated for use according to this disclosure. For example, cells such as adoptive T cells can be homologous. The term “homogeneous” can refer to a state of being derived from, originating from, or being a member of the same species with genetically identical genes, particularly in terms of antigens or immune responses. These include identical twins with matching MHC types. Thus, “homogeneous transplantation” refers to the transfer of cells from a donor to a recipient that is genetically identical to the donor or immunologically sufficiently compatible to allow transplantation without undesirable adverse immunogenic responses (e.g., responses that contribute to the interpretation of the results of the immune screening described herein).

[0477] Syngeneic transplantation can be “autologous” if the transferred cells are obtained from and transplanted into the same subject. “Autologous transplantation” refers to the collection and re-infusion or transplantation of the subject’s own cells or organs. The exclusive or complementary use of autologous cells can eliminate or reduce many of the adverse effects of administering cells back to the host, particularly the graft’s response to the host.

[0478] If the transferred cells are obtained from different members of the same species and transplanted into different members of the same species, but their major histocompatibility complex (MHC) antigens are sufficiently matched to avoid an adverse immunogenic response, then syngeneic transplantation can be considered “matched allogeneic.” The degree of MHC mismatch can be determined using standard tests known and used in the art. For example, at least six major MHC genes in humans have been identified as important in transplantation biology. HLA-A, HLA-B, and HLA-C encode HLA class I proteins, while HLA-DR, HLA-DQ, and HLA-DP encode HLA class II proteins. Genes within each of these groups exhibit high polymorphism, as reflected in the numerous HLA alleles or variants found in the human population, and inter-individual differences within these groups are correlated with the strength of the immune response against the transplanted cells. Standard methods for determining the degree of MHC matching examine alleles within the HLA-B and HLA-DR or HLA-A, HLA-B, and HLA-DR groups. Therefore, at least four, or even five or six, MHC antigens within two or three HLA groups can be tested separately. In serological MHC tests, antibodies against each HLA antigen type react with cells from a subject (e.g., a donor) to determine the presence or absence of certain MHC antigens that react with the antibody. This is compared to the response profile of other subjects (e.g., recipients). The reaction of antibodies with MHC antigens is typically determined by incubating the antibody with the cells and then adding complement to induce cell lysis (i.e., a lymphocytotoxicity test). The reaction is examined based on the amount of cells lysed in the reaction and the graded response (see, for example, Mickelson and Petersdorf (1999), Hematopoietic Cell Transplantation, Thomas, ED et al., eds., pp. 28–37, Blackwell Scientific, Malden, Mass.). Other cell-based assays include flow cytometry or enzyme-linked immunosorbent assays (ELISA) using labeled antibodies. Molecular methods for determining MHC types are well known and typically employ synthetic probes and / or primers to detect specific gene sequences encoding HLA proteins. Synthetic oligonucleotides can be used as hybridization probes to detect restriction fragment length polymorphisms associated with specific HLA types (Vaughn (2002) Methods in Molecular Biology, MHC Protocol 210:45-60).Alternatively, primers can be used to amplify HLA sequences (e.g., by polymerase chain reaction or ligation chain reaction), and the products can be further examined by direct DNA sequencing, restriction fragment polymorphism analysis (RFLP), or hybridization with a range of sequence-specific oligonucleotide primers (SSOP) (Petersdorf et al. (1998) Blood 92:3515-3520; Morishima et al. (2002) Blood 99:4200-4206; and Middleton and Williams (2002) Molecular Biology Methods MHC Protocol 210:67-112).

[0479] If the transferred cells and the recipient's cells differ in terms of a defined locus, such as a single gene locus, typically through inbreeding, then syngeneic transplantation can be considered "syngeneic." The term "syngeneic" refers to a member derived from, originating from, or belonging to the same species, where members are genetically identical except for small genetic regions, typically a single genetic locus (i.e., a single gene). "Syngeneic transplantation" refers to the transfer of cells or organs from a donor to a recipient, where the recipient is genetically identical to the donor except for a single gene locus. For example, CD45 exists in several allelic forms, and syngeneic mouse strains exist, where mouse strains differ in whether they express the CD45.1 or CD45.2 allelic versions.

