KIR3DL3 as HHLA2 receptor, anti-HHLA2 antibodies and uses thereof

Abstract

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

Description

[0001] Rights Statement

[0002] This invention was made with government support under Grant No. P01 AI056299 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0003] Immune checkpoints, such as CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, butyrophilin and A2aR, as well as a variety of other immune checkpoints, negatively regulate the immune response progression based on the complexity and combinatorial interactions between multiple inputs. Inhibitors of immune checkpoints can regulate the immune response of some subjects, but immune checkpoint expression and the interaction with natural binding partners vary between different subjects and within different tissues of the subject. Therefore, there is a great need in the art to identify new checkpoints for intervention. HHLA2 is a newly identified B7 family member that regulates T cell function. HHLA2 is identified as a specific ligand for TMIGD2, and the HHLA2 / TMIGD2 interaction selectively co-stimulates human T cell growth and cytokine production via the AKT-dependent signaling cascade (Zhu et al. (2013) Nat. Comm. 4: 2043; Janakiram et al. (2015) Clin. Cancer Res. 21: 2359–2366). Several studies have shown that a second, uncharacterized receptor for HHLA2 on activated T cells plays a co-inhibitory function (Zhao et al. (2013) Proc. Natl. Acad. Sci. USA 110:9879–9884; Xiao and Freeman et al. (2015) Clin. Cancer Res 21:2201–2203; Wang et al. (2014) J. Immunol. 192:126.11). HHLA2 is expressed on various human cancers, and its co-inhibitory function makes it a candidate for cancer immunotherapy. Summary of the Invention

[0004] The present invention is based at least in part on the following discovery, that is, HHLA2, a member of the B7 gene family, is widely expressed in various tumors and antigen-presenting cells, and is regarded as both an activating ligand and an inhibitory ligand of T cells. TMIGD2 expressed in naive T cells is an activating receptor of HHLA2 and transduces co-stimulatory signals after T cell antigen receptor (TCR) engagement. TMIGD2 is downregulated after repeated TCR stimulation. The putative inhibitory receptor of HHLA2 may be upregulated on activated T cells to regulate T cell activation. The present invention is based at least in part on the following discovery, that is, HHLA2 binds to the receptor KIR3DL3 on T cells and NK cells, and the result of the HHLA2:KIR3DL3 interaction is that T cell activation is suppressed. Based on the observation that HHLA2 is highly expressed in tumors and can act as a checkpoint ligand, a group of anti-HHLA2 human monoclonal antibodies (mAbs) as candidate immune checkpoint inhibitors are generated. Blocking and non-blocking anti-HHLA2 mAbs were identified by evaluating the binding of soluble human HHLA2-mIgG2a to TMIGD2-transfected 300.19 mouse pre-B leukemia cells or to KIR3DL3-transfected 300.19 mouse pre-B leukemia cells. Anti-HHLA2 mAbs that blocked HHLA2 binding to both TMIGD2 and KIR3DL3 in T cell assays, or more selectively blocked binding to KIR3DL3 but not to TMIGD2, were shown to be checkpoint inhibitor antibodies.

[0005] In one aspect, a monoclonal antibody or an antigen-binding fragment thereof is provided, wherein the monoclonal antibody comprises: a) a heavy chain sequence that is at least about 95% identical to a heavy chain sequence selected from the group consisting of the sequences listed in Table 2; and / or b) a light chain sequence that is at least about 95% identical to a light chain sequence selected from the group consisting of the sequences listed in Table 2.

[0006] In another aspect, a monoclonal antibody or antigen-binding fragment thereof is provided, wherein the monoclonal antibody comprises: a) a heavy chain CDR sequence that is at least about 95% identical 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 that is at least about 95% identical to a light chain CDR sequence selected from the group consisting of the sequences listed in Table 2.

[0007] In another aspect, a monoclonal antibody or an antigen-binding fragment thereof is provided, 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.

[0008] In another aspect, a monoclonal antibody or an antigen-binding fragment thereof is provided, 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 of the sequences listed in Table 2.

