KIR3DL3 AS AN HHLA2 RECEPTOR, ANTI-HHLA2 ANTIBODIES AND THEIR USES

Abstract

The present invention is based, in part, on the discovery of monoclonal antibodies, and antigen-binding fragments thereof, that bind specifically to HHLA2, as well as immunoglobulins, polypeptides, nucleic acids thereof, and methods for using such antibodies for diagnostic, prognostic, and therapeutic purposes.

Description

[0001] KIR3DL3 AS AN HHLA2 RECEPTOR, ANTI-HHLA2 ANTIBODIES, AND USES

[0002] THEREOF

[0003] Statement of Rights

[0004] This invention was made with government support under grant number P01

[0005] AI056299 awarded by The National Institutes of Health. The government has certain rights in the invention.

[0006] Background of the Invention

[0007] Immune checkpoints, such as CTLA-4, PD-l, VISTA, B7-H2, B7-H3, PD-L1, B7- H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-l, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, butyrophilins, and A2aR, and many more, negatively regulate immune response progression based on complex and combinatorial interactions between numerous inputs. Inhibitors of immune checkpoints can modulate immune responses in some subjects, but immune checkpoint expression and interactions with natural binding partners vary between subjects and within tissues of a subject.

[0008] Accordingly, a great need exists in the art to identify new immune checkpoints for use in interventions. HHLA2 is a newly identified B7 family member that modulates T-cell functions. HHLA2 was identified as a specific ligand for TMIGD2 and the

[0009] HHLA2 / TMIGD2 interaction selectively costimulates human T-cell growth and cytokine production via an AKT-dependent signaling cascade (Zhu et al. (2013) Nat. Comm. 4:2043; Janakiram et al. (2015) Clin. Cancer Res. 21 :2359-2366). A second uncharacterized receptor for HHLA2 on activated T cells that exerts a coinhibitory function was suggested by several studies (Zhao et al. (2013) Proc. Natl. Acad. Sci. USA 110:9879-9884; Xiao and Freeman et al. (2015) Clin. Cancer Res. 21 :2201-2203; Wang et al. (2014) J Immunol. 192: 126.11). HHLA2 is expressed on a variety of human cancers, and its co-inhibitory function makes it a candidate for cancer immunotherapy.

[0010] Summary of the Invention

[0011] The present invention is based, at least in part, on the discovery that HHLA2, a B7 gene family member, is broadly expressed in a variety of tumors and antigen presenting cells and has been implicated as both an activating and inhibitory ligand for T cells.

[0012] TMIGD2 expressed in naive T cells is an activating receptor for HHLA2 and transduces co- stimulatory signals following T cell antigen receptor (TCR) engagement. TMIGD2 is downregulated following repeated TCR stimulation. It is possible that a putative inhibitory receptor for HHLA2 is upregulated on activated T cells to modulate T cell activation. The present invention is based, at least in part, on the discovery that HHLA2 binds KIR3DL3, a receptor on T and NK cells, and a consequence of the HHLA2:KIR3DL3 interaction is inhibition of T cell activation. Based on the observations that HHLA2 is highly expressed in tumors and can serve as a checkpoint ligand, a panel of anti-HHLA2 human monoclonal antibodies (mAbs) were generated as candidate immune checkpoint inhibitor agents.

[0013] Blocking and non-blocking anti-HHLA2 mAbs were identified by evaluating soluble human HHLA2-mIgG2a binding to TMIGD2 transfected 300.19 mouse pre-B leukemic cells or to KIR3DL3 transfected 300.19 mouse pre-B leukemic cells. Anti-HHLA2 mAbs that block HHLA2 binding to both TMIGD2 and KIR3DL3 or more selectively block KIR3DL3 but not TMIGD2 were shown to be checkpoint inhibitor antibodies in T cell assays.

[0014] In one aspect, a monoclonal antibody, or antigen-binding fragment thereof, wherein the monoclonal antibody comprises a) a heavy chain sequence with at least about 95% identity to a heavy chain sequence selected from the group consisting of the sequences listed in Table 2; and / or b) a light chain sequence with at least about 95% identity to a light chain sequence selected from the group consisting of the sequences listed in Table 2, is provided.

[0015] In another aspect, a monoclonal antibody, or antigen-binding fragment thereof, wherein the monoclonal antibody comprises a) a heavy chain CDR sequence with at least about 95% identity to a heavy chain CDR sequence selected from the group consisting of the sequences listed in Table 2; and / or b) a light chain CDR sequence with at least about 95% identity to a light chain CDR sequence selected from the group consisting of the sequences listed in Table 2, is provided.

[0016] In still another aspect, a monoclonal antibody, or antigen-binding fragment thereof, wherein the monoclonal antibody comprises a) a heavy chain sequence selected from the group consisting of the sequences listed in Table 2; and / or b) a light chain sequence selected from the group consisting of the sequences listed in Table 2, is provided.

[0017] In yet another aspect, a monoclonal antibody, or antigen-binding fragment thereof, wherein the monoclonal antibody comprises a) a heavy chain CDR sequence selected from the group consisting of the sequences listed in Table 2; and / or b) a light chain CDR sequence selected from the group consisting the sequences listed in Table 2, is provided.

[0018] Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in one embodiment, the monoclonal antibody, or antigen-binding fragment thereof, is chimeric, humanized, composite, murine, or human. In another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab’)2), Fab’, dsFv, scFv, sc(Fv)2, and diabodies fragments. In still another embodiment, the monoclonal antibody, or antigen binding fragment thereof, is obtainable from hybridoma _ deposited under deposit accession number _ . In yet another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, inhibits a) the binding of HHLA2 to TMIGD2, b) the binding of HHLA2 to KIR3DL3, or c) the binding of HHLA2 to TMIGD2 and the binding of HHLA2 to KIR3DL3. HHLA2 mAbs that block HHLA2 binding to KIR3DL3 in T cell activation assays were shown to be checkpoint blockers. In another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, specifically binds HHLA2.

[0019] In another aspect, an immunoglobulin heavy and / or light chain selected from the group consisting of immunoglobulin heavy and light chain sequences listed in Table 2, is provided.

[0020] In still another aspect, an isolated nucleic acid molecule that hybridizes, under stringent conditions, with the complement of a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2, or a sequence with at least about 95% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2, is provided.

[0021] In yet another aspect, a vector comprising the isolated nucleic acid described herein, is provided.

[0022] In another aspect, a host cell which comprises the isolated nucleic acid described herein, comprises the vector decribed herein, expresses the antibody, or antigen-binding fragment thereof, described herein, or is accessible under deposit accession number _ , is provided.

[0023] In still another aspect, a device or kit comprising at least one monoclonal antibody, or antigen-binding fragment thereof, described herein, the device or kit optionally comprising a label to detect the at least one monoclonal antibody, or antigen-binding fragment thereof, or a complex comprising the monoclonal antibody, or antigen-binding fragment thereo, is provided.

[0024] In yet another aspect, a method of producing at least one monoclonal antibody, or antigen-binding fragment thereof, described herein, which method comprises the steps of:

[0025] (i) culturing a transformed host cell which has been transformed by a nucleic acid comprising a sequence encoding at least one monoclonal antibody according to any one of claims 1-9 under conditions suitable to allow expression of said monoclonal antibody, or antigen-binding fragment thereof; and (ii) recovering the expressed monoclonal antibody, or antigen-binding fragment thereof, is provided.

[0026] In another aspect, a method of detecting the presence or level of an HHLA2 polypeptide comprising obtaining a sample and detecting said polypeptide in the sample by use of at least one monoclonal antibody, or antigen-binding fragment thereof, described herein.

[0027] As described above, certain embodiments are applicable to any method described herein. For example, in one embodiment, the at least one monoclonal antibody, or antigen binding fragment thereof, forms a complex with an HHLA2 polypeptide and the complex is detected in the form of an enzyme linked immunosorbent assay (ELISA), radioimmune assay (RIA), immunochemically, Western blot, or using an intracellular flow assay.

[0028] In another aspect, a method for monitoring the progression of a disorder associated with aberrant HHLA2 expression in a subject, the method comprising a) detecting in a subject sample at a first point in time the level of HHLA2 using at least one monoclonal antibody, or antigen-binding fragment thereof, described herein; b) repeating step a) at a subsequent point in time; and c) comparing the level of HHLA2 detected in steps a) and b) to monitor the progression of the disorder in the subject, is provided.

[0029] As described above, certain embodiments are applicable to any method described herein. For example, in one embodiment, between the first point in time and the subsequent point in time, the subject has undergone treatment to ameliorate the disorder.

[0030] In another aspect, a method for predicting the clinical outcome of a subject afflicted with a disorder associated with aberrant HHLA2 expression, the method comprising a) determining the level of HHLA2 in a subject sample using at least one monoclonal antibody, or antigen-binding fragment thereof, described herein; b) determining the level of HHLA2 in a sample from a control subject having a good clinical outcome using the at least one monoclonal antibody, or antigen-binding fragment thereof; and c) comparing the level of HHLA2 in the subject sample and in the sample from the control subject; wherein a significantly higher level of HHLA2 in the subject sample as compared to the level in the sample from the control subject is an indication that the subject has a poor clinical outcome, is provided.

[0031] In still another aspect, a method of assessing the efficacy of a therapy for a disorder associated with aberrant HHLA2 expression in a subject, the method comprising a) determining the level of HHLA2 using at least one monoclonal antibody, or antigen binding fragment thereof, described herein, in a first sample obtained from the subject prior to providing at least a portion of the therapy to the subject, and b) determining the level of HHLA2 in a second sample obtained from the subject following provision of the portion of the therapy, wherein a significantly lower level of HHLA2 in the second sample, relative to the first sample, is an indication that the therapy is efficacious for inhibiting the disorder in the subject, is provided.

[0032] In yet another aspect, a method of assessing the efficacy of a test compound for inhibiting a disorder associated with aberrant HHLA2 expression in a subject, the method comprising a) determining the level of HHLA2 using at least one monoclonal antibody, or antigen-binding fragment thereof, described herein, in a first sample obtained from the subject and exposed to the test compound; and b) determining the level of HHLA2 in a second sample obtained from the subject, wherein the second sample is not exposed to the test compound, and a significantly lower level of HHLA2, relative to the second sample, is an indication that the test compound is efficacious for inhibiting the disorder in the subject, is provided.

[0033] As described above, certain embodiments are applicable to any method described herein. For example, in one embodiment, the first and second samples are portions of a single sample obtained from the subject or portions of pooled samples obtained from the subject. In another embodiment, the disorder is a cancer. In yet another embodiment, the cancer is selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, Acute myeloid leukemia, head and neck carcinoma, liver cancer, ovarian cancer, prostate cancer, uterine cancer, gliomas, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B cell lymphoma, and a cancer infiltrated with immune cells expressing a receptor to HHLA2. In another embodiment, the sample comprises cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject. In still another embodiment, the significantly higher level of HHLA2 comprises an at least twenty percent increase between the level of HHLA2 in the subject sample relative to the normal level of HHLA2 in the sample from the control subject. In another embodiment, the significantly lower level of HHLA2 comprises an at least twenty percent decrease of the level of HHLA2. In yet another embodiment, the subject is a human.

[0034] In yet another aspect, a method of treating a subject afflicted with cancer comprising administering to the subject at least one monoclonal antibody, or antigen-binding fragment thereof, described herein, is provided.

[0035] As described above, certain embodiments are applicable to any method described herein. For example, in one embodiment, the at least one monoclonal antibody, or antigen binding fragment thereof, is conjugated to a cytotoxic agent. In another embodiment, the cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, a biologic agent, a toxin, and a radioactive isotope. In yet another embodiment, the at least one monoclonal antibody, or antigen-binding fragment thereof, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of a tumor of the cancer. In another embodiment, the at least one monoclonal antibody, or antigen-binding fragment thereof, is administered in a pharmaceutically acceptable formulation. In still another embodiment, the method described herein, further comprising administering to the subject a therapeutic agent or regimen for treating cancer. In yet another embodiment, the method described herein, further comprising administering to the subject an additional therapy selected from the group consisting of immunotherapy, checkpoint blockade, cancer vaccines, chimeric antigen receptors, chemotherapy, radiation, target therapy, and surgery. In another embodiment, cancer cells and / or tumor immune infiltrating cells in the subject express HHLA2. In yet another embodiment, the cancer is selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, Acute myeloid leukemia, head and neck carcinoma, liver cancer, ovarian cancer, prostate cancer, uterine cancer, gliomas, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B cell lymphoma, and a cancer infiltrated with immune cells expressing a receptor to HHLA2. In another embodiment, the cancer is selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), head and neck carcinoma, liver cancer, ovarian cancer, prostate cancer, and uterine cancer. In still 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 another embodiment, the subject is a mammal. In yet another embodiment, the mammal is a humanized mouse or a human. In still another embodiment, the mammal is a human.

[0036] In another aspect, a method of modulating an immune response by inhibiting the interaction between HHLA2 and its binding inhibitor receptor, KIRDL3, is provided.

[0037] In still another aspect, a method of modulating an immune response by selectively inhibiting the interaction between HHLA2 and its binding inhibitor receptor, KIR3DL3, without blocking or significantly inhibiting the interaction between HHLA2 and its binding stimulatory receptor, TMIGD2, is provided.

[0038] As described above, certain embodiments are applicable to any method described herein. For example, in one embodiment, the interaction between HHLA2 and KIRDL3 is blocked for use in checkpoint blockade cancer immunotherapy. In another embodiment, the interaction between HHLA2 and KIRDL3 is inhibited or blocked using an anti-HHLA2 antibody. In still another embodiment, the anti-HHLA2 antibody is a checkpoint inhibitor of T cell activation for cancer immunotherapy.

[0039] For any figure showing a bar histogram, curve, or other data associated with a legend, the bars, curve, or other data presented from left to right for each indication correspond directly and in order to the boxes from top to bottom, or from left to right, of the legend.

[0040] Brief Description of Figures

[0041] Figure 1A shows binding affinity data for Anti-HHLA2 mAbs on HHLA2 transfected 300.19 mouse pre-B cell leukemic cell line by flow cytomtery

[0042] Figure IB shows Anti-HHLA2 mAh blockade of TMIGD2-human IgG binding to HHLA2 transfected 300.19 mouse pre-B cell leukemic cell line by flow cytomtery

[0043] Figure 2 shows Western blot data of protein from nine human tumor cell lines probed with anti-HHLA2 mAh 8D2 (IHC mAh).

[0044] Figure 3 shows HHLA2 mRNA expression compared to other checkpoint inhibitors in normal kidney versus clear cell renal carcinoma (ccRCC).

[0045] Figure 4 depicts HHLA2 expression in various cancers from the TCGA database.

[0046] Figure 5 A shows HHLA2 immunohistochemistry (IHC) results on negative control cells (300.19), HHLA-2 transfected 300.19 cells (positive control), OCl-Lyl cells (negative tumor) and HDLM2 (positive Hodgkon’s lymphoma cell line) HHLA2 control cells. Figure 5B shows HHLA2 expression in normal kidney.

[0047] Figure 5C shows a representative image of HHLA2 expression in a ccRCC from a microarray (TMA).

[0048] Figure 5D shows a representative image of lack of HHLA2 expression in a different ccRCC from a tumor microarray (TMA).

[0049] Figure 6A shows screening results of a representative set of - 300 plasma membrane clones in duplicate, as well as a confirmatory screen showing HHLA2 binding to KIR3DL3. A total 5682 cell surface receptor membrane clones were screened

[0050] Figure 6B shows selective binding of HHLA2 to KIR3DL3

[0051] Figure 6C shows gene ID and NCBI accession information for relevant biomarkers.

[0052] Figure 7 shows a schematic of a Jurkat NFAT receptor gene assay.

[0053] Figure 8 shows a CAR-T cell model.

[0054] Figure 9 shows that inhibitory B7 family members that can be expressed by tumors.

[0055] Figure 10 shows a model for HHLA2 interaction with two receptors (stimulatory and inhibitory HHLA2 receptors) to regulate T-cell functions. Concomitant with T-cell receptor (TCR) signaling, TMIGD2 on naive T cells interacts with HHLA2 on APCs and co-stimulates T-cell proliferation and cytokine production via a pathway involving ART phosphorylation. With repetitive T-cell activation, expression of stimulatory receptor TMIGD2 is gradually lost, allowing expression of a the inhibitory receptor KIR3DL3 to become dominant. HHLA2 on APCs or tumor cells can interact with this second receptor and exert a co-inhibitory function. This figure is adapted from Xiao and Freeman el al. (2015) Clin. Cancer Res. 21 :2201-2203.

[0056] Figure 11 shows HHLA2 + ve (i.e., lung cancer tissues that expressed HHLA2 based on immunohistochemistry (IHC) staining with an HHLA2 mAh) patient stratification in non-small cell lung cancer. The percentage of HHLA2 expression in PD-L1 positive and negative non-small cell lung cancers was calculated based on the HHLA2 and PD-L1 immunostaining study (Cheng et al. (2018) Clin. Cancer Res. 24:1954-1964).

[0057] Figures 12A and 12B show expression of TMIGD2 (Figure 12A) or KIR3DL3 (Figure 12B) on transfected 293T Cells. TMIGD2 or KIR3DL3 cDNA in pEF-Puro expression vector was transiently transfected in 293T cells and stained 48-72 hours later with (1) TMIGD2 mAh (R&D systems catalog #M B83162; clone #953743) followed by goat-anti-mouse IgG F(ab)2-PE (R&D Systems Catlaog F0102B or (2) KIR3DL3-PE conjugated mAb (R&D Systems catalog# FAB8919R, done #1 136B), respectively, and detected by flow cytometry.

[0058] Figures 12C and 12D show HHLA2-Fc binding to TMIGD2 (Figure 12C) or KIR3DL3 (Figure 12D) transfected 293T cells. HHLA2-mIgG2a binding to 293T cells transiently transfected with TMIGD2 or KIR3DL3 was detected using a PE-labeled Fab2 goat anti-mouse IgG2a antibody (absorbed for cross-reactivity with human Ig, Southern Biotech Catalog # 1082-09 ) by flow cytometry.

[0059] Figures 13A and 13B show HHLA2 mAb binding to human and cynomolgus monkey HHLA2. Different concentrations of HHLA2 mAbs were incubated with either human or cynomolgus monkey HHLA2 -transfected 300.19 pre-B cells for 30 minutes at 4°C. HHLA2 mAb binding to transfected 300.19 cells was detected with a PE-labeled goat anti-mouse IgG (H+L) by flow cytometry.

[0060] Figure 14 shows a schematic of TMIGD2 / HHLA2 T cell co-stimulation assay. TMIGD2 Jurkat T cell NFAT-luciferase reporter gene cells were stimulated with CHO cells transfected with anti-CD3 scFV or anti-CD3 scFV + HHLA2.

[0061] Figure 15 shows HHLA2 expression in CHO-anti-CD3 scFV Cells. HHLA2 expression on CHO cells (clone #28) transfected with anti-CD3 scFV + HHLA2 was detected with PE-conjugated 6F10 HHLA2 mAb by flow cytometry.

[0062] Figure 16 shows TMIGD2 expression in Jurkat NF AT reporter cells. TMIGD2 expression in TMIGD2-transfected Jurkat NFAT reporter cells (clone #62) is shown.

[0063] Figure 17 shows ITHLA2 mAb blockade of TMIGD2-mediated T cell co stimulation. FfHLA2-TCR-CHO cells were seeded at 2 x 104cells / well density in CHOK1 growth medium in a white opaque bottom 96-well plate. Cells attached to the plate after overnight incubation at 37°C with 5% CO2. The next day, medium was carefully removed from each well, anti HITLA2 antibody in 50 mΐ Jurkat cell medium was added, and E1HLA2- TCR-CHO cells were incubated for one hour before the addition of MIGD2 NFAT Jurkat reporter cell line at 4-5 x 104cells / well in 50 mΐ Jurkat cell medium. The plate well was mixed and incubated for approximately 3-6 hours. To develop the luciferase signal, 100 mΐ of the ONE-Step™ Luciferase Assay System (BPS Bioscience, Cat. #60690) was added to each well, according to the manufacturer’s recommended protocol. Luminescence was read using a luminometer. Figure 18 shows a schematic of KIR3DL3 / HHLA2 checkpoint T cell assay.

[0064] KIR3DL3 Jurkat T cell IL-2 promoter luciferase reporter gene cells were stimulated with CHO cells transfected with anti-CD3 scFV or anti-CD3 scFV + HHLA2.

[0065] Figure 19 shows KIR3DL3 expression in Jurkat-IL-2 reporter clones. KIR3DL3 expression was detected in KIR3DL3 -transfected Jurkat IL-2 reporter cell clones 1-6, 1-7 and 2-12.

[0066] Figures 20A-20C show HHLA2 mAh checkpoint blockade of KIR3DL3 -mediated T cell inhibition. HHLA2-TCR-CHO cells were seeded at 2 x 104cells / well density in CHOK1 growth medium in a white opaque bottom 96-well plate. Cells attached to the plate after overnight incubation at 37°C with 5% CO2. The next day, medium was carefully removed from each well, anti HHLA2 antibody in 50 mΐ Jurkat cell medium was added, and HHLA2-TCR-CHO cells were incubated for one hour before the addition of

[0067] KIR3DL3_IL2_Jurkat reporter cell line at 4-5 x 104cells / well in 50 mΐ Jurkat cell medium, plus 2 pg / mL anti CD28 antibody (BPS Bioscience #100186) (final concentration at 1 pg / mL in 100 pL assay mixture per well. The plate well was mixed and incubated for approximately 5 hours. To develop the luciferase signal, 100 mΐ of the ONE-Step™

[0068] Luciferase Assay System (BPS Bioscience, Cat. #60690) was added to each well, according to recommended protocol. Luminescence was read using a luminometer.

[0069] Figures 21A-21C show titration of HHLA2 mAh in Jurkat KIR3DL3 inhibition assay. Different concentrations of HHLA2 mAbs 2C4 and 6F10 were evaluated in Jurkat IL-2 reporter luciferase assay using Jurkat IL-2 luciferase clones 1-6, 1-7 and 2-12.

[0070] Figure 22 shows KIR3DL3 -selective HHLA2 mAh 2C4 does not block TMIGD2- mediated co-stimulation. HHLA2 mAh 2C4 at a concentration 30 pg / ml was evaluated in Jurkat parental NFAT reporter cells. HHLA2-TCR-CHO cells were seeded at 2 x 104cells / well density in CHOK1 growth medium in a white opaque bottom 96-well plate.

[0071] Cells attached to the plate after overnight incubation at 37°C with 5% CO2. The next day, medium was carefully removed from each well, anti HHLA2 antibody in 50 mΐ Jurkat cell medium was added, and HHLA2-TCR-CHO cells were incubated for one hour before the addition of Jurkat parental NFAT Jurkat reporter cell line at 4-5 x 104cells / well in 50 mΐ Jurkat cell medium. The plate well was mixed and incubated for approximately 3-6 hours. To develop the luciferase signal, 100 mΐ of the ONE-Step™ Luciferase Assay System (BPS Bioscience, Cat. #60690) was added to each well, according to recommended protocol. Luminescence was read using a luminometer. Figures 23A-23C show how HHLA2 mAbs in humanized SRG-15 mouse tumor model (has both T and NK cells) can be evaluated. HHLA2 mAbs are administered to humanized SRG-15 mouse bearing HHLA2-expressing tumor cells, and tumor growth inhibition is evaluated. The figures are adapted from Hemdler-Brandstetter D el al. (2017)

[0072] 114:E9626-E9634.

[0073] Figures 24 A and 24B show how HHLA2 mAbs in cynomolgus monkey T cell model can be evaluated. Cynomolgus monkeys are administered HHLA2 mAbs and are immunized with KLH, and T cell-dependent antibody and cell-mediated responses are evaluated. NK cytotoxicity is evaluated ex vivo.

[0074] Detailed Description of the Invention

[0075] The present invention is based, at least in part, on the discovery that HHLA2, a B7 gene family member, is broadly expressed in a variety of tumors and antigen presenting cells and has been implicated as both an activating and inhibitory ligand for T cells.

[0076] TMIGD2 expressed in naive T cells is an activating receptor for HHLA2 and transduces co- stimulatory signals following T cell antigen receptor (TCR) engagement. TMIGD2 is downregulated following repeated TCR stimulation. Based on the observations that HHLA2 is highly expressed in tumors and can serve as a checkpoint ligand, a panel of anti- HHLA2 human monoclonal antibodies (mAbs) were generated as candidate immune checkpoint inhibitor therapeutics. Given that the same ligand binding domains of B7 family members B7-1 and B7-2 are known to bind both activating and inhibitory receptors ( e.g ., CD28 and CTLA-4), anti-HHLA2 monoclonal antibodies that block TMIGD2 binding are believed to serve as good candidates for blocking binding to its putative inhibitory receptor. Evaluating soluble hHHLA2-mIgG2a binding to TMIGD2 transfected 300.19 mouse pre-B leukemic cells, both blocking and non-blocking anti-HHLA2 mAbs were identified. Anti-HHLA2 mAbs listed in Table 2 (e.g., 6F10, 4D1, 4E5 and 2G2) that blocked TMIGD2 binding and also bound HHLA2 transfected 300.19 cells with relative EC50 binding affinities of 0.25, 0.44 and 0.21 ug / ml (nanomolar range), respectively. Non- blocking antibodies listed in Table 2 (e.g, 1C8 and 6D10) bound HHLA2 with relative binding affinities of 0.63 and 22.49 ug / ml, respectively. The variable region heavy and light chain gene sequences for these candidate therapeutic anti-HHLA2 antibodies are described herein. Anti-HHLA2 mAbs 1C8 and 6D10 were identified as good formalin-fixed paraffin- embedded immunohistochemistry or Western blotting reagents. HHLA2 in primary tumors from the TCGA database shows high expression in lung, renal, pancreatic and colorectal cancers and in AML and intermediate levels in head and neck, liver, ovarian, prostate and uterine cancers. HHLA2 mRNA expression in cancer is higher than corresponding normal tissues.

[0077] Screening a cell surface expressed human plasma protein library of > 4500 full- length clones covering more than 3,500 different plasma membrane proteins with soluble human HHLA2-mIgG2a identified KIR3DL3 (killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 3) as a new receptor for HHLA2. The cytoplasmic tail of KIR3DL3 contains an ITIM motif comprised of the sequence“VTYAQL” indicating an inhibitory receptor for HHLA2 that can serve as a checkpoint receptor target for cancer immunotherapy. Selectivity of HHLA2 binding to KIR3DL3 was demonstrated because no binding against other KIRs receptors was observed using a panel of 14 KIR receptors (i.e., KIR3DL3, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5, KIR3DL1, KIR3DS1, and KIR3DS1).

[0078] Since HHLA2 is expressed at high levels in multiple types of tumors, the anti- HHLA2 mAb check-point inhibitor therapeutics may increase the pool of patients that respond to check-point inhibitor treatment. Furthermore, patients who develop resistance to PD-l therapy may express HHLA2 as an alternative immune evasion strategy and HHLA2 blockade may offer an avenue to overcome resistance to PD-l immunotherapy.

[0079] Accordingly, the present invention provides monoclonal antibodies, and antigen binding fragments thereof, that specifically bind to HHLA2, as well as immunoglobulins, polypeptides, nucleic acids thereof, and methods of using such antibodies for diagnostic, prognostic, and therapeutic purposes. Definitions

[0080] The articles“a” and“an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

[0081] The term“altered amount” of a marker refers to increased or decreased copy number of a marker and / or increased or decreased nucleic acid level of a particular marker gene or genes in a sample, as compared to that of the marker in a control sample. The term “altered amount” of a marker also includes an increased or decreased protein level of a marker in a sample, as compared to the protein level of the marker in a normal, control sample.

[0082] The term“altered activity” of a marker refers to an activity of a marker which is increased or decreased in a disease state, e.g., in a biological sample, as compared to the activity of the marker in a normal, control sample. Altered activity of a marker may be the result of, for example, altered expression of the marker, altered protein level of the marker, altered structure of the marker, or, e.g, an altered interaction with other proteins involved in the same or different pathway as the marker, or altered interaction with transcriptional activators or inhibitors.

[0083] The term“altered structure” of a marker refers to the presence of mutations or allelic variants within the marker gene or maker protein, e.g, mutations which affect expression or activity of the marker, as compared to the normal or wild-type gene or protein. For example, mutations include, but are not limited to substitutions, deletions, or addition mutations. Mutations may be present in the coding or non-coding region of the marker.

[0084] The term“activating receptor” includes immune cell receptors that bind antigen, complexed antigen (e.g, in the context of MHC polypeptides), or bind to antibodies. Such activating receptors include T cell receptors (TCR), B cell receptors (BCR), cytokine receptors, LPS receptors, complement receptors, and Fc receptors.

[0085] T cell receptors are present on T cells and are associated with CD3 polypeptides. T cell receptors are stimulated by antigen in the context of MHC polypeptides (as well as by polyclonal T cell activating reagents). T cell activation via the TCR results in numerous changes, e.g, protein phosphorylation, membrane lipid changes, ion fluxes, cyclic nucleotide alterations, RNA transcription changes, protein synthesis changes, and cell volume changes.

[0086] The term“chimeric antigen receptor” or“CAR” refers to engineered T cell receptors (TCR) having a desired antigen specificity. T lymphocytes recognize specific antigens through interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, naive T cells are dependent on professional antigen-presenting cells (APCs) that provide additional co-stimulatory signals. TCR activation in the absence of co stimulation can result in unresponsiveness and clonal anergy. To bypass immunization, different approaches for the derivation of cytotoxic effector cells with grafted recognition specificity have been developed. CARs have been constructed that consist of binding domains derived from natural ligands or antibodies specific for cell-surface components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric antigen receptors link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. Since the first reports on chimeric antigen receptors, this concept has steadily been refined and the molecular design of chimeric receptors has been optimized and routinely use any number of well-known binding domains, such as scFV, Fav, and another protein binding fragments described herein.

[0087] Generally, CARs are one type of“cell therapy” ( e.g ., T cell therapy) contemplated for use according to the present invention. Although numerous representative embodiments of agents and methods for modulating immune cell activity by modulating the HHLA2 pathway, such as modulating the interaction between HHLA2 and a HHLA2 natural binding partner, such as TMIGD2 and / or KIR3DL3, immune cell-based therapies and methods are also encompassed. For example, T cells engineered to have a knockout, knockdown, or increased expression of TMIGD2 and / or KIR3DL3 are contemplated.

[0088] Similarly, immune cells or other cells engineered to have a knockout, knockdown, or increased expression of a HHLA2 ligand, such as TMIGD2 and / or KIR3DL3, are also contemplated.

[0089] B cell receptors are present on B cells. B cell antigen receptors are a complex between membrane Ig (mlg) and other transmembrane polypeptides (e.g., Iga and IgP).

[0090] The signal transduction function of mlg is triggered by crosslinking of receptor

[0091] polypeptides by oligomeric or multimeric antigens. B cells can also be activated by anti immunoglobulin antibodies. Upon BCR activation, numerous changes occur in B cells, including tyrosine phosphorylation.

[0092] Fc receptors are found on many cells which participate in immune responses. Fc receptors (FcRs) are cell surface receptors for the Fc portion of immunoglobulin

[0093] polypeptides (Igs). Among the human FcRs that have been identified so far are those which recognize IgG (designated Fey R), IgE (Fes Rl), IgA (Fca), and polymerized IgM / A (Fcpa R). FcRs are found in the following cell types: Fes R I (mast cells), Fes R.II (many leukocytes), Fca R (neutrophils), and Fcpa R (glandular epithelium, hepatocytes) (Hogg,

[0094] N. (1988) Immunol. Today 9: 185-86). The widely studied FcyRs are central in cellular immune defenses, and are responsible for stimulating the release of mediators of inflammation and hydrolytic enzymes involved in the pathogenesis of autoimmune disease (Unkeless, J. C. et al. (1988) Annu. Rev. Immunol. 6:251-81). The FcyRs provide a crucial link between effector cells and the lymphocytes that secrete Ig, since the

[0095] macrophage / monocyte, polymorphonuclear leukocyte, and natural killer (NK) cell FcyRs confer an element of specific recognition mediated by IgG. Human leukocytes have at least three different receptors for IgG: h Fey RI (found on monocytes / macrophages), hFcy RII (on monocytes, neutrophils, eosinophils, platelets, possibly B cells, and the K562 cell line), and Fey III (on NK cells, neutrophils, eosinophils, and macrophages).

[0096] With respect to T cells, transmission of a costimulatory signal to a T cell involves a signaling pathway that is not inhibited by cyclosporin A. In addition, a costimulatory signal can induce cytokine secretion ( e.g ., IL-2 and / or IL-10) in a T cell and / or can prevent the induction of unresponsiveness to antigen, the induction of anergy, or the induction of cell death (deletion) in the T cell.

[0097] The term“activity,” when used with respect to a polypeptide, e.g., HHLA2 and / or a HHLA2 natural binding partner, such as TMIGD2 and / or KIR3DL3, includes activities that are inherent in the structure of the protein. For example, with regard to a HHLA2 ligand, the term“activity” includes the ability to modulate immune cell inhibition by modulating an inhibitory signal in an immune cell (e.g, by engaging a natural receptor on an immune cell). Those of skill in the art will recognize that when an activating form of the HHLA2 ligannd polypeptide binds to an inhibitory receptor, an inhibitory signal is generated in the immune cell.