[0480] In contrast, a “mismatched allotype” refers to a member of the same species that is derived from, originates from, or is intended to possess different major histocompatibility complex (MHC) antigens (i.e., proteins), as typically determined by standard assays used in the art, such as a serological or molecular analysis defining a number of MHC antigens sufficient to elicit an adverse immunogenic response. A “partial mismatch” refers to a partial match of the MHC antigens tested between members, typically between donor and recipient. For example, a “half-mismatch” means that 50% of the tested MHC antigens exhibit different MHC antigen types between the two members. A “full or complete” mismatch means that all tested MHC antigens are different between the two members.

[0481] Similarly, by contrast, "xenogeneic" refers to a species that is derived from, originates from, or is a member of a different species, such as humans and rodents, humans and pigs, humans and chimpanzees, etc. "Xenotransplantation" refers to the transfer of cells or organs from a donor to a recipient, where the recipient is a different species from the donor.

[0482] Additionally, cells can be obtained from a single source or multiple sources (e.g., a single subject or multiple subjects). Multiple means at least two (e.g., more than one). In yet another embodiment, the non-human mammal is a mouse. The animal from which the cell type of interest is obtained can be an adult, a newborn (e.g., less than 48 hours old), immature, or in utero. The cell type of interest can be primary cancer cells, cancer stem cells, established cancer cell lines, immortalized primary cancer cells, etc. In some embodiments, the host subject's immune system can be engineered or otherwise selected to be immunely compatible with the transplanted cancer cells. For example, in one embodiment, the subject can be "humanized" to be compatible with human cancer cells. The term "humanization of the immune system" refers to the survival of an animal, such as a mouse, which includes human HSC lineage cells and human acquired and innate immune cells, without rejection by the host animal, thereby allowing the artificial blood and the reconstruction of acquired and innate immunity in the host animal. Acquired immune cells include T cells and B cells. Innate immune cells include macrophages, granulocytes (basophils, eosinophils, neutrophils), dendritic cells (DCs), NK cells, and mast cells.Representative non-restrictive examples include SCID-hu, Hu-PBL-SCID, Hu-SRC-SCID, NSG (NOD-SCID IL2r-γ (ineffective) lack of innate immune system, B cells, T cells and cytokine signaling), and NOG (NOD-SCID) IL2r-γ (truncated), BRG (BALB / c-Rag2 (ineffective) IL2r-γ (ineffective)) and H2dRG (stock-H2d-Rag2 (ineffective) IL2r-γ (ineffective)) mice (see, for example, Shultz et al. (2007) Nature Review Immunology 7:118; Pearson et al. (2008) Current Protocols in Immunology 15:21; Brehm et al. (2010) Clin Immunology 135:84-98; McCune et al. (1988) Science 241:1632-16) 39. U.S. Patent 7,960,175 and U.S. Patent Publication 2006 / 0161996) and related null mutants of immune-related genes such as Rag1 (lacking B and T cells), Rag2 (lacking B and T cells), TCRα (lacking T cells), perforin (cD8+ T cells lacking cytotoxic function), FoxP3 (lacking functional CD4+ T regulatory cells), IL2rg or Prfl, and mutants or knockouts of HHLA2, KIR3DL3, TMIGD2, PD-1, PD-L1, Tim3, and / or 2B4, allow for the effective implantation of human immune cells and / or the provision of compartment-specific models in immunocompromised animals such as mice (see, for example, PCT Publication WO2013 / 062134). Additionally, NSG-CD34+ (NOD-SCIDIL2r-γ (null)CD34+) humanized mice can be used to study human gene and tumor activity in animal models such as mice.