[0009] Also provided are various embodiments that can be applied to any aspect of the 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 a diabody fragment. In yet 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 are shown to be checkpoint blockers. In another embodiment, the monoclonal antibody or antigen-binding fragment thereof specifically binds to HHLA2.

[0010] In another aspect, an immunoglobulin heavy chain and / or light chain is provided, which is selected from the group consisting of the immunoglobulin heavy chain sequences and light chain sequences listed in Table 2.

[0011] In yet another aspect, an isolated nucleic acid molecule is provided that hybridizes under stringent conditions to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2 or to the complement of a sequence having at least about 95% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2.

[0012] In yet another aspect, a vector comprising the isolated nucleic acid described herein is provided.

[0013] In another aspect, a host cell is provided that comprises the isolated nucleic acid described herein, comprises the vector described herein, expresses the antibody or antigen-binding fragment thereof described herein, or is accessible under deposit accession number ______.

[0014] In yet another aspect, a device or kit is provided, comprising at least one monoclonal antibody or antigen-binding fragment thereof as described herein, optionally comprising a label for detecting the at least one monoclonal antibody or antigen-binding fragment thereof, or a complex comprising the monoclonal antibody or antigen-binding fragment thereof.

[0015] In yet another aspect, a method of producing at least one monoclonal antibody or antigen-binding fragment thereof as described herein is provided, the method comprising the steps of: (i) culturing a transformed host cell that has been transformed with a nucleic acid comprising a sequence encoding the monoclonal antibody under conditions suitable for allowing expression of at least one monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; and (ii) recovering the expressed monoclonal antibody or antigen-binding fragment thereof.

[0016] In another aspect, a method of detecting the presence or level of an HHLA2 polypeptide comprises obtaining a sample and detecting the polypeptide in the sample by using at least one monoclonal antibody or antigen-binding fragment thereof described herein.

[0017] As described above, certain embodiments are applicable to any of the methods 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 by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blotting, or using intracellular flow cytometry.

[0018] In another aspect, a method for monitoring the progression of a condition associated with aberrant HHLA2 expression in a subject is provided, the method comprising: a) detecting the level of HHLA2 in a sample from the subject at a first time point using at least one monoclonal antibody or antigen-binding fragment thereof described herein; b) repeating step a) at a subsequent time point; and c) comparing the levels of HHLA2 detected in steps a) and b) to monitor the progression of the condition in the subject.

[0019] As described above, certain embodiments are applicable to any of the methods described herein.For example, in one embodiment, between a first time point and a subsequent time point, the subject undergoes treatment to improve the condition.

[0020] In another aspect, a method for predicting clinical outcome in a subject suffering from a disorder associated with aberrant HHLA2 expression is provided, the method comprising: a) determining the level of HHLA2 in a sample from the subject 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 with 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 sample from the subject with the level in a sample from the control subject; wherein a significantly higher level of HHLA2 in the sample from the subject compared to the level in the sample from the control subject indicates that the subject has a poor clinical outcome.

[0021] In yet another aspect, a method of assessing the efficacy of a therapy for a condition associated with aberrant HHLA2 expression in a subject is provided, the method comprising: a) determining the level of HHLA2 in a first sample obtained from the subject using at least one monoclonal antibody or antigen-binding fragment thereof described herein before 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 after providing the portion of the therapy, wherein a significantly lower level of HHLA2 in the second sample relative to the first sample indicates that the therapy is effective in inhibiting the condition in the subject.

[0022] In yet another aspect, a method for assessing the efficacy of a test compound for inhibiting a condition associated with aberrant HHLA2 expression in a subject is provided, the method comprising: a) determining the level of HHLA2 in a first sample obtained from the subject and exposed to the test compound using at least one monoclonal antibody or antigen-binding fragment thereof described herein; and b) determining the level of HHLA2 in a second sample obtained from the subject, wherein the second sample was not exposed to the test compound, and a significantly lower level of HHLA2 relative to the second sample indicates that the test compound is effective for inhibiting the condition in the subject.