[0098] The term“inhibitory signal” refers to a signal transmitted via an inhibitory receptor (e.g, HHLA2, KLRB1, CTLA4, PD-l, and the like) for a polypeptide on a immune cell. Such a signal antagonizes a signal via an activating receptor (e.g, via a TCR, CD3, BCR, TMIGD2, or Fc polypeptide) and can result in, e.g., inhibition of second messenger generation; an inhibition of proliferation; an inhibition of effector function in the immune cell, e.g, reduced phagocytosis, reduced antibody production, reduced cellular cytotoxicity, the failure of the immune cell to produce mediators, (such as cytokines (e.g, IL-2) and / or mediators of allergic responses); or the development of anergy.

[0099] The amount of a biomarker in a subject is“significantly” higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal or control level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount. Alternatively, the amount of the biomarker in the subject can be considered“significantly” higher or lower than the normal and / or control amount if the amount is at least about two, and preferably at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

[0100] 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 in between, such as 5%-l00%, higher or lower, respectively, than the normal and / or control amount of the biomarker. Such significant modulation values can be applied to any metric described herein, such as altered level of expression, altered activity, changes in cancer cell hyperproliferative growth, changes in cancer cell death, changes in biomarker inhibition, changes in test agent binding, and the like.

[0101] The“amount” of a marker, e.g., expression or copy number of a marker or MCR, or protein level of a marker, in a subject is“significantly” higher or lower than the normal amount of a marker, if the amount of the marker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least twice, and more preferably three, four, five, ten or more times that amount. Alternately, the amount of the marker in the subject can be considered “significantly” higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the marker.

[0102] The term“altered level of expression” of a marker refers to an expression level or copy number of a marker in a test sample e.g, a sample derived from a subject suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the marker or

[0103] chromosomal region in a control sample (e.g, sample from a healthy subject not having the associated disease) and preferably, the average expression level or copy number of the marker or chromosomal region in several control samples. The altered level of expression is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the marker in a control sample (e.g, sample from a healthy subject not having the associated disease) and preferably, the average expression level or copy number of the marker in several control samples.

[0104] The term“immunotherapy” refers to a form of targeted therapy that may comprise, for example, the use of cancer vaccines and / or sensitized antigen presenting cells. For example, an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in immunomodulatory therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site. The immunotherapy can involve passive immunity for short-term protection of a host, achieved by the

[0105] administration of pre-formed antibody directed against a cancer antigen or disease antigen ( e.g ., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense

[0106] polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and / or pathology of a tumor or cancer. As described above, immunotherapy against immune checkpoint targets, such as HHLA2, TMIGD2, KIR3DL3, and the like are useful.

[0107] Unless otherwise specified here within, the terms“antibody” and“antibodies” broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody. An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term“inactivating antibodies” refers to antibodies that do not induce the complement system.

[0108] The term“antibody” as used herein also includes an“antigen-binding portion” of an antibody (or simply“antibody portion”). The term“antigen-binding portion”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen ( e.g ., HHLA2 polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two

[0109] Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al ., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g ., Bird et al. (1988) Science 242:423-426; and Huston el al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term“antigen binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab , Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g. , Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121- 1123).

[0110] Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov, S.M., et al. (1994) Mol. Immunol. 31 : 1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

[0111] Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies may also be fully human. In one embodiment, antibodies of the present invention bind specifically or substantially specifically to HHLA2 polypeptides or fragments thereof. The terms “monoclonal antibodies” and“monoclonal antibody composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and“polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

[0112] The term“body fluid” refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). The terms“cancer” or“tumor” or“hyperproliferative disorder” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g. , fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,

[0113] leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,

[0114] craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g. , acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,

[0115] promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal 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, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma ( e.g ., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0116] The terms“CDR”, and its plural“CDRs”, refer to a complementarity determining region (CDR) of which three make up the binding character of a light chain variable region (CDR-L1, CDR-L2 and CDR-L3) and three make up the binding character of a heavy chain variable region (CDR-H1, CDR-H2 and CDR-H3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to by Rabat, Chothia, contact or any other boundary definitions. Despite differing boundaries, each of these systems has some degree of overlap in what constitutes the so called“hypervariable regions” within the variable sequences. CDR definitions according to these systems may therefore differ in length and boundary areas with respect to the adjacent framework region. See for example Rabat, Chothia, and / or MacCallum et al. , (Rabat et al ., in“Sequences of Proteins of Immunological Interest,” 5thEdition, U.S. Department of Health and Human Services, 1992; Chothia et al. (1987) J. Mol. Biol. 196, 901; and MacCallum et al. , J. Mol. Biol. (1996) 262, 732, each of which is incorporated by reference in its entirety).

[0117] As used herein, the term“classifying” includes“to associate” or“to categorize” a sample with a disease state. In certain instances,“classifying” is based on statistical evidence, empirical evidence, or both. In certain embodiments, the methods and systems of classifying use of a so-called training set of samples having known disease states. Once established, the training data set serves as a basis, model, or template against which the features of an unknown sample are compared, in order to classify the unknown disease state of the sample. In certain instances, classifying the sample is akin to diagnosing the disease state of the sample. In certain other instances, classifying the sample is akin to

[0118] differentiating the disease state of the sample from another disease state.

[0119] As used herein, the term“coding region” refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term “noncoding region” refers to regions of a nucleotide sequence that are not translated into amino acids ( e.g ., 5' and 3' untranslated regions).

[0120] “Complement [to]” or“complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In one embodiment, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In another embodiment, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0121] As used herein, the term“composite antibody” refers to an antibody which has variable regions comprising germline or non-germline immunoglobulin sequences from two or more unrelated variable regions. Additionally, the term“composite, human antibody” refers to an antibody which has constant regions derived from human germline or non- germline immunoglobulin sequences and variable regions comprising human germline or non-germline sequences from two or more unrelated human variable regions. A composite, human antibody is useful as an effective component in a therapeutic agent according to the present invention since the antigenicity of the composite, human antibody in the human body is lowered. The term“control” refers to any reference standard suitable to provide a comparison to the expression products in the test sample. In one embodiment, the control comprises obtaining a“control sample” from which expression product levels are detected and compared to the expression product levels from the test sample. Such a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells / tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells / tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells / tissues obtained from a depository. In another preferred embodiment, the control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy). It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention. In one embodiment, the control may comprise normal or non-cancerous cell / tissue sample. In another preferred embodiment, the control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome. In the former case, the specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level. In another preferred embodiment, the control may comprise normal cells, cells from patients treated with combination chemotherapy, and cells from patients having benign cancer. In another embodiment, the control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population. Such a population may comprise normal subjects, cancer patients who have not undergone any treatment (z.e., treatment naive), cancer patients undergoing standard of care therapy, or patients having benign cancer. In another preferred embodiment, the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of 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;

[0122] determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control. In particularly preferred embodiments, the control comprises a control sample which is of the same lineage and / or type as the test sample. In another embodiment, the control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer. In one embodiment a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome. In another preferred embodiment, a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome. As demonstrated by the data below, the methods of the invention are not limited to use of a specific cut-point in comparing the level of expression product in the test sample to the control.

[0123] As used herein, the term“Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. Suitable native- sequence Fc regions for use in the antibodies of the present invention include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0124] As used herein,“Fc receptor” or“FcR” describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRII A (an “activating receptor”) and Fey RUB (an“inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine- based inhibition motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev.

[0125] Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.

[0126] Immunol. 9: 457-92 (1991); Capel et al. , Immunomethods 4: 25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term“FcR” herein.

[0127] A molecule is“fixed” or“affixed” to a substrate if it is covalently or non-covalently associated with the substrate such the substrate can be rinsed with a fluid ( e.g . standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.

[0128] As used herein,“framework” or“FR” residues are those variable-domain residues other than the HVR residues as herein defined.

[0129] “Function-conservative variants” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A“function- conservative variant” also includes a polypeptide which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.

[0130] As used herein, the term“heterologous antibody” is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence

[0131] corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal. “Homologous” as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5'- ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, 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 of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

[0132] As used herein, the term“host cell” is intended to refer to a cell into which a nucleic acid of the present invention, such as a recombinant expression vector of the present invention, 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 the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0133] The term“humanized antibody”, as used herein, is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences ( e.g ., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo ), for example in the CDRs. The term“humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. A humanized mouse, as used herein, is a mouse carrying functioning human genes ( e.g ., HHLA2, TMIGD2, 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. The nude mouse and severe combined immunodeficiency (SCID) mouse may be used for this purpose. The NCG mouse, NOG mouse and the NSG mouse may be used to engraft human cells and tissues more efficiently than other models. Such humanized mouse models may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

[0134] As used herein, the term“hypervariable region,”“HVR,” or“HV,” refers to the regions of an antibody -variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (Ll, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu el al. (2000) Immunity 13, 37-45; Johnson and Wu in Methods in Molecular Biology 248, 1-25 (Lo, ed., Human Press, Totowa, NJ, 2003)). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain (see, e.g, Hamers- Casterman et al. (1993) Nature 363:446-448 (1993) and Sheriff et al. (1996) Nature Struct. Biol. 3, 733-736).

[0135] 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; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0136] As used herein, the term“immune disorder” includes immune diseases, conditions, and predispositions to, including, but not limited to, cancer, chronic inflammatory disease and disorders (including, e.g, Crohn's disease, inflammatory bowel disease, reactive arthritis, and Lyme disease), insulin-dependent diabetes, organ specific autoimmunity (including, e.g, multiple sclerosis, Hashimoto's thyroiditis, autoimmune uveitis, and Grave's disease), contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions (including, e.g, asthma and allergy including, but not limited to, allergic rhinitis and gastrointestinal allergies such as food allergies), eosinophilia, conjunctivitis, glomerular nephritis, systemic lupus erythematosus, scleroderma, certain pathogen susceptibilities such as helminthic (including, e.g, leishmaniasis) and certain viral infections (including, e.g. , HIV and bacterial infections such as tuberculosis and

[0137] lepromatous leprosy) and malaria.

[0138] As used herein, the term“immune response” includes T cell mediated and / or B cell mediated immune responses. Exemplary immune responses include T cell responses, e.g. , cytokine production, and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly effected by T cell activation, e.g. , antibody production (humoral responses) and activation of cytokine responsive cells, e.g. , macrophages.

[0139] The term“immunotherapeutic agent” can include any molecule, peptide, antibody or other agent which can stimulate a host immune system to generate an immune response to a tumor or cancer in the subject. Various immunotherapeutic agents are useful in the compositions and methods described herein.

[0140] 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 down-modulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well-known in the art and include, without limitation, CTLA-4, PD-l, VISTA, B7-H2, B7-H3, PD-L1, B7- H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-l, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR (see, for example, WO 2012 / 177624). The term further encompasses biologically active protein fragment, as well as nucleic acids encoding full-length immune checkpoint proteins and biologically active protein fragments thereof. In some embodiment, the term further encompasses any fragment according to homology descriptions provided herein.

[0141] Immune checkpoints and their sequences are well-known in the art and

[0142] representative embodiments are described below. For example, the term“PD-l” refers to a member of the immunoglobulin gene superfamily that functions as a coinhibitory receptor having PD-L1 and PD-L2 as known ligands. PD-l was previously identified using a subtraction cloning based approach to select for genes upregulated during TCR-induced activated T cell death. PD-l is a member of the CD28 / CTLA-4 family of molecules based on its ability to bind to PD-L1. Like CTLA-4, PD-l is rapidly induced on the surface of T- cells in response to anti-CD3 (Agata et al. 25 (1996) Int. Immunol. 8:765). In contrast to CTLA-4, however, PD-l is also induced on the surface of B-cells (in response to anti-IgM). PD-l is also expressed on a subset of thymocytes and myeloid cells (Agata et al. (1996) supra; Nishimura et al. (1996) Int. Immunol. 8:773).

[0143] As used herein, the term“inhibiting” and grammatical equivalents thereof refer decrease, limiting, and / or blocking a particular action, function, or interaction. In one embodiment, the term refers to reducing the level of a given output or parameter to a quantity ( e.g ., background staining, HHLA2 signaling, HHLA2 immunoinhibitory function, and the like) which 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

[0144] corresponding control. A reduced level of a given output or parameter need not, although it may, mean an absolute absence of the output or parameter. The invention does not require, and is not limited to, methods that wholly eliminate the output or parameter. The given output or parameter can be determined using methods well-known in the art, including, without limitation, immunohistochemical, molecular biological, cell biological, clinical, and biochemical assays, as discussed herein and in the examples. The opposite terms “promoting,”“increasing,” and grammatical equivalents thereof refer to the increase in the level of a given output or parameter that is the reverse of that described for inhibition or decrease.

[0145] As used herein, the term“interaction”, when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules with one another (e.g, binding of HHLA2 to TMIGD2 or binding of HHLA2 to KIR3DL3). Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules. The activity may be a direct activity of one or both of the molecules, (e.g, signal transduction). Alternatively, one or both molecules in the interaction may be prevented from binding their ligand, and thus be held inactive with respect to ligand binding activity (e.g, binding its ligand and triggering or inhibiting an immune response). To inhibit such an interaction results in the disruption of the activity of one or more molecules involved in the interaction. To enhance such an interaction is to prolong or increase the likelihood of said physical contact, and prolong or increase the likelihood of said activity.

[0146] The term“neoadjuvant therapy” refers to a treatment given before the primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy. As used herein, the term an“isolated antibody” is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities ( e.g ., an isolated antibody that specifically binds to HHLA2 and is substantially free of antibodies that do not bind to HHLA2). An isolated antibody that specifically binds to a HHLA2 may, however, have cross-reactivity to other B7 family proteins, respectively, from different species. For example, in some embodiments, the antibody maintains specific binding affinity for at least two species, such as human and other animals, such as non-rodent animals, or other mammal or non-mammal species. However, in some embodiments, the antibody maintains higher or indeed specific affinity and selectivity for human HHLA2. In addition, an isolated antibody is typically substantially free of other cellular material and / or chemicals. In one embodiment of the present invention, a combination of“isolated” monoclonal antibodies having different specificities to human HHLA2 are combined in a well-defined composition.

[0147] As used herein, an“isolated protein” refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An“isolated” or“purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of a target polypeptide (e.g., immunoglobulin) or fragment thereof, in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations of target protein or fragment thereof, having less than about 30% (by dry weight) of non-target protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-target protein, still more preferably less than about 10% of non-target protein, and most preferably less than about 5% non-target protein. When antibody, polypeptide, peptide or fusion protein or fragment thereof, e.g, a biologically active fragment thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. As used herein, the term“isotype” refers to the antibody class ( e.g ., IgM or IgGl) that is encoded by heavy chain constant region genes.

[0148] As used herein, the term“KD” is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. The binding affinity of antibodies of the disclosed invention may be measured or determined by standard antibody-antigen assays, for example, competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA.

[0149] As used herein, a“kit” is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. a probe, for specifically detecting or modulating the expression of a marker of the present invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.

[0150] As used herein, the term“monoclonal antibody”, refers to an antibody which displays a single binding specificity and affinity for a particular epitope. Accordingly, the term“human monoclonal antibody” refers to an antibody which displays a single binding specificity and which has variable and constant regions derived from human germline or non-germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g. , a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0151] A“marker” is a gene whose altered level of expression in a tissue or cell from its expression level in normal or healthy tissue or cell is associated with a disease state, such as cancer. A“marker nucleic acid” is a nucleic acid (e.g, mRNA, cDNA) encoded by or corresponding to a marker of the present invention. Such marker nucleic acids include DNA (e.g, cDNA) comprising the entire or a partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing or the complement of such a sequence. The marker nucleic acids also include RNA comprising the entire or a partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing or the complement of such a sequence, wherein all thymidine residues are replaced with uridine residues. A“marker protein” is a protein encoded by or corresponding to a marker of the present invention. A marker protein comprises the entire or a partial sequence of any of the sequences set forth in the Sequence Listing. In some embodiments, the overall HHLA2 is used as a marker. In other embodiments, a fragment of HHLA2 is used as a marker. The terms“protein” and “polypeptide” are used interchangeably. As used herein, the term“modulate” includes up-regulation and down-regulation, e.g ., enhancing or inhibiting a response.

[0152] The“normal” level of expression of a marker is the level of expression of the marker in cells of a subject, e.g. , a human patient, not afflicted with a disease or disorder related to aberrant marker levels. An“over-expression” or“significantly higher level of expression” of a marker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least twice, and more preferably three, four, five or ten times the expression level of the marker in a control sample (e.g, sample from a healthy subjects not having the marker associated disease) and preferably, the average expression level of the marker in several control samples. A“significantly lower level of expression” of a marker refers to an expression level in a test sample that is at least twice, and more preferably three, four, five or ten times lower than the expression level of the marker in a control sample (e.g, sample from a healthy subject not having the marker associated disease) and preferably, the average expression level of the marker in several control samples.

[0153] Such“significance” levels can also be applied to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, and the like.

[0154] The term“pre-determined” biomarker amount and / or activity measurement(s) may be a biomarker amount and / or activity measurement(s) used to, by way of example only, evaluate a subject that may be selected for a particular treatment, evaluate a response to a treatment such as one or more modulators of the HHLA2 pathway, such as a modulator of HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3, either alone or in combination with one or more immunotherapies, and / or evaluate the disease state. A pre-determined biomarker amount and / or activity measurement(s) may be determined in populations of patients with or without cancer. The pre-determined biomarker amount and / or activity measurement(s) can be a single number, equally applicable to every patient, or the pre-determined biomarker amount and / or activity measurement(s) can vary according to specific subpopulations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and / or activity measurement(s) of the individual. Furthermore, the pre-determined biomarker amount and / or activity can be determined for each subject individually. In one embodiment, the amounts determined and / or compared in a method described herein are based on absolute measurements. In another embodiment, the amounts determined and / or compared in a method described herein are based on relative measurements, such as ratios ( e.g ., cell ratios or serum biomarker normalized to the expression of housekeeping or otherwise generally constant biomarker). The pre-determined biomarker amount and / or activity measurement(s) can be any suitable standard. For example, the pre-determined biomarker amount and / or activity measurement(s) can be obtained from the same or a different human for whom a patient selection is being assessed. In one embodiment, the pre-determined biomarker amount and / or activity measurement(s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time. In addition, the control can be obtained from an assessment of another human or multiple humans, e.g., selected groups of humans, if the subject is a human. In such a manner, the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g, other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same condition(s) and / or of the same ethnic group.

[0155] The term“predictive” includes the use of a biomarker nucleic acid and / or protein status, e.g, over- or under- activity, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy, for determining the likelihood of response of a cancer to immunomodulatory therapy, such as HHLA2 pathway modulator therapy (e.g, modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3, either alone or in combination with an immunotherapy, such as an immune checkpoint inhibition therapy). Such predictive use of the biomarker may be confirmed by, e.g, (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 at J. Biotechnol., 86:289-301, or qPCR), overexpression or underexpression of a biomarker nucleic acid (e.g, by ISH, Northern Blot, or qPCR), increased or decreased biomarker protein (e.g, by IHC) and / or biomarker target, or increased or decreased activity, e.g, in more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed human cancers types or cancer samples; (2) its absolute or relatively modulated presence or absence in a biological sample, e.g, a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g. a human, afflicted with cancer; (3) its absolute or relatively modulated presence or absence in clinical subset of patients with cancer (e.g, those responding to a particular immunomodulatory therapy (e.g., HHLA2 pathway modulator therapy (e.g, modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 AND / OR KIR3DL3, either alone or in combination with an immunotherapy) or those developing resistance thereto).

[0156] The terms“prevent,”“preventing,”“prevention,”“prophylactic treatment,” and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.

[0157] The term“prognosis” includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis of cancer in an individual. For example, the prognosis can be surgery, development of a clinical subtype of cancer (e.g., solid tumors, such as lung cancer, melanoma, and renal cell carcinoma), development of one or more clinical factors, development of intestinal cancer, or recovery from the disease.

[0158] The term“response to therapy” (e.g, HHLA2 pathway modulator therapy (e.g, modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3, either alone or in combination with an immunotherapy, such as an immune checkpoint inhibition therapy) relates to any response to therapy (e.g, HHLA2 pathway modulator therapy (e.g, modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3, either alone or in combination with an immunotherapy, such as an immune checkpoint inhibition therapy), and, for cancer, preferably to a change in cancer cell numbers, tumor mass, and / or volume after initiation of neoadjuvant or adjuvant chemotherapy. Hyperproliferative disorder response may be assessed, for example for efficacy or in a neoadjuvant or adjuvant situation, where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation. Responses may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like

[0159] “pathological complete response” (pCR),“clinical complete remission” (cCR),“clinical partial remission” (cPR),“clinical stable disease” (cSD),“clinical progressive disease” (cPD) or other qualitative criteria. Assessment of hyperproliferative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, e.g, after a few hours, days, weeks or preferably after a few months. A typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and / or the tumor bed. This is typically three months after initiation of neoadjuvant therapy. In some embodiments, clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy. The shorthand for this formula is CBR=CR+PR+SD over 6 months. In some embodiments, the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating the response to cancer therapies are related to“survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related);“recurrence- free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g, time of diagnosis or start of treatment) and end point (e.g, death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence. For example, in order to determine appropriate threshold values, a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any immunomodulatory therapy. The outcome measurement may be pathologic response to therapy given in the neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free survival can be monitored over a period of time for subjects following immunomodulatory therapy for whom biomarker measurement values are known. In certain embodiments, the doses administered are standard doses known in the art for cancer therapeutic agents. The period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. The term“resistance” refers to an acquired or natural resistance of a cancer sample or a mammal to an immunomodulatory therapy (i.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a 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, to 2- fold, 3-fold, 4-fold, 5-fold, lO-fold, l5-fold, 20-fold or more. The reduction in response can be measured by comparing with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal who is known to have no resistance to the therapeutic treatment. A typical acquired resistance to chemotherapy is called“multidrug resistance.” The multidrug resistance can be mediated by P-glycoprotein or can be mediated by other mechanisms, or it can occur when a mammal is infected with a multi-drug-resistant microorganism or a combination of microorganisms. The determination of resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician, for example, can be measured by cell proliferative assays and cell death assays as described herein as“sensitizing.” In some embodiments, the term“reverses resistance” means that the use of a second agent in combination with a primary cancer therapy ( e.g ., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance (e.g., p<0.05) when compared to tumor volume of untreated tumor in the circumstance where the primary cancer therapy (e.g, chemotherapeutic or radiation therapy) alone is unable to produce a statistically significant decrease in tumor volume compared to tumor volume of untreated tumor. This generally applies to tumor volume measurements made at a time when the untreated tumor is growing log rhythmically.

[0160] The terms“response” or“responsiveness” refers to response to therapy. For example, an anti-cancer response includes reduction of tumor size or inhibiting tumor growth. The terms can also refer to an improved prognosis, for example, as reflected by an increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death without evidence of recurrence, or an increased overall survival, which is the period from treatment to death from any cause. To respond or to have a response means there is a beneficial endpoint attained when exposed to a stimulus. Alternatively, a negative or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus. It will be appreciated that evaluating the likelihood that a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response (i.e., will exhibit a lack of response or be non-responsive).

[0161] The term“tolerance” or“unresponsiveness” includes refractivity of cells, such as immune cells, to stimulation, e.g ., stimulation via an activating receptor or a cytokine. Unresponsiveness can occur, e.g. , because of exposure to immunosuppressants or exposure to high doses of antigen. Several independent methods can induce tolerance. One mechanism is referred to as“anergy,” which is defined as a state where cells persist in vivo as unresponsive cells rather than differentiating into cells having effector functions. Such refractivity is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy in T cells is characterized by lack of cytokine production, e.g. , IL-2. T cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, reexposure of the cells to the same antigen (even if reexposure occurs in the presence of a costimulatory polypeptide) results in failure to produce cytokines and, thus, failure to proliferate. Anergic T cells can, however, proliferate if cultured with cytokines (e.g, IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using an indicator cell line. Alternatively, a reporter gene construct can be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5’ IL-2 gene enhancer or by a multimer of the AP1 sequence that can be found within the enhancer (Kang el al. (1992) Science 257: 1134). Another mechanism is referred to as“exhaustion.” T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections and cancer. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells.

[0162] As used herein, the term“nucleic acid molecule” is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double- stranded, but preferably is double-stranded DNA. As used herein, the term“isolated nucleic acid molecule” in reference to nucleic acids encoding antibodies or antibody portions (e.g, VH, VL, CDR3) that bind to HHLA2 (e.g., mAbs 2G2, 4D1, 8A12, 8D2, 1C8, 2C4, 6D10, 4E5, and 6F10 and polyclonal antibodies), is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than HHLA2, which other sequences may naturally flank the nucleic acid in human genomic DNA.

[0163] A nucleic acid is“operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.

[0164] An“over-expression” or“significantly higher level of expression” of a marker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least twice, and more preferably

[0165] 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,

[0166] 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the expression activity or level of the marker in a control sample ( e.g ., sample from a healthy subject not having the marker associated disease) and preferably, the average expression level of the marker in several control samples. A“significantly lower level of expression” of a marker refers to an expression level in a test sample that is at least twice, and more preferably 2.1, 2.2, 2.3,

[0167] 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12

[0168] 13, 14, 15, 16, 17, 18, 19, 20 times or more lower than the expression level of the marker in a control sample (e.g., sample from a healthy subject not having the marker associated disease) and preferably, the average expression level of the marker in several control samples.

[0169] Such antibodies, described herein, can be used in any one of well-known immunoassay forms, including, without limitation, a radioimmunoassay, a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay. General techniques to be used in performing the various immunoassays noted above and other variations of the techniques, such as in situ proximity ligation assay (PLA), fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA), ELISA, etc. alone or in combination or alternatively with NMR, MALDI-TOF, LC-MS / MS, are known to those of ordinary skill in the art.

[0170] Such reagents can also be used to monitor protein levels in a cell or tissue, e.g., white blood cells or lymphocytes, as part of a clinical testing procedure, e.g. , in order to monitor an optimal dosage of an inhibitory agent. Detection can be facilitated by coupling (e.g, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, b-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include

[0171] streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 1 25 I, 1 31 I, 35 S or 3 H.

[0172] Such reagents can also be used with any number of biological samples. Biological samples can be collected from a variety of sources from a patient including a body fluid sample, cell sample, or a tissue sample comprising nucleic acids and / or proteins. In a preferred embodiment, the subject and / or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin embedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow. In one embodiment, the sample is serum, plasma, or urine. In another embodiment, the sample is serum.

[0173] The samples can be collected from individuals repeatedly over a longitudinal period of time (e.g, once or more on the order of days, weeks, months, annually, biannually, etc.). Obtaining numerous samples from an individual over a period of time can be used to verify results from earlier detections and / or to identify an alteration in biological pattern as a result of, for example, disease progression, drug treatment, etc. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three month intervals according to the present invention. In addition, the biomarker amount and / or activity measurements of the subject obtained over time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject’s own values, as an internal, or personal, control for long-term monitoring.

[0174] Samples can contain live cells / tissue, fresh frozen cells, fresh tissue, biopsies, fixed cells / tissue, cells / tissue embedded in a medium, such as paraffin, histological slides, or any combination thereof.

[0175] Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and / or analysis of biomarker measurement(s). Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides ( e.g ., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids.

[0176] The sample preparation can also isolate molecules that are bound in non-covalent complexes to other protein (e.g., carrier proteins). This process may isolate those molecules bound to a specific carrier protein (e.g, albumin), or use a more general process, such as the release of bound molecules from all carrier proteins via protein denaturation, for example using an acid, followed by removal of the carrier proteins.

[0177] Removal of undesired proteins (e.g. , high abundance, uninformative, or

[0178] undetectable proteins) from a sample can be achieved using high affinity reagents, high molecular weight filters, ultracentrifugation and / or electrodialysis. High affinity reagents include antibodies or other reagents (e.g, aptamers) that selectively bind to high abundance proteins. Sample preparation could also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques. Molecular weight filters include membranes that separate molecules on the basis of size and molecular weight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and microfiltration.

[0179] The terms“polypeptide fragment” or“fragment”, when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions may occur at the amino-terminus, internally, or at the carboxyl-terminus of the reference polypeptide, or alternatively both. Fragments typically are 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. They can be, for example, at least and / or including 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,

[0180] 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,

[0181] 1340 or more long so long as they are less than the length of the full-length polypeptide. Alternatively, they can be no longer than and / or excluding such a range so long as they are less than the length of the full-length polypeptide.

[0182] The term“probe” refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a marker. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0183] As used herein, the term“rearranged” refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH and VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer / nonamer homology element.

[0184] As used herein, the term“recombinant host cell” (or simply“host cell”), is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term“host cell” as used herein.

[0185] As used herein, the term“recombinant human antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal ( e.g ., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the antibody, e.g ., from a transfectoma, (c) antibodies isolated from a recombinant,

[0186] combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0187] The term“costimulate,” as used with reference to activated immune cells, includes the ability of a costimulatory polypeptide to provide a second, non-activating receptor mediated signal (a“costimulatory signal“) that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion, e.g., in a T cell that has received a T cell-receptor-mediated signal. Immune cells that have received a cell-receptor mediated signal, e.g, via an activating receptor are referred to herein as“activated immune cells.”

[0188] The term“costimulatory receptor” includes receptors which transmit a

[0189] costimulatory signal to a immune cell, e.g, CD28. As used herein, the term“inhibitory receptors” includes receptors which transmit a negative signal to an immune cell (e.g, CTLA4, KIR3DL3 or PD-l). An inhibitory signal as transduced by an inhibitory receptor can occur even if a costimulatory receptor (such as CD28) is not present on the immune cell and, thus, is not simply a function of competition between inhibitory receptors and costimulatory receptors for binding of costimulatory polypeptides (Fallarino et al. (1998) . / . Exp. Med. 188:205). Transmission of an inhibitory signal to an immune cell can result in unresponsiveness or anergy or programmed cell death in the immune cell. Preferably transmission of an inhibitory signal operates through a mechanism that does not involve apoptosis. As used herein the term“apoptosis” includes programmed cell death which can be characterized using techniques which are known in the art. Apoptotic cell death can be characterized, e.g, by cell shrinkage, membrane blebbing and chromatin condensation culminating in cell fragmentation. Cells undergoing apoptosis also display a characteristic pattern of internucleosomal DNA cleavage. Depending upon the form of the polypeptide that binds to a receptor, a signal can either be transmitted ( e.g ., by a multivalent form of HHLA2 and / or KIR3DL3 polypeptide) or a signal can be inhibited (e.g., by a soluble, monovalent form of a HHLA2 and / or KIR3DL3), for instance by competing with activating forms of HHLA2 and / or KIR3DL3 for binding to one or more natural binding partners. However, there are instances in which a soluble polypeptide can be stimulatory. The effects of a modulatory agent can be easily demonstrated using routine screening assays as described herein.

[0190] The terms“high,”“low,”“intermediate,” and“negative” in connection with cellular biomarker expression refers to the amount of the biomarker expressed relative to the cellular expression of the biomarker by one or more reference cells. Biomarker expression can be determined according to any method described herein including, without limitation, an analysis of the cellular level, activity, structure, and the like, of one or more biomarker genomic nucleic acids, ribonucleic acids, and / or polypeptides. In one embodiment, the terms refer to a defined percentage of a population of cells expressing the biomarker at the highest, intermediate, or lowest levels, 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 in between, inclusive, of a population of cells that either highly express or weakly express the biomarker. The term“low” excludes cells that do not detectably express the biomarker, since such cells are“negative” for biomarker expression. The term “intermediate” includes cells that express the biomarker, but at levels lower than the population expressing it at the“high” level. In another embodiment, the terms can also refer to, or in the alternative refer to, cell populations of biomarker expression identified by qualitative or statistical plot regions. For example, cell populations sorted using flow cytometry can be discriminated on the basis of biomarker expression level by identifying distinct plots based on detectable moiety analysis, such as based on mean fluorescence intensities and the like, according to well-known methods in the art. Such plot regions can be refined according to number, shape, overlap, and the like based on well-known methods in the art for the biomarker of interest. In still another embodiment, the terms can also be determined according to the presence or absence of expression for additional biomarkers. As described above, the term“response” is generally related to for example, determining the effects on progression, efficacy, or outcome of a clinical intervention. In some embodiments, responses relate directly to a change in tumor mass and / or volume after initiation of clinical intervention ( e.g ., administration of an anti-HHLA2 monoclonal antibody, such as 2G2, 4D1, 8A12, 8D2, 1C8, 2C4, 6D10, 4E5, or 6F10 and polyclonal antibodies). For example, hyperproliferative disorder responses may be assessed according to the size of a tumor after systemic intervention compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like“pathological complete response” (pCR),“clinical complete remission” (cCR),“clinical partial remission” (cPR), “clinical stable disease” (cSD),“clinical progressive disease” (cPD) or other qualitative criteria. Assessment may be done early after the onset of the clinical intervention, e.g., after a few hours, days, weeks or preferably after a few months. A typical endpoint for response assessment is upon termination of the clinical intervention or upon surgical removal of residual tumor cells and / or the tumor bed.