[0483] As used herein, “obtained from a source of biological material” means any conventional method of acquiring or dispensing biological material from a donor. For example, biological material can be obtained from solid tumors, blood samples such as peripheral blood or cord blood, or other bodily fluids such as bone marrow or amniotic fluid. Methods for obtaining such samples are well known to those skilled in the art. In this disclosure, samples can be fresh (i.e., obtained from the donor without freezing). Furthermore, samples can be further processed to remove irrelevant or unwanted components before expansion. Samples can also be obtained from a preserved stock. For example, in the case of cell lines or fluids such as peripheral blood or cord blood, samples can be extracted from such cell lines or fluid banks that are cryogenically or otherwise preserved. Such samples can be obtained from any suitable donor.

[0484] The obtained cell population can be used directly or frozen for later use. Various media and protocols for cryopreservation are known in the art. Typically, the cryopreservation medium will consist of approximately 5-10% DMSO, 10-90% serum albumin, and 50-90% culture medium. Other additives that can be used to preserve cells include, for example, but not limited to, disaccharides such as trehalose (Scheinkonig et al. (2004) Bone Marrow Transplant. 34:531-536) or plasma expanders such as hetastarch (i.e., hydroxyethylstarch). In some embodiments, isotonic buffer solutions such as phosphate-buffered saline can be used. An exemplary cryopreservation composition has a cell culture medium containing 4% HSA, 7.5% dimethyl sulfoxide (DMSO), and 2% hydroxyethylstarch. Other compositions and methods for cryopreservation are well known in the art and have been described (see, for example, Broxmeyer et al. (2003) Proceedings of the National Academy of Sciences 100:645-650). The cells were stored at a final temperature below approximately -135°C.

[0485] It can be 0.1 x 10 per kilogram of subject body weight. 6 0.2x10 6 0.3x10 6 0.4x10 6 0.5x10 6 0.6x10 6 0.7x10 6 0.8x10 6 0.9x10 6 1.0x10 6 5.0x10 6 1.0x10 7 5.0x10 7 1.0x10 8 5.0x10 8 Cells are administered in any range or value between the two, or more. The number of cells transplanted can be adjusted based on the desired transplantation level over a given time period. Typically, 1x10 cells may be transplanted if necessary. 5 Approximately 1x10 9 Cells / kg body weight, approximately 1x10 6 Approximately 1x10 8 One cell / kg body weight or approximately 1x10 7 Cells per kg body weight or more. In one embodiment, at least about 0.1 x 10⁻⁶ cells were transplanted relative to an average-sized mouse. 6 0.5x106 1.0x10 6 2.0x10 6 3.0x10 6 4.0x10 6 Or 5.0x10 6 The total number of cells is effective.

[0486] Cells can also be administered before, simultaneously with, or after other anticancer agents.

[0487] As described above, the administration of agents such as cells can be accomplished using methods commonly known in the art, including but not limited to intravascular, intracerebral, extra-gastric, intraperitoneal, intravenous, epidural, intraspinal, intrasternal, intra-articular, intrasynovial, intrasheath, intra-arterial, intracardiac or intramuscular, intravenous, subcutaneous, specific tissue (e.g., focal transplantation), femoral medullary cavity, spleen, renal capsule in fetal liver pattern, etc.

[0488] The implantation of transplanted cells can be assessed using any of a variety of methods, such as, but not limited to, tumor volume, cytokine levels, administration time, and flow cytometry analysis of cells of interest obtained from the subject at one or more time points post-transplantation. For example, time-based analyses at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 days can signal the time of tumor collection. Any such metric can be a variable adjusted based on well-known parameters to determine the impact of variables on the response to anticancer immunotherapy. Furthermore, transplanted cells can be co-transplanted with other agents, such as cytokines, extracellular matrix, and cell culture supports.

[0489] IX. Subjects

[0490] In some embodiments, the subjects for therapeutic or immunomodulatory purposes using this disclosure (e.g., anti-KIR3DL3 antibody or its antigen-binding fragment) are mammals (e.g., mice, humanized mice, rats, primates, non-human mammals, livestock such as dogs, cats, cattle, horses, etc.), and preferably humans. In another embodiment, the subject is an animal model of cancer. For example, the animal model may be an orthotopic xenograft animal model of human cancer.