[0023] As described above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, the first sample and the second sample are portions of a single sample obtained from a subject or portions of a pooled sample obtained from a subject. In another embodiment, the condition is cancer. In yet another embodiment, the cancer is selected from the group consisting of lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancers infiltrating immune cells expressing a receptor for HHLA2. In another embodiment, the sample comprises cells, serum, peri-cancer tissue, and / or intratumoral tissue obtained from a subject. In yet another embodiment, a significantly higher level of HHLA2 comprises an increase of at least 20% in the level of HHLA2 in the subject sample relative to the normal level of HHLA2 in a sample from a control subject. In another embodiment, a significantly lower level of HHLA2 comprises a decrease of at least 20% in the level of HHLA2. In yet another embodiment, the subject is a human.

[0024] In yet another aspect, a method of treating a subject suffering from cancer is provided, comprising administering to the subject at least one monoclonal antibody or antigen-binding fragment thereof described herein.

[0025] As described above, certain embodiments are applicable to any of the methods 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 biological agent, a toxin, and a radioisotope. In yet another embodiment, the at least one monoclonal antibody or antigen-binding fragment thereof reduces the number of proliferating cells in cancer and / or reduces the volume or size of a cancer tumor. In another embodiment, the at least one monoclonal antibody or antigen-binding fragment thereof is administered in a pharmaceutically acceptable dosage form. In yet another embodiment, the methods described herein further comprise administering to the subject a therapeutic agent or regimen for treating cancer. In yet another embodiment, the methods described herein further comprise administering to the subject an additional therapy selected from the group consisting of: immunotherapy, checkpoint blockade, cancer vaccines, chimeric antigen receptors, chemotherapy, radiation, targeted therapy, and surgery. In another embodiment, the cancer cells and / or tumor immune-infiltrating cells in the subject express HHLA2. In yet another embodiment, the cancer is selected from the group consisting of lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancer infiltrating immune cells expressing receptors for HHLA2. In another embodiment, the cancer is selected from the group consisting of lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), head and neck cancer, liver cancer, ovarian cancer, prostate cancer, and uterine cancer. In yet another embodiment, the subject is an animal model for 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 yet another embodiment, the mammal is a human.

[0026] In another aspect, a method for modulating an immune response by inhibiting the interaction between HHLA2 and its binding inhibitor receptor KIRDL3 is provided.

[0027] In yet another aspect, a method for modulating an immune response by selectively inhibiting the interaction between HHLA2 and its binding inhibitory receptor KIR3DL3 without blocking or significantly inhibiting the interaction between HHLA2 and its binding stimulatory receptor TMIGD2 is provided.

[0028] As described above, certain embodiments are applicable to any of the methods 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, an anti-HHLA2 antibody is used to inhibit or block the interaction between HHLA2 and KIRDL3. In yet another embodiment, the anti-HHLA2 antibody is a checkpoint inhibitor of T cell activation for use in cancer immunotherapy.

[0029] For any figure showing a bar graph, curve, or other data associated with a legend, the bar, curve, or other data presented from left to right for each indication corresponds directly and sequentially to the top to bottom or left to right boxes of the legend. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A Shown are binding affinity data for anti-HHLA2 mAbs on the HHLA2-transfected 300.19 mouse pre-B cell leukemia cell line obtained by flow cytometry.

[0031] Figure 1B Shown is the blocking of TMIGD2-human IgG binding to the HHLA2-transfected 300.19 mouse pre-B cell leukemia cell line by anti-HHLA2 mAb as determined by flow cytometry.

[0032] Figure 2 Shown are Western blot data of proteins from nine human tumor cell lines probed with anti-HHLA2 mAb 8D2 (IHC mAb).

[0033] Figure 3 Shown are HHLA2 mRNA expression in normal kidney versus clear cell renal cell carcinoma (ccRCC) compared with other checkpoint inhibitors.

[0034] Figure 4 HHLA2 expression in various cancers from the TCGA database is depicted.