[0191] As used herein, the term“specific binding” refers to antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity (KD) of approximately less than 107M, such as approximately less than 108M, 109M or 1010M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using human HHLA2 as the analyte and the antibody as the ligand, and binds to the predetermined antigen with an affinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-,

[0192] 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 lO.O-fold or greater than its affinity for binding to a non-specific antigen (e.g, BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases“an antibody recognizing an antigen” and“an antibody specific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

[0193] As used herein,“subject” refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a disease or disorder related to aberrant marker levels. The term“subject” is interchangeable with“patient”. The term“non-human animal” includes all vertebrates, e.g, mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. The language“substantially free of chemical precursors or other chemicals” includes preparations of antibody, polypeptide, peptide or fusion protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includes preparations of antibody, polypeptide, peptide or fusion protein having less than about 30% (by dry weight) of chemical precursors or non antibody, polypeptide, peptide or fusion protein chemicals, more preferably less than about 20% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, still more preferably less than about 10% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, and most preferably less than about 5% chemical precursors or non- antibody, polypeptide, peptide or fusion protein chemicals.

[0194] As used herein, the term“survival” includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related);“recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point ( e.g . time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0195] A“transcribed polynucleotide” or“nucleotide transcript” is a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a marker of the present invention and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.

[0196] As used herein, the term“T cell” includes CD4+ T cells and CD8+ T cells. The term T cell also includes both T helper 1 type T cells and T helper 2 type T cells. The term “antigen presenting cell” includes professional antigen presenting cells (e.g, B

[0197] lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen presenting cells (e.g, keratinocytes, endothelial cells, astrocytes, fibroblasts,

[0198] oligodendrocytes) . Conventional T cells, also known as Tconv or Teffs, have effector functions ( e.g ., cytokine secretion, cytotoxic activity, anti-self-recognization, and the like) to increase immune responses by virtue of their expression of one or more T cell receptors. Tcons or Teffs are generally defined as any T cell population that is not a Treg and include, for example, naive T cells, activated T cells, memory T cells, resting Tcons, or Tcons that have differentiated toward, for example, the Thl or Th2 lineages. In some embodiments, Teffs are a subset of non-Treg T cells. In some embodiments, Teffs are CD4+ Teffs or CD8+ Teffs, such as CD4+ helper T lymphocytes (e.g., ThO, Thl, Tfh, or Thl 7) and CD8+ cytotoxic T lymphocytes. As described further herein, cytotoxic T cells are CD8+ T lymphocytes. “Naive Tcons” are CD4+T cells that have differentiated in bone marrow, and successfully underwent a positive and negative processes of central selection in a thymus, but have not yet been activated by exposure to an antigen. Naive Tcons are commonly characterized by surface expression of L-selectin (CD62L), absence of activation markers such as CD25, CD44 or CD69, and absence of memory markers such as CD45RO. Naive Tcons are therefore believed to be quiescent and non-dividing, requiring interleukin-7 (IL- 7) and interleukin- 15 (IL- 15) for homeostatic survival (see, at least WO 2010 / 101870).

[0199] The presence and activity of such cells are undesired in the context of suppressing immune responses. Unlike Tregs, Tcons are not anergic and can proliferate in response to antigen- based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol. Sci. 356:625-637). In tumors, exhausted cells can present hallmarks of anergy.

[0200] As used herein, the term“unrearranged” or“germline configuration” in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.

[0201] As used herein, the term“vector” refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector is a“plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g, non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as“recombinant expression vectors” or simply“expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification,“plasmid” and“vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors ( e.g ., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0202] For nucleic acids, the term“substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more of the nucleotides, and more preferably at least about 97%, 98%, 99% or more of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

[0203] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity= # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be

[0204] accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0205] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available on the world wide web at the GCG company website), using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11 17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444 453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package

[0206] (available on the world wide web at the GCG company website), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0207] The nucleic acid and protein sequences of the present invention can further be used as a“query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 10. BLAST nucleotide searches can be performed with the NBLAST program, score=l00,

[0208] wordlength=l2 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al ., (1997) Nucleic Acids Res. 25(l7):3389 3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs ( e.g ., XBLAST and NBLAST) can be used (available on the world wide web at the NCBI website).

[0209] The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is“isolated” or“rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well-known in the art (see, F. Ausubel, et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)).

[0210] The term“determining a suitable treatment regimen for the subject” is taken to mean the determination of a treatment regimen ( i.e ., a single therapy or a combination of different therapies that are used for the prevention and / or treatment of the cancer in the subject) for a subject that is started, modified and / or ended based or essentially based or at least partially based on the results of the analysis according to the present invention. One example is determining whether to provide targeted therapy against a cancer to provide immunomodulatory therapy (e.g, HHLA2 pathway modulator therapy (e.g, modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3)). Another example is starting an adjuvant therapy after surgery whose purpose is to decrease the risk of recurrence, another would be to modify the dosage of a particular chemotherapy. The determination can, in addition to the results of the analysis according to the present invention, be based on personal characteristics of the subject to be treated. In most cases, the actual determination of the suitable treatment regimen for the subject will be performed by the attending physician or doctor.

[0211] II. Monoclonal Antibodies. Immunoglobulins and Polypeptides

[0212] The present invention relates, in part, to isolated monoclonal antibodies or fragments thereof that are directed against HHLA2 (such as monoclonal antibodies and polyclonal antibodies listed herein). Such molecules, in part, are characterized in that they exhibit the ability to recognize HHLA2 protein in diagnostic assays, such as

[0213] immunohistochemical (IHC), Western blot, intercellular flow, ELISA, and the like. Such molecules, in part, are characterized in that they exhibit the ability to inhibit HHLA2 binding to receptors, such as receptors expressed on T-cells (e.g. TMIGD2, and KIR3DL3)

[0214] The term“HHLA2”, also known as human endogenous retrovirus-H long terminal repeat-associating protein 2, HERV-H LTR-associating 2, B7y, B7H7, B7-H5, B7-H7, refers to a member of the B7 family. HHLA2 protein has limited expression in normal human tissues but is widely expressed in human cancers. The HHLA2 protein is a membrane protein with three Ig-like domains (IgV-IgC-IgV), whereas other members of the B7 family generally have only two Ig domains (IgV-IgC). HHLA2 protein in normal human tissues is expressed in the epithelium of kidney, gut, gallbladder, and breast as well as placental trophoblast cells. In the immune system, HHLA2 protein is constitutively expressed on human monocytes / macrophages. HHLA2 regulates human T-cell functions including, for example, HHLA2 inhibits T-cell proliferation and cytokine production, and increases T-cell production and cytokine production. HHLA2 is expressed in higher levels in a wide range of human cancers from the colorectal, renal, lung, pancreas, ovary, and prostate. HHLA2 is also expressed in human cancers of thyroid, melanoma, liver, bladder, colon, kidney, breast, and esophagus.

[0215] HHLA2 structures and functions, are well-known in the art as described above (see, for example, Xiao et al. (2015) Clin. Cancer Res. 21 :2201-2203, Janakiram et al. (2015) Clin. Cancer Res. 21 :2359-2366, Mager et al. (1999) Genomics 21 :2359-2366, Flajnik et al. (2012) Immunogenet. 64:571-590, Zhao et al. (2013) Proc. Natl. Acad. Sci. U.S.A.

[0216] 110:9879-9884, and Zhu et al. (2013) Nat. Commun. 4:2043).

[0217] The term“HHLA2” is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human HHLA2 cDNA and human HHLA2 protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Human HHLA2 variants include variant 1

[0218] (NM_007072.3 and NP_009003.l, which represents the longest transcript and encodes the longest isoform a), variant 2 (NM_00l282556.l and NP_00l269485. l, which represents the use of an alternate promoter and differs in the 5' UTR, compared to variant 1), vaiant 3 (NM_001282557.1 and NR_001269486.1, which represents the use of an alternate promoter and differs in the 5' UTR, compared to variant 1), variant 4 (NM_00l282558.l and

[0219] NP 001269487.1, which encodes isoform b, represents the use of an alternate promoter, differs in the 5' UTR and lacks an alternate in-frame exon in the 3' coding region, compared to variant 1, resulting a shorter isoform than isoform a), and variant 5 (NM_00l282559. l and NP 001269488.1, which encodes isoform c, represents the use of an alternate promoter, and has multiple differences compared to variant 2, resulting in a distinct 5' UTR and causing translation initiation at an alternate start codon, compared to variant 1, resulting in a distinct N-terminus and a shorter isoform than isoform a). Nucleic acid and polypeptide sequences of HHLA2 orthologs in organisms other than humans are well-known and include, for example, frog HHLA2 (NM_00l 128644.1 and NP_00l 122116.1).

[0220] Representative sequences of HHLA2 orthologs are presented below in Table 1.

[0221] Anti-HHLA2 antibodies suitable for detecting HHLA2 protein are well-known in the art and include, for example, antibodies Cat #: abl07l 19 and ab2l4327 (abeam), antibodies PA5-24146 and PA5-6313 (ThermoFisher Scientific), antibodies MAB80841, AF8084, FAB80841R, FAB80841T, and MAB8084 (R&D systems), antibody

[0222] AP52042PU-N (Origene), antibodies NBP2-49187, MAB80842, H00011148-B01P, and NBP2-32420 (Novus Biologicals), antibody GTX51981 (GeneTex), antibody HPA055478 (Atlas Antibodies), antibodies LS-C321945, LS-C308228, LS-C246742, LS-C246743, LS- C246744, LS-C236210, and LS-C249186 (LifeSpan Biosiences), etc. Moreover, multiple siRNA, shRNA, CRISPR constructs for reducing HHLA2 expression can be found in the commercial product lists of the above-referenced companies, such as shRNA product #

[0223] TL312462, TF312462, TR312462, TG312462, and TL312462V, siRNA product #

[0224] SR323358 from Origene Technologies, SiRNA product # Ϊ009616, i0096l6a, i0096l6b, i0096l6c, i0096l6d, iV0096l6, iV0096l6a, iV0096l6b, iV0096l6c, iV0096l6d, iAAV0096l600, iAAV0096l60l, iAAV0096l602, iAAV0096l603, iAAV0096l604, iAAV0096l605, iAAV0096l606, iAAV0096l607, iAAV0096l608, and iAAV0096l609, CRISPR product # K0950321, K0950301, K0950302, K0950303, K0950304, K0950305, K0950306, K0950307, K0950308, and K0950311 (abm), siRNA product # sc-78498, shRNA product # sc-78498-V and sc-78498-SH, CRISPR product # sc-4l 1576, sc-4l 1576- HDR, sc-4l 1576-NIC, sand c-4l 1576-NIC-2 (Santa Cruz Biotechnology), etc. It is to be noted that the term can further be used to refer to any combination of features described herein regarding HHLA2 molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe an HHLA2 molecule of the present invention.

[0225] The term“HHLA2 pathway” includes HHLA2 and interactions of HHLA2 with one or more of its natural binding partners, such as TMIGD2 and KIR3DL3.

[0226] The term“TMIGD2” refers to transmembrane and immunoglobulin domain containing 2, CD28H, IGPR1, and IGPR-l, which is a membrane protein having -10% amino acid identity with CD28, CTLA-4, ICOS, and PD-l. TMIGD2 has one extracellular IgV-like domain, a transmembrane region, and a proline-rich cytoplasmic domain with two tyrosine signaling motifs. TMIGD2 protein is constitutively expressed on all naive T cells and the majority of natural killer (NK) cells, but not on T regulatory cells or B cells.

[0227] TMIGD2 expression is slowly lost with repetitive stimulation of T cells. Consistent with this, TMIGD2 is expressed on only about half of memory T cells, and TMIGD2-negative T cells have a terminally-differentiated, senescent phenotype. TMIGD2 has also been shown to be expressed in endothelial and epithelial cells and function to reduce cell migration and promote capillary tube formation during angiogenesis.

[0228] TMIGD2 structures and functions, are well-known in the art as described above (see, for example, Xiao et al. (2015) Clin. Cancer Res. 21 :2201-2203, Janakiram et al. (2015) Clin. Cancer Res. 21 :2359-2366, Zhu et al. (2013) Nat. Commun. 4:2043, and Rahimi (2012) Cell 23: 1646-1656).

[0229] The term“TMIGD2” is intended to include fragments, variants (e.g, allelic variants), and derivatives thereof. Representative human TMIGD2 cDNA and human TMIGD2 protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Human TMIGD2 isoforms include isoform 1 (NM_144615.2 and NP_653216.2), isoform 2 (NM_001169126.1 and NP_00l 162597.1; which uses an alternate in-frame splice site in the 3' coding region, compared to variant 1, resulting a shorter isoform, compared to isoform 1), and isoform 3 (NM_00l308232. l and NP_00l295161.1, which lacks an alternate in-frame exon in the 5' coding region compared to variant 1, resulting a shorter isoform, compared to isoform 1). Nucleic acid and polypeptide sequences of TMIGD2 orthologs in organisms other than humans are well-known and include, for example, chimpanzee TMIGD2

[0230] (XM_009434393.2 and XP_009432668.2, and XM_00l 138228.4 and XP_00l 138228.3), and cattle TMIGD2 (XM_005208980.3 and XP 005209037.1, XM_005208979.3 and XP_005209036.l, and XM_002688933.5 and XP_002688979. l). Representative sequences of TMIGD2 orthologs are presented below in Table 1.

[0231] Anti-TMIGD2 antibodies suitable for detecting TMIGD2 protein are well-known in the art and include, for example, antibodies Cat # MAB8316, MAB83162, FAB8316R, FAB83162R, FAB83162G, FAB83162N, FAB83162S, FAB83162T, FAB83162U, and FAB83162V (R&D systems), antibody TA326695 (Origene), antibodies PA5-52787, and PA5-38055 (ThermoFisher Scientific), antibodies MAB 83161, and NBP1-81164 (Novus Biologicals), etc.. Moreover, multiple siRNA, shRNA, CRISPR constructs for reducing TMIGD2 expression can be found in the commercial product lists of the above-referenced companies, such as shRNA product # TF317829, TG317829, TL317829, TR317829, and TL317829V, siRNA product # SR314913, and CRISPR products # KN204938,

[0232] KN204938LP, KN204938RB, and KN204938BN from Origene Technologies, siRNA products # Ϊ024914, i0249l4a, i0249l4b, i0249l4c, i0249l4d, iV0249l4, iV0249l4a, iV0249l4b, iV0249l4c, iV0249l4d, iAAV0249l400, iAAV0249l40l, iAAV0249l402, iAAV0249l403, iAAV0249l404, iAAV0249l405, iAAV0249l406, iAAV0249l407, iAAV0249l408, and iAAV0249l409, and CRISPR products # K2409321, K2409301, K2409302, K2409303, K2409304, K2409305, K2409306, K2409307, K2409308, and K2409311 (Abm), siRNA product # sc-97757, shRNA products # sc-97757-SH, and sc- 97757-V, and CRISPR products # sc-414261, SC-414261-HDR, sc-414261 -NIC, and sc- 414261 -NIC-2 (Santa Cruz Biotechnology), shRNA products # SH888208, and SH874720 (Vigene Biosciences), etc.. Moreover, multiple CRISPR constructs for increasing TMIGD2 expression can be found in the commercial product lists of the above-referenced companies, such as CRISPR products # K2409378, K2409377, K2409376, K2409375, K2409374, K2409373, K2409372, and K2409371 (Abm), CRISPR products # SC-414261- ACT, sc-414261 -ACT-2, sc-414261 -LAC, and sc-414261 -LAC-2 (Santa Cruz

[0233] Biotechnology), etc.. It is to be noted that the term can further be used to refer to any combination of features described herein regarding TMIGD2 molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe an TMIGD2 molecule of the present invention.

[0234] Interactions between TMIGD2 and HHLA2 as well as their functions, are well- known in the art as described above (see, for example, Xiao et al. (2015) Clin. Cancer Res. 21 :2201-2203 and Janakiram et al. (2015) Clin. Cancer Res. 21 :2359-2366).

[0235] The term“KIR3DL3”, also known as Killer cell immunoglobulin-like receptor 3DL3, CD158Z, KIR3DL7, KIR44, KIRC1, KIR2DS2, killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 3, refers to a member of a

[0236] transmembrane glycoprotein family expressed by natural killer cells and subsets of T cells. The killer cell immunoglobulin-like receptor (KIR) genes are polymorphic and highly homologous and they are found in a cluster on chromosome l9ql3.4 within the 1 Mb leukocyte receptor complex (LRC). The gene content of the KIR gene cluster varies among haplotypes, although several "framework" genes are found in all haplotypes (KIR3DL3, KIR3DP1, KIR3DL4, KIR3DL2). The KIR proteins are classified by the number of extracellular immunoglobulin domains (2D or 3D) and by whether they have a long (L) or short (S) cytoplasmic domain. KIR proteins with the long cytoplasmic domain transduce inhibitory signals upon ligand binding via an immune tyrosine-based inhibitory motif (ITIM), while KIR proteins with the short cytoplasmic domain lack the ITIM motif and instead associate with the TYRO protein tyrosine kinase binding protein to transduce activating signals. The ligands for several KIR proteins are subsets of HLA class I molecules; thus, KIR proteins are thought to play an important role in regulation of the immune response. This gene is one of the "framework" loci that is present on all haplotypes. The KIR3DL3 protein has an N-terminal signal sequence, 3 Ig domains, a transmembrane region lacking a positively charged residue, and a long cytoplasmic tail containing an immunoreceptor tyrosine-based inhibitory motif (ITIM). KIR3DL3 lacks the stalk region found in other KIRs.

[0237] KIR3DL3 structures and functions, are well-known in the art as described above (see, for example, Hsu et al. (2002) Immunol Rev. 190:40-52, Trompeter el al. (2005) J. Immunol. 174:4135-4143, Trundley et al. (2006) Immunogenet. 57:904-916, and Jones et al. (2006) Immunogenet. 58:614-627).

[0238] The term“KIR3DL3” is intended to include fragments, variants (e.g, allelic variants), and derivatives thereof. Representative human KIR3DL3 cDNA and human KIR3DL3 protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). For example, at least one human KIR3DL3 isoform is known: human KIR3DL3 (NM 153443.4) is encodable by the transcript (NP 703144.3). Nucleic acid and polypeptide sequences of KIR3DL3 orthologs in organisms other than humans are well-known and include, for example, chimpanzee KIR3DL3 (XM 003316679.3 and XP 003316727.3), Rhesus monkey KIR3DL3

[0239] (NM_00l 104552.2 and NP_00l098022.l), mouse KIR3DL3 (NM_00l310690.1 and NR_001297619.1, NM_177749.4 and NR_808417.2, NM_l77748.2 and NP_8084l6. l), and rat KIR3DL3 (NM_181479.2 and NP_852l44.l). Representative sequences of KIR3DL3 orthologs are presented below in Table 1.

[0240] Anti-KIR3DL3 antibodies suitable for detecting KIR3DL3 protein are well-known in the art and include, for example, antibodies Cat #: FAB8919R, MAB8919, FAB8919G, FAB8919N, FAB8919S, FAB8919T, FAB8919U, and FAB8919V (R&D systems), antibody AP52374PU-N (Origene), antibody PA5-26178 (ThermoFisher Scientific), antibodies OAAB05761, OAAF08125, OAAN04122, OACA09134, OACA09135, OACD04988, and OASG01190 (Aviva Systems Biology), etc.. Moreover, multiple siRNA, shRNA, CRISPR constructs for reducing KIR3DL3 expression can be found in the commercial product lists of the above-referenced companies, such as shRNA products # TF303684, TR303684, TG303684, TL303684, TL303684V, siRNA products # SR314516, and CRISPR products # KN224383, KN224383BN, KN224383RB, and KN224383LP from Origene Technologies, siRNA products # iOl 1627, iOl l627a, iO 11627b, iOl l627c, iO 11627d, i V011627, i V011627a, i V011627b, i V011627c, iV0H627d, iAAVOl 162700, iAAVOl 162701, iAAVOl 162702, iAAVOl 162703, iAAVOl 162704, iAAVOl 162705, iAAVOl 162706, iAAVOl 162707, iAAVOl 162708, and iAAVOl 162709, and CRISPR products # Kl 151421, K1151401, K1151402, K1151403, K1151404, K1151405,

[0241] K 1151406, K 1151407, K 1151408, and K 1151411 (Abm), siRNA product # sc-60892, shRNA products # sc-60892-SH, and sc-60892-V, and CRISPR products # sc-406227, sc- 406227-KO-2, SC-406227-HDR-2, sc-406227-NIC, and sc-406227-NIC-2 (Santa Cruz Biotechnology), etc.. It is to be noted that the term can further be used to refer to any combination of features described herein regarding KIR3DL3 molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe an KIR3DL3 molecule of the present invention. The term“peripheral blood cell subtypes” refers to cell types normally found in the peripheral blood including, but is not limited to, eosinophils, neutrophils, T cells, monocytes, NK cells, granulocytes, and B cells.

[0242] The term“recombinant human antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal ( e.g ., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0243] The term“sample” used for detecting or determining the presence or level of at least one biomarker is typically whole blood, plasma, serum, saliva, urine, stool (e.g, feces), tears, and any other bodily fluid (e.g, as described above under the definition of“body fluids”), or a tissue sample (e.g, biopsy) such as a small intestine, colon sample, or surgical resection tissue. In certain instances, the method of the present invention further comprises obtaining the sample from the individual prior to detecting or determining the presence or level of at least one marker in the sample.

[0244] An“RNA interfering agent” as used herein, is defined as any agent which interferes with or inhibits expression of a target biomarker gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target biomarker gene of the present invention, or a fragment thereof, short interfering RNA (siRNA), and small molecules which interfere with or inhibit expression of a target biomarker nucleic acid by RNA interference (RNAi). “RNA interference (RNAi)” is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target biomarker nucleic acid results in the sequence specific degradation or specific post- transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene ( see Coburn, G. and Cullen, B. (2002) J. of Virology 76(l8):9225), thereby inhibiting expression of the target biomarker nucleic acid. In one embodiment, the RNA is double stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs. siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid molecules, e.g, synthetic siRNAs, shRNAs, or other RNA interfering agents, to inhibit or silence the expression of target biomarker nucleic acids. As used herein,“inhibition of target biomarker nucleic acid expression” or“inhibition of marker gene expression” includes any decrease in expression or protein activity or level of the target biomarker nucleic acid or protein encoded by the target biomarker nucleic acid. The decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target biomarker nucleic acid or the activity or level of the protein encoded by a target biomarker nucleic acid which has not been targeted by an RNA interfering agent.

[0245] In addition to RNAi, genome editing can be used to modulate the copy number or genetic sequence of a biomarker of interest, such as constitutive or induced knockout or mutation of a biomarker of interest, such as a HHLA2 pathway component like HHLA2, TMIGD2, and / or KIR3DL3. For example, the CRISPR-Cas system can be used for precise editing of genomic nucleic acids (e.g, for creating non-functional or null mutations). In such embodiments, the CRISPR guide RNA and / or the Cas enzyme may be expressed. For example, a vector containing only the guide RNA can be administered to an animal or cells transgenic for the Cas9 enzyme. Similar strategies may be used (e.g, designer zinc finger, transcription activator-like effectors (TALEs) or homing meganucleases). Such systems are well-known in the art (see, for example, U.S. Pat. No. 8,697,359; Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Pat. Publ. 2014 / 0087426 and 2012 / 0178169; Boch et al. (2011) Nat. Biotech. 29: 135-136; Boch et al. (2009) Science 326:1509-1512; Moscou and

[0246] Bogdanove (2009) Science 326: 1501; Weber et al. (2011) PLoS One 6:el9722; Li et al. (2011 ) Nucl. Acids Res. 39:6315-6325; Zhang et al. (2011 ) Nat. Biotech. 29: 149-153;

[0247] Miller et al. (201 1 ) Nat. Biotech. 29: 143-148; Lin et al. (2014 ) Nucl. Acids Res. 42:e47). Such genetic strategies can use constitutive expression systems or inducible expression systems according to well-known methods in the art.

[0248] “Pi wi -interacting RNA (piRNA)” is the largest class of small non-coding RNA molecules. piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, particularly those in spermatogenesis. They are distinct from microRNA (miRNA) in size (26-31 nt rather than 21-24 nt), lack of sequence conservation, and increased complexity. However, like other small RNAs, piRNAs are thought to be involved in gene silencing, specifically the silencing of transposons. The majority of piRNAs are antisense to transposon sequences, suggesting that transposons are the piRNA target. In mammals it appears that the activity of piRNAs in transposon silencing is most important during the development of the embryo, and in both C. elegans and humans, piRNAs are necessary for

[0249] spermatogenesis. piRNA has a role in RNA silencing via the formation of an RNA-induced silencing complex (RISC).

[0250] “Aptamers” are oligonucleotide or peptide molecules that bind to a specific target molecule. “Nucleic acid aptamers” are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.“Peptide aptamers” are artificial proteins selected or engineered to bind specific target molecules. These proteins consist of one or more peptide loops of variable sequence displayed by a protein scaffold. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection. The“Affimer protein”, an evolution of peptide aptamers, is a small, highly stable protein engineered to display peptide loops which provides a high affinity binding surface for a specific target protein. It is a protein of low molecular weight, 12-14 kDa, derived from the cysteine protease inhibitor family of cystatins. Aptamers are useful in

[0251] biotechnological and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.

[0252] “Short interfering RNA” (siRNA), also referred to herein as“small interfering RNA” is defined as an agent which functions to inhibit expression of a target biomarker nucleic acid, e.g., by RNAi. An siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced within a host cell. 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 in length, and more preferably about 19, 20, 21, or 22 nucleotides in length, and may contain a 3’ and / or 5’ overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Preferably the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).

[0253] In another embodiment, an siRNA is a small hairpin (also called stem loop) RNA (shRNA). In one embodiment, these shRNAs are composed of a short (e.g, 19-25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and the analogous sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow. These shRNAs may be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA Apr;9(4):493-50l incorporated by reference herein).

[0254] RNA interfering agents, e.g, siRNA molecules, may be administered to a patient having or at risk for having cancer, to inhibit expression of a biomarker gene which is overexpressed in cancer and thereby treat, prevent, or inhibit cancer in the subject.

[0255] The term“small molecule” is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides,

[0256] peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g, polyketides) (Cane et al. 1998. Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.

[0257] The term“selective modulator” or“selectively modulate” as applied to a

[0258] biologically active agent refers to the agent's ability to modulate the target, such as a cell population, signaling activity, etc. as compared to off-target cell population, signaling activity, etc. via direct or interact interaction with the target. For example, an agent that selectively inhibits the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and KIR3DL3, over another interaction between HHLA2 and another binding partner, and / or such interaction(s) on a cell population of interest may have an activity against the HHLA2 pathway modulator therapy (e.g., modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and KIR3DL3, interaction that 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%,

[0259] 140%, 150%, 160%, 170%, 180%, 190%, 2x (times) or more than the agent's activity against at least one other binding partner (e.g, at least about 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 15c, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, lOOx, l05x, l lOx, l20x, l25x, l50x, 200x, 250x, 300x, 350x, 400x, 450x, 500x, 600x, 700x,

[0260] 800x, 900x, lOOOx, l500x, 2000x, 2500x, 3000x, 3500x, 4000x, 4500x, 5000x, 5500x, 6000x, 6500x, 7000x, 7500x, 8000x, 8500x, 9000x, 9500x, lOOOOx, or greater, or any range in between, inclusive). Such metrics are typically expressed in terms of relative amounts of agent required to reduce the interaction / activity by half.

[0261] More generally, the term“selective” refers to a preferential action or function. The term“selective” can be quantified in terms of the preferential effect in a particular target of interest relative to other targets. For example, a measured variable (e.g, modulation of Tregs / Bregs versus other cells, such as other immune cells like Tcons) can be 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1- fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5- fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, lO-fold, l l-fold, l2-fold, l3-fold,

[0262] 14-fold, 15-fold, l6-fold, l7-fold, l8-fold, l9-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, lOO-fold, or greater or any range in between inclusive (e.g, 50% to 16-fold), different in a target of interest versus unintended or undesired targets. The same fold analysis can be used to confirm the magnitude of an effect in a given tissue, cell population, measured variable, measured effect, and the like, such as the Tregs:Tcons ratio, Bregs:Tcons ratio, hyperproliferative cell growth rate or volume, Tregs / Bregs proliferation rate or number, and the like.

[0263] By contrast, the term“specific” refers to an exclusionary action or function. For example, specific modulation of the HHLA2-TMIGD2 and HHLA2-KIR3DL3 interactions refers to the exclusive modulation of the HHLA2 / TMIDG2 and HHLA2 / KIR3DL3 interactions, repectively, and not modulation of HHLA2 with another ligand. In another example, specific binding of an antibody to a predetermined antigen refers to the ability of the antibody to bind to the antigen of interest without binding to other antigens. Typically, the antibody binds with an affinity (KD) of approximately less than 1 x 107M, such as approximately less than 108M, 109M, 1010M, 1011M, or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using an antigen of interest as the analyte and the antibody as the ligand, and binds to the predetermined antigen with an affinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or lO.O-fold or greater than its affinity for binding to a non-specific antigen ( e.g ., BSA, casein) other than the predetermined antigen or a closely-related antigen. In addition, KD is the inverse of KA.

[0264] The phrases“an antibody recognizing an antigen” and“an antibody specific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

[0265] The term“sensitize” means to alter cells, such as cancer cells or tumor cells, in a way that allows for more effective treatment with a therapy (e.g., HHLA2 pathway modulator therapy (e.g, modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and KIR3DL3), either alone or in combination with an immunotherapy, such as an immune checkpoint inhibition therapy). In some embodiments, normal cells are not affected to an extent that causes the normal cells to be unduly injured by the therapy (e.g, HHLA2 pathway modulator therapy (e.g, modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and KIR3DL3), either alone or in combination with an immunotherapy, such as an immune checkpoint inhibition therapy). An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94: 161-173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69: 615-632; Weisenthal L M, In: Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwood Academic Publishers, 1993: 415-432; Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). The sensitivity or resistance may also be measured in animal by measuring the tumor size reduction over a period of time, for example, 6 months for human and 4-6 weeks for mouse. A composition or a method sensitizes response to a therapeutic treatment if the increase in treatment sensitivity or the reduction 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, to 2-fold, 3-fold, 4-fold, 5-fold, lO-fold, l5-fold, 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of an immunomodulatory can be equally applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g, resistant cells) to the therapy.

[0266] The term“synergistic effect” refers to the combined effect of two or more therapeutic agents, such as two or more HHLA2 pathway modulators, a HHLA2 pathway modulator and an immunotherapy, HHLA2 pathway modulators either alone or in combination with an immunotherapy, such as an immune checkpoint inhibition therapy, and the like, can be greater than the sum of the separate effects of the anticancer agents alone.

[0267] The term“subject” refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a condition of interest (e.g, cancer). The term“subject” is interchangeable with“patient.”

[0268] The term“survival” includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related);“recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0269] The term“therapeutic effect” refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human. The phrase“therapeutically- effective amount” means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. In certain embodiments, a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like. For example, certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment.

[0270] The terms“therapeutically-effective amount” and“effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 and the ED50. Compositions that exhibit large therapeutic indices are preferred. In some embodiments, the LD50 (lethal dosage) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for the agent relative to no administration of the agent. Similarly, the EDso (i.e., the concentration which achieves a half-maximal inhibition of symptoms) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. Also, similarly, the ICso (i.e., the concentration which achieves half-maximal cytotoxic or cytostatic effect on cancer cells) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. In some embodiments, cancer cell growth in an assay can be inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. Cancer cell death can be promoted by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease in cancer cell numbers and / or a solid malignancy can be achieved.

[0271] The term“substantially free of chemical precursors or other chemicals” includes preparations of antibody, polypeptide, peptide or fusion protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includes preparations of antibody, polypeptide, peptide or fusion protein having less than about 30% (by dry weight) of chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, more preferably less than about 20% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, still more preferably less than about 10% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, and most preferably less than about 5% chemical precursors or non- antibody, polypeptide, peptide or fusion protein chemicals.

[0272] A“transcribed polynucleotide” or“nucleotide transcript” is a polynucleotide ( e.g . an mRNA, hnRNA, cDNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti- miRNA, or a miRNA binding site, or a variant thereof or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a marker of the present invention and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.

[0273] The term“vector” refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector is a“plasmid“, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g, non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as“recombinant expression vectors” or simply“expression vectors“. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification,“plasmid” and“vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors ( e.g ., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0274] There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

[0275] GENETIC CODE

[0276] Alanine (Ala, A) GCA, GCC, GCG, GCT

[0277] Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT

[0278] Asparagine (Asn, N) AAC, AAT

[0279] Aspartic acid (Asp, D) GAC, GAT

[0280] Cysteine (Cys, C) TGC, TGT

[0281] Glutamic acid (Glu, E) GAA, GAG

[0282] Glutamine (Gln, Q) CAA, CAG

[0283] Glycine (Gly, G) GGA, GGC, GGG, GGT

[0284] Histidine (His, H) CAC, CAT

[0285] Isoleucine (He, I) ATA, ATC, ATT

[0286] Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG

[0287] Lysine (Lys, K) AAA, AAG

[0288] Methionine (Met, M) ATG

[0289] Phenylalanine (Phe, F) TTC, TTT

[0290] Proline (Pro, P) CCA, CCC, CCG, CCT

[0291] Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT

[0292] Threonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG

[0293] Tyrosine (Tyr, Y) TAC, TAT

[0294] Valine (Val, V) GTA, GTC, GTG, GTT

[0295] Termination signal (end) TAA, TAG, TGA

[0296] An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0297] In view of the foregoing, the nucleotide sequence of a DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) can be used to derive the polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Thus, description and / or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and / or disclosure of a polypeptide amino acid sequence herein should be considered to also include description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0298] Finally, nucleic acid and amino acid sequence information for nucleic acid and polypeptide molecules useful in the present invention are well-known in the art and readily available on publicly available databases, such as the National Center for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases are provided in Table 1 below.