[0491] In another embodiment of the methods covered in this disclosure, the subject has not received treatment such as chemotherapy, radiation therapy, targeted therapy, and / or anti-immune checkpoint therapy. In yet another embodiment, the subject has not received treatment such as chemotherapy, radiation therapy, targeted therapy, and / or anti-immune checkpoint therapy.

[0492] In some embodiments, the subject has undergone surgery to remove cancerous or precancerous tissue. In other embodiments, the cancerous tissue has not yet been removed; for example, the cancerous tissue may be located in an inoperable area of ​​the body, such as in life-threatening tissues or in areas where surgery would pose a significant risk of harm to the patient.

[0493] The methods covered in this disclosure can be used to determine the responsiveness to KIR3DL3 therapy and / or the type of cancer in the treated subject, such as those described herein.

[0494] X. Sample collection, preparation and separation

[0495] In some embodiments, the amount and / or activity of a biomarker in a sample from a subject is measured against a predetermined control (standard) sample. The sample from the subject is typically from diseased tissue, such as cancer cells or other tissues. The control sample may be from the same subject or from different subjects. The control sample is typically a normal, disease-free sample. However, in some embodiments, such as for disease staging or for assessing treatment efficacy, the control sample may be from diseased tissue. The control sample may be a combination of samples from several different subjects. In some embodiments, the amount and / or activity of a biomarker from a subject is measured against a predetermined level. This predetermined level is typically obtained from a normal sample. As described herein, a “predetermined” amount and / or activity measurement may be used to assess, by way of example only, the following: assessment of subjects who may be selected for treatment (e.g., based on the number of genomic mutations and / or the number of genomic mutations in nonfunctional proteins that cause DNA repair genes); assessment of response to anti-KIR3DL3 antibody therapy; and / or assessment of response to anti-KIR3DL3 antibody therapy using one or more other anticancer therapies. Predetermined biomarker levels and / or activity measurements can be determined in patient populations with or without cancer. The predetermined biomarker levels and / or activity measurements can be a single number equally suitable for each patient, or they can vary depending on a specific subgroup of patients. Subject age, weight, height, and other factors may affect the individual's predetermined biomarker levels and / or activity measurements. Further...

Claims

1. An anti-KIR3DL3 monoclonal antibody or its antigen-binding fragment, comprising: a) Heavy chain variable region (VH), comprising the CDR-H1 amino acid sequence as shown in SEQ ID NO: 67, the CDR-H2 amino acid sequence as shown in SEQ ID NO: 69, and the CDR-H3 amino acid sequence as shown in SEQ ID NO: 71; and b) Light chain variable region (VL), which includes the CDR-L1 amino acid sequence as shown in SEQ ID NO: 73, the CDR-L2 amino acid sequence as shown in SEQ ID NO: 75, and the CDR-L3 amino acid sequence as shown in SEQ ID NO:

77.

2. The anti-KIR3DL3 monoclonal antibody or its antigen-binding fragment according to claim 1, comprising: a) The VH sequence, which has at least 95% identity with SEQ ID NO: 79; and b) A VL sequence that is at least 95% identical to SEQ ID NO:

81.

3. The anti-KIR3DL3 monoclonal antibody or its antigen-binding fragment according to claim 1, comprising: a) A VH sequence as shown in SEQ ID NO: 79; and b) The VL sequence as shown in SEQ ID NO:

81.

4. The anti-KIR3DL3 monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the monoclonal antibody or its antigen-binding fragment is chimeric, humanized, complexed, mouse, or human.

5. The anti-KIR3DL3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-KIR3DL3 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) comprising an effector domain; (d) comprising an Fc domain; and / or (e) selected from the group consisting of: Fv, Fav, F(ab')2), Fab', dsFv, scFv, sc(Fv)2 and biantibody fragments.

6. The anti-KIR3DL3 monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the anti-KIR3DL3 monoclonal antibody or its antigen-binding fragment inhibits the binding of HHLA2 to KIR3DL3.