[0035] Figure 5A Shown are HHLA2 immunohistochemistry (IHC) results for negative control cells (300.19), HHLA-2 transfected 300.19 cells (positive cells), OC1-Ly1 cells (negative tumor), and HDLM2 (positive Hodgkin lymphoma cell line) HHLA2 control cells.

[0036] Figure 5B HHLA2 expression in normal kidney is shown.

[0037] Figure 5C Representative images of HHLA2 expression in ccRCC from microarray (TMA) are shown.

[0038] Figure 5D Representative images showing the lack of HHLA2 expression in different ccRCCs from tumor microarrays (TMA).

[0039] Figure 6A Shown are the results of duplicate screening of a representative panel of approximately 300 plasma membrane clones and a confirmatory screen showing HHLA2 binding to KIR3DL3. A total of 5682 cell surface receptor membrane clones were screened.

[0040] Figure 6B Selective binding of HHLA2 to KIR3DL3 is shown.

[0041] Figure 6C Gene IDs and NCBI accession information of relevant biomarkers are shown.

[0042] Figure 7 Schematic diagram of the Jurkat NFAT receptor gene assay is shown.

[0043] Figure 8 A CAR-T cell model is shown.

[0044] Figure 9 Shown are inhibitory B7 family members that may be expressed by tumors.

[0045] Figure 10 A model showing the interaction of HHLA2 with two receptors (stimulatory and inhibitory HHLA2 receptors) to regulate T cell function. Along 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 pathways involving AKT phosphorylation. In the case of repetitive T cell activation, the expression of the stimulatory receptor TMIGD2 is gradually lost, allowing the expression of 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 et al. (2015) Clinical Cancer Research 21:2201–2203.

[0046] Figure 11 The stratification of patients with HHLA2+ve (i.e., lung cancer tissue expressing HHLA2 based on immunohistochemistry (IHC) staining with an HHLA2 mAb) in non-small cell lung cancer is shown. The percentage of HHLA2 expression in PD-L1-positive and -negative non-small cell lung cancers was calculated based on HHLA2 and PD-L1 immunostaining studies (Cheng et al. (2018) Clin Cancer Res 24:1954-1964).

[0047] Figure 12A and Figure 12B The expression of TMIGD2 on transfected 293T cells is shown. Figure 12A ) or KIR3DL3( Figure 12B ) expression. TMIGD2 or KIR3DL3 cDNA in the pEF-Puro expression vector was transiently transfected into 293T cells and stained 48–72 h later with: (1) TMIGD2 mAb (R&D systems, catalog #MAB83162; clone #953743), followed by goat-anti-mouse IgG F(ab) 2- PE (Andy Biotech, catalog F0102B); or (2) KIR3DL3-PE conjugated mAb (Andy Biotech, catalog #FAB8919R; clone #1136B), and detected by flow cytometry.

[0048] Figure 12C and Figure 12D HHLA2-Fc and TMIGD2 ( Figure 12C ) or KIR3DL3( Figure 12D Binding of HHLA2-mIgG2a to 293T cells transiently transfected with TMIGD2 or KIR3DL3 was detected by flow cytometry using a PE-labeled Fab2 goat anti-mouse IgG2a antibody (absorbed due to cross-reactivity with human Ig, Southern Biotech, catalog #1082-09).

[0049] Figure 13A and Figure 13B Binding of HHLA2 mAbs to human and cynomolgus monkey HHLA2 is shown. Various concentrations of HHLA2 mAbs were incubated with 300.19 pre-B cells transfected with human or cynomolgus monkey HHLA2 for 30 minutes at 4°C. Binding of HHLA2 mAbs to transfected 300.19 cells was detected by flow cytometry using PE-conjugated goat anti-mouse IgG (H+L).

[0050] Figure 14 Schematic diagram of the TMIGD2 / HHLA2 T cell co-stimulation assay is shown. TMIGD2 Jurkat T cell NFAT-luciferase reporter cells were stimulated with CHO cells transfected with anti-CD3 scFV or anti-CD3 scFV+HHLA2.