[0299] Table 1 SEP ID NO: 1 Human HHEA2 Variant 1 cDNA Sequence (NM 007072.3. CDS region from position 415-1659)

[0300] 1 agttctcttc aagtcatgta atcgactttt ttgaattagt tttcagtttc attttgtttt 61 ccctaattca agttgggaac acttcatttt ccccaattca agttgggaac acttccttgg 121 tatttccttg ctacatggac tttagcaaat gctactttac tctccttcca gctactcagg 181 aggctgaggc aggagaatcg cttgaacccg ggaggcggag gttacagtga gccttttcct 241 agttttactg ttggaagcct aactcacagg agagattatg caatacagtc ctgaagtcaa 301 gggaggagag catgtaggag aatactaacc ctgcacagat tgtgatggtg atgtggaata 361 tactaaagcc tagaacgcac ctcctctgca tgactaatat gttctgcaca agacatgaag 421 gcacagacag cactgtcttt cttcctcatt ctcataacat ctctgagtgg atctcaaggc 481 atattccctt tggctttctt catttatgtt cctatgaatg aacaaatcgt cattggaaga 541 cttgatgaag atataattct cccttcttca tttgagaggg gatccgaagt cgtaatacac 601 tggaagtatc aagatagcta taaggttcac agttactaca aaggcagtga ccatttggaa 661 agccaagatc ccagatatgc aaacaggaca tcccttttct ataatgagat tcaaaatggg 721 aatgcgtcgc tatttttcag aagagtaagc cttctggacg aaggaattta cacctgctat 781 gtaggaacag caattcaagt gattacaaac aaagtggtgc taaaggtggg agtttttctc 841 acacccgtga tgaagtatga aaagaggaac acaaacagct tcttaatatg cagcgtgtta 901 agtgtttatc ctcgtccaat tatcacgtgg aaaatggaca acacacctat ctctgaaaac 961 aacatggaag aaacagggtc tttggattct ttttctatta acagcccact gaatattaca 1021 ggatcaaatt catcttatga atgtacaatt gaaaattcac tgctgaagca aacatggaca 1081 gggcgctgga cgatgaaaga tggccttcat aaaatgcaaa gtgaacacgt ttcactctca 1141 tgtcaacctg taaatgatta tttttcacca aaccaagact tcaaagttac ttggtccaga 1201 atgaaaagtg ggactttctc tgtcctggct tactatctga gctcctcaca aaatacaatt 1261 atcaatgaat cccgattctc atggaacaaa gagctgataa accagagtga cttctctatg 1321 aatttgatgg atcttaatct ttcagacagt ggggaatatt tatgcaatat ttcttcggat 1381 gaatatactt tacttaccat ccacacagtg catgtagaac cgagccaaga aacagcttcc 1441 cataacaaag gcttatggat tttggtgccc tctgcgattt tggcagcttt tctgctgatt 1501 tggagcgtaa aatgttgcag agcccagcta gaagccagga ggagcagaca ccctgctgat 1561 ggagcccaac aagaaagatg ttgtgtccct cctggtgagc gctgtcccag tgcacccgat 1621 aatggcgaag aaaatgtgcc tctttcagga aaagtatagg aaatgagaga agactgtgac 1681 aactcatgac ctgcatcctt aatatccagt gacttcatct cccctttctt caccacaatt 1741 ccaggcaatg gcctgtcgga gcagacaatt ctaccactgc aaagagttgt aaccattttc 1801 tggtatcaca tttatttttc aagacatact tttcaagaca tcattcactg acccactacc 1861 tgcattgagt ataaatgcct ggatgttaag gattccaatt taactttgaa aagaactgtc 1921 tcattcattt acatttctgt tacagtcagc ccaggaggtt acagtgagct ctccactaag 1981 aatctggaag aaatgcatca ctaggggttg attcccaatc tgatcaactg ataatgggtg 2041 agagagcagg taagagccaa agtcacctta gtggaaaggt taaaaaccag agcctggaaa 2101 ccaagatgat tgatttgaca aggtatttta gtctagtttt atatgaacgg ttgtatcagg 2161 gtaaccaact cgatttggga tgaatcttag ggcaccaaag actaagacag tatctttaag 2221 attgctaggg aaaagggccc tatgtgtcag gcctctgagc ccaagccaag catcgcatcc 2281 cctgtgattt gcacgtatac atccagatgg cctaaagtaa ctgaagatcc acaaaagaag 2341 taaaaatagc cttaactgat gacattccac cattgtgatt tgttcctgcc ccaccctaac 2401 tgatcaatgt actttgtaat ctcccccacc cttaagaagg tactttgtaa tcttccccac 2461 ccttaagaag gttctttgta attctcccca cccttgagaa tgtactttgt gagatccacc 2521 ctgcccacaa aacattgctc ttaacttcac cgcctaaccc aaaacctata agaactaatg 2581 ataatccatc acccttcgct gactctcttt tcggactcag cccacctgca cccaggtgaa 2641 ataaacagct ttattgctca cacaaaaaaa aaaaaaaaa

[0301] SEP ID NO: 2 Human HHLA2 Variant 1 Amino Acid Sequence (NP 009003.1)

[0302] 1 MKAQTALSFF LILITSLSGS QGIFPLAFFI YVPMNEQIVI GRLDEDIILP SSFERGSEW 61 IHWKYQDSYK VHSYYKGSDH LESQDPRYAN RTSLFYNEIQ NGNASLFFRR VSLLDEGIYT 121 CYVGTAIQVI TNKWLKVGV FLTPVMKYEK RNTNSFLICS VLSVYPRPII TWKMDNTPIS 181 ENNMEETGSL DSFSINSPLN ITGSNSSYEC TIENSLLKQT WTGRWTMKDG LHKMQSEHVS 241 LSCQPWDYF SPNQDFKVTW SRMKSGTFSV LAYYLSSSQN TIINESRFSW NKELINQSDF 301 SMNLMDLNLS DSGEYLCNIS SDEYTLLTIH TVHVEPSQET ASHNKGLWIL VPSAILAAFL 361 LIWSVKCCRA QLEARRSRHP ADGAQQERCC VPPGERCPSA PDNGEENVPL SGKV SEP ID NO: 3 Human HHEA2 Variant 2 cDNA Sequence (NM 001282556.1.

[0303] CDS region from position 224-1468)

[0304] 1 aaatcaaacg taccttggac tttactctct gagaaactca tagctgaatt caatgtttat

[0305] 61 tcttatggac tacttagcat ttgactagac ggtatgaatt tctaagtaag cacatataga

[0306] 121 actggatgcc cttgtggtac atctcaaggc tgatttgaaa gcttgagaga ccatcaagaa

[0307] 181 ttggatttgg ggaagagcat gactaatatg ttctgcacaa gacatgaagg cacagacagc

[0308] 241 actgtctttc ttcctcattc tcataacatc tctgagtgga tctcaaggca tattcccttt

[0309] 301 ggctttcttc atttatgttc ctatgaatga acaaatcgtc attggaagac ttgatgaaga

[0310] 361 tataattctc ccttcttcat ttgagagggg atccgaagtc gtaatacact ggaagtatca

[0311] 421 agatagctat aaggttcaca gttactacaa aggcagtgac catttggaaa gccaagatcc

[0312] 481 cagatatgca aacaggacat cccttttcta taatgagatt caaaatggga atgcgtcgct

[0313] 541 atttttcaga agagtaagcc ttctggacga aggaatttac acctgctatg taggaacagc

[0314] 601 aattcaagtg attacaaaca aagtggtgct aaaggtggga gtttttctca cacccgtgat

[0315] 661 gaagtatgaa aagaggaaca caaacagctt cttaatatgc agcgtgttaa gtgtttatcc

[0316] 721 tcgtccaatt atcacgtgga aaatggacaa cacacctatc tctgaaaaca acatggaaga

[0317] 781 aacagggtct ttggattctt tttctattaa cagcccactg aatattacag gatcaaattc

[0318] 841 atcttatgaa tgtacaattg aaaattcact gctgaagcaa acatggacag ggcgctggac

[0319] 901 gatgaaagat ggccttcata aaatgcaaag tgaacacgtt tcactctcat gtcaacctgt

[0320] 961 aaatgattat ttttcaccaa accaagactt caaagttact tggtccagaa tgaaaagtgg

[0321] 1021 gactttctct gtcctggctt actatctgag ctcctcacaa aatacaatta tcaatgaatc

[0322] 1081 ccgattctca tggaacaaag agctgataaa ccagagtgac ttctctatga atttgatgga

[0323] 1141 tcttaatctt tcagacagtg gggaatattt atgcaatatt tcttcggatg aatatacttt

[0324] 1201 acttaccatc cacacagtgc atgtagaacc gagccaagaa acagcttccc ataacaaagg

[0325] 1261 cttatggatt ttggtgccct ctgcgatttt ggcagctttt ctgctgattt ggagcgtaaa

[0326] 1321 atgttgcaga gcccagctag aagccaggag gagcagacac cctgctgatg gagcccaaca

[0327] 1381 agaaagatgt tgtgtccctc ctggtgagcg ctgtcccagt gcacccgata atggcgaaga

[0328] 1441 aaatgtgcct ctttcaggaa aagtatagga aatgagagaa gactgtgaca actcatgacc

[0329] 1501 tgcatcctta atatccagtg acttcatctc ccctttcttc accacaattc caggcaatgg

[0330] 1561 cctgtcggag cagacaattc taccactgca aagagttgta accattttct ggtatcacat

[0331] 1621 ttatttttca agacatactt ttcaagacat cattcactga cccactacct gcattgagta

[0332] 1681 taaatgcctg gatgttaagg attccaattt aactttgaaa agaactgtct cattcattta

[0333] 1741 catttctgtt acagtcagcc caggaggtta cagtgagctc tccactaaga atctggaaga

[0334] 1801 aatgcatcac taggggttga ttcccaatct gatcaactga taatgggtga gagagcaggt

[0335] 1861 aagagccaaa gtcaccttag tggaaaggtt aaaaaccaga gcctggaaac caagatgatt

[0336] 1921 gatttgacaa ggtattttag tctagtttta tatgaacggt tgtatcaggg taaccaactc

[0337] 1981 gatttgggat gaatcttagg gcaccaaaga ctaagacagt atctttaaga ttgctaggga

[0338] 2041 aaagggccct atgtgtcagg cctctgagcc caagccaagc atcgcatccc ctgtgatttg

[0339] 2101 cacgtataca tccagatggc ctaaagtaac tgaagatcca caaaagaagt aaaaatagcc

[0340] 2161 ttaactgatg acattccacc attgtgattt gttcctgccc caccctaact gatcaatgta

[0341] 2221 ctttgtaatc tcccccaccc ttaagaaggt actttgtaat cttccccacc cttaagaagg

[0342] 2281 ttctttgtaa ttctccccac ccttgagaat gtactttgtg agatccaccc tgcccacaaa

[0343] 2341 acattgctct taacttcacc gcctaaccca aaacctataa gaactaatga taatccatca

[0344] 2401 cccttcgctg actctctttt cggactcagc ccacctgcac ccaggtgaaa taaacagctt

[0345] 2461 tattgctcac acaaaaaaaa aaaaaaaa

[0346] SEP ID NO: 4 _ Human HHLA2 Variant 2 Amino Acid Sequence

[0347] (NP 001269485.11

[0348] 1 MKAQTALSFF LILITSLSGS QGIFPLAFFI YVPMNEQIVI GRLDEDIILP SSFERGSEW 61 IHWKYQDSYK VHSYYKGSDH LESQDPRYAN RTSLFYNEIQ NGNASLFFRR VSLLDEGIYT 121 CYVGTAIQVI TNKWLKVGV FLTPVMKYEK RNTNSFLICS VLSVYPRPII TWKMDNTPIS 181 ENNMEETGSL DSFSINSPLN ITGSNSSYEC TIENSLLKQT WTGRWTMKDG LHKMQSEHVS 241 LSCQPWDYF SPNQDFKVTW SRMKSGTFSV LAYYLSSSQN TIINESRFSW NKELINQSDF 301 SMNLMDLNLS DSGEYLCNIS SDEYTLLTIH TVHVEPSQET ASHNKGLWIL VPSAILAAFL 361 LIWSVKCCRA QLEARRSRHP ADGAQQERCC VPPGERCPSA PDNGEENVPL SGKV

[0349] SEP ID NO: 5 Human HHEA2 Variant 3 cDNA Sequence (NM 001282557.1.

[0350] CDS region from position 155-1399)

[0351] 1 agtttactct acatcatagc agagaaaatg gacaaaacac agctgttttg catgtaggag 61 aatactaacc ctgcacagat tgtgatggtg atgtggaata tactaaagcc tagaacgcac 121 ctcctctgca tgactaatat gttctgcaca agacatgaag gcacagacag cactgtcttt 181 cttcctcatt ctcataacat ctctgagtgg atctcaaggc atattccctt tggctttctt 241 catttatgtt cctatgaatg aacaaatcgt cattggaaga cttgatgaag atataattct 301 cccttcttca tttgagaggg gatccgaagt cgtaatacac tggaagtatc aagatagcta 361 taaggttcac agttactaca aaggcagtga ccatttggaa agccaagatc ccagatatgc 421 aaacaggaca tcccttttct ataatgagat tcaaaatggg aatgcgtcgc tatttttcag 481 aagagtaagc cttctggacg aaggaattta cacctgctat gtaggaacag caattcaagt 541 gattacaaac aaagtggtgc taaaggtggg agtttttctc acacccgtga tgaagtatga 601 aaagaggaac acaaacagct tcttaatatg cagcgtgtta agtgtttatc ctcgtccaat 661 tatcacgtgg aaaatggaca acacacctat ctctgaaaac aacatggaag aaacagggtc 721 tttggattct ttttctatta acagcccact gaatattaca ggatcaaatt catcttatga 781 atgtacaatt gaaaattcac tgctgaagca aacatggaca gggcgctgga cgatgaaaga 841 tggccttcat aaaatgcaaa gtgaacacgt ttcactctca tgtcaacctg taaatgatta 901 tttttcacca aaccaagact tcaaagttac ttggtccaga atgaaaagtg ggactttctc 961 tgtcctggct tactatctga gctcctcaca aaatacaatt atcaatgaat cccgattctc 1021 atggaacaaa gagctgataa accagagtga cttctctatg aatttgatgg atcttaatct 1081 ttcagacagt ggggaatatt tatgcaatat ttcttcggat gaatatactt tacttaccat 1141 ccacacagtg catgtagaac cgagccaaga aacagcttcc cataacaaag gcttatggat 1201 tttggtgccc tctgcgattt tggcagcttt tctgctgatt tggagcgtaa aatgttgcag 1261 agcccagcta gaagccagga ggagcagaca ccctgctgat ggagcccaac aagaaagatg 1321 ttgtgtccct cctggtgagc gctgtcccag tgcacccgat aatggcgaag aaaatgtgcc 1381 tctttcagga aaagtatagg aaatgagaga agactgtgac aactcatgac ctgcatcctt 1441 aatatccagt gacttcatct cccctttctt caccacaatt ccaggcaatg gcctgtcgga 1501 gcagacaatt ctaccactgc aaagagttgt aaccattttc tggtatcaca tttatttttc 1561 aagacatact tttcaagaca tcattcactg acccactacc tgcattgagt ataaatgcct 1621 ggatgttaag gattccaatt taactttgaa aagaactgtc tcattcattt acatttctgt 1681 tacagtcagc ccaggaggtt acagtgagct ctccactaag aatctggaag aaatgcatca 1741 ctaggggttg attcccaatc tgatcaactg ataatgggtg agagagcagg taagagccaa 1801 agtcacctta gtggaaaggt taaaaaccag agcctggaaa ccaagatgat tgatttgaca 1861 aggtatttta gtctagtttt atatgaacgg ttgtatcagg gtaaccaact cgatttggga 1921 tgaatcttag ggcaccaaag actaagacag tatctttaag attgctaggg aaaagggccc 1981 tatgtgtcag gcctctgagc ccaagccaag catcgcatcc cctgtgattt gcacgtatac 2041 atccagatgg cctaaagtaa ctgaagatcc acaaaagaag taaaaatagc cttaactgat 2101 gacattccac cattgtgatt tgttcctgcc ccaccctaac tgatcaatgt actttgtaat 2161 ctcccccacc cttaagaagg tactttgtaa tcttccccac ccttaagaag gttctttgta 2221 attctcccca cccttgagaa tgtactttgt gagatccacc ctgcccacaa aacattgctc 2281 ttaacttcac cgcctaaccc aaaacctata agaactaatg ataatccatc acccttcgct 2341 gactctcttt tcggactcag cccacctgca cccaggtgaa ataaacagct ttattgctca 2401 cacaaaaaaa aaaaaaaaa

[0352] SEP ID NO: 6 _ Human HHLA2 Variant 3 Amino Acid Sequence

[0353] (NP 001269486.11

[0354] 1 MKAQTALSFF LILITSLSGS QGIFPLAFFI YVPMNEQIVI GRLDEDIILP SSFERGSEW 61 IHWKYQDSYK VHSYYKGSDH LESQDPRYAN RTSLFYNEIQ NGNASLFFRR VSLLDEGIYT 121 CYVGTAIQVI TNKWLKVGV FLTPVMKYEK RNTNSFLICS VLSVYPRPII TWKMDNTPIS 181 ENNMEETGSL DSFSINSPLN ITGSNSSYEC TIENSLLKQT WTGRWTMKDG LHKMQSEHVS 241 LSCQPWDYF SPNQDFKVTW SRMKSGTFSV LAYYLSSSQN TIINESRFSW NKELINQSDF 301 SMNLMDLNLS DSGEYLCNIS SDEYTLLTIH TVHVEPSQET ASHNKGLWIL VPSAILAAFL 361 LIWSVKCCRA QLEARRSRHP ADGAQQERCC VPPGERCPSA PDNGEENVPL SGKV

[0355] SEP ID NO: 7 Human HHEA2 Variant 4 cDNA Sequence (NM 001282558.1.

[0356] CDS region from position 302-1495)

[0357] 1 aaatcaaacg taccttggac tttactctct gagaaactca tagctgaatt caatgtttat

[0358] 61 tcttatggac tacttagcat ttgactagac ggtatgaatt tctaagtaag cacatataga

[0359] 121 actggatgcc cttgtggtac atctcaaggc tgatttgaaa gcttgagaga ccatcaagaa

[0360] 181 ttggatttgg ggaagagcat gtaggagaat actaaccctg cacagattgt gatggtgatg

[0361] 241 tggaatatac taaagcctag aacgcacctc ctctgcatga ctaatatgtt ctgcacaaga 301 catgaaggca cagacagcac tgtctttctt cctcattctc ataacatctc tgagtggatc 361 tcaaggcata ttccctttgg ctttcttcat ttatgttcct atgaatgaac aaatcgtcat 421 tggaagactt gatgaagata taattctccc ttcttcattt gagaggggat ccgaagtcgt 481 aatacactgg aagtatcaag atagctataa ggttcacagt tactacaaag gcagtgacca 541 tttggaaagc caagatccca gatatgcaaa caggacatcc cttttctata atgagattca 601 aaatgggaat gcgtcgctat ttttcagaag agtaagcctt ctggacgaag gaatttacac 661 ctgctatgta ggaacagcaa ttcaagtgat tacaaacaaa gtggtgctaa aggtgggagt 721 ttttctcaca cccgtgatga agtatgaaaa gaggaacaca aacagcttct taatatgcag 781 cgtgttaagt gtttatcctc gtccaattat cacgtggaaa atggacaaca cacctatctc 841 tgaaaacaac atggaagaaa cagggtcttt ggattctttt tctattaaca gcccactgaa 901 tattacagga tcaaattcat cttatgaatg tacaattgaa aattcactgc tgaagcaaac 961 atggacaggg cgctggacga tgaaagatgg ccttcataaa atgcaaagtg aacacgtttc 1021 actctcatgt caacctgtaa atgattattt ttcaccaaac caagacttca aagttacttg 1081 gtccagaatg aaaagtggga ctttctctgt cctggcttac tatctgagct cctcacaaaa 1141 tacaattatc aatgaatccc gattctcatg gaacaaagag ctgataaacc agagtgactt 1201 ctctatgaat ttgatggatc ttaatctttc agacagtggg gaatatttat gcaatatttc 1261 ttcggatgaa tatactttac ttaccatcca cacagtgcat gtagaaccga gccaagaaac 1321 agcttcccat aacaaaggct tatggatttt ggtgccctct gcgattttgg cagcttttct 1381 gctgatttgg agcgtaaaat gttgcagaga aagatgttgt gtccctcctg gtgagcgctg 1441 tcccagtgca cccgataatg gcgaagaaaa tgtgcctctt tcaggaaaag tataggaaat 1501 gagagaagac tgtgacaact catgacctgc atccttaata tccagtgact tcatctcccc 1561 tttcttcacc acaattccag gcaatggcct gtcggagcag acaattctac cactgcaaag 1621 agttgtaacc attttctggt atcacattta tttttcaaga catacttttc aagacatcat 1681 tcactgaccc actacctgca ttgagtataa atgcctggat gttaaggatt ccaatttaac 1741 tttgaaaaga actgtctcat tcatttacat ttctgttaca gtcagcccag gaggttacag 1801 tgagctctcc actaagaatc tggaagaaat gcatcactag gggttgattc ccaatctgat 1861 caactgataa tgggtgagag agcaggtaag agccaaagtc accttagtgg aaaggttaaa 1921 aaccagagcc tggaaaccaa gatgattgat ttgacaaggt attttagtct agttttatat 1981 gaacggttgt atcagggtaa ccaactcgat ttgggatgaa tcttagggca ccaaagacta 2041 agacagtatc tttaagattg ctagggaaaa gggccctatg tgtcaggcct ctgagcccaa 2101 gccaagcatc gcatcccctg tgatttgcac gtatacatcc agatggccta aagtaactga 2161 agatccacaa aagaagtaaa aatagcctta actgatgaca ttccaccatt gtgatttgtt 2221 cctgccccac cctaactgat caatgtactt tgtaatctcc cccaccctta agaaggtact 2281 ttgtaatctt ccccaccctt aagaaggttc tttgtaattc tccccaccct tgagaatgta 2341 ctttgtgaga tccaccctgc ccacaaaaca ttgctcttaa cttcaccgcc taacccaaaa 2401 cctataagaa ctaatgataa tccatcaccc ttcgctgact ctcttttcgg actcagccca 2461 cctgcaccca ggtgaaataa acagctttat tgctcacaca aaaaaaaaaa aaaaa

[0362] SEP ID NO: 8 _ Human HHLA2 Variant 4 Amino Acid Sequence

[0363] (NP 001269487.11

[0364] 1 MKAQTALSFF LILITSLSGS QGIFPLAFFI YVPMNEQIVI GRLDEDIILP SSFERGSEW 61 IHWKYQDSYK VHSYYKGSDH LESQDPRYAN RTSLFYNEIQ NGNASLFFRR VSLLDEGIYT 121 CYVGTAIQVI TNKWLKVGV FLTPVMKYEK RNTNSFLICS VLSVYPRPII TWKMDNTPIS 181 ENNMEETGSL DSFSINSPLN ITGSNSSYEC TIENSLLKQT WTGRWTMKDG LHKMQSEHVS 241 LSCQPWDYF SPNQDFKVTW SRMKSGTFSV LAYYLSSSQN TIINESRFSW NKELINQSDF 301 SMNLMDLNLS DSGEYLCNIS SDEYTLLTIH TVHVEPSQET ASHNKGLWIL VPSAILAAFL 361 LIWSVKCCRE RCCVPPGERC PSAPDNGEEN VPLSGKV

[0365] SEP ID NO: 9 Human HHEA2 Variant 5 cDNA Sequence (NM 001282559.1.

[0366] CDS region from position 232-1284)

[0367] 1 aaatcaaacg taccttggac tttactctct gagaaactca tagctgaatt caatgtttat

[0368] 61 tcttatggac tacttagcat ttgactagac ggtatgaatt tctaagtaag cacatataga

[0369] 121 actggatgcc cttgtggtac atctcaaggc tgatttgaaa gcttgagaga ccatcaagaa

[0370] 181 ttggatttgg ggaagagcat gtaggagaat actaaccctg cacagattgt gatggtgatg

[0371] 241 tggaatatac taaagcctag aacgcacctc ctctgcatga ctaatatgtt ctgcacaaga

[0372] 301 catgaaggca cagacagcac tgtctttctt cctcattctc ataacatctc tgagtggatc

[0373] 361 tcaagaagag taagccttct ggacgaagga atttacacct gctatgtagg aacagcaatt

[0374] 421 caagtgatta caaacaaagt ggtgctaaag gtgggagttt ttctcacacc cgtgatgaag 481 tatgaaaaga ggaacacaaa cagcttctta atatgcagcg tgttaagtgt ttatcctcgt 541 ccaattatca cgtggaaaat ggacaacaca cctatctctg aaaacaacat ggaagaaaca 601 gggtctttgg attctttttc tattaacagc ccactgaata ttacaggatc aaattcatct 661 tatgaatgta caattgaaaa ttcactgctg aagcaaacat ggacagggcg ctggacgatg 721 aaagatggcc ttcataaaat gcaaagtgaa cacgtttcac tctcatgtca acctgtaaat 781 gattattttt caccaaacca agacttcaaa gttacttggt ccagaatgaa aagtgggact 841 ttctctgtcc tggcttacta tctgagctcc tcacaaaata caattatcaa tgaatcccga 901 ttctcatgga acaaagagct gataaaccag agtgacttct ctatgaattt gatggatctt 961 aatctttcag acagtgggga atatttatgc aatatttctt cggatgaata tactttactt 1021 accatccaca cagtgcatgt agaaccgagc caagaaacag cttcccataa caaaggctta 1081 tggattttgg tgccctctgc gattttggca gcttttctgc tgatttggag cgtaaaatgt 1141 tgcagagccc agctagaagc caggaggagc agacaccctg ctgatggagc ccaacaagaa 1201 agatgttgtg tccctcctgg tgagcgctgt cccagtgcac ccgataatgg cgaagaaaat 1261 gtgcctcttt caggaaaagt ataggaaatg agagaagact gtgacaactc atgacctgca 1321 tccttaatat ccagtgactt catctcccct ttcttcacca caattccagg caatggcctg 1381 tcggagcaga caattctacc actgcaaaga gttgtaacca ttttctggta tcacatttat 1441 ttttcaagac atacttttca agacatcatt cactgaccca ctacctgcat tgagtataaa 1501 tgcctggatg ttaaggattc caatttaact ttgaaaagaa ctgtctcatt catttacatt 1561 tctgttacag tcagcccagg aggttacagt gagctctcca ctaagaatct ggaagaaatg 1621 catcactagg ggttgattcc caatctgatc aactgataat gggtgagaga gcaggtaaga 1681 gccaaagtca ccttagtgga aaggttaaaa accagagcct ggaaaccaag atgattgatt 1741 tgacaaggta ttttagtcta gttttatatg aacggttgta tcagggtaac caactcgatt 1801 tgggatgaat cttagggcac caaagactaa gacagtatct ttaagattgc tagggaaaag 1861 ggccctatgt gtcaggcctc tgagcccaag ccaagcatcg catcccctgt gatttgcacg 1921 tatacatcca gatggcctaa agtaactgaa gatccacaaa agaagtaaaa atagccttaa 1981 ctgatgacat tccaccattg tgatttgttc ctgccccacc ctaactgatc aatgtacttt 2041 gtaatctccc ccacccttaa gaaggtactt tgtaatcttc cccaccctta agaaggttct 2101 ttgtaattct ccccaccctt gagaatgtac tttgtgagat ccaccctgcc cacaaaacat 2161 tgctcttaac ttcaccgcct aacccaaaac ctataagaac taatgataat ccatcaccct 2221 tcgctgactc tcttttcgga ctcagcccac ctgcacccag gtgaaataaa cagctttatt 2281 gctcacacaa aaaaaaaaaa aaaa

[0375] SEQ ID NO: 10 _ Human HHLA2 Variant 5 Amino Acid Sequence

[0376] (NP 001269488.11

[0377] 1 MVMWNILKPR THLLCMTNMF CTRHEGTDST VFLPHSHNIS EWISRRVSLL DEGIYTCYVG 61 TAIQVITNKV VLKVGVFLTP VMKYEKRNTN SFLICSVLSV YPRPIITWKM DNTPISENNM 121 EETGSLDSFS INSPLNITGS NSSYECTIEN SLLKQTWTGR WTMKDGLHKM QSEHVSLSCQ 181 PWDYFSPNQ DFKVTWSRMK SGTFSVLAYY LSSSQNTIIN ESRFSWNKEL INQSDFSMNL 241 MDLNLSDSGE YLCNISSDEY TLLTIHTVHV EPSQETASHN KGLWILVPSA ILAAFLLIWS 301 VKCCRAQLEA RRSRHPADGA QQERCCVPPG ERCPSAPDNG EENVPLSGKV

[0378] SEP ID NO: 11 Human TMIGD2 Isoform 1 cDNA Sequence (NM 144615.2

[0379] CDS region from position 47-895)

[0380] 1 ggaagtctgt caactgggag ggggagaggg gggtgatggg ccaggaatgg ggtccccggg 61 catggtgctg ggcctcctgg tgcagatctg ggccctgcaa gaagcctcaa gcctgagcgt 121 gcagcagggg cccaacttgc tgcaggtgag gcagggcagt caggcgaccc tggtctgcca 181 ggtggaccag gccacagcct gggaacggct ccgtgttaag tggacaaagg atggggccat 241 cctgtgtcaa ccgtacatca ccaacggcag cctcagcctg ggggtctgcg ggccccaggg 301 acggctctcc tggcaggcac ccagccatct caccctgcag ctggaccctg tgagcctcaa 361 ccacagcggg gcgtacgtgt gctgggcggc cgtagagatt cctgagttgg aggaggctga 421 gggcaacata acaaggctct ttgtggaccc agatgacccc acacagaaca gaaaccggat 481 cgcaagcttc ccaggattcc tcttcgtgct gctgggggtg ggaagcatgg gtgtggctgc 541 gatcgtgtgg ggtgcctggt tctggggccg ccgcagctgc cagcaaaggg actcaggtaa 601 cagcccagga aatgcattct acagcaacgt cctataccgg ccccgggggg ccccaaagaa 661 gagtgaggac tgctctggag aggggaagga ccagaggggc cagagcattt attcaacctc 721 cttcccgcaa ccggcccccc gccagccgca cctggcgtca agaccctgcc ccagcccgag 781 accctgcccc agccccaggc ccggccaccc cgtctctatg gtcagggtct ctcctagacc 841 aagccccacc cagcagccga ggccaaaagg gttccccaaa gtgggagagg agtgagagat 901 cccaggagac ctcaacagga ccccacccat aggtacacac aaaaaagggg ggatcgaggc 961 cagacacggt ggctcacgcc tgtaatccca gcagtttggg aagccgaggc gggtggaaca 1021 cttgaggtca ggggtttgag accagcctgg cttgaacctg ggaggcggag gttgcagtga 1081 gccgagattg cgccactgca ctccagcctg ggcgacagag tgagactccg tctcaaaaaa 1141 aacaaaaagc aggaggattg ggagcctgtc agccccatcc tgagaccccg tcctcatttc 1201 tgtaatgatg gatctcgctc ccactttccc ccaagaacct aataaaggct tgtgaagaaa 1261 aagcaaaaaa aaaaaaaaaa aa

[0381] SEP ID NO: 12 _ Human TMIGD2 Isoform 1 Amino Acid Sequence

[0382] (NP 653216.21

[0383] 1 MGSPGMVLGL LVQIWALQEA SSLSVQQGPN LLQVRQGSQA TLVCQVDQAT AWERLRVKWT 61 KDGAILCQPY ITNGSLSLGV CGPQGRLSWQ APSHLTLQLD PVSLNHSGAY VCWAAVEIPE 121 LEEAEGNITR LFVDPDDPTQ NRNRIASFPG FLFVLLGVGS MGVAAIVWGA WFWGRRSCQQ 181 RDSGNSPGNA FYSNVLYRPR GAPKKSEDCS GEGKDQRGQS IYSTSFPQPA PRQPHLASRP 241 CPSPRPCPSP RPGHPVSMVR VSPRPSPTQQ PRPKGFPKVG EE

[0384] SEP ID NO: 13 Human TMIGD2 Isoform 2 cDNA Sequence (NM 001169126.1.