7. The anti-KIR3DL3 monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the anti-KIR3DL3 monoclonal antibody or its antigen-binding fragment specifically binds to KIR3DL3.

8. An anti-KIR3DL3 bispecific antibody or its antigen-binding fragment, comprising: a) Heavy chain variable region (VH), comprising the CDR-H1 amino acid sequence as shown in SEQ ID NO: 67, the CDR-H2 amino acid sequence as shown in SEQ ID NO: 69, and the CDR-H3 amino acid sequence as shown in SEQ ID NO:

71. b) Light chain variable region (VH), which includes the CDR-L1 amino acid sequence as shown in SEQ ID NO: 73, the CDR-L2 amino acid sequence as shown in SEQ ID NO: 75, and the CDR-L3 amino acid sequence as shown in SEQ ID NO:

77.

9. The anti-KIR3DL3 bispecific antibody or its antigen-binding fragment according to claim 8, comprising: a) The VH sequence, which has at least 95% identity with the amino acid sequence of SEQ ID NO: 79; and b) The VL sequence, which has at least 95% identity with the amino acid sequence of SEQ ID NO:

81.

10. The anti-KIR3DL3 bispecific antibody or its antigen-binding fragment according to claim 8, comprising: a) A VH sequence as shown in SEQ ID NO: 79; and b) The VL sequence as shown in SEQ ID NO:

81.

11. The anti-KIR3DL3 bispecific antibody or its antigen-binding fragment according to any one of claims 8 to 10, wherein the anti-KIR3DL3 bispecific antibody or its antigen-binding fragment is chimeric, humanized, complex, mouse, or human.

12. The anti-KIR3DL3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 8 to 10, wherein the anti-KIR3DL3 bispecific 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) comprising an effector domain; (d) comprising an Fc domain; and / or (e) selected from the group consisting of: Fv, Fav, F(ab')2), Fab', dsFv, scFv, sc(Fv)2 and the bispecific antibody fragment.

13. The anti-KIR3DL3 bispecific antibody or its antigen-binding fragment according to any one of claims 8 to 10, wherein the anti-KIR3DL3 bispecific antibody or its antigen-binding fragment inhibits (a) the binding of HHLA2 to KIR3DL3 and (b) the binding of PD-1 to PD-L1 and / or PD-L2.

14. The anti-KIR3DL3 bispecific antibody or its antigen-binding fragment according to any one of claims 8 to 10, wherein the anti-KIR3DL3 bispecific antibody or its antigen-binding fragment specifically binds to KIR3DL3 and PD-1.

15. An isolated nucleic acid molecule encoding an anti-KIR3DL3 monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1 to 14.

16. A vector comprising the nucleic acid according to claim 15.

17. A host cell comprising the nucleic acid according to claim 15.

18. An apparatus or kit comprising an anti-KIR3DL3 monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof according to any one of claims 1 to 14.

19. A method for generating an anti-KIR3DL3 monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof, the method comprising the following steps: (i) culturing transformed host cells that have been transformed with nucleic acids under conditions suitable for allowing the expression of the anti-KIR3DL3 antibody or its antigen-binding fragment, the nucleic acid comprising a sequence encoding an anti-KIR3DL3 monoclonal antibody, bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 14; and (ii) recovering the expressed anti-KIR3DL3 antibody, bispecific antibody or its antigen-binding fragment.

20. Use of any anti-KIR3DL3 monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1 to 14 in the manufacture of a kit for a method of detecting the presence or level of a KIR3DL3 peptide, said method comprising detecting said peptide in a sample.

21. The use according to claim 20, wherein the anti-KIR3DL3 monoclonal antibody, bispecific antibody or antigen-binding fragment thereof forms a complex with the KIR3DL3 polypeptide, and the complex is detected by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical method, Western blotting or intracellular flow assay.