[0051] Figure 15HHLA2 expression in CHO-anti-CD3 scFV cells is shown. HHLA2 expression on CHO cells transfected with anti-CD3 scFV+HHLA2 (clone #28) was detected by flow cytometry using PE-conjugated 6F10 HHLA2 mAb.

[0052] Figure 16 TMIGD2 expression in Jurkat NFAT reporter cells is shown. TMIGD2 expression in TMIGD2-transfected Jurkat NFAT reporter cells (clone #62) is shown.

[0053] Figure 17 HHLA2 mAb blockade of TMIGD2-mediated T cell co-stimulation is shown. HHLA2-TCR-CHO cells were plated at 2 x 10 4 The cells were seeded at a density of 10 cells / well. After overnight incubation at 37°C with 5% CO2, the cells adhered to the plate. The next day, the culture medium was carefully removed from each well, and anti-HHLA2 antibody in 50 μl Jurkat cell culture medium was added, and the HHLA2-TCR-CHO cells were incubated for 1 hour, after which they were plated at 4-5 x 10 in 50 μl Jurkat cell culture medium. 4 Add MIGD2 NFAT Jurkat reporter cell line to each well. Mix the wells and incubate for approximately 3-6 hours. To visualize the luciferase signal, add 100 μl of ONE-Step TM A luciferase assay system (BPS Bioscience, catalog #60690) was added to each well and luminescence was read using a luminometer.

[0054] Figure 18 Schematic diagram of the KIR3DL3 / HHLA2 checkpoint T cell assay is shown. KIR3DL3 Jurkat T cell IL-2 promoter luciferase reporter cells were stimulated with CHO cells transfected with anti-CD3 scFV or anti-CD3 scFV+HHLA2.

[0055] 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.

[0056] Figures 20A-20CHHLA2 mAb checkpoint blockade of KIR3DL3-mediated T cell inhibition is shown. HHLA2-TCR-CHO cells were plated at 2 x 10 in CHOK1 growth medium in white opaque bottom 96-well plates. 4 The cells were seeded at a density of 10 cells / well. After overnight incubation at 37°C with 5% CO2, the cells were attached to the plate. The next day, the culture medium was carefully removed from each well and anti-HHLA2 antibodies in 50 μl Jurkat cell culture medium were added, and the HHLA2-TCR-CHO cells were incubated for 1 hour, followed by 4-5 x 10 in 50 μl Jurkat cell culture medium plus 2 μg / mL anti-CD28 antibodies (BPS Biosciences, #100186) (final concentration of 1 μg / mL / well in 100 μL assay mixture). 4 Add KIR3DL3_IL2_Jurkat reporter cell line to each well. Mix the wells and incubate for approximately 5 hours. To visualize the luciferase signal, add 100 μl of ONE-Step TM A luciferase assay system (BPS Biosciences, catalog #60690) was added to each well and luminescence was read using a luminometer.

[0057] Figures 21A-21C Titers of HHLA2 mAbs in the Jurkat KIR3DL3 inhibition assay are shown. Different concentrations of HHLA2 mAbs 2C4 and 6F10 were evaluated in a Jurkat IL-2 reporter luciferase assay using Jurkat IL-2 luciferase clones 1-6, 1-7, and 2-12.

[0058] Figure 22 KIR3DL3-selective HHLA2 mAb 2C4 does not block TMIGD2-mediated co-stimulation. HHLA2 mAb 2C4 was evaluated at a concentration of 30 μg / ml in Jurkat parental NFAT reporter cells. HHLA2-TCR-CHO cells were plated in white opaque bottom 96-well plates in CHOK1 growth medium at 2 x 10 4 The cells were seeded at a density of 10 cells / well. After overnight incubation at 37°C with 5% CO2, the cells adhered to the plate. The next day, the culture medium was carefully removed from each well, and anti-HHLA2 antibody in 50 μl Jurkat cell culture medium was added, and the HHLA2-TCR-CHO cells were incubated for 1 hour, after which they were plated at 4-5 x 10 in 50 μl Jurkat cell culture medium. 4Add Jurkat parental NFAT Reporter cell line to each well. Mix the wells and incubate for approximately 3-6 hours. To visualize the luciferase signal, add 100 μl ONE-Step TM A luciferase assay system (BPS Biosciences, catalog #60690) was added to each well and luminescence was read using a luminometer.