[0385] CDS region from position 47-883)

[0386] 1 ggaagtctgt caactgggag ggggagaggg gggtgatggg ccaggaatgg ggtccccggg 61 catggtgctg ggcctcctgg tgcagatctg ggccctgcaa gaagcctcaa gcctgagcgt 121 gcagcagggg cccaacttgc tgcaggtgag gcagggcagt caggcgaccc tggtctgcca 181 ggtggaccag gccacagcct gggaacggct ccgtgttaag tggacaaagg atggggccat 241 cctgtgtcaa ccgtacatca ccaacggcag cctcagcctg ggggtctgcg ggccccaggg 301 acggctctcc tggcaggcac ccagccatct caccctgcag ctggaccctg tgagcctcaa 361 ccacagcggg gcgtacgtgt gctgggcggc cgtagagatt cctgagttgg aggaggctga 421 gggcaacata acaaggctct ttgtggaccc agatgacccc acacagaaca gaaaccggat 481 cgcaagcttc ccaggattcc tcttcgtgct gctgggggtg ggaagcatgg gtgtggctgc 541 gatcgtgtgg ggtgcctggt tctggggccg ccgcagctgc cagcaaaggg actcaggaaa 601 tgcattctac agcaacgtcc tataccggcc ccggggggcc ccaaagaaga gtgaggactg 661 ctctggagag gggaaggacc agaggggcca gagcatttat tcaacctcct tcccgcaacc 721 ggccccccgc cagccgcacc tggcgtcaag accctgcccc agcccgagac cctgccccag 781 ccccaggccc ggccaccccg tctctatggt cagggtctct cctagaccaa gccccaccca 841 gcagccgagg ccaaaagggt tccccaaagt gggagaggag tgagagatcc caggagacct 901 caacaggacc ccacccatag gtacacacaa aaaagggggg atcgaggcca gacacggtgg 961 ctcacgcctg taatcccagc agtttgggaa gccgaggcgg gtggaacact tgaggtcagg 1021 ggtttgagac cagcctggct tgaacctggg aggcggaggt tgcagtgagc cgagattgcg 1081 ccactgcact ccagcctggg cgacagagtg agactccgtc tcaaaaaaaa caaaaagcag 1141 gaggattggg agcctgtcag ccccatcctg agaccccgtc ctcatttctg taatgatgga 1201 tctcgctccc actttccccc aagaacctaa taaaggcttg tgaagaaaaa gcaaaaaaaa 1261 aaaaaaaaaa

[0387] SEP ID NO: 14 _ Human TMIGD2 Isoform 2 Amino Acid Sequence

[0388] (NP 001162597.11

[0389] 1 MGSPGMVLGL LVQIWALQEA SSLSVQQGPN LLQVRQGSQA TLVCQVDQAT AWERLRVKWT 61 KDGAILCQPY ITNGSLSLGV CGPQGRLSWQ APSHLTLQLD PVSLNHSGAY VCWAAVEIPE 121 LEEAEGNITR LFVDPDDPTQ NRNRIASFPG FLFVLLGVGS MGVAAIVWGA WFWGRRSCQQ 181 RDSGNAFYSN VLYRPRGAPK KSEDCSGEGK DQRGQSIYST SFPQPAPRQP HLASRPCPSP 241 RPCPSPRPGH PVSMVRVSPR PSPTQQPRPK GFPKVGEE

[0390] SEP ID NO: 15 Human TMIGD2 Isoform 3 cDNA Sequence (NM 001308232.1.

[0391] CDS region from position 47-5351

[0392] 1 ggaagtctgt caactgggag ggggagaggg gggtgatggg ccaggaatgg ggtccccggg

[0393] 61 catggtgctg ggcctcctgg tgcagatctg ggatgacccc acacagaaca gaaaccggat

[0394] 121 cgcaagcttc ccaggattcc tcttcgtgct gctgggggtg ggaagcatgg gtgtggctgc

[0395] 181 gatcgtgtgg ggtgcctggt tctggggccg ccgcagctgc cagcaaaggg actcaggtaa

[0396] 241 cagcccagga aatgcattct acagcaacgt cctataccgg ccccgggggg ccccaaagaa 301 gagtgaggac tgctctggag aggggaagga ccagaggggc cagagcattt attcaacctc 361 cttcccgcaa ccggcccccc gccagccgca cctggcgtca agaccctgcc ccagcccgag 421 accctgcccc agccccaggc ccggccaccc cgtctctatg gtcagggtct ctcctagacc 481 aagccccacc cagcagccga ggccaaaagg gttccccaaa gtgggagagg agtgagagat 541 cccaggagac ctcaacagga ccccacccat aggtacacac aaaaaagggg ggatcgaggc 601 cagacacggt ggctcacgcc tgtaatccca gcagtttggg aagccgaggc gggtggaaca 661 cttgaggtca ggggtttgag accagcctgg cttgaacctg ggaggcggag gttgcagtga 721 gccgagattg cgccactgca ctccagcctg ggcgacagag tgagactccg tctcaaaaaa 781 aacaaaaagc aggaggattg ggagcctgtc agccccatcc tgagaccccg tcctcatttc 841 tgtaatgatg gatctcgctc ccactttccc ccaagaacct aataaaggct tgtgaagaaa 901 aagcaaaaaa aaaaaaaa

[0397] SEP ID NO: 16 _ Human TMIGD2 Isoform 3 Amino Acid Sequence

[0398] (NP 001295161.11

[0399] 1 MGSPGMVLGL LVQIWDDPTQ NRNRIASFPG FLFVLLGVGS MGVAAIVWGA WFWGRRSCQQ 61 RDSGNSPGNA FYSNVLYRPR GAPKKSEDCS GEGKDQRGQS IYSTSFPQPA PRQPHLASRP 121 CPSPRPCPSP RPGHPVSMVR VSPRPSPTQQ PRPKGFPKVG EE

[0400] SEP ID NO: 17 Human KIR3DL3 cDNA Sequence (NM 153443.4. CDS region from position 51-1283)

[0401] 1 tgctgctgaa ctgagctggg gcgcagccgc ctgtctgcac cggcagcacc atgtcgctca 61 tggtcgtcag catggcgtgt gttgggttct tcttgctgga ggggccctgg ccacatgtgg 121 gtggtcagga caagcccttc ctctctgcct ggcccggcac tgtggtgtct gaaggacaac 181 atgtgactct tcagtgtcgc tctcgtcttg ggtttaatga attcagtctg tccaaagaag 241 acgggatgcc tgtccctgag ctctacaaca gaatattccg gaacagcttt ctcatgggcc 301 ctgtgacccc agcacatgca gggacctaca gatgttgcag ttcacaccca cactccccca 361 ctgggtggtc ggcacccagc aaccctgtgg tgatcatggt cacaggagtc cacagaaaac 421 cttccctcct ggcccaccca ggtcccctgg tgaaatcagg agagacggtc atcctgcaat 481 gttggtcaga tgtcaggttt gagcgcttcc ttctgcacag agaggggatc actgaggacc 541 ccttgcgcct cgttggacag ctccacgatg cgggttccca ggtcaactat tccatgggtc 601 ccatgacacc tgcccttgca gggacctaca gatgctttgg ttctgtcact cacttaccct 661 atgagttgtc ggctcccagt gaccctctgg acatcgtggt cgtaggtcta tatgggaaac 721 cttctctctc agcccagccg ggccccacgg ttcaggcagg agagaatgtg accttgtcct 781 gcagctcccg gagcttgttt gacatttacc atctatccag ggaggcggag gccggtgaac 841 ttaggctcac tgcagtgctg agggtcaatg gaacattcca ggccaacttc cctctgggcc 901 ctgtgaccca cggagggaac tacagatgct tcggctcttt ccgtgccctg ccccatgcgt 961 ggtcagaccc gagtgaccca ctgcccgttt ctgtcacagg taactccaga aacctgcacg 1021 ttctgattgg gacctcagtg gtcatcatcc cctttgctat cctcctcttc tttctccttc 1081 atcgctggtg tgccaacaaa aagaatgctg ttgtaatgga ccaagagcct gcagggaaca 1141 gaacagtgaa cagggaggac tctgatgaac aagaccctca ggaggtgaca tacgcacagt 1201 tgaatcactg cgttttcaca cagagaaaaa tcactcgccc ttctcagagg cccaagacac 1261 ccccaacaga taccagcgtg taacacggaa cttccaaatg ctgagcgcag atccaaagtt 1321 gtcttctgtc cactagcacc acagtcaggc cttgatggga tcttctaggg agacaatagc 1381 cctgtctcaa aaccgggttg ccagctccca tgtaccagca gctggactct gaaggcgtga 1441 gtctgcatct tagggcatcg ctcttcctca caccacgaat ctgaacatgc ctctctcttg 1501 cttacaaatg tctaaggtcc ccactgcctg ctggagagaa aacacacttg cttagcccac 1561 aattctccat ttcacttgac ccctgcccac ctctccaacc taactggctt acttcctagt 1621 ctacttgagg ctgcgatcac actgaggaac tcacaattcc aaacatataa gaggctccct 1681 cttaacacgg cacttagata cgtgctattc cacctttcct cag

[0402] SEP ID NO: 18 Human KIR3DL3 Amino Acid Sequence (NP 703144.31

[0403] 1 MSLMWSMAC VGFFLLEGPW PHVGGQDKPF LSAWPGTWS EGQHVTLQCR SRLGFNEFSL 61 SKEDGMPVPE LYNRIFRNSF LMGPVTPAHA GTYRCCSSHP HSPTGWSAPS NPWIMVTGV 121 HRKPSLLAHP GPLVKSGETV ILQCWSDVRF ERFLLHREGI TEDPLRLVGQ LHDAGSQWY 181 SMGPMTPALA GTYRCFGSVT HLPYELSAPS DPLDIVWGL YGKPSLSAQP GPTVQAGENV 241 TLSCSSRSLF DIYHLSREAE AGELRLTAVL RWGTFQANF PLGPVTHGGN YRCFGSFRAL 301 PHAWSDPSDP LPVSVTGNSR NLHVLIGTSV VIIPFAILLF FLLHRWCANK KNAWMDQEP 361 AGNRTWRED SDEQDPQEVT YAQLNHCVFT QRKITRPSQR PKTPPTDTSV

[0404] * Included in Table 1 are RNA nucleic acid molecules ( e.g ., thymines replaced with uridines), nucleic acid molecules encoding orthologs of the encoded proteins, as well as DNA or RNA nucleic acid sequences comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the nucleic acid sequence of any SEQ ID NO listed in Table 1, or a portion thereof. Such nucleic acid molecules can have a function of the full-length nucleic acid as described further herein.

[0405] * Included in Table 1 are orthologs of the proteins, as well as polypeptide molecules comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with an amino acid sequence of any SEQ ID NO listed in Table 1, or a portion thereof. Such polypeptides can have a function of the full-length polypeptide as described further herein.

[0406] * Included in Table 1 are other known HHLA2, TMIGD2, and KIR3DL3 nucleic acid and amino acid sequences.

[0407] In addition to being stimulatory receptors (i.e. transmitting a costimulatory signal to an immune cell), HHLA2 polypeptides are inhibitory receptors capable of transmitting an inhibitory signal to an immune cell to thereby inhibit immune cell effector function, or are capable of promoting costimulation of immune cells, e.g., when bound to inhibitory receptors or stimulatory receptors. HHLA2 bind to one or more receptors, e.g, TMIGD2, KIR3DL3, and / or other polypeptides on T-cells.

[0408] The term“HHLA2 activity,” includes the ability of a HHLA2 polypeptide to modulate an inhibitory signal in an activated immune cell, e.g, by engaging a natural HHLA2 ligand on a T-cell. Modulation of an inhibitory signal in an immune cell results in modulation of proliferation of, and / or cytokine secretion by, an immune cell. Thus, the term“HHLA2 activity” includes the ability of a HHLA2 polypeptide to bind its natural ligand(s), the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate the immune response. In some embodiments, a condition such as cancer is responsive to HHLA2 blockade alone. In other embodiments, a condition such as cancer is responsive to HHLA2 blockade alone, but is significantly or synergistically more responsive when treated with HHLA2 blockade and another therapy in combination. Many conditions responsive to HHLA2 blockade alone or in combination include, without limitation, 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, gastric, head and neck, renal, prostate, gynecologic, colorectal, ovary, and hematologic cancers.

[0409] Preferred B7 polypeptides are capable of providing costimulatory or inhibitory signals to immune cells to thereby promote or inhibit immune cell responses. For example, B7 family members that bind to costimulatory receptors increase T cell activation and proliferation, while B7 family members that bind to inhibitory receptors reduce

[0410] costimulation. Moreover, the same B7 family member may increase or decrease T cell costimulation. For example, when bound to a costimulatory receptor, HHLA2 can induce costimulation of immune cells or when bound to an inhibitory receptor, HHLA2 can inhibit immune cells. When bound to an inhibitory receptor, HHLA2 can transmit an inhibitory signal to an immune cell. Preferred B7 family members include HHLA2, B7-1, B7-2, B7h, PD-L1 or PD-L2 and soluble fragments or derivatives thereof. In one embodiment, B7 family members bind to one or more receptors on an immune cell, e.g., TMIGD2,

[0411] KIR3DL3, CTLA4, CD28, ICOS, PD-l and / or other receptors, and, depending on the receptor, have the ability to transmit an inhibitory signal or a costimulatory signal to an immune cell, preferably a T cell.

[0412] Modulation of a costimulatory signal results in modulation of effector function of an immune cell. Thus, the term“HHLA2 ligand activity” includes the ability of a HHLA2 ligand polypeptide to bind its natural receptor(s) (e.g. HHLA2), the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate the immune response.

[0413] It is demonstrated herein that the HHLA2 pathway is a negative regulator or a positive regulator of immune function, such that modulating the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3 can modulate immune function. HHLA2 binding to TMIGD2 can be a positive regulator of immune function. HHLA2 binding to inhibitory receptors is a negative regulator of immune function. HHLA2 binding to TMIGD2 is believed to be similar to HHLA2 binding to inhibitory receptors. Therefore, inhibiting HHLA2 binding to TMIGD2 is believed to inhibit HHLA2 binding to inhibitory receptors. Thus, the agents of the present invention described herein that are HHLA2 pathway modulators (e.g, modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3) modulate the interaction between HHLA2 and one or more natural binding partners, whether directly or indirectly, can upregulate or downregulate the immune system and, thereby, upregulate or downregulate an immune response. Agents that modulate such an interaction can do so either directly or indirectly.

[0414] The interaction between HHLA2 and one or more HHLA2 natural binding partners, such as TMIGD2 and / or KIR3DL3, results in the delivery of a co-stimulatory or co- inhibitory immune signal. Thus, in one embodiment, agents which directly block such an interaction(s) (e.g, anti-HHLA2, anti-TMIGD2, and / or anti-KIR3DL3 blocking antibodies) can prevent inhibitory or stimulatory signaling and upregulate or downregulate an immune response. Alternatively, agents that indirectly block the interaction(s) can prevent inhibitory signaling and upregulate an immune response. Exemplary agents for

[0415] upregulating an immune response include antibodies against HHLA2 or KIR3DL3 that block the interaction between HHLA2 and KIR3DL3; a non-activating form of HHLA2 or KIR3DL3 (e.g, a dominant negative polypeptide), small molecules or peptides that block the interaction between HHLA2 and KIR3DL3; fusion proteins (e.g, the extracellular portion of HHLA2 or KIR3DL3 fused to the Fc portion of an antibody or immunoglobulin) that bind to HHLA2 or KIR3DL3, respectively, and inhibit the interaction between HHLA2 and KIR3DL3; nucleic acid molecules and / or genetic modifications that block HHLA2 and / or KIR3DL3 transcription or translation; a non-activating form of a natural HHLA2 ligand, and a soluble form of a natural KIR3DL3 ligand.

[0416] In other exemplary embodiments, agents that promote the binding of a HHLA2 polypeptide to one or more natural binding partners, such as KIR3DL3 polypeptide, promote an inhibitory signal to an immune cell. Agents that modulate such an interaction can do so either directly or indirectly. Thus, in one embodiment, agents which directly enhance the interaction between HHLA2 and KIR3DL3 (HHLA2 agonists and / or KIR3DL3 agonists) can promote inhibitory signaling and downregulate an immune response.

[0417] Alternatively, agents that block KIR3DL3 binding to other targets increase the effective concentration of KIR3DL3 available to bind to HHLA2. Exemplary agents for

[0418] downregulating an immune response include antibodies against HHLA2 or KIR3DL3 that activate or promote the interaction between HHLA2 and KIR3DL3; small molecules or peptides that activate or promote the interaction between HHLA2 and KIR3DL3; and blocking antibodies that bind natural binding partners of HHLA2 and KIR3DL3 other than HHLA2 and KIR3DL3, respectively. These relationships also apply for HHLA2 and TMIGD2 interactions.

[0419] Additional agents useful in the methods of the present invention include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and / or activate or inhibit protein biomarkers of the present invention, including the biomarkers listed in Table 1, or fragments thereof; RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and / or activity of the biomarkers of the present invention, including the biomarkers listed in Table 1, or fragments thereof.

[0420] Isolated monoclonal antibodies or fragments thereof that are directed against HHLA2 are provided. In some embodiments, mAbs produced by hybridomas have been deposited at the American Type Culture Collection (ATCC), in accordance with the terms of Budapest Treaty, on _ , under deposit numbers _ .

[0421] Since it is well-known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity / affmity of an antibody for an antigen, the recombinant monoclonal antibodies of the present invention prepared as set forth above preferably comprise the heavy and light chain CDR3s of variable regions of the present invention ( e.g ., including the sequences of Table 2, or portions thereof). The antibodies further can comprise the CDR2s of variable regions of the present invention (e.g., including the sequences of Table 2, or portions thereof). The antibodies further can comprise the CDRls of variable regions of the present invention (e.g, including the sequences of Table 2, or portions thereof). In other embodiments, the antibodies can comprise any combinations of the CDRs.

[0422] The CDR1, 2, and / or 3 regions of the engineered antibodies described above can comprise the exact amino acid sequence(s) as those of variable regions of the present invention (e.g, including the sequences of Table 2, or portions thereof) disclosed herein. However, the ordinarily skilled artisan will appreciate that some deviation from the exact CDR sequences may be possible while still retaining the ability of the antibody to bind HHLA2 effectively (e.g., conservative sequence modifications). Accordingly, in another embodiment, the engineered antibody may be composed of one or more CDRs that are, for example, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to one or more CDRs of the present invention (e.g., including the sequences of Table 2, or portions thereof).

[0423] The structural features of known, non-human or human antibodies (e.g, a mouse or a non-rodent anti-human HHLA2 antibody) can be used to create structurally related human anti-human HHLA2 antibodies that retain at least one functional property of the antibodies of the present invention, such as binding of HHLA2 (such as mAh 8A12 and polyclonal antibodies 1.2 and 2.2). Another functional property includes inhibiting binding of the original known, non-human or human antibodies in a competition ELISA assay.

[0424] In some embodiments, monoclonal antibodies capable of binding human HHLA2 (such as mAh 8A12 and polyclonal antibodies 1.2 and 2.2) are provided, comprising a heavy chain wherein the variable domain comprises at least a CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical from the group of heavy chain variable domain CDRs presented in Table 2.

[0425] Similarly, monoclonal antibodies capable of binding human HHLA2 (such as mAh 8A12 and polyclonal antibodies 1.2 and 2.2), comprising a light chain wherein the variable domain comprises at least a CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical from the group of light chain variable domain CDRs presented in Table 2, are also provided.

[0426] Monoclonal antibodies capable of binding human HHLA2 (such as mAh 8A12 and polyclonal antibodies 1.2 and 2.2), comprising a heavy chain wherein the variable domain comprises at least a CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical from the group of heavy chain variable domain CDRs presented in Table 2; and comprising a light chain wherein the variable domain comprises at least a CDR having a sequence that is at least 80%, 85%,

[0427] 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical from the group of light chain variable domain CDRs presented in Table 2, are also provided.

[0428] A skilled artisan will note 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 conservative amino acid substitutions within a given CDR. The monoclonal antibodies of the present invention can comprise a heavy chain, wherein the variable domain comprises at least a CDR having a sequence selected from the group consisting of the heavy chain variable domain CDRs presented in Table 2 and a light chain, wherein the variable domain comprises at least a CDR having a sequence selected from the group consisting of the light chain variable domain CDRs presented in Table 2.

[0429] Such monoclonal antibodies can comprise a light chain, wherein the variable domain comprises at least a CDR having a sequence selected from the group consisting of CDR-L1, CDR-L2, and CDR-L3, as described herein; and / or a heavy chain, wherein the variable domain comprises at least a CDR having a sequence selected from the group consisting of CDR-H1, CDR-H2, and CDR-H3, as described herein. In some

[0430] embodiments, the monoclonal antibodies capable of binding human HHLA2 comprises or consists of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, as described herein.

[0431] The heavy chain variable domain of the monoclonal antibodies of the present invention can comprise or consist of the vH amino acid sequence set forth in Table 2 and / or the light chain variable domain of the monoclonal antibodies of the present invention can comprise or consist of the VK amino acid sequence set forth in Table 2.

[0432] The monoclonal antibodies of the present invention can be produced and modified by any technique well-known in the art. For example, such monoclonal antibodies can be murine or non-rodent antibodies, such as those obtainable from the hybridoma deposited on

[0433] _ with the ATCC as deposit _ . Similarly, such monoclonal antibodies can be chimeric, preferably chimeric mouse / human antibodies. In some embodiments, the monoclonal antibodies are humanized antibodies such that the variable domain comprises human acceptor frameworks regions, and optionally human constant domain where present, and non-human donor CDRs, such as mouse or non-rodent CDRs as defined above.

[0434] The present invention further provides fragments of said monoclonal antibodies which include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 and diabodies; and multispecific antibodies formed from antibody fragments. For example, a number of immunoinhibitory molecules, such as HHLA2, PD-L2, PD-L1, CTLA-4, KIR3DL3, and the like, can be detected in a bispecific or multispecific manner in order to efficiently characterize the expression of such molecules.

[0435] Other fragments of the monoclonal antibodies of the present invention are also contemplated. For example, individual immunoglobulin heavy and / or light chains are provided, wherein the variable domains thereof comprise at least a CDR presented in Table 2. In one embodiment, the immunoglobulin heavy chain comprises at least a CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical from the group of heavy chain or light chain variable domain CDRs presented in Table 2. In another embodiment, an immunoglobulin light chain comprises at least a CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical from the group of light chain or heavy chain variable domain CDRs described herein ( e.g ., presented in Table 2)·

[0436] In some embodiments, the immunoglobulin heavy and / or light chain comprises a variable domain comprising at least one of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR- H2, or CDR-H3 described herein. Such immunoglobulin heavy chains can comprise or consist of at least one of CDR-H1, CDR-H2, and CDR-H3. Such immunoglobulin light chains can comprise or consist of at least one of CDR-L1, CDR-L2, and CDR-L3.

[0437] In other embodiments, an immunoglobulin heavy and / or light chain according to the present invention comprises or consists of a vH or VK variable domain sequence, respectively, provided in Table 2.

[0438] The present invention further provides polypeptides which have a sequence selected from the group consisting of vH variable domain, VK variable domain, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences described herein.

[0439] Antibodies, immunoglobulins, and polypeptides of the invention can be use in an isolated (e.g., purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).

[0440] Table 2: Identification and sequencing of the leader and variable regions of anti-HHLA2 monoclonal antibodies including mAbs 8A12, 6D10, 6F10, 8D2, 2G2, 2C4, 4D1, 1C8, and

[0441] 4E5

[0442] 8A12 Heavy Chain Variable (vH) DNA and Amino Acid Sequences*

[0443] MHC2554HC .1 ;M13 499.5.8A12.11.10.5

[0444] CDR Analysis

[0445] GFTFNTNV...._IRTKTNNYAT_VGAMDY

[0446] Amino Acid Sequence in FASTA format (MHC2554HC .1\;M13F)

[0447] > MHC2554HC .1\ ; M13F

[0448] LOCUS 8A12_vH 402 bp DNA linear FEATURES Location / Qualifiers

[0449] J_segment 370..402

[0450] / label=FWR4

[0451] V_segment 358..369

[0452] / label=CDR3

[0453] V_region 235..357

[0454] / label=FWR3

[0455] V_segment 211..234

[0456] / label=CDR2

[0457] V_region 154..210

[0458] / label=FWR2

[0459] V_segment 133..153

[0460] / label=CDRl

[0461] V_region 58..132

[0462] / label=FWRl

[0463] sig_peptide 1..57

[0464] / label=LS

[0465] CDS 1..402

[0466] / label=8A12 vH

[0467] Nucleotide Sequence in FASTA format (MHC2554HC .1\;M13F)

[0468] > MHC2554HC .1\ ; M13F . 499.5.8A12.11.10.5

[0469] ORIGIN

[0470] 1 ATGCTGTTGG GGCTGAAGTG GATTTTCTTT GTTGTTTTTT ATCAAGGTGT GCATTGTGAG 61 GTGCAACTTG TTGAGACTGG TGGAGGATTG GTGCAGCCTA AAGGGTCATT GAAACTCTCA 121 TGTGCAGCCT CTGGATTCAC CTTCAACACC AATGTCATGA ACTGGGTCCG CCAGGCTCCA 181 GGAAAGGGTT TGGAATGGGT TGGTCGCATA AGAACTAAAA CTAATAATTA TGCAACATAT 241 TATGCCGATT CAGTGAAAGG CAGGTTCACC ATCTCCAGAG ATGATTCACA AAGTATGCTC 301 TATCTGCAAA TGAACAACTT GAAAACTGAG GACACAGCCA CGTATTTCTG TGTGGGAGCT 361 ATGGACTACT GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CA (SEQ ID NO : 20)

[0471] Signal Peptide (base pairs 1-57) :

[0472] 1 ATGCTGTTGG GGCTGAAGTG GATTTTCTTT GTTGTTTTTT ATCAAGGTGT GCATTGT 57 (SEQ ID NO: 21)

[0473] / translation="MLLGLKWIFFWFYQGVHC" (SEQ ID NO: 22)

[0474] Framework 1 (base pairs 58-132) :

[0475] 58 GAG GTGCAACTTG TTGAGACTGG TGGAGGATTG GTGCAGCCTA AAGGGTCATT

[0476] GAAACTCTCA TGTGCAGCCT CT 132 (SEQ ID NO: 23)

[0477] / translation="EVQLVETGGGLVQPKGSLKLSCAAS" (SEQ ID NO: 24) CDR-H1 (base pairs 133-153) :

[0478] 133 GGATTCAC CTTCAACACC AAT 153 (SEQ ID NO: 25)

[0479] / translation="GFTFNTN" (SEQ ID NO: 26)

[0480] Framework 2 (base pairs 154-210) :

[0481] 154 GTCATGA ACTGGGTCCG CCAGGCTCCA GGAAAGGGTT TGGAATGGGT TGGTCGCATA 210 (SEQ ID NO: 27)

[0482] / translation="VMNWVRQAPGKGLEWVGRI" (SEQ ID NO: 28)

[0483] CDR-H2 (base pairs 211-234) :

[0484] 211 AGAACTAAAA CTAATAATTA TGCA 234 (SEQ ID NO: 29)

[0485] / translation="RTKTNNYA" (SEQ ID NO: 30)

[0486] Framework 3 (base pairs 235-357) :

[0487] 235 ACATAT TATGCCGATT CAGTGAAAGG CAGGTTCACC ATCTCCAGAG ATGATTCACA AAGTATGCTC TATCTGCAAA TGAACAACTT GAAAACTGAG GACACAGCCA CGTATTTCTG TGTGGGA 357 (SEQ ID NO: 31)

[0488] / translation="TYYADSVKGRFTI SRDDSQSMLYLQMNNLKTEDTATYFCVG" (SEQ ID NO: 32)

[0489] CDR-H3 (base pairs 358-369) :

[0490] 358 GCT ATGGACTAC 369 (SEQ ID NO: 33)

[0491] / translation="AMDY" (SEQ ID NO: 34)

[0492] Framework 4 (base pairs 370-402):

[0493] 370 T GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CA 402 (SEQ ID NO: 35)

[0494] / translation="WGQGTSVTVSS" (SEQ ID NO: 36)

[0495] MHC2554HC .1 499.5.8A12.11.10.5

[0496]

[0497] 8A12 Light Chain Variable DNA and Amino Acid Sequences*

[0498]

[0499] MHC2554LC .2 ;M13. 499.5.8A12.11.10.5 CDR Analysis

[0500] ESVDNSGINF .. RAS . QQSYKDPPT

[0501] Amino Acid Sequence in FASTA format (MHC2554LC .2\;M13F)

[0502] > MHC2554LC .2\;M13F

[0503] LOCUS 8A12_vL 393 bp DNA linear

[0504] FEATURES Location / Qualifiers

[0505] J_segment 364..393

[0506] / label=FWR4

[0507] V_segment 337..363

[0508] / label=CDR3

[0509] V_region 241..336

[0510] / label=FWR3

[0511] V_segment 220..240

[0512] / label=CDR2

[0513] V_region 175..219

[0514] / label=FWR2

[0515] V_segment 130..174

[0516] / label=CDRl

[0517] V region 61..129

[0518] / label=FWRl

[0519] sig peptide 1..60

[0520] / label=LS

[0521] CDS 1..393

[0522] / label=8A12 vL

[0523] 8A12_vL

[0524] / translation="METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATVSCRASESVDNSGINF IHWYQQKPGQSPKLLLYRASNLKSGIPARFSGSGSRTDFTLTINPVETGDVATYYCQQSYKDPPTFGTGT KLELK" (SEQ ID NO: 37)

[0525] Nucleotide Sequence in FASTA format (MHC2554LC .2\;M13F)

[0526] > MHC2554LC .2\;M13F . 499.5.8A12.11.10.5

[0527] ORIGIN

[0528] 1 ATGGAGACAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG TTCCACAGGT 61 GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTGGGGCA GAGGGCCACC 121 GTCTCCTGCA GAGCCAGCGA AAGTGTTGAT AATTCTGGCA TAAATTTTAT ACACTGGTAC 181 CAGCAGAAAC CAGGACAGTC ACCCAAACTC CTCCTCTATC GTGCATCCAA CCTAAAATCT 241 GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAAT

[0529] 301 CCTGTGGAGA CTGGTGATGT TGCAACCTAT TACTGTCAGC AAAGTTATAA GGATCCTCCT 361 ACGTTCGGTA CTGGGACCAA GCTGGAGCTG AAG (SEQ ID NO: 38)

[0530] Signal Peptide (base pairs 1-60) :

[0531] 1 ATGGAGACAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG TTCCACAGGT 60 (SEQ ID NO: 39)

[0532] / translation="METDTLLLWVLLLWVPGSTG" (SEQ ID NO: 40)

[0533] Framework 1 (base pairs 61-129) :

[0534] 61 GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTGGGGCA GAGGGCCACC GTCTCCTGC 129 (SEQ ID NO: 41)

[0535] / translation="DIVLTQSPASLAVSLGQRATVSC" (SEQ ID NO: 42)

[0536] CDR-Ll (base pairs 130-174) :

[0537] 130 A GAGCCAGCGA AAGTGTTGAT AATTCTGGCA TAAATTTTAT ACAC 174 (SEQ ID NO: 43)

[0538] / translation="RASESVDNSGINFIH" (SEQ ID NO: 44)

[0539] Framework 2 (base pairs 175-219) :

[0540] 175 TGGTAC CAGCAGAAAC CAGGACAGTC ACCCAAACTC CTCCTCTAT 219 (SEQ ID NO: 45)

[0541] / translation="WYQQKPGQSPKLLLY" (SEQ ID NO: 46)

[0542] CDR-L2 (base pairs 220-240) :

[0543] 220 C GTGCATCCAA CCTAAAATCT 240 (SEQ ID NO: 47)

[0544] / translation="RASNLKS" (SEQ ID NO: 48)

[0545] Framework 3 (base pairs 241-336) :

[0546] 241 GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAAT CCTGTGGAGA CTGGTGATGT TGCAACCTAT TACTGT 336 (SEQ ID NO: 49)

[0547] / translation="GIPARFSGSGSRTDFTLTINPVETGDVATYYC" (SEQ ID NO: 50)

[0548] CDR-L3 (base pairs 337-363) :

[0549] 337 CAGC AAAGTTATAA GGATCCTCCT ACG 363 (SEQ ID NO: 51)

[0550] / translation="QQSYKDPPT" (SEQ ID NO: 52)

[0551] Framework 4 (base pairs 364-393):

[0552] 364 TTCGGTA CTGGGACCAA GCTGGAGCTG AAG 393 (SEQ ID NO: 53)

[0553] / translation="FGTGTKLELK" (SEQ ID NO: 54)

[0554] MHC2554LC .2. 499.5.8A12.11.10.5

[0555]

[0556] 6D10 Heavy Chain Variable (vH) DNA and Amino Acid Sequences*

[0557] MHC2555HC .1 ;M13. 499.5.6D10.11.11.7

[0558] CDR Analysis

[0559] GFTFNTNV...._IRTKTNNYAT_VGAMDY

[0560] Amino Acid Sequence in FASTA format (MHC2555HC .1\;M13F) > MHC2555HC .1\ ; M13F 499.5.6D10.11.11.7

[0561] LOCUS 6D10_vH 402 bp DNA linear

[0562] FEATURES Location / Qualifiers

[0563] J_segment 370..402

[0564] / label=FWR4

[0565] V_segment 358..369

[0566] / label=CDR3

[0567] V_region 235..357

[0568] / label=FWR3

[0569] V_segment 211..234

[0570] / label=CDR2

[0571] V_region 154..210

[0572] / label=FWR2

[0573] V_segment 133..153

[0574] / label=CDRl

[0575] V_region 58..132

[0576] / label=FWRl

[0577] sig_peptide 1..57

[0578] / label=LS

[0579] CDS 1..402

[0580] / label=6D10 vH

[0581] 6D10 vH / translation="MLLGLKWIFFWFYQGVHCEVQLVETGGGLVQPKGSLKLSCAASGFTFNTNVMNWV RQAPGKGLEWVARIRTKTNNYATYYADSVKDRFTI FRDDSQSILYLQMNNLKTEDTAMYYCVGAMDYWGQ

[0582] GTSVTVSS" (SEQ ID NO: 55)

[0583] Nucleotide Sequence in FASTA format (MHC2555HC .1\;M13F)

[0584] 499.5.6D10.11.11.7

[0585]