22. Use of the anti-KIR3DL3 monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1 to 14 in a kit for manufacturing a method for predicting the response to a therapy targeting KIR3DL3, said method comprising: a) Use the aforementioned anti-KIR3DL3 monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof to determine the levels of KIR3DL3 and / or HHLA2 in the subject's sample; b) Using the aforementioned anti-KIR3DL3 monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof, determine the levels of KIR3DL3 and / or HHLA2 in samples from at least one control subject who has responded well to a KIR3DL3-targeting therapy; and c) Compare the levels of KIR3DL3 and / or HHLA2 in the subject's sample with the levels of KIR3DL3 and / or HHLA2 in the sample from the control subject; The fact that the levels of KIR3DL3 and / or HHLA2 in the subject's sample are the same as or higher than the levels of KIR3DL3 and / or HHLA2 in the sample from at least one control subject indicates that the subject will respond to the therapy targeting KIR3DL3.

23. The use according to claim 22, wherein the therapy targeting KIR3DL3 comprises an anti-KIR3DL3 monoclonal antibody, a bispecific antibody, or an antigen-binding fragment thereof according to any one of claims 1 to 14.

24. Use of the anti-KIR3DL3 monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1 to 14 in a kit for manufacturing a method for predicting the response to a therapy targeting KIR3DL3, said method comprising: a) Measure the levels of KIR3DL3 and / or HHLA2 in the subject samples; b) Determine the levels of KIR3DL3 and / or HHLA2 in samples from at least one control subject who has responded well to therapy targeting KIR3DL3; and c) Compare the levels of KIR3DL3 and / or HHLA2 in the subject's sample with the levels of KIR3DL3 and / or HHLA2 in the sample from the control subject; The fact that the levels of KIR3DL3 and / or HHLA2 in the subject's sample are the same as or higher than the levels of KIR3DL3 and / or HHLA2 in the sample from at least one control subject indicates that the subject will respond to the therapy targeting KIR3DL3.

25. The use according to claim 22, wherein the subject sample is a portion of a single sample obtained from a subject or a portion of a combined sample obtained from a subject.

26. The use according to claim 22, wherein the KIR3DL3-targeting therapy blocks (a) HHLA2 with KIR3DL3; and / or (b) the interaction and / or signal transduction between PD-1 and PD-L1 and / or PD-L2.

27. The use according to claim 22, wherein the subject sample comprises cells, serum, peritumoral tissue and / or intratumoral tissue obtained from a subject.

28. The use according to claim 27, wherein the cell is a T cell or a natural killer (NK) cell.

29. Use of the anti-KIR3DL3 monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1 to 14 in a medicament for manufacturing a method for treating a subject with cancer expressing HHLA2 and / or KIR3DL3, said method comprising administering the anti-KIR3DL3 monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof to said subject, wherein, The cancer is selected from kidney cancer, leukemia, or lymphoma.

30. The use according to claim 29, wherein the anti-KIR3DL3 monoclonal antibody, bispecific 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 in the cancer; and / or (c) activates T cells and / or NK cells.

31. The use according to claim 29, wherein the anti-KIR3DL3 monoclonal antibody, bispecific antibody, or antigen-binding fragment thereof is administered in a pharmaceutically acceptable formulation.

32. The use according to claim 29, wherein the method further comprises administering to the subject a therapeutic agent or regimen for treating cancer.

33. The use according to claim 29, wherein the method further comprises administering to the subject an additional therapy selected from the group consisting of: immunotherapy, checkpoint blockade, cancer vaccines, chimeric antigen receptors, chemotherapy, radiation, targeted therapy, and surgery.

34. The use according to claim 33, wherein the chimeric antigen receptor targets CD19.

35. The use according to claim 29, wherein the subject is an animal model of cancer.

36. The use according to claim 35, wherein the animal model is a mouse model, and optionally the mouse model is a humanized mouse model.

37. The use according to claim 29, wherein the subject is a mammal.

38. The use according to claim 37, wherein the mammal is a humanized mouse or a human.

39. The use according to claim 37, wherein the mammal is a human.

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