[0059] Figures 23A-23C Shown is how HHLA2 mAbs can be evaluated in a humanized SRG-15 mouse tumor model (with both T cells and NK cells). HHLA2 mAbs were administered to humanized SRG-15 mice bearing HHLA2-expressing tumor cells, and tumor growth inhibition was assessed. Figure adapted from Herndler-Brandstetter D et al. (2017) 114: E9626-E9634.

[0060] Figure 24A and Figure 24B This study demonstrates how HHLA2 mAbs can be evaluated in a cynomolgus monkey T cell model. Cynomolgus monkeys were administered HHLA2 mAbs and immunized with KLH, and T cell-dependent antibody and cell-mediated responses were assessed. NK cell cytotoxicity was assessed ex vivo. DETAILED DESCRIPTION

[0061] The present invention is based at least in part on the discovery that HHLA2, as a member of the B7 gene family, is widely expressed in various tumors and antigen-presenting cells and is considered to be both an activating ligand and an inhibitory ligand of T cells. TMIGD2 expressed in primary T cells is an activating receptor of HHLA2 and transduces co-stimulatory signals after T cell antigen receptor (TCR) engagement. TMIGD2 is lowered after repeated TCR stimulation. Based on the observation that HHLA2 is highly expressed in tumors and can serve as a checkpoint ligand, a group of anti-HHLA2 human monoclonal antibodies (mAbs) as candidate immune checkpoint inhibitor therapeutic agents are generated. In view of the fact that the same ligand binding domains of known B7 family members B7-1 and B7-2 bind to both activating receptors and inhibitory receptors (e.g., CD28 and CTLA-4), it is believed that the anti-HHLA2 monoclonal antibodies that block TMIGD2 binding serve as good candidates for blocking the binding of their inferred inhibitory receptors. Both blocking and non-blocking anti-HHLA2 mAbs were identified by evaluating the binding of soluble hHHLA2-mIgG2a to TMIGD2-transfected 300.19 mouse pre-B leukemia cells. The anti-HHLA2 mAbs listed in Table 2 (e.g., 6F10, 4D1, 4E5, and 2G2) blocked TMIGD2 binding and also bound to HHLA2-transfected 300.19 cells with relative EC50 binding affinities of 0.25, 0.44, and 0.21 ug / ml (nanomolar range), respectively. The non-blocking antibodies listed in Table 2 (e.g., 1C8 and 6D10) bound to HHLA2 with relative binding affinities of 0.63 and 22.49 ug / ml, respectively. The variable region heavy and light chain gene sequences of these candidate therapeutic anti-HHLA2 antibodies are described herein.

[0062] Anti-HHLA2 mAbs 1C8 and 6D10 have been identified as good reagents for formalin-fixed, paraffin-embedded immunohistochemistry or Western blotting. HHLA2 expression in primary tumors from the TCGA database is high in lung, renal, pancreatic, and colorectal cancers, as well as in AML, and is moderate in head and neck cancer, liver cancer, ovarian cancer, prostate cancer, and uterine cancer. HHLA2 mRNA expression is higher in cancers than in corresponding normal tissues.

[0063] Screening a cell-surface expressed human plasma protein library of >4500 full-length clones covering over 3500 different plasma membrane proteins with soluble human HHLA2-mIgG2a identified KIR3DL3 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 3) as a novel receptor for HHLA2. The cytoplasmic tail of KIR3DL3 contains an ITIM motif consisting of the sequence "VTYAQL," indicating an inhibitory receptor for HHLA2 that could serve as a checkpoint receptor target for cancer immunotherapy. The binding selectivity of HHLA2 to KIR3DL3 was demonstrated, as no binding to other KIR 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).