[0586] 1 ATGCTGTTGG GGCTGAAGTG GATTTTCTTT GTTGTTTTTT ATCAAGGTGT GCATTGTGAG 61 GTGCAGCTTG TTGAGACTGG TGGAGGATTG GTGCAGCCTA AAGGGTCATT GAAACTCTCA 121 TGTGCAGCCT CTGGATTCAC CTTCAATACC AATGTCATGA ACTGGGTCCG CCAGGCTCCA 181 GGAAAGGGTT TGGAATGGGT TGCTCGCATA AGAACTAAAA CTAATAATTA TGCAACATAT 241 TATGCCGATT CAGTGAAAGA CAGGTTCACC ATCTTCAGAG ATGATTCACA AAGCATTCTC 301 TATCTGCAAA TGAACAACTT GAAAACTGAG GACACAGCCA TGTATTACTG TGTGGGAGCT 361 ATGGACTACT GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CA(SEQ ID NO: 56)

[0587] Signal Peptide (base pairs 1-57) :

[0588] 1 ATGCTGTTGG GGCTGAAGTG GATTTTCTTT GTTGTTTTTT ATCAAGGTGT GCATTGT 57 (SEQ ID NO: 57)

[0589] / translation=" MLLGLKWIFFVVFYQGVHC" (SEQ ID NO: 58)

[0590] Framework 1 (base pairs 58-132) :

[0591] 58 GAG GTGCAGCTTG TTGAGACTGG TGGAGGATTG GTGCAGCCTA AAGGGTCATT

[0592] GAAACTCTCA TGTGCAGCCT CT 132 (SEQ ID NO: 59)

[0593] / translation="EVQLVETGGGLVQPKGSLKLSCAAS" (SEQ ID NO: 60)

[0594] CDR-H1 (base pairs 133-153) :

[0595] 133 GGATTCAC CTTCAATACC AAT 153 (SEQ ID NO: 61)

[0596] / translation="GFTFNTN" (SEQ ID NO: 62)

[0597] Framework 2 (base pairs 154-210) :

[0598] 154 GTCATGA ACTGGGTCCG CCAGGCTCCA GGAAAGGGTT TGGAATGGGT TGCTCGCATA 210 (SEQ ID NO: 63)

[0599] / translation="VMNWVRQAPGKGLEWVARI" (SEQ ID NO: 64)

[0600] CDR-H2 (base pairs 211-234) :

[0601] 211 AGAACTAAAA CTAATAATTA TGCA 234 (SEQ ID NO: 65)

[0602] / translation="RTKTNNYA" (SEQ ID NO: 66)

[0603] Framework 3 (base pairs 235-357) :

[0604] 235 ACATAT TATGCCGATT CAGTGAAAGA CAGGTTCACC ATCTTCAGAG ATGATTCACA AAGCATTCTC TATCTGCAAA TGAACAACTT GAAAACTGAG GACACAGCCA TGTATTACTG TGTGGGA 357 (SEQ ID NO: 67)

[0605] / translation="TYYADSVKDRFTI FRDDSQSILYLQMNNLKTEDTAMYYCVG" (SEQ ID NO:

[0606] 68)

[0607] CDR-H3 (base pairs 358-369) :

[0608] 358 GCT ATGGACTAC 369 (SEQ ID NO: 69)

[0609] / translation="AMDY" (SEQ ID NO: 70) Framework 4 (base pairs 370-402):

[0610] 370 T GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CA 402 (SEQ ID NO: 71) / translation="WGQGTSVTVSS" (SEQ ID NO: 72)

[0611] MHC2555HC .1 ;M13. 499.5.6D10.11.11.7

[0612]

[0613] 6D10 Light Chain Variable (vL) DNA and Amino Acid Sequences*

[0614] MHC2555LC .1 ;M13. 499.5.6D10.11.11.7

[0615] CDR Analysis

[0616] ESVDNYGISF .._RAS ..QQSSKDPPT

[0617] Amino Acid Sequence in FASTA format (MHC2555LC .1\;M13F) > MHC2555LC .1\ ; M13F . 499.5.6D10.11.11.7

[0618] LOCUS 6D10_vL 393 bp DNA linear

[0619] FEATURES Location / Qualifiers

[0620] J_segment 364..393

[0621] / label=FWR4

[0622] V_segment 337..363

[0623] / label=CDR3

[0624] V_region 241..336

[0625] / label=FWR3

[0626] V_segment 220..240

[0627] / label=CDR2

[0628] V_region 175..219

[0629] / label=FWR2

[0630] V_segment 130..174

[0631] / label=CDRl

[0632] V_region 61..129

[0633] / label=FWRl

[0634] sig_peptide 1..60

[0635] / label=LS

[0636] CDS 1..393 / label=6P10 vL

[0637] 6D10_vL

[0638] / translation="

[0639] METPTLLLWVLLLWVPGSTGPIVLTQS PASLAVSLGQRAT I SCRASESVDNYGI S FMHWYQQKPGQPPKl· LIYRASNLKSGIPARFSGSGSRTPFTLTINPVETGPVATYYCQQSSKPPPTFGTGTKLELK" (SEQ IP NO: 73)

[0640] Nucleotide Sequence in FASTA format (MHC2555LC .1\;M13F)

[0641] > MHC2555LC .1\ ; M13F . 499.5.6P10.11.11.7

[0642] ORIGIN

[0643] 1 ATGGAGACAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG TTCCACAGGT

[0644] 61 GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTAGGGCA GAGGGCCACC

[0645] 121 ATCTCCTGCA GAGCCAGCGA GAGTGTTGAT AATTATGGCA TTAGTTTTAT GCACTGGTAC

[0646] 181 CAGCAGAAAC CAGGACAGCC ACCCAAACTC CTCATCTATC GTGCATCCAA CCTAAAATCT

[0647] 241 GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAAT

[0648] 301 CCTGTGGAGA CTGGTGATGT TGCTACCTAT TACTGTCAGC AAAGTAGTAA GGATCCTCCT

[0649] 361 ACGTTCGGTA CTGGGACCAA GCTAGAGCTG AAA (SEQ IP NO: 74)

[0650] Signal Peptide (base pairs 1-60) :

[0651] 1 ATGGAGACAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG TTCCACAGGT 60 (SEQ IP NO: 75)

[0652] / translation="METQTLLLWVLLLWVPGSTG" (SEQ IP NO: 76)

[0653] Framework 1 (base pairs 61-129) :

[0654] 61 GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTAGGGCA GAGGGCCACC ATCTCCTGC 129 (SEQ IP NO: 77)

[0655] / translation="QIVLTQSPASLAVSLGQRATI SC" (SEQ IP NO: 78)

[0656] CPR-L1 (base pairs 130-174) :

[0657] 130 A GAGCCAGCGA GAGTGTTGAT AATTATGGCA TTAGTTTTAT GCAC 174 (SEQ IP NO: 79)

[0658] / translation="RASESVQNYGISFMH" (SEQ IP NO: 80)

[0659] Framework 2 (base pairs 175-219) :

[0660] 175 TGGTAC CAGCAGAAAC CAGGACAGCC ACCCAAACTC CTCATCTAT 219 (SEQ IP NO: 81)

[0661] / translation="WYQQKPGQPPKLLIY" (SEQ IP NO: 82)

[0662] CPR-L2 (base pairs 220-240) :

[0663] 220 C GTGCATCCAA CCTAAAATCT 240 (SEQ IP NO: 83)

[0664] / translation="RASNLKS" (SEQ IP NO: 84) Framework 3 (base pairs 241-336) :

[0665] 241 GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAAT CCTGTGGAGA CTGGTGATGT TGCTACCTAT TACTGT 336 (SEQ ID NO: 85)

[0666] / translation="GIPARFSGSGSRTDFTLTINPVETGDVATYYC" (SEQ ID NO: 86)

[0667] CDR-I3 (base pairs 337-363) :

[0668] 337 CAGC AAAGTAGTAA GGATCCTCCT ACG 363 (SEQ ID NO: 87)

[0669] / translation="QQSSKDPPT" (SEQ ID NO: 88)

[0670] Framework 4 (base pairs 364-393):

[0671] 364 TTCGGTA CTGGGACCAA GCTAGAGCTG AAA 393 (SEQ ID NO: 89)

[0672] / translation="FGTGTKLELK" (SEQ ID NO: 90)

[0673] MHC2555LC .1 499.5.6D10.11.11.7

[0674]

[0675] 6F10 Heavy Chain Variable DNA and Amino Acid Sequences’’

[0676]

[0677] MHC2557HCO .1 ; Ml3. 499.5.6F10. A4.10

[0678] CDR Analysis

[0679] GYTFTTYT ...._INPSSGYT .._ ARHPWDSDY

[0680] Amino Acid Sequence in FASTA format (MHC2557HCO .1\ ; M13F)

[0681] > MHC2557HCO.1\;M13F. 499.5.6F10.A4.10

[0682] LOCUS 6F10_vH 405 bp DNA linear

[0683] FEATURES Location / Qualifiers

[0684] J segment 373..405

[0685] / label=FWR4

[0686] V_segment 352..372

[0687] / label=CDR3

[0688] V_region 229..351

[0689] / label=FWR3

[0690] V segment 211..228 / label=CDR2

[0691] V_region 154..210

[0692] / label=FWR2

[0693] V_segment 133..153

[0694] / label=CDRl

[0695] V_region 58..132

[0696] / label=FWRl

[0697] sig_peptide 1..57

[0698] / label=LS

[0699] CDS 1..405

[0700] / label=6F10 vH

[0701] 6F10_vH

[0702] / translation="MERHWIFLFLLSVTAGVHSQVHLQQSAAELARPGASVKMSCKASGYTFTTYTMHWV KQRPGQGl·EWIGHINPSSGYTDYNQKFKDKTTl·TADKSSSTAYMQl·NSl·TSEDSAVYYCARHPWDSDYWG QGTTLTVSS" (SEQ ID NO: 91)

[0703] Nucleotide Sequence in FASTA format (MHC2557HCO .1\ ; M13F)

[0704] > MHC2557HCO.1\;M13F. 499.5.6F10. A4.10

[0705] ORIGIN

[0706] 1 ATGGAAAGGC ACTGGATCTT TCTCTTCCTG TTGTCAGTAA CTGCAGGTGT CCACTCCCAG 61 GTCCACCTGC AGCAGTCTGC AGCTGAACTG GCAAGACCTG GGGCCTCAGT GAAGATGTCC 121 TGCAAGGCTT CTGGCTACAC CTTTACTACC TACACGATGC ACTGGGTAAA ACAGAGGCCT 181 GGACAGGGTC TGGAATGGAT TGGACACATT AATCCTAGCA GTGGATATAC TGATTACAAT 241 CAGAAATTCA AGGACAAGAC CACATTGACT GCAGACAAAT CCTCCAGTAC AGCCTACATG 301 CAACTGAACA GCCTGACATC TGAGGACTCT GCGGTCTATT ACTGTGCAAG ACACCCCTGG 361 GACTCGGACT ACTGGGGCCA AGGCACCACT CTCACAGTCT CCTCA (SEQ ID NO: 92)

[0707] Signal Peptide (base pairs 1-57) :

[0708] 1 ATGGAAAGGC ACTGGATCTT TCTCTTCCTG TTGTCAGTAA CTGCAGGTGT CCACTCC 57 (SEQ ID NO: 93)

[0709] / translation="MERHWIFLFLLSVTAGVHS" (SEQ ID NO: 94)

[0710] Framework 1 (base pairs 58-132) :

[0711] 58 CAG GTCCACCTGC AGCAGTCTGC AGCTGAACTG GCAAGACCTG GGGCCTCAGT

[0712] GAAGATGTCC TGCAAGGCTT CT 132 (SEQ ID NO: 95)

[0713] / translation="QVHLQQSAAELARPGASVKMSCKAS" (SEQ ID NO: 96)

[0714] CDR-H1 (base pairs 133-153) :

[0715] 133 GGCTACAC CTTTACTACC TAC 153 (SEQ ID NO: 97)

[0716] / translation="GYTFTTY" (SEQ ID NO: 98)

[0717] Framework 2 (base pairs 154-210) :

[0718] 154 ACGATGC ACTGGGTAAA ACAGAGGCCT

[0719] 181 GGACAGGGTC TGGAATGGAT TGGACACATT 210 (SEQ ID NO: 99)

[0720] / translation="TMHWVKQRPGQGLEWIGHI" (SEQ ID NO: 100) CDR-H2 (base pairs 211-228) :

[0721] 211 AATCCTAGCA GTGGATAT 228 (SEQ ID NO: 101)

[0722] / translation="NPSSGY" (SEQ ID NO: 102)

[0723] Framework 3 (base pairs 229-351) :

[0724] 229 AC TGATTACAAT CAGAAATTCA AGGACAAGAC CACATTGACT GCAGACAAAT

[0725] CCTCCAGTAC AGCCTACATG CAACTGAACA GCCTGACATC TGAGGACTCT GCGGTCTATT ACTGTGCAAG A 351 (SEQ ID NO: 103)

[0726] / translation="TDYNQKFKDKTTLTADKSSSTAYMQLNSLTSEDSAVYYCAR" (SEQ ID NO: 104)

[0727] CDR-H3 (base pairs 352-372) :

[0728] 352 CACCCCTGG GACTCGGACT AC 372 (SEQ ID NO: 105)

[0729] / translation="HPWDSDY" (SEQ ID NO: 106)

[0730] Framework 4 (base pairs 373-405):

[0731] 373 TGGGGCCA AGGCACCACT CTCACAGTCT CCTCA 405 (SEQ ID NO: 107)

[0732] / translation="WGQGTTLTVSS" (SEQ ID NO: 108)

[0733] MHC2557HCO .1 499.5.6F10. A4.10

[0734]

[0735] 6F10 Light Chain Variable DNA and Amino Acid Sequences*

[0736]

[0737] MHC2557LC .1;M13.

[0738] CDR Analysis

[0739] ENIDSY ..AAT ..QHYYITPFT

[0740] Amino Acid Sequence in FASTA format (MHC2557LC .1\;M13F)

[0741] > MHC2557LC .1\ ; M13F . 499.5.6F10.A4.10

[0742] LOCUS 6F10_vL 381 bp DNA linear

[0743] FEATURES Location / Qualifiers

[0744] J_segment 352..381

[0745] / label=FWR4

[0746] V_segment 325..351

[0747] / label=CDR3

[0748] V_region 229..324 / label=FWR3

[0749] V_segment 208..228

[0750] / label=CDR2

[0751] V_region 163..207

[0752] / label=FWR2

[0753] V_segment 130..162

[0754] / label=CDRl

[0755] V_region 61..129

[0756] / label=FWRl

[0757] sig_peptide 1..60

[0758] / label=LS

[0759] CDS 1..381

[0760] / label=6F10 vL

[0761] 6F10_vL

[0762] / translation="MSVPTQLLGLLLLWLTDARCDIQMTQSPASLSASVGETVTITCRASENIDSYLAWY

[0763] Nucleotide Sequence in FASTA format (MHC2557LC .1\;M13F)

[0764] > MHC2557LC .1\ ; M13F . 499.5.6F10. A4.10

[0765] ORIGIN

[0766] 1 ATGAGTGTGC CCACTCAGCT CCTGGGGTTG CTGCTGCTGT GGCTTACAGA TGCCAGATGT

[0767] 61 GACATCCAGA TGACTCAGTC TCCAGCTTCC CTGTCTGCAT CTGTGGGAGA AACTGTCACC

[0768] 121 ATCACATGTC GAGCAAGTGA GAATATTGAC AGTTATTTAG CATGGTATCA GCAGAAACAG

[0769] 181 GGAAGATCTC CTCAGCTCCT GGTCTATGCT GCAACAAACT TAGCAGATGG TGTGCCATCA

[0770] 241 AGGTTCAGTG GCAGTGGATC AGGCACACAG TTTTCTCTCC AGATCAACCG CCTGCAGTCT

[0771] 301 GAAGATGTTG CGAGATATTA CTGTCAACAT TATTATATTA CTCCATTCAC GTTCGGCTCG

[0772] 361 GGGACAAAAT TGGAAATAGC A (SEQ ID NO: 110)

[0773] Signal Peptide (base pairs 1-60) :

[0774] 1 ATGAGTGTGC CCACTCAGCT CCTGGGGTTG CTGCTGCTGT GGCTTACAGA TGCCAGATGT 60 (SEQ ID NO: 111)

[0775] / translation="MSVPTQLLGLLLLWLTDARC" (SEQ ID NO: 112)

[0776] Framework 1 (base pairs 61-129) :

[0777] 61 GACATCCAGA TGACTCAGTC TCCAGCTTCC CTGTCTGCAT CTGTGGGAGA AACTGTCACC ATCACATGT 129 (SEQ ID NO: 113)

[0778] / translation="DIQMTQSPASLSASVGETVTITC" (SEQ ID NO: 114)

[0779] CDR-L1 (base pairs 130-162) :

[0780] 130 C GAGCAAGTGA GAATATTGAC AGTTATTTAG CA 162 (SEQ ID NO: 115)

[0781] / translation="RASENIDSYLA" (SEQ ID NO: 116)

[0782] Framework 2 (base pairs 163-207) : 163 TGGTATCA GCAGAAACAG GGAAGATCTC CTCAGCTCCT GGTCTAT 207 (SEQ ID NO: 117)

[0783] / translation="WYQQKQGRSPQLLVY" (SEQ ID NO: 118)

[0784] CDR-I2 (base pairs 208-228) :

[0785] 208 GCT GCAACAAACT TAGCAGAT 228 (SEQ ID NO: 119)

[0786] / translation="AATNLAD" (SEQ ID NO: 120)

[0787] Framework 3 (base pairs 229-324) :

[0788] 229 GG TGTGCCATCA AGGTTCAGTG GCAGTGGATC AGGCACACAG TTTTCTCTCC

[0789] AGATCAACCG CCTGCAGTCT GAAGATGTTG CGAGATATTA CTGT 324 (SEQ ID NO: 121) / translation="GVPSRFSGSGSGTQFSLQINRLQSEDVARYYC" (SEQ ID NO: 122)

[0790] CDR-L3 (base pairs 325-351) :

[0791] 325 CAACAT TATTATATTA CTCCATTCAC G 351 (SEQ ID NO: 123)

[0792] / translation="QHYYITPFT" (SEQ ID NO: 124)

[0793] Framework 4 (base pairs 352-381):

[0794] 352 TTCGGCTCG GGGACAAAAT TGGAAATAGC A 381 (SEQ ID NO: 125)

[0795] / translation="FGSGTKLEIA" (SEQ ID NO: 126)

[0796] MHC2557LC .1 499.5.6F10.A4.10

[0797]

[0798] 8D2 Heavy Chain Variable (vH) DNA and Amino Acid Sequences*

[0799] MHC2558HC .1 ; Ml3. 499.8D2.6.8

[0800] CDR Analysis

[0801] GFTFNTNV...._IRTKTNNYAT_VGAMDY

[0802] Amino Acid Sequence in FASTA format (MHC2558HC .1\;M13F)

[0803] > MHC2558HC .1\;M13F . 499.8D2.6.8

[0804] LOCUS 8D2_vH 402 bp DNA linear

[0805] FEATURES Location / Qualifiers

[0806] J_segment 370..402

[0807] / label=FWR4

[0808] V_segment 358..369

[0809] / label=CDR3 V_region 235..357

[0810] / label=FWR3

[0811] V_segment 211..234

[0812] / label=CDR2

[0813] V_region 154..210

[0814] / label=FWR2

[0815] V_segment 133..153

[0816] / label=CDRl

[0817] V_region 58..132

[0818] / label=FWRl

[0819] sig_peptide 1..57

[0820] / label=LS

[0821] CDS 1..402

[0822] / label=8D2 vH

[0823] 8D2_vH

[0824] / translation="MLLGLKWIFFWFYQGVHCEVQLVETGGGLVQPKGSLKLSCAASGFTFNTNVMNWV RQAPGKGLEWVGRIRTKTNNYATYYADSVKGRFTI SRDDSQSMLYLQMNNLKTEDTATYFCVGAMDYWGQ

[0825] GTSVTVSS" (SEQ ID NO: 127)

[0826] Nucleotide Sequence in FASTA format (MHC2558HC .1\;M13F)

[0827] > MHC2558HC .1\;M13F . 499.8D2.6.8

[0828] ORIGIN

[0829] 1 ATGCTGTTGG GGCTGAAGTG GATTTTCTTT GTTGTTTTTT ATCAAGGTGT GCATTGTGAG 61 GTGCAACTTG TTGAGACTGG TGGAGGATTG GTGCAGCCTA AAGGGTCATT GAAACTCTCA 121 TGTGCAGCCT CTGGATTCAC CTTCAACACC AATGTCATGA ACTGGGTCCG CCAGGCTCCA 181 GGAAAGGGTT TGGAATGGGT TGGTCGCATA AGAACTAAAA CTAATAATTA TGCAACATAT 241 TATGCCGATT CAGTGAAAGG CAGGTTCACC ATCTCCAGAG ATGATTCACA AAGTATGCTC 301 TATCTGCAAA TGAACAACTT GAAAACTGAG GACACAGCCA CGTATTTCTG TGTGGGAGCT 361 ATGGACTACT GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CA (SEQ ID NO: 128)

[0830] Signal Peptide (base pairs 1-57) :

[0831] 1 ATGCTGTTGG GGCTGAAGTG GATTTTCTTT GTTGTTTTTT ATCAAGGTGT GCATTGT 57

[0832] (SEQ ID NO: 129)

[0833] / translation="MLLGLKWIFFWFYQGVHC" (SEQ ID NO: 130)

[0834] Framework 1 (base pairs 58-132) :

[0835] 58 GAG GTGCAACTTG TTGAGACTGG TGGAGGATTG GTGCAGCCTA AAGGGTCATT

[0836] GAAACTCTCA TGTGCAGCCT CT 132 (SEQ ID NO: 131)

[0837] / translation="EVQLVETGGGLVQPKGSLKLSCAAS" (SEQ ID NO: 132)

[0838] CDR-H1 (base pairs 133-153) :

[0839] 133 GGATTCAC CTTCAACACC AAT 153 (SEQ ID NO: 133)

[0840] / translation="GFTFNTN" (SEQ ID NO: 134)

[0841] Framework 2 (base pairs 154-210) : 154 GTCATGA ACTGGGTCCG CCAGGCTCCA GGAAAGGGTT TGGAATGGGT TGGTCGCATA 210 (SEQ ID NO: 135)

[0842] / translation="VMNWVRQAPGKGLEWVGRI" (SEQ ID NO: 136)

[0843] CDR-H2 (base pairs 211-234) :

[0844] 211 AGAACTAAAA CTAATAATTA TGCA 234 (SEQ ID NO: 137)

[0845] / translation="RTKTNNYA" (SEQ ID NO: 138)

[0846] Framework 3 (base pairs 235-357) :

[0847] 235 ACATAT TATGCCGATT CAGTGAAAGG CAGGTTCACC ATCTCCAGAG ATGATTCACA AAGTATGCTC TATCTGCAAA TGAACAACTT GAAAACTGAG GACACAGCCA CGTATTTCTG TGTGGGA 357 (SEQ ID NO: 139)

[0848] / translation="TYYADSVKGRFTI SRDDSQSMLYLQMNNLKTEDTATYFCVG" (SEQ ID NO: 140)

[0849] CDR-H3 (base pairs 358-369) :

[0850] 358 GCT ATGGACTAC 369 (SEQ ID NO: 141)

[0851] / translation="AMDY" (SEQ ID NO: 142)

[0852] Framework 4 (base pairs 370-402):

[0853] 370 T GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CA 402 (SEQ ID NO: 143)

[0854] / translation="WGQGTSVTVSS" (SEQ ID NO: 144)

[0855] MHC2558HC .1 499.8D2.6.8

[0856]

[0857] 8D2 Light Chain Variable DNA and Amino Acid Sequences*

[0858]

[0859] MHC2558LC .2 ;M13. 499.8D2.6.8

[0860] CDR Analysis

[0861] ESVDNSGINF .._RAS ..QQSYKDPPT

[0862] Amino Acid Sequence in FASTA format (MHC2558LC .2\;M13F)

[0863] > MHC2558LC .2\;M13F . 499.8D2.6.8

[0864] LOCUS 8D2_vL 393 bp DNA linear

[0865] FEATURES Location / Qualifiers J_segment 364..393

[0866] / label=FWR4

[0867] V_segment 337..363

[0868] / label=CDR3

[0869] V_region 241..336

[0870] / label=FWR3

[0871] V_segment 220..240

[0872] / label=CDR2

[0873] V_region 175..219

[0874] / label=FWR2

[0875] V_segment 130..174

[0876] / label=CDRl

[0877] V_region 61..129

[0878] / label=FWRl

[0879] sig_peptide 1..60

[0880] / label=LS

[0881] CDS 1..393

[0882] / label=8D2 vL

[0883] IHWYQQKPGQSPKLLLYRASNLKSGIPARFSGSGSRTDFTLTINPVETGDVATYYCQQSYKDPPTFGTGT

[0884] KLELK" (SEQ ID NO: 145)

[0885] Nucleotide Sequence in FASTA format (MHC2558LC .2\;M13F)

[0886] > MHC2558LC .2\;M13F . 499.8D2.6.8

[0887] ORIGIN

[0888] 1 ATGGAGACAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG TTCCACAGGT 61 GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTGGGGCA GAGGGCCACC 121 GTCTCCTGCA GAGCCAGCGA AAGTGTTGAT AATTCTGGCA TAAATTTTAT ACACTGGTAC 181 CAGCAGAAAC CAGGACAGTC ACCCAAACTC CTCCTCTATC GTGCATCCAA CCTAAAATCT 241 GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAAT 301 CCTGTGGAGA CTGGTGATGT TGCAACCTAT TACTGTCAGC AAAGTTATAA GGATCCTCCT 361 ACGTTCGGTA CTGGGACCAA GCTGGAGCTG AAG (SEQ ID NO: 146)

[0889] Signal Peptide (base pairs 1-60) :

[0890] 1 ATGGAGACAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG TTCCACAGGT 60 (SEQ ID NO: 147)

[0891] / translation="METDTLLLWVLLLWVPGSTG" (SEQ ID NO: 148)

[0892] Framework 1 (base pairs 61-129) :

[0893] 61 GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTGGGGCA GAGGGCCACC GTCTCCTGC 129 (SEQ ID NO: 149)

[0894] / translation="DIVLTQSPASLAVSLGQRATVSC" (SEQ ID NO: 150) CDR-L1 (base pairs 130-174) :

[0895] 130 A GAGCCAGCGA AAGTGTTGAT AATTCTGGCA TAAATTTTAT ACAC 174 (SEQ ID NO: 151)

[0896] / translation="RASESVDNSGINFIH" (SEQ ID NO: 152)

[0897] Framework 2 (base pairs 175-219) :

[0898] 175 CTGGTAC CAGCAGAAAC CAGGACAGTC ACCCAAACTC CTCCTCTAT 219 (SEQ ID NO: 153)

[0899] / translation="WYQQKPGQSPKLLLY" (SEQ ID NO: 154)

[0900] CDR-L2 (base pairs 220-240) :

[0901] 220 C GTGCATCCAA CCTAAAATCT 240 (SEQ ID NO: 155)

[0902] / translation="RASNLKS" (SEQ ID NO: 156)

[0903] Framework 3 (base pairs 241-336) :

[0904] 241 GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAAT CCTGTGGAGA CTGGTGATGT TGCAACCTAT TACTGT 336 (SEQ ID NO: 157)

[0905] / translation="GIPARFSGSGSRTDFTLTINPVETGDVATYYC" (SEQ ID NO: 158)

[0906] CDR-L3 (base pairs 337-363) :

[0907] 337 CAGC AAAGTTATAA GGATCCTCCT ACG 363 (SEQ ID NO: 159)

[0908] / translation="QQSYKDPPT" (SEQ ID NO: 160)

[0909] Framework 4 (base pairs 364-393):

[0910] 364 TTCGGTA CTGGGACCAA GCTGGAGCTG AAG 393 (SEQ ID NO: 161)

[0911] / translation="FGTGTKLELK" (SEQ ID NO: 162)

[0912] MHC2558LC .2 499.8D2.6.8

[0913]

[0914] 2G2 Heavy Chain Variable (vH) DNA and Amino Acid Sequences*

[0915] MHC2561HC .1 ;M13. 499.5.2G2.13.3.10

[0916] CDR Analysis

[0917] GLTFSSYA...._ISSGGSHT .._TRLGRAFDY

[0918] Amino Acid Sequence in FASTA format (MHC2561HC .1\;M13F) > MHC2561HC .1\;M13F . 499.5.2G2.13.3.10

[0919] LOCUS 2G2_vH 405 bp DNA linear

[0920] FEATURES Location / Qualifiers

[0921] J_segment 373..405

[0922] / label=FWR4

[0923] V_segment 352..372

[0924] / label=CDR3

[0925] V_region 229..351

[0926] / label=FWR3

[0927] V_segment 211..228

[0928] / label=CDR2

[0929] V_region 154..210

[0930] / label=FWR2

[0931] V_segment 133..153

[0932] / label=CDRl

[0933] V_region 58..132

[0934] / label=FWRl

[0935] sig_peptide 1..57

[0936] / label=LS

[0937] CDS 1..405

[0938] / label=2G2 vH

[0939] 2G2_vH

[0940] / translation="MNFGLSLIFLVLVLKGVQCEVMLVESGGGLVKPGGSLKLSCAVSGLTFSSYAMSWV RQTPEKRLEWVATISSGGSHTYYPDSVKGRFIISRDNAKNTLYLQMNSLRSEDTAMYYCTRLGRAFDYWG QGTTLTVSS" (SEQ ID NO: 163)

[0941] Nucleotide Sequence m FASTA format (MHC2561HC .1\;M13F)

[0942] > MHC2561HC .1\;M13F . 499.5.2G2.13.3 10

[0943] ORIGIN

[0944] 1 ATGAACTTCG GGCTCAGCTT GATTTTCCTT GTCCTTGTTT TAAAAGGTGT CCAGTGTGAA 61 GTGATGCTGG TGGAGTCAGG GGGAGGCTTA GTGAAGCCTG GAGGATCCCT GAAACTCTCC 121 TGTGCAGTCT CTGGATTAAC TTTTAGTAGT TATGCCATGT CTTGGGTTCG CCAGACTCCG 181 GAGAAGAGGC TGGAGTGGGT CGCAACCATT AGTAGTGGTG GTAGTCACAC CTACTATCCA 241 GACAGTGTGA AGGGGCGATT CATCATTTCT AGAGACAATG CCAAGAACAC CCTGTACCTG 301 CAAATGAACA GTCTGAGGTC TGAGGACACG GCCATGTATT ACTGTACAAG ACTGGGACGG 361 GCCTTTGACT ACTGGGGCCA AGGCACCACT CTCACAGTCT CCTCA (SEQ ID NO: 164)

[0945] Signal Peptide (base pairs 1-57) :

[0946] 1 ATGAACTTCG GGCTCAGCTT GATTTTCCTT GTCCTTGTTT TAAAAGGTGT CCAGTGT 57 (SEQ ID NO: 165)

[0947] / translation="MNFGLSLIFLVLVLKGVQC" (SEQ ID NO: 166)

[0948] Framework 1 (base pairs 58-132) : 58 GAA GTGATGCTGG TGGAGTCAGG GGGAGGCTTA GTGAAGCCTG GAGGATCCCT

[0949] GAAACTCTCC TGTGCAGTCT CT 132 (SEQ ID NO: 167)

[0950] / translation="EVMLVESGGGLVKPGGSLKLSCAVS" (SEQ ID NO: 168)

[0951] CDR-H1 (base pairs 133-153) :

[0952] 133 GGATTAAC TTTTAGTAGT TAT 153 (SEQ ID NO: 169)

[0953] / translation="GLTFSSY" (SEQ ID NO: 170)

[0954] Framework 2 (base pairs 154-210) :

[0955] 154 GCCATGT CTTGGGTTCG CCAGACTCCG GAGAAGAGGC TGGAGTGGGT CGCAACCATT 210 (SEQ ID NO: 171)

[0956] / translation="AMSWVRQTPEKRLEWVATI" (SEQ ID NO: 172)

[0957] CDR-H2 (base pairs 211-228) :

[0958] 211 AGTAGTGGTG GTAGTCAC 228 (SEQ ID NO: 173)

[0959] / translation="SSGGSH" (SEQ ID NO: 174)

[0960] Framework 3 (base pairs 229-351) :

[0961] 229 AC CTACTATCCA GACAGTGTGA AGGGGCGATT CATCATTTCT AGAGACAATG

[0962] CCAAGAACAC CCTGTACCTG CAAATGAACA GTCTGAGGTC TGAGGACACG GCCATGTATT ACTGTACAAG A 351 (SEQ ID NO: 175)

[0963] / translation="TYYPDSVKGRFI I SRDNAKNTLYLQMNSLRSEDTAMYYCTR" (SEQ ID NO: 176)

[0964] CDR-H3 (base pairs 352-372) :

[0965] 352 CTGGGACGG GCCTTTGACT AC 372 (SEQ ID NO: 177)

[0966] / translation="LGRAFDY" (SEQ ID NO: 178)

[0967] Framework 4 (base pairs 373-405):

[0968] 373 TGGGGCCA AGGCACCACT CTCACAGTCT CCTCA 405 (SEQ ID NO: 179)

[0969] / translation="WGQGTTLTVSS" (SEQ ID NO: 180)

[0970] MHC2561HC.1 499.5.2G2.13.3.10

[0971]

[0972] 2G2 Light Chain Variable (vL) DNA and Amino Acid Sequences*

[0973] MHC2561LC .1 ;M13. 499.5.2G2.13.3.10 CDR Analysis

[0974] QDVSTA . WAS. QQDYSTPWT

[0975] Amino Acid Sequence in FASTA format (MHC2561LC .1\;M13F)

[0976] > MHC2561LC .1\ ; M13 . 499.5.2G2.13.3.10

[0977] LOCUS 2G2_vL 381 bp DNA linear

[0978] FEATURES Location / Qualifiers

[0979] J_segment 352..381

[0980] / label=FWR4

[0981] V_segment 325..351

[0982] / label=CDR3

[0983] V_region 229..324

[0984] / label=FWR3

[0985] V_segment 208..228

[0986] / label=CDR2

[0987] V_region 163..207

[0988] / label=FWR2

[0989] V_segment 130..162

[0990] / label=CDRl

[0991] V_region 61..129

[0992] / label=FWRl

[0993] sig_peptide 1..60

[0994] / label=LS

[0995] CDS 1..381

[0996] / label=2G2 vL

[0997] 2G2_vL

[0998] / translation="MESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWY QQKPGQSPKVLIYWASTRHTGVPDRFTGSGSGTDFTLTI SSVQAEDLALYYCQQDYSTPWTFGGGTKLEI

[0999] K" (SEQ ID NO: 181)

[1000] Nucleotide Sequence in FASTA format (MHC2561LC .1\;M13F)

[1001] > MHC2561LC .1\ ; M13F . 499.5.2G2.13.3.10

[1002] ORIGIN

[1003] 1 ATGGAGTCAC AGATTCAGGT CTTTGTATTC GTGTTTCTCT GGTTGTCTGG TGTTGACGGA 61 GACATTGTGA TGACCCAGTC TCACAAATTC ATGTCCACAT CAGTAGGAGA CAGGGTCAGC 121 ATCACCTGCA AGGCCAGTCA GGATGTGAGT ACTGCTGTAG CCTGGTATCA ACAAAAGCCA 181 GGGCAATCTC CTAAAGTTCT GATTTACTGG GCATCCACCC GGCACACTGG AGTCCCTGAT 241 CGCTTCACAG GCAGTGGATC TGGGACAGAT TTTACTCTCA CCATCAGCAG TGTGCAGGCT 301 GAAGACCTGG CACTTTATTA CTGTCAGCAA GATTATAGCA CTCCGTGGAC GTTCGGTGGA 361 GGCACCAAGC TGGAAATCAA A (SEQ ID NO: 182)

[1004] Signal Peptide (base pairs 1-60) : 1 ATGGAGTCAC AGATTCAGGT CTTTGTATTC GTGTTTCTCT GGTTGTCTGG TGTTGACGGA 60 (SEQ ID NO: 183)

[1005] / translation="MESQIQVFVFVFLWLSGVDG" (SEQ ID NO: 184)

[1006] Framework 1 (base pairs 61-129) :

[1007] 61 GACATTGTGA TGACCCAGTC TCACAAATTC ATGTCCACAT CAGTAGGAGA CAGGGTCAGC ATCACCTGC 129 (SEQ ID NO: 185)

[1008] / translation="DIVMTQSHKFMSTSVGDRVSITC" (SEQ ID NO: 186)

[1009] CDR-Ll (base pairs 130-162) :

[1010] 130 A AGGCCAGTCA GGATGTGAGT ACTGCTGTAG CC 162 (SEQ ID NO: 187)

[1011] / translation="KASQDVSTAVA" (SEQ ID NO: 188)

[1012] Framework 2 (base pairs 163-207) :

[1013] 163 TGGTATCA ACAAAAGCCA GGGCAATCTC CTAAAGTTCT GATTTAC 207 (SEQ ID NO: 189)

[1014] / translation="WYQQKPGQSPKVLIY" (SEQ ID NO: 190)

[1015] CDR-L2 (base pairs 208-228) :

[1016] 208 TGG GCATCCACCC GGCACACT 228 (SEQ ID NO: 191)

[1017] / translation="WASTRHT" (SEQ ID NO: 192)

[1018] Framework 3 (base pairs 229-324) :

[1019] 229 GG AGTCCCTGAT CGCTTCACAG GCAGTGGATC TGGGACAGAT TTTACTCTCA

[1020] CCATCAGCAG TGTGCAGGCT GAAGACCTGG CACTTTATTA CTGT 324 (SEQ ID NO: 193)

[1021] / translation="GVPDRFTGSGSGTDFTLTISSVQAEDLALYYC" (SEQ ID NO: 194)

[1022] CDR-L3 (base pairs 325-351) :

[1023] 325 CAGCAA GATTATAGCA CTCCGTGGAC G 351 (SEQ ID NO: 195)

[1024] / translation="QQDYSTPWT" (SEQ ID NO: 196)

[1025] Framework 4 (base pairs 352-381):

[1026] 352 TTCGGTGGA GGCACCAAGC TGGAAATCAA A 381 (SEQ ID NO: 197)

[1027] / translation="FGGGTKLEIK" (SEQ ID NO: 198)

[1028] MHC2561LC.1 499.5.2G2.13.3.10

[1029] 2C4 Heavy Chain Variable (vH) DNA and Amino Acid Sequences*

[1030] MHC2562HC .1;M13. 499.5.2C4.E6.6.8.1

[1031] CDR Analysis

[1032] GFSFTDYN .... _IDPYYGRI .. _ATGAYTSGYSWFAY

[1033] Amino Acid Sequence in FASTA format (MHC2562HC .1\;M13F)

[1034] > MHC2562HC .1\;M13F . 499.5.2C4. E6.6.8.1

[1035] LOCUS 2C4_vH 420 bp DNA linear

[1036] FEATURES Location / Qualifiers

[1037] J_segment 388..420

[1038] / label=FWR4

[1039] V_segment 352..387

[1040] / label=CDR3

[1041] V_region 229..351

[1042] / label=FWR3

[1043] V_segment 211..228

[1044] / label=CDR2

[1045] V_region 154..210

[1046] / label=FWR2

[1047] V_segment 133..153

[1048] / label=CDRl

[1049] V_region 58..132

[1050] / label=FWRl

[1051] sig_peptide 1..57

[1052] / label=LS

[1053] CDS 1..420

[1054] / label=2C4 vH

[1055] 2C4_vH

[1056] / translation="MGWTWIFILILSVTTGVHSEVHLQQSGPELEKPGVSVKISCKASGFSFTDYNMNWV KQSSGKSLEWIGNIDPYYGRINYNQKFKGKATLSVDKSSSTAYMHLKSLTSEDSAVYYCATGAYTSGYSW

[1057] FAYWGQGTLVTVSA" (SEQ ID NO: 199)

[1058] Nucleotide Sequence in FASTA format (MHC2562HC .1\;M13F)

[1059] > MHC2562HC .1\;M13F . 499.5.2C4. E6.6 .8.1

[1060] ORIGIN

[1061] 1 ATGGGATGGA CCTGGATCTT TATTTTAATC CTGTCAGTAA CTACAGGTGT CCACTCTGAG 61 GTCCACCTGC AGCAGTCTGG ACCTGAGCTG GAGAAGCCTG GCGTTTCAGT GAAGATATCC 121 TGCAAGGCTT CTGGTTTCTC ATTCACTGAC TACAACATGA ACTGGGTGAA ACAGAGCAGT 181 GGAAAGAGCC TTGAGTGGAT TGGAAATATT GATCCTTACT ATGGACGTAT TAACTATAAC 241 CAGAAATTCA AGGGCAAGGC CACATTGAGT GTAGACAAAT CCTCCAGCAC AGCCTACATG 301 CACCTCAAGA GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAC TGGGGCCTAC 361 ACCTCGGGCT ACTCCTGGTT TGCTTACTGG GGCCAAGGGA CTCTGGTCAC TGTCTCTGCA (SEQ ID NO: 200)

[1062] Signal Peptide (base pairs 1-57) : 1 ATGGGATGGA CCTGGATCTT TATTTTAATC CTGTCAGTAA CTACAGGTGT CCACTCT 57 (SEQ ID NO: 201)

[1063] / translation="MGWTWIFILILSVTTGVHS" (SEQ ID NO: 202)

[1064] Framework 1 (base pairs 58-132) :

[1065] 58 GAG GTCCACCTGC AGCAGTCTGG ACCTGAGCTG GAGAAGCCTG GCGTTTCAGT

[1066] GAAGATATCC TGCAAGGCTT CT 132 (SEQ ID NO: 203)

[1067] / translation="EVHLQQSGPELEKPGVSVKISCKAS" (SEQ ID NO: 204)

[1068] CDR-H1 (base pairs 133-153) :

[1069] 133 GGTTTCTC ATTCACTGAC TAC 153 (SEQ ID NO: 205)

[1070] / translation="GFSFTDY" (SEQ ID NO: 206)

[1071] Framework 2 (base pairs 154-210) :

[1072] 154 AACATGA ACTGGGTGAA ACAGAGCAGT GGAAAGAGCC TTGAGTGGAT TGGAAATATT 210 (SEQ ID NO: 207)

[1073] / translation="NMNWVKQSSGKSLEWIGNI" (SEQ ID NO: 208)

[1074] CDR-H2 (base pairs 211-228) :

[1075] 211 GATCCTTACT ATGGACGT 228 (SEQ ID NO: 209)

[1076] / translation^'DPYYGR" (SEQ ID NO: 210)

[1077] Framework 3 (base pairs 229-351) :

[1078] 229 AT TAACTATAAC CAGAAATTCA AGGGCAAGGC CACATTGAGT GTAGACAAAT

[1079] CCTCCAGCAC AGCCTACATG CACCTCAAGA GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAC T 351 (SEQ ID NO: 211)

[1080] / translation=" INYNQKFKGKATLSVDKSSSTAYMHLKSLTSEDSAVYYCAT" (SEQ ID NO:

[1081] 212)

[1082] CDR-H3 (base pairs 352-387) :

[1083] 352 GGGGCCTAC ACCTCGGGCT ACTCCTGGTT TGCTTAC 387 (SEQ ID NO: 213)

[1084] / translation="GAYTSGYSWFAY" (SEQ ID NO: 214)

[1085] Framework 4 (base pairs 388-420):

[1086] 388 TGG GGCCAAGGGA CTCTGGTCAC TGTCTCTGCA 420 (SEQ ID NO: 215)

[1087] / translation="WGQGTLVTVSA" (SEQ ID NO: 216)

[1088] MHC2562HC.1 499.5.2C4. E6.6.8.1

[1089] 11

[1090] 2C4 Light Chain Variable (vL) DNA and Amino Acid Sequences*

[1091] MHC2562LC .2 ;M13. 499.5.2C4.E6.6.8.1

[1092] CDR Analysis

[1093] SSVSSSY ..RTS ..QQWSGYPFT

[1094] Amino Acid Sequence in FASTA format (MHC2562LC .2\;M13F)

[1095] > MHC2562LC .2\;M13F . 499.5.2C4. E6.6.8.1

[1096] LOCUS 2C4_vL 390 bp DNA linear

[1097] FEATURES Location / Qualifiers

[1098] J_segment 361..390

[1099] / label=FWR4

[1100] V_segment 334..360

[1101] / label=CDR3

[1102] V_region 238..333

[1103] / label=FWR3

[1104] V segment 217..237

[1105] / label=CDR2

[1106] V region 172..216

[1107] / label=FWR2

[1108] V_segment 136..171

[1109] / label=CDRl

[1110] V region 67..135

[1111] / label=FWRl

[1112] sig peptide 1..66

[1113] / label=LS

[1114] CDS 1..390

[1115] / label=2C4 vL

[1116] 2C4_vL

[1117] / translation="MGLQVQVISFLLISVTVIMSRGENVLTQSPGIMAASLGEKVTMTCSASSSVSSSYL HWYQQRSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGSGTK

[1118] LEIK" (SEQ ID NO: 217)

[1119] Nucleotide Sequence in FASTA format (MHC2562LC .2\;M13F)

[1120] > MHC2562LC .2\;M13F . 499.5.2C4. E6.6.8.1

[1121] ORIGIN

[1122] 1 ATGGGTTTAC AGGTGCAGGT TATCAGCTTC CTGTTAATCA GTGTCACAGT CATAATGTCC 61 AGAGGAGAAA ATGTGCTCAC CCAGTCTCCA GGAATAATGG CTGCCTCTCT GGGGGAGAAG 121 GTCACCATGA CCTGCAGTGC CAGCTCAAGT GTAAGTTCCA GTTACTTGCA CTGGTACCAG 181 CAGAGGTCAG GCGCTTCCCC CAAACCCTTG ATTCATAGGA CATCCAACCT GGCTTCTGGT 241 GTCCCAGCTC GCTTCAGTGG CAGTGGGTCT GGGACCTCTT ACTCTCTCAC AATCAGCAGC 301 GTGGAGGCTG AAGATGATGC AACTTATTAC TGCCAGCAGT GGAGTGGTTA CCCATTCACG 361 TTCGGCTCGG GGACAAAGTT GGAAATAAAA (SEQ ID NO: 218)

[1123] Signal Peptide (base pairs 1-66) :

[1124] 1 ATGGGTTTAC AGGTGCAGGT TATCAGCTTC CTGTTAATCA GTGTCACAGT CATAATGTCC AGAGGA 66 (SEQ ID NO: 219)

[1125] / translation="MGLQVQVISFLLI SVTVIMSRG" (SEQ ID NO: 220)

[1126] Framework 1 (base pairs 67-135) :

[1127] 67 GAAA ATGTGCTCAC CCAGTCTCCA GGAATAATGG CTGCCTCTCT GGGGGAGAAG

[1128] GTCACCATGA CCTGC 135 (SEQ ID NO: 221)

[1129] / translation="ENVLTQSPGIMAASLGEKVTMTC" (SEQ ID NO: 222)

[1130] CDR-Ll (base pairs 136-171) :

[1131] 136 AGTGC CAGCTCAAGT GTAAGTTCCA GTTACTTGCA C 171 (SEQ ID NO: 223)

[1132] / translation="SASSSVSSSYLH" (SEQ ID NO: 224)

[1133] Framework 2 (base pairs 172-216) :

[1134] 172 TGGTACCAG CAGAGGTCAG GCGCTTCCCC CAAACCCTTG ATTCAT 216 (SEQ ID NO: 225)

[1135] / translation="WYQQRSGASPKPLIH" (SEQ ID NO: 226)

[1136] CDR-L2 (base pairs 217-237) :

[1137] 217 AGGA CATCCAACCT GGCTTCT 237 (SEQ ID NO: 227)

[1138] / translation="RTSNLAS" (SEQ ID NO: 228)

[1139] Framework 3 (base pairs 238-333) :

[1140] 238 GGT GTCCCAGCTC GCTTCAGTGG CAGTGGGTCT GGGACCTCTT ACTCTCTCAC

[1141] AATCAGCAGC GTGGAGGCTG AAGATGATGC AACTTATTAC TGC 333 (SEQ ID NO: 229)

[1142] / translation="GVPARFSGSGSGTSYSLTISSVEAEDDATYYC" (SEQ ID NO: 230)

[1143] CDR-L3 (base pairs 334-360) :

[1144] 334 CAGCAGT GGAGTGGTTA CCCATTCACG 360 (SEQ ID NO: 231)

[1145] / translation="QQWSGYPFT" (SEQ ID NO: 232)

[1146] Framework 4 (base pairs 361-390):

[1147] 361 TTCGGCTCGG GGACAAAGTT GGAAATAAAA 390 (SEQ ID NO: 233)

[1148] / translation="FGSGTKLEIK" (SEQ ID NO: 234)

[1149] MHC2562LC.2 499.5.2C4. E6.6.8.1

[1150]

[1151] 4D1 Heavy Chain Variable (vH) DNA and Amino Acid Sequences*

[1152] MHC2563HC .1 ;M13. 499.5.4D1.5.16

[1153] CDR Analysis

[1154] GYTFTDYA.... _ISTYYGDT .. _ARGNYDDWYFNV

[1155] Amino Acid Sequence in FASTA format (MHC2563HC .1\;M13F)

[1156] > MHC2563HC .1\;M13F . 499.5.4D1.5.16

[1157] LOCUS 4Dl_vH 414 bp DNA linear

[1158] FEATURES Location / Qualifiers

[1159] J_segment 382..414

[1160] / label=FWR4

[1161] V_segment 352..381

[1162] / label=CDR3

[1163] V_region 229..351

[1164] / label=FWR3

[1165] V_segment 211..228

[1166] / label=CDR2

[1167] V_region 154..210

[1168] / label=FWR2

[1169] V_segment 133..153

[1170] / label=CDRl

[1171] V_region 58..132

[1172] / label=FWRl

[1173] sig_peptide 1..57

[1174] / label=LS

[1175] CDS 1..414

[1176] / label=4Dl vH

[1177] 4Dl_vH

[1178] / translation="MDWSCI IFFLVATATGVHSQVQLQQSGAELVRPGVSVKISCKGSGYTFTDYAMHWV KQSHAKSLEWIGSISTYYGDTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGNYDDWYFN VWGAGTTVTVSS" (SEQ ID NO: 235)

[1179] Nucleotide Sequence in FASTA format (MHC2563HC .1\;M13F)

[1180] > MHC2563HC .1\;M13F . 499.5.4D1.5.16

[1181] ORIGIN

[1182] 1 ATGGATTGGA GCTGTATCAT CTTCTTTCTG GTAGCAACAG CTACAGGTGT GCACTCCCAG 61 GTCCAGCTGC AGCAGTCTGG GGCTGAACTG GTGAGGCCTG GGGTCTCAGT GAAGATTTCC 121 TGCAAGGGTT CTGGCTACAC ATTCACTGAT TATGCTATGC ACTGGGTGAA GCAGAGTCAT 181 GCAAAGAGTC TAGAGTGGAT TGGAAGTATT AGTACTTACT ATGGTGATAC TAATTACAAC 241 CAGAAATTCA AGGGCAAGGC CACAATGACT GTAGACAAAT CCTCCAGCAC AGCCTATATG 301 GAACTTGCCA GACTGACATC TGAGGATTCT GCCATCTATT ACTGTGCAAG AGGTAATTAC 361 GACGACTGGT ACTTCAATGT CTGGGGCGCA GGGACCACGG TCACCGTCTC CTCA (SEQ ID NO: 236)

[1183] Signal Peptide (base pairs 1-57) :

[1184] 1 ATGGATTGGA GCTGTATCAT CTTCTTTCTG GTAGCAACAG CTACAGGTGT GCACTCC 57 (SEQ ID NO: 237)

[1185] / translation="MDWSCIIFFLVATATGVHS" (SEQ ID NO: 238)

[1186] Framework 1 (base pairs 58-132) :

[1187] 58 CAG GTCCAGCTGC AGCAGTCTGG GGCTGAACTG GTGAGGCCTG GGGTCTCAGT

[1188] GAAGATTTCC TGCAAGGGTT CT 132 (SEQ ID NO: 239)

[1189] / translation="QVQLQQSGAELVRPGVSVKISCKGS" (SEQ ID NO: 240)

[1190] CDR-H1 (base pairs 133-153) :

[1191] 133 GGCTACAC ATTCACTGAT TAT 153 (SEQ ID NO: 241)

[1192] / translation="GYTFTDY" (SEQ ID NO: 242)

[1193] Framework 2 (base pairs 154-210) :

[1194] 154 GCTATGC ACTGGGTGAA GCAGAGTCAT GCAAAGAGTC TAGAGTGGAT TGGAAGTATT 210 (SEQ ID NO: 243)

[1195] / translation="AMHWVKQSHAKSLEWIGSI" (SEQ ID NO: 244)

[1196] CDR-H2 (base pairs 211-228) :

[1197] 211 AGTACTTACT ATGGTGAT 228 (SEQ ID NO: 245)

[1198] / translation="STYYGD" (SEQ ID NO: 246)

[1199] Framework 3 (base pairs 229-351) :

[1200] 229 AC TAATTACAAC CAGAAATTCA AGGGCAAGGC CACAATGACT GTAGACAAAT

[1201] CCTCCAGCAC AGCCTATATG GAACTTGCCA GACTGACATC TGAGGATTCT GCCATCTATT ACTGTGCAAG A 351 (SEQ ID NO: 247)

[1202] / translation="TNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCAR" (SEQ ID NO: 248)

[1203] CDR-H3 (base pairs 352-381) :

[1204] 352 GGTAATTAC GACGACTGGT ACTTCAATGT C 381 (SEQ ID NO: 249)

[1205] / translation="GNYDDWYFNV" (SEQ ID NO: 250)

[1206] Framework 4 (base pairs 382-414):

[1207] 382 TGGGGCGCA GGGACCACGG TCACCGTCTC CTCA 414 (SEQ ID NO: 251)

[1208] / translation="WGAGTTVTVSS" (SEQ ID NO: 252)

[1209] MHC2563HC.1 499.5.4D1.5.16

[1210]

[1211]

[1212] 4D1 Light Chain Variable (vL) DNA and Amino Acid Sequences*

[1213] MHC2563LC .1 ;M13. 499.5.4D1.5.16

[1214] CDR Analysis

[1215] QDISGY ..STS ..LQYASSPYT

[1216] Amino Acid Sequence in FASTA format (MHC2563LC .1\;M13F) > MHC2563LC .1\;M13F . 499.5.4D1.5.16

[1217] LOCUS 4Dl_vL 381 bp DNA linear

[1218] FEATURES Location / Qualifiers

[1219] J_segment 352..381

[1220] / label=FWR4

[1221] V_segment 325..351

[1222] / label=CDR3

[1223] V_region 229..324

[1224] / label=FWR3

[1225] V_segment 208..228

[1226] / label=CDR2

[1227] V_region 163..207

[1228] / label=FWR2

[1229] V_segment 130..162

[1230] / label=CDRl

[1231] V_region 61..129

[1232] / label=FWRl

[1233] sig_peptide 1..60

[1234] / label=LS

[1235] CDS 1..381

[1236] / label=4Dl vL

[1237] 4D1 vL / translation="MRIPAHVFGFLLLWFPGARCDIQMTQSPSSLSASLGERVSLTCRASQDI SGYLSWL QQKPDGTIKRLIYSTSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASSPYTFGGGAKLEI

[1238] KR" (SEQ ID NO: 253)

[1239] Nucleotide Sequence in FASTA format (MHC2563LC .1\;M13F)

[1240] > MHC2563LC .1\;M13F . 499.5.4D1.5.16

[1241] ORIGIN

[1242] 1 ATGAGGATTC CTGCTCACGT TTTTGGCTTC TTGTTGCTCT GGTTTCCAGG TGCCAGATGT

[1243] 61 GACATCCAAA TGACCCAGTC TCCATCTTCC TTATCTGCCT CTCTGGGAGA AAGAGTCAGT

[1244] 121 CTCACTTGTC GGGCAAGTCA GGATATTAGT GGTTACTTAA GCTGGCTTCA GCAGAAACCA

[1245] 181 GATGGAACTA TTAAACGTCT GATTTATAGC ACATCCACTT TAGATTCTGG TGTCCCAAAA

[1246] 241 AGGTTCAGTG GCAGTAGGTC TGGGTCAGAT TATTCTCTCA CCATCAGCAG CCTAGAGTCT

[1247] 301 GAAGATTTTG CAGACTATTA CTGTCTACAA TATGCTAGTT CTCCGTACAC GTTCGGAGGG

[1248] 361 GGGGCCAAGC TGGAAATAAA A (SEQ ID NO: 254)

[1249] Signal Peptide (base pairs 1-60) :

[1250] 1 ATGAGGATTC CTGCTCACGT TTTTGGCTTC TTGTTGCTCT GGTTTCCAGG TGCCAGATGT

[1251] 60 (SEQ ID NO: 255)

[1252] / translation="MRIPAHVFGFLLLWFPGARC" (SEQ ID NO: 256)

[1253] Framework 1 (base pairs 61-129) :

[1254] 61 GACATCCAAA TGACCCAGTC TCCATCTTCC TTATCTGCCT CTCTGGGAGA AAGAGTCAGT CTCACTTGT 129 (SEQ ID NO: 257)

[1255] / translation="DIQMTQSPSSLSASLGERVSLTC" (SEQ ID NO: 258)

[1256] CDR-L1 (base pairs 130-162) :

[1257] 130 C GGGCAAGTCA GGATATTAGT GGTTACTTAA GC 162 (SEQ ID NO: 259)

[1258] / translation="RASQDISGYLS" (SEQ ID NO: 260)

[1259] Framework 2 (base pairs 163-207) :

[1260] 163 TGGCTTCA GCAGAAACCA GATGGAACTA TTAAACGTCT GATTTAT 207 (SEQ ID NO: 261)

[1261] / translation="WLQQKPDGT IKRLIY" (SEQ ID NO: 262)

[1262] CDR-L2 (base pairs 208-228) :

[1263] 208 AGC ACATCCACTT TAGATTCT 228 (SEQ ID NO: 263)

[1264] / translation="STSTLDS" (SEQ ID NO: 264)

[1265] Framework 3 (base pairs 229-324) :

[1266] 229 GG TGTCCCAAAA AGGTTCAGTG GCAGTAGGTC TGGGTCAGAT TATTCTCTCA

[1267] CCATCAGCAG CCTAGAGTCT GAAGATTTTG CAGACTATTA CTGT 324 (SEQ ID NO: 265) / translation="GVPKRFSGSRSGSDYSLTISSLESEDFADYYC" (SEQ ID NO: 266)

[1268] CDR-L3 (base pairs 325-351) :

[1269] 325 CTACAA TATGCTAGTT CTCCGTACAC G 351 (SEQ ID NO: 267)

[1270] / translation="LQYASSPYT" (SEQ ID NO: 268)

[1271] Framework 4 (base pairs 352-381): 352 TTCGGAGGG GGGGCCAAGC TGGAAATAAA A 381 (SEQ ID NO: 269) / translation="FGGGAKLEIK" (SEQ ID NO: 270)

[1272] MHC2563LC.1 499.5.4D1.5.16

[1273]

[1274] 1C8 Heavy Chain Variable (vH) DNA and Amino Acid Sequences*

[1275] MHC2564HC .1;M13. 499.5.1C8.9.12.2

[1276] CDR Analysis

[1277] GFNIKDTY ...._IDPANGKT .._AWLLPYYFDY

[1278] Amino Acid Sequence in FASTA format (MHC2564HC .1\;M13F) > MHC2564HC .1\;M13F . 499.5.1C8.9.12.2

[1279] LOCUS 1C8_VH 408 bp DNA linear

[1280] FEATURES Location / Qualifiers

[1281] J_segment 376..408

[1282] / label=FWR4

[1283] V_segment 352..375

[1284] / label=CDR3

[1285] V_region 229..351

[1286] / label=FWR3

[1287] V_segment 211..228

[1288] / label=CDR2

[1289] V_region 154..210

[1290] / label=FWR2

[1291] V_segment 133..153

[1292] / label=CDRl

[1293] V_region 58..132

[1294] / label=FWRl

[1295] sig_peptide 1..57

[1296] / label=LS

[1297] CDS 1..408

[1298] / label=lC8 vH

[1299] 1C8 vH / translation="MKCSWI IFFLMAVVTGVNSEVQLQQSGAELVQPGASVKLSCTASGFNIKDTYMHWV KQRPEQGl·EWIGRIDPANGKTIFDPKFQVKATITADTSSNTVYl·Hl·SSl·TSEDTAIYYCAWl·l·PYYFDYW GQGTTLTVSS" (SEQ ID NO: 271)

[1300] Nucleotide Sequence in FASTA format (MHC2564HC .1\;M13F)

[1301] > MHC2564HC .1\;M13F . 499.5.1C8.9.12.2

[1302] ORIGIN

[1303] 1 ATGAAATGCA GCTGGATTAT CTTCTTCCTG ATGGCTGTGG TTACAGGGGT CAATTCAGAG 61 GTTCAGCTGC AGCAGTCTGG GGCAGAACTT GTGCAGCCAG GGGCCTCAGT CAAGTTGTCC 121 TGTACAGCTT CTGGCTTCAA TATTAAAGAC ACCTATATGC ACTGGGTAAA ACAGAGGCCT 181 GAACAGGGCC TGGAGTGGAT TGGAAGGATT GATCCTGCGA ATGGTAAAAC TATTTTTGAC 241 CCGAAGTTCC AGGTCAAGGC CACTATAACT GCCGACACAT CCTCCAACAC AGTCTACCTG 301 CATCTCAGCA GCCTGACATC TGAGGACACT GCCATCTATT ACTGTGCTTG GTTACTTCCT 361 TACTACTTTG ACTACTGGGG CCAAGGCACC ACTCTCACAG TCTCCTCA (SEQ ID NO: 272)

[1304] Signal Peptide (base pairs 1-57) :

[1305] 1 ATGAAATGCA GCTGGATTAT CTTCTTCCTG ATGGCTGTGG TTACAGGGGT CAATTCA 57 (SEQ ID NO: 273)

[1306] / translation="MKCSWI IFFLMAVVTGVNS" (SEQ ID NO: 274)

[1307] Framework 1 (base pairs 58-132) :

[1308] 58 GAG GTTCAGCTGC AGCAGTCTGG GGCAGAACTT GTGCAGCCAG GGGCCTCAGT

[1309] CAAGTTGTCC TGTACAGCTT CT 132 (SEQ ID NO: 275)

[1310] / translation="EVQLQQSGAELVQPGASVKLSCTAS" (SEQ ID NO: 276)

[1311] CDR-H1 (base pairs 133-153) :

[1312] 133 GGCTTCAA TATTAAAGAC ACC 153 (SEQ ID NO: 277)

[1313] / translation="GFNIKDT" (SEQ ID NO: 278)

[1314] Framework 2 (base pairs 154-210) :

[1315] 154 TATATGC ACTGGGTAAA ACAGAGGCCT GAACAGGGCC TGGAGTGGAT TGGAAGGATT 210 (SEQ ID NO: 279)

[1316] / translation="YMHWVKQRPEQGLEWIGRI" (SEQ ID NO: 280)

[1317] CDR-H2 (base pairs 211-228) :

[1318] 211 GATCCTGCGA ATGGTAAA 228 (SEQ ID NO: 281)

[1319] / translation="DPANGK" (SEQ ID NO: 282)

[1320] Framework 3 (base pairs 229-351) :

[1321] 229 AC TATTTTTGAC CCGAAGTTCC AGGTCAAGGC CACTATAACT GCCGACACAT

[1322] CCTCCAACAC AGTCTACCTG CATCTCAGCA GCCTGACATC TGAGGACACT GCCATCTATT ACTGTGCTTG G 351 (SEQ ID NO: 283)

[1323] / translation="T IFDPKFQVKATITADTSSNTVYLHLSSLTSEDTAIYYCAW" (SEQ ID NO:

[1324] 284)

[1325] CDR-H3 (base pairs 352-375) :

[1326] 352 TTACTTCCT TACTACTTTG ACTAC 375 (SEQ ID NO: 285)

[1327] / translation="LLPYYFDY" (SEQ ID NO: 286) Framework 4 (base pairs 376-408):

[1328] 376 TGGGG CCAAGGCACC ACTCTCACAG TCTCCTCA 408 (SEQ ID NO: 287) / translation="WGQGTTLTVSS" (SEQ ID NO: 288)

[1329] MHC2564HC.1 499.5.1C8.9.12.2

[1330]

[1331] 1C8 Light Chain Variable (vL) DNA and Amino Acid Sequences*

[1332] MHC2564LCB .4; Ml3. 499.5.1C8.9.12.2

[1333] CDR Analysis

[1334] SSISSSN ..GTS ..QKWSHYPLT

[1335] Amino Acid Sequence in FASTA format (MHC2564LCB .4\;M13F) > MHC2564LCB .4\ ; M13 F . 499.5.1C8.9.12.2

[1336] LOCUS 1C8_VL 390 bp DNA linear

[1337] FEATURES Location / Qualifiers

[1338] J_segment 361..390

[1339] / label=FWR4

[1340] V_segment 334..360

[1341] / label=CDR3

[1342] V_region 238..333

[1343] / label=FWR3

[1344] V_segment 217..237

[1345] / label=CDR2

[1346] V_region 172..216

[1347] / label=FWR2

[1348] V_segment 136..171

[1349] / label=CDRl

[1350] V_region 67..135

[1351] / label=FWRl

[1352] sig peptide 1..66 / label=LS

[1353] CDS 1..390

[1354] / label=lC8_vL

[1355] 1C8_VL

[1356] / translation="MDFHVQIFSFMLI SVTVMLSSGEIVLTQSPAVMAASPGEKVTITCSVSSSISSSNL HWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQKWSHYPLTFGAGTK

[1357] LELK" (SEQ ID NO: 289)

[1358] Nucleotide Sequence in FASTA format (MHC2564LCB .4\;M13F)

[1359] 499.5.1C8.9.12.2

[1360]

[1361] 1 ATGGATTTTC ATGTGCAGAT TTTCAGCTTC ATGCTAATCA GTGTCACAGT CATGTTGTCC

[1362] 61 AGTGGGGAAA TTGTACTCAC CCAGTCTCCA GCAGTCATGG CTGCATCTCC AGGGGAGAAG

[1363] 121 GTCACCATCA CCTGCAGCGT CAGTTCAAGT ATAAGTTCCA GCAACTTGCA CTGGTACCAG

[1364] 181 CAGAAGTCAG GAACCTCGCC CAAACTCTGG ATTTATGGCA CATCCAACCT GGCTTCTGGA

[1365] 241 GTCCCTGTTC GCTTCAGTGG CAGTGGATCT GGGACCTCTT ATTCTCTCAC AATCAGCAGC

[1366] 301 ATGGAGGCTG AAGATGCTGC CACTTATTAC TGTCAAAAGT GGAGTCATTA CCCGCTCACG

[1367] 361 TTCGGTGCTG GGACCAAGCT GGAGCTGAAA (SEQ ID NO: 290)

[1368] Signal Peptide (base pairs 1-66) :

[1369] 1 ATGGATTTTC ATGTGCAGAT TTTCAGCTTC ATGCTAATCA GTGTCACAGT CATGTTGTCC AGTGGG 66 (SEQ ID NO: 291)

[1370] / translation="MDFHVQIFSFMLI SVTVMLSSG" (SEQ ID NO: 292)

[1371] Framework 1 (base pairs 67-135) :

[1372] 67 GAAA TTGTACTCAC CCAGTCTCCA GCAGTCATGG CTGCATCTCC AGGGGAGAAG

[1373] GTCACCATCA CCTGC 135 (SEQ ID NO: 293)

[1374] / translation="EIVLTQSPAVMAASPGEKVTITC" (SEQ ID NO: 294)

[1375] CDR-L1 (base pairs 136-171) :

[1376] 136 AGCGT CAGTTCAAGT ATAAGTTCCA GCAACTTGCA C 171 (SEQ ID NO: 295)

[1377] / translation="SVSSSISSSNLH" (SEQ ID NO: 296)

[1378] Framework 2 (base pairs 172-216) :

[1379] 172 TGGTACCAG CAGAAGTCAG GAACCTCGCC CAAACTCTGG ATTTAT 216 (SEQ ID NO: 297)

[1380] / translation="WYQQKSGTSPKLWIY" (SEQ ID NO: 298)

[1381] CDR-L2 (base pairs 217-237) :

[1382] 217 GGCA CATCCAACCT GGCTTCT 237 (SEQ ID NO: 299)

[1383] / translation="GTSNLAS" (SEQ ID NO: 300)

[1384] Framework 3 (base pairs 238-333) :

[1385] 238 GGA GTCCCTGTTC GCTTCAGTGG CAGTGGATCT GGGACCTCTT ATTCTCTCAC

[1386] AATCAGCAGC ATGGAGGCTG AAGATGCTGC CACTTATTAC TGT 333 (SEQ ID NO: 301)

[1387] / translation="GVPVRFSGSGSGTSYSLTISSMEAEDAATYYC" (SEQ ID NO: 302) CDR-L3 (base pairs 334-360) :

[1388] 334 CAAAAGT GGAGTCATTA CCCGCTCACG 360 (SEQ ID NO: 303)

[1389] / translation="QKWSHYPLT" (SEQ ID NO: 304)

[1390] Framework 4 (base pairs 361-390):

[1391] 361 TTCGGTGCTG GGACCAAGCT GGAGCTGAAA 390 (SEQ ID NO: 305) / translation="FGAGTKLELK" (SEQ ID NO: 306)

[1392] MHC2564LCB .4 499.5.1C8.9.12.2

[1393]

[1394] 4E5 Heavy Chain Variable DNA and Amino Acid Sequences’’

[1395]

[1396] MHC2556HCN .1 ; Ml 3. 499.5.4E5.20.22

[1397] CDRAnalysis

[1398] GYTFTTYT .... _INPSSGYT .. _ARHPWDSNY

[1399] AminoAcidSequenceinFASTAformat (MHC2556HCN .1 \ ; M13F)

[1400] >MHC2556HCN .1\ ; M13F

[1401] LOCUS 4E5_vH 405 bp DNA linear

[1402] FEATURES Location / Qualifiers

[1403] J segment 373..405

[1404] / label=FWR4

[1405] V_segment 352..372

[1406] / label=CDR3

[1407] V_region 229..351

[1408] / label=FWR3

[1409] V_segment 211..228

[1410] / label=CDR2

[1411] V_region 154..210

[1412] / label=FWR2

[1413] V_segment 133..153

[1414] / label=CDRl

[1415] V_region 58..132

[1416] / label=FWRl sig_peptide 1..57

[1417] / label=LS

[1418] CDS 1..405

[1419] / label=4E5 vH

[1420] 4E5_vH

[1421] / translation="MERHWIFLFLLSVTAGVHSQVQLQQSAAELARPGASVKMSCKASGYTFTTYTMHWV KQRPGQGl·EWIGHINPSSGYTEYNQKFKDKTTl·TADKSSSTAHMQl·SSl·TSEDSAVYYCARHPWDSNYWG QGTTLTVSS" (SEQ ID NO: 307)

[1422] NucleotideSequenceinFASTAformat (MHC2556HCN.1\;M13F)

[1423] >MHC2556HCN .1\ ; M13F

[1424] ORIGIN

[1425] 1 ATGGAAAGGC ACTGGATCTT TCTCTTCCTG TTGTCAGTAA CTGCAGGTGT CCACTCCCAG 61 GTCCAGCTGC AGCAGTCTGC AGCTGAACTG GCAAGACCTG GGGCCTCAGT GAAGATGTCC 121 TGCAAGGCTT CTGGCTACAC CTTTACTACC TACACGATGC ACTGGGTAAA ACAGAGGCCT 181 GGACAGGGTC TGGAGTGGAT TGGACACATT AATCCTAGCA GTGGATATAC TGAGTACAAT 241 CAGAAATTCA AGGACAAGAC CACACTGACT GCAGACAAAT CCTCCAGCAC AGCCCACATG 301 CAACTGAGCA GCCTAACATC TGAGGACTCT GCGGTCTATT ACTGTGCAAG ACACCCCTGG 361 GACTCGAACT ACTGGGGCCA AGGCACCACT CTCACAGTCT CCTCA (SEQ ID NO: 308)

[1426] Signal Peptide (base pairs 1-57) :

[1427] 1 ATGGAAAGGC ACTGGATCTT TCTCTTCCTG TTGTCAGTAA CTGCAGGTGT CCACTCC 57 (SEQ ID NO: 309)

[1428] / translation="MERHWIFLFLLSVTAGVHS" (SEQ ID NO: 310)

[1429] Framework 1 (base pairs 58-132) :

[1430] 58 CAG GTCCAGCTGC AGCAGTCTGC AGCTGAACTG GCAAGACCTG GGGCCTCAGT

[1431] GAAGATGTCC TGCAAGGCTT CT 132 (SEQ ID NO: 311)

[1432] / translation="QVQLQQSAAELARPGASVKMSCKAS" (SEQ ID NO: 312)

[1433] CDR-H1 (base pairs 133-153) :

[1434] 133 GGCTACAC CTTTACTACC TAC 153 (SEQ ID NO: 313)

[1435] / translation="GYTFTTY" (SEQ ID NO: 314)

[1436] Framework 2 (base pairs 154-210) :

[1437] 154 ACGATGC ACTGGGTAAA ACAGAGGCCT GGACAGGGTC TGGAGTGGAT TGGACACATT 210 (SEQ ID NO: 315)

[1438] / translation="TMHWVKQRPGQGLEWIGHI" (SEQ ID NO: 316)

[1439] CDR-H2 (base pairs 211-228) :

[1440] 211 AATCCTAGCA GTGGATAT 228 (SEQ ID NO: 317)

[1441] / translation="NPSSGY" (SEQ ID NO: 318)

[1442] Framework 3 (base pairs 229-351) : 229 AC TGAGTACAAT CAGAAATTCA AGGACAAGAC CACACTGACT GCAGACAAAT

[1443] CCTCCAGCAC AGCCCACATG CAACTGAGCA GCCTAACATC TGAGGACTCT GCGGTCTATT ACTGTGCAAG A 351 (SEQ ID NO: 319)

[1444] / translation="TEYNQKFKDKTTLTADKSSSTAHMQLSSLTSEDSAVYYCAR" (SEQ ID NO: 320)

[1445] CDR-H3 (base pairs 352-372) :

[1446] 352 CACCCCTGG GACTCGAACT AC 372 (SEQ ID NO: 321)

[1447] / translation="HPWDSNY" (SEQ ID NO: 322)

[1448] Framework 4 (base pairs 373-405):

[1449] 373 TGGGGCCA AGGCACCACT CTCACAGTCT CCTCA 405 (SEQ ID NO: 323)

[1450] / translation="WGQGTTLTVSS" (SEQ ID NO: 324)

[1451] MHC2556HCN.1 499.5.4E5.20.22

[1452]

[1453] 4E5 Light Chain Variable (vL) DNA and Amino Acid Sequences*

[1454] MHC2556LCN.2 ;M13. 499.5.4E5.20.22

[1455] CDR Analysis

[1456] ENIDSY ..AAT ..QHYYITPFT

[1457] Amino Acid Sequence in FASTA format (MHC2556LCN .2 \ ; M13F)

[1458] > MHC2556LCN .2 \ ; M13 F

[1459] LOCUS 4E5_vL 381 bp DNA linear

[1460] FEATURES Location / Qualifiers

[1461] J_segment 352..381

[1462] / label=FWR4

[1463] V_segment 325..351

[1464] / label=CDR3

[1465] V region 229..324

[1466] / label=FWR3 V_segment 208..229

[1467] / label=CDR2

[1468] V_region 163..207

[1469] / label=FWR2

[1470] V_segment 130..162

[1471] / label=CDRl

[1472] V_region 61..129

[1473] / label=FWRl

[1474] sig_peptide 1..60

[1475] / label=LS

[1476] CDS 1..381

[1477] / label=4E5 vL

[1478] 4E5_vL

[1479] / translation="MSVPTQLLGLLLLWLTDARCDIQMTQSPASLSASVGETVTITCRASENIDSYLAWY QQKQGRSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDVARYYCQHYYITPFTFGSGTKLEI

[1480] K" (SEQ ID NO: 325)

[1481] Nucleotide Sequence in FASTA format (MHC2556LCN .2 \ ; M13F)

[1482] > MHC2556LCN .2 \ ; M13 F

[1483] ORIGIN

[1484] 1 ATGAGTGTGC CCACTCAGCT CCTGGGGTTG CTGCTGCTGT GGCTTACAGA TGCCAGATGT 61 GACATCCAGA TGACTCAGTC TCCAGCTTCC CTGTCTGCAT CTGTGGGAGA AACTGTCACC 121 ATCACATGTC GAGCAAGTGA GAATATTGAC AGTTATTTAG CATGGTATCA GCAGAAACAG 181 GGAAGATCTC CTCAGCTCCT GGTCTATGCT GCAACAAACT TAGCAGATGG TGTGCCATCA 241 AGGTTCAGTG GCAGTGGATC AGGCACACAG TATTCTCTCA AGATCAACAG CCTGCAGTCT 301 GAAGATGTTG CGAGATATTA CTGTCAACAT TATTATATTA CTCCATTCAC GTTCGGCTCG 361 GGGACAAAGT TGGAAATAAA A (SEQ ID NO: 326)

[1485] Signal Peptide (base pairs 1-60) :

[1486] 1 ATGAGTGTGC CCACTCAGCT CCTGGGGTTG CTGCTGCTGT GGCTTACAGA TGCCAGATGT 60 (SEQ ID NO: 327)

[1487] / translation="MSVPTQLLGLLLLWLTDARC" (SEQ ID NO: 328)

[1488] Framework 1 (base pairs 61-129) :

[1489] 61 GACATCCAGA TGACTCAGTC TCCAGCTTCC CTGTCTGCAT CTGTGGGAGA AACTGTCACC ATCACATGT 129 (SEQ ID NO: 329)

[1490] / translation="DIQMTQSPASLSASVGETVTITC" (SEQ ID NO: 330)

[1491] CDR-L1 (base pairs 130-162) :

[1492] 130 C GAGCAAGTGA GAATATTGAC AGTTATTTAG CA 162 (SEQ ID NO: 331)

[1493] / translation="RASENIDSYLA" (SEQ ID NO: 332) Framework 2 (base pairs 163-207) :

[1494] 163 TGGTATCA GCAGAAACAG GGAAGATCTC CTCAGCTCCT GGTCTAT 207 (SEQ ID NO: 333)

[1495] / translation="WYQQKQGRSPQLLVY" (SEQ ID NO: 334)

[1496] CDR-L2 (base pairs 208-228) :

[1497] 208 GCT GCAACAAACT TAGCAGAT 228 (SEQ ID NO: 335)

[1498] / translation="AATNLAD" (SEQ ID NO: 336)

[1499] Framework 3 (base pairs 229-324) :

[1500] 229 GG TGTGCCATCA AGGTTCAGTG GCAGTGGATC AGGCACACAG TATTCTCTCA

[1501] AGATCAACAG CCTGCAGTCT GAAGATGTTG CGAGATATTA CTGT 324 (SEQ ID NO: 337) / translation="GVPSRFSGSGSGTQYSLKINSLQSEDVARYYC" (SEQ ID NO: 338)

[1502] CDR-L3 (base pairs 325-351) :

[1503] 325 CAACAT TATTATATTA CTCCATTCAC G 351 (SEQ ID NO: 339)

[1504] / translation="QHYYITPFT" (SEQ ID NO: 340)

[1505] Framework 4 (base pairs 352-381):

[1506] 352 TTCGGCTCG GGGACAAAGT TGGAAATAAA A 381 (SEQ ID NO: 341)

[1507] / translation="FGSGTKLEIK" (SEQ ID NO: 342)

[1508] MHC2556LCN.2 499.5.4E5.20.22

[1509]

[1510] * CDR definitions and protein sequence numbering according to Kabat. CDR amino acid sequences are underlined in order of CDR1, CDR2, and CDR3, respectively.

[1511] Table 3: Summary of Anti-HHLA2 mAb binding and ligand blocking characteristics

[1512] l: Binding to HHLA2 transfected 300.19 mouse pre-B cell leukemic cell line by flow cytometry

[1513] 2: Blockade of HHLA2-mIgG2a binding to TMIGD2 transfected 300.19 mouse pre-B cell leukemic cell line by flow cytometry

[1514] 3:not determined: 8Al2-not measurable due to low binding; 4E5 -culture supernatants

[1515] III Nucleic Acids. Vectors and Recombinant Host Cells

[1516] A further object of the invention relates to nucleic acid sequences encoding monoclonal antibodies and fragments thereof, immunoglobulins, and polypeptides of the present invention.

[1517] For example, in a particular embodiment, the present invention relates, in part, to a nucleic acid sequence encoding the vH domain of mAb 8A12, or the vL domain of mAb 8A12. In another particular embodiment, the present invention relates, in part, to a nucleic acid sequence encoding the vH domain or the vL domain of at least one effective anti- HHLA2 mAb isolated from the polyclonal antibodies 1.2 and / or 2.2.

[1518] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[1519] The terms“vector”,“cloning vector” and“expression vector” mean the vehicle by which a DNA or RNA sequence ( e.g . a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Thus, a further object of the invention relates to a vector comprising a nucleic acid of the present invention.

[1520] Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said polypeptide upon

[1521] administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason J O et al. 1985) and enhancer (Gillies S D et al. 1983) of immunoglobulin H chain and the like.

[1522] Any expression vector for animal cell can be used. Examples of suitable vectors include pAGEl07 (Miyaji H et al. 1990), pAGEl03 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSGl beta d2-4-(Miyaji H et al. 1990) and the like. Other representative examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pETC, pcDNA, pBR, and the like. Representative examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO 94 / 19478.

[1523] A further object of the present invention relates to a cell which has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention. The term“transformation” means the introduction of a“foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been“transformed.”

[1524] The nucleic acids of the present invention may be used to produce a recombinant polypeptide of the invention in a suitable expression system. The term“expression system” means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

[1525] 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, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli , Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g, Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g, produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as“DHFR gene”) is defective (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11. l6Ag.20 cell (ATCC CRL 1662, hereinafter referred to as“YB2 / 0 cell”), and the like. The YB2 / 0 cell is preferred, since ADCC activity of chimeric or humanized antibodies is enhanced when expressed in this cell.

[1526] The present invention also relates to a method of producing a recombinant host cell expressing an antibody or a polypeptide of the invention according to the invention, said method comprising the steps consisting of (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and / or secrete said antibody or polypeptide. Such recombinant host cells can be used for the production of antibodies and polypeptides of the invention.

[1527] In another aspect, the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein. Thus, the polynucleotides of this embodiment can be used for isolating, detecting, and / or quantifying nucleic acids comprising such polynucleotides. For example, polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library. Preferably, the cDNA library comprises at least 80% full- length sequences, preferably, at least 85% or 90% full-length sequences, and, more preferably, at least 95% full-length sequences. The cDNA libraries can be normalized to increase the representation of rare sequences. Low or moderate stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions can optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences. Optionally, polynucleotides of this invention will encode at least a portion of an antibody encoded by the polynucleotides described herein. The polynucleotides of this invention embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding an antibody of the present invention. See, e.g, Ausubel, supra; Colligan, supra, each entirely incorporated herein by reference.

[1528] IV. Methods of Producing Antibodies

[1529] Antibodies and fragments thereof, immunoglobulins, and polypeptides of the present invention may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.

[1530] Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies or polypeptides, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, Calif.) and following the manufacturer's instructions. Alternatively, antibodies and other polypeptides of the present invention can be synthesized by recombinant DNA techniques as is well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

[1531] In particular, the present invention further relates to a method of producing an antibody or a polypeptide of the invention, which method comprises the steps consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said antibody or polypeptide; and (ii) recovering the expressed antibody or polypeptide.

[1532] Antibodies and other polypeptides of the present invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification. See, e.g. , Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g, Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference. Chimeric antibodies (e.g., mouse-human chimeras or non-rodent-human chimeras) of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. The CH domain of a human chimeric antibody can be any region which belongs to human immunoglobulin, such as the IgG class or a subclass thereof, such as IgGl, IgG2, IgG3 and IgG4. Similarly, the CL of a human chimeric antibody can be any region which belongs to Ig, such as the kappa class or lambda class chimeric and humanized monoclonal antibodies, comprising both human and non human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by 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

[1533] Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger el al. PCT Application WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.

[1534] 139:3521-3526; Sun et al. ( 1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al.

[1535] (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559); Morrison, S. L. (1985) Science 229: 1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) , / . Immunol. 141 :4053-4060.

[1536] In addition, humanized antibodies can be made according to standard protocols such as those disclosed in U.S. Patent 5,565,332. In another embodiment, antibody chains or specific binding pair members can be produced by recombination between vectors comprising nucleic acid molecules encoding a fusion of a polypeptide chain of a specific binding pair member and a component of a replicable generic display package and vectors containing nucleic acid molecules encoding a second polypeptide chain of a single binding pair member using techniques known in the art, e.g, as described in U.S. Patents 5,565,332, 5,871,907, or 5,733,743. Humanized antibodies of the present invention can be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell. The humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type).

[1537] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well-known in the art (See, e.g ., Riechmann L. et al. 1988; Neuberger M S. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka G M et al. (1994); Roguska M A. et al. (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO

[1538] 96 / 02576).

[1539] Similarly, bispecific or multispecific antibodies described herein can be made according to standard procedures. For example, triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific or multispecific antibodies. Examples of bispecific and multispecific antibodies produced by a hybrid hybridoma or a trioma are disclosed in U.S. Patent 4,474,893. Such antibodies can also be constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and Staerz and Bevan (1986) Immunol. Today 7:241). Alternatively, such antibodiescan also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling the desired antibodies. They can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and Fv sequences. The antibody component can bind to a polypeptide or a fragment thereof of one or more biomarkers of the invention, including one or more immunoinhibitory biomarkers described herein. In addition, methods for producing antibody fragments are well-known. For example, Fab fragments of the present invention can be obtained by treating an antibody which specifically reacts with human HHLA2 (such as mAh 8A12 and polyclonal antibodies 1.2 and 2.2) with a protease such as papain. Also, Fabs can be produced by inserting DNA encoding Fabs of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote (as appropriate) to express the Fabs.

[1540] Similarly, F(ab')2 fragments of the present invention can be obtained treating an antibody which specifically reacts with HHLA2 with a protease, pepsin. Also, the F(ab')2 fragment can be produced by binding Fab' described below via a thioether bond or a disulfide bond.

[1541] Fab' fragments of the present invention can be obtained treating F(ab')2 which specifically reacts with human HHLA2 with a reducing agent, dithiothreitol. Also, the Fab' fragments can be produced by inserting DNA encoding a Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.

[1542] In addition, scFvs of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well-known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) from a donor scFv fragment, and grafting them onto a human scFv fragment framework of known three dimensional structure (see, e.g ., W098 / 45322; WO 87 / 02671; U.S. Pat. No. 5,859,205; U.S. Pat. No. 5,585,089; U.S. Pat. No. 4,816,567; ER0173494).

[1543] V. Modification of Antibodies. Immunoglobulins and Polypeptides

[1544] Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. It is known that when a humanized antibody is produced by simply grafting only CDRs in VH and VL of an antibody derived from a non human animal in FRs of the VH and VL of a human antibody, the antigen binding activity is reduced in comparison with that of the original antibody derived from a non-human animal. It is considered that several amino acid residues of the VH and VL of the non human antibody, not only in CDRs but also in FRs, are directly or indirectly associated with the antigen binding activity. Hence, substitution of these amino acid residues with different amino acid residues derived from FRs of the VH and VL of the human antibody would reduce binding activity and can be corrected by replacing the amino acids with amino acid residues of the original antibody derived from a non-human animal.

[1545] Modifications and changes may be made in the structure of the antibodies of the present invention, and in the DNA sequences encoding them, and still obtain a functional molecule that encodes an antibody and polypeptide with desirable characteristics. For example, certain amino acids may be substituted by other amino acids in a protein structure without appreciable loss of activity. Since the interactive capacity and nature of a protein define the protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA encoding sequence, while nevertheless obtaining a protein with like properties. It is thus contemplated that various changes may be made in the antibodies sequences of the invention, or corresponding DNA sequences which encode said polypeptides, without appreciable loss of their biological activity.

[1546] In one embodiment, amino acid changes may be achieved by changing codons in the DNA sequence to encode conservative substitutions based on 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 sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code (see genetic code chart above).

[1547] As described above, an important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[1548] In making the changes in the amino sequences of polypeptide, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these 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); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (<RTI 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[1549] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein.

[1550] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their

[1551] hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well-known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[1552] Another type of amino acid modification of the antibody of the invention may be useful for altering the original glycosylation pattern of the antibody to, for example, increase stability. By“altering” is meant deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically N-linked.“N-linked” refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagines-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in W087 / 05330.

[1553] Similarly, removal of any carbohydrate moieties present on the antibody may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N- acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described by Sojahr H. et al. (1987) and by Edge, A S. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura, N R. et al. (1987).

[1554] Other modifications can involve the formation of immunoconjugates. For example, in one type of covalent modification, antibodies or proteins are covalently linked to one of a variety of non proteinaceous polymers, e.g ., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in ET.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4, 179,337.

[1555] Conjugation of antibodies or other proteins of the present invention with

[1556] heterologous agents can be made using a variety of bifunctional protein coupling agents including but not limited to N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl)cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido

[1557] compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6 diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2, 4-dinitrobenzene). For example, carbon labeled l-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (WO 94 / 11026).

[1558] In another aspect, the present invention features antibodies that specifically bind HHLA2 conjugated to a therapeutic moiety, such as a cytotoxin, a drug, and / or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as “immunotoxins.” A cytotoxin or cytotoxic agent includes any agent that is detrimental to ( e.g ., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g, mechlorethamine, thioepa

[1559] chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g, dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti -mitotic agents (e.g, vincristine and vinblastine). An antibody of the present invention can be conjugated to a radioisotope, e.g, radioactive iodine, to generate cytotoxic radiopharmaceuticals for treating a related disorder, such as a cancer.

[1560] Conjugated anti-HHLA2 antibodies can be used diagnostically or prognostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g, to determine the efficacy of a given treatment regimen or to select patients most likely to response to an immunotherapy. For example, cells can be permeabilized in a flow cytometry assay to allow antibodies that bind HHLA2 (such as mAb 8A12 and polyclonal antibodies 1.2 and 2.2) to target its recognized intracellular epitope and allow detection of the binding by analyzing signals emanating from the conjugated molecules. Detection can ...

Claims

What is claimed is:

1. A monoclonal antibody, or antigen-binding fragment thereof, wherein the monoclonal antibody comprises:a) a heavy chain sequence with at least about 95% identity to a heavy chain sequence selected from the group consisting of the sequences listed in Table 2; and / orb) a light chain sequence with at least about 95% identity to a light chain sequence selected from the group consisting of the sequences listed in Table 2.

2. A monoclonal antibody, or antigen-binding fragment thereof, wherein the monoclonal antibody comprises:a) a heavy chain CDR sequence with at least about 95% identity to a heavy chain CDR sequence selected from the group consisting of the sequences listed in Table 2; and / or b) a light chain CDR sequence with at least about 95% identity to a light chain CDR sequence selected from the group consisting of the sequences listed in Table 2.

3. A monoclonal antibody, or antigen-binding fragment thereof, wherein the monoclonal antibody comprises:a) a heavy chain sequence selected from the group consisting of the sequences listed in Table 2; and / orb) a light chain sequence selected from the group consisting of the sequences listed in Table 2.

4. A monoclonal antibody, or antigen-binding fragment thereof, wherein the monoclonal antibody comprises:a) a heavy chain CDR sequence selected from the group consisting of the sequences listed in Table 2; and / orb) a light chain CDR sequence selected from the group consisting the sequences listed in Table 2.

5. The monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-4, wherein the monoclonal antibody, or antigen-binding fragment thereof, is chimeric, humanized, composite, murine, or human.

6. The monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-5, wherein the monoclonal antibody, or antigen-binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab’)2), Fab’, dsFv, scFv, sc(Fv)2, and diabodies fragments.

7. The monoclonal antibody, or antigen-binding fragment thereof, of any one of claims1-6, wherein said monoclonal antibody, or antigen-binding fragment thereof, is obtainable from hybridoma _ deposited under deposit accession number _ .

8. The monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-7, wherein the monoclonal antibody, or antigen-binding fragment thereof, inhibits a) the binding of HHLA2 to TMIGD2, b) the binding of HHLA2 to KIR3DL3, or c) the binding of HHLA2 to TMIGD2 and the binding of HHLA2 to KIR3DL3.

9. The monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-8, wherein the monoclonal antibody, or antigen-binding fragment thereof, specifically binds HHLA2.

10. An immunoglobulin heavy and / or light chain selected from the group consisting of immunoglobulin heavy and light chain sequences listed in Table 2.

11. An isolated nucleic acid molecule that hybridizes, under stringent conditions, with the complement of a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2, or a sequence with at least about 95% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2.

12. A vector comprising the isolated nucleic acid of claim 11.

13. A host cell which comprises the isolated nucleic acid of claim 11, comprises the vector of claim 12, expresses the antibody, or antigen-binding fragment thereof, of any one of claims 1-9, or is accessible under deposit accession number _ .

14. A device or kit comprising at least one monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1-9, said device or kit optionally comprising a label to detect the at least one monoclonal antibody, or antigen-bindingfragment thereof, or a complex comprising the monoclonal antibody, or antigen-binding fragment thereof.

15. A method of producing at least one monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1-9, which method comprises the steps of: (i) culturing a transformed host cell which has been transformed by a nucleic acid comprising a sequence encoding at least one monoclonal antibody according to any one of claims 1-9 under conditions suitable to allow expression of said monoclonal antibody, or antigen-binding fragment thereof; and (ii) recovering the expressed monoclonal antibody, or antigen-binding fragment thereof.

16. A method of detecting the presence or level of an HHLA2 polypeptide comprising obtaining a sample and detecting said polypeptide in the sample by use of at least one monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1-9.

17. The method of claim 16, wherein the at least one monoclonal antibody, or antigen binding fragment thereof, forms a complex with an HHLA2 polypeptide and the complex is detected in the form of an enzyme linked immunosorbent assay (ELISA), radioimmune assay (RIA), immunochemically, Western blot, or using an intracellular flow assay.

18. A method for monitoring the progression of a disorder associated with aberrant HHLA2 expression in a subject, the method comprising:a) detecting in a subject sample at a first point in time the level of HHLA2 using at least one monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1-9;b) repeating step a) at a subsequent point in time; andc) comparing the level of HHLA2 detected in steps a) and b) to monitor the progression of the disorder in the subject.

19. The method of claim 18, wherein between the first point in time and the subsequent point in time, the subject has undergone treatment to ameliorate the disorder.

20. A method for predicting the clinical outcome of a subject afflicted with a disorder associated with aberrant HHLA2 expression, the method comprising:a) determining the level of HHLA2 in a subject sample using at least one monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1-9;b) determining the level of HHLA2 in a sample from a control subject having a good clinical outcome using the at least one monoclonal antibody, or antigen-binding fragment thereof; andc) comparing the level of HHLA2 in the subject sample and in the sample from the control subject;wherein a significantly higher level of HHLA2 in the subject sample as compared to the level in the sample from the control subject is an indication that the subject has a poor clinical outcome.

21. A method of assessing the efficacy of a therapy for a disorder associated with aberrant HHLA2 expression in a subject, the method comprising:a) determining the level of HHLA2 using at least one monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1-9, in a first sample obtained from the subject prior to providing at least a portion of the therapy to the subject, andb) determining the level of HHLA2 in a second sample obtained from the subject following provision of the portion of the therapy,wherein a significantly lower level of HHLA2 in the second sample, relative to the first sample, is an indication that the therapy is efficacious for inhibiting the disorder in the subject.

22. A method of assessing the efficacy of a test compound for inhibiting a disorder associated with aberrant HHLA2 expression in a subject, the method comprising:a) determining the level of HHLA2 using at least one monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1-9, in a first sample obtained from the subject and exposed to the test compound; andb) determining the level of HHLA2 in a second sample obtained from the subject, wherein the second sample is not exposed to the test compound, and a significantly lower level of HHLA2, relative to the second sample, is an indication that the test compound is efficacious for inhibiting the disorder in the subject.

23. The method of claim 22, wherein the first and second samples are portions of a single sample obtained from the subject or portions of pooled samples obtained from the subject.

24. The method of any one of claims 18-23, wherein the disorder is a cancer.

25. The method of claim 24, wherein the cancer is selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, Acute myeloid leukemia, head and neck carcinoma, liver cancer, ovarian cancer, prostate cancer, uterine cancer, gliomas, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B cell lymphoma, and a cancer infiltrated with immune cells expressing a receptor to HHLA2.

26. The method of any one of claims 16-25, wherein the sample comprises cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject.

27. The method of claim 20, wherein said significantly higher level of HHLA2 comprises an at least twenty percent increase between the level of HHLA2 in the subject sample relative to the normal level of HHLA2 in the sample from the control subject.

28. The method of any one of claims 21-26, wherein said significantly lower level of HHLA2 comprises an at least twenty percent decrease of the level of HHLA2.

29. The method of any one of claims 18-28, wherein the subject is a human.

30. A method of treating a subject afflicted with cancer comprising administering to the subject at least one monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1-9.

31. The method of claim 30, wherein the at least one monoclonal antibody, or antigen binding fragment thereof, is conjugated to a cytotoxic agent.

32. The method of claim 31, wherein the cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, a biologic agent, a toxin, and a radioactive isotope.

33. The method of any one of claims 30-32, wherein the at least one monoclonal antibody, or antigen-binding fragment thereof, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of a tumor of the cancer.

34. The method of any one of claims 30-33, wherein the at least one monoclonal antibody, or antigen-binding fragment thereof, is administered in a pharmaceutically acceptable formulation.

35. The method of any one of claims 30-34, further comprising administering to the subject a therapeutic agent or regimen for treating cancer.

36. The method of any one of claims 30-35, further comprising administering to the subject an additional therapy selected from the group consisting of immunotherapy, checkpoint blockade, cancer vaccines, chimeric antigen receptors, chemotherapy, radiation, target therapy, and surgery.

37. The method of any one of claims 30-36, wherein cancer cells and / or tumor immune infiltrating cells in the subject express HHLA2.

38. The method of any one of claims 30-37, wherein the cancer is selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, Acute myeloid leukemia, head and neck carcinoma, liver cancer, ovarian cancer, prostate cancer, uterine cancer, gliomas, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B cell lymphoma, and a cancer infiltrated with immune cells expressing a receptor to HHLA2.

39. The method of claim 38, wherein the cancer is selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), head and neck carcinoma, liver cancer, ovarian cancer, prostate cancer, and uterine cancer.

40. The method of any one of claims 30-39, wherein the subject is an animal model of cancer.

41. The method of claim 40, wherein the animal model is a mouse model, optionally wherein the mouse model is a humanized mouse model.

42. The method of any one of claims 30-41, wherein the subject is a mammal.

43. The method of claim 42, wherein the mammal is a humanized mouse or a human.

44. The method of claim 43, wherein the mammal is a human.

45. A method of modulating an immune response by inhibiting the interaction between HHL A2 and its binding inhibitor recept, KIRDL3.

46. The method of claim 45, wherein the interaction between HHLA2 and KIRDL3 is blocked for use in checkpoint blockade cancer immunotherapy.

47. The method of claim 45 or 46, wherein the interaction between HHLA2 and KIRDL3 is inhibited or blocked using an anti-HHLA2 antibody.

48. The method of claim 47, wherein the anti-HHLA2 antibody is a checkpoint inhibitor of T cell activation for cancer immunotherapy.

49. A method of modulating an immune response by selectively inhibiting the interaction between HHLA2 and its binding inhibitor receptor, KIR3DL3, without blocking or significantly inhibiting the interaction between HHLA2 and its binding stimulatory receptor, TMIGD2.