[0064] Because HHLA2 is expressed at high levels in many tumor types, anti-HHLA2 mAb checkpoint inhibitor therapy could increase the pool of patients who respond to checkpoint inhibitor therapy. Furthermore, patients who develop resistance to PD-1 therapy may express HHLA2 as an alternative immune escape strategy, and HHLA2 blockade could provide a means to overcome resistance to PD-1 immunotherapy.

[0065] Thus, the present invention provides monoclonal antibodies and antigen-binding fragments thereof that specifically bind to HHLA2, as well as immunoglobulins, polypeptides, and nucleic acids thereof, and methods of using such antibodies for diagnostic, prognostic, and therapeutic purposes.

[0066] I. Definition

[0067] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0068] The term "altered amount" of a marker refers to an increase or decrease in the number of copies of the marker in a sample compared to the marker in a control sample, and / or an increase or decrease in the nucleic acid level of one or more specific marker genes. The term "altered amount" of a marker also includes an increase or decrease in the protein level of the marker in a sample compared to the protein level of the marker in a normal control sample.

[0069] The term "altered activity" of a marker refers to an increase or decrease in the activity of a marker in a disease state, for example, in a biological sample, compared to the activity of the marker in a normal control sample. The altered activity of a marker can be the result of, for example, altered expression of the marker, altered protein levels of the marker, altered structure of the marker, or altered interactions with other proteins involved in the same or different pathways as the marker, or altered interactions with transcriptional activators or repressors.

[0070] The term "altered structure" of a marker refers to the presence of a mutation or allelic variant within a marker gene or protein, e.g., a mutation that affects the expression or activity of the marker compared to the normal or wild-type gene or protein. For example, mutations include, but are not limited to, substitution, deletion, or addition mutations. Mutations can occur in either the coding or non-coding regions of the marker.

[0071] The term "activating receptor" includes immune cell receptors that bind antigen, complexed antigen (e.g., in the case of MHC polypeptides), or bind antibodies. Such activating receptors include T cell receptors (TCRs), B cell receptors (BCRs), cytokine receptors, LPS receptors, complement receptors, and Fc receptors.

[0072] The T cell receptor is present on T cells and associates with the CD3 polypeptide. The T cell receptor is stimulated by antigen (and by polyclonal T cell activation reagents) in the presence of MHC polypeptides. T cell activation via TCR produces a variety of changes, such as protein phosphorylation, membrane lipid changes, ion flux, cyclic nucleotide changes, RNA transcription changes, protein synthesis changes, and cell volume changes.

[0073] The term "chimeric antigen receptor" or "CAR" refers to an engineered T cell receptor (TCR) with desired antigen specificity. T lymphocytes recognize specific antigens by the interaction of T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or class II molecules. For initial activation and clonal expansion, naive T cells rely on professional antigen presenting cells (APCs) that provide additional costimulatory signals. TCR activation may lead to anergy and clonal non-response in the absence of costimulation. In order to avoid immune effects, different methods for deriving cytotoxic effector cells with grafted recognition specificity have been developed. CAR consisting of a binding domain of an antibody specific for the cell surface component of the CD3 complex associated with natural ligands or TCR has been constructed. When antigen is combined, such chimeric antigen receptors are connected to the endogenous signal transduction pathways in effector cells and generate activation signals similar to those initiated by the TCR complex. Since the first reports of chimeric antigen receptors, the concept has been gradually refined, and the molecular design of chimeric receptors has been optimized and routinely uses any number of well-known binding domains, such as scFv, Fav, and other protein binding fragments described herein.

[0074] Typically, CAR is a type of "cell therapy" (e.g., T cell therapy) envisioned for use according to the present invention. Although there are multiple representative embodiments of reagents and methods for regulating immune cell activity by regulating HHLA2 pathways, such as regulating the interaction between HHLA2 and HHLA2 natural binding partners (such as TMIGD2 and / or KIR3DL3), immune cell-based therapies and methods are also encompassed. For example, it is encompassed that engineered to have knockout, knockdown, or increased expression of TMIGD2 and / or KIR3DL3 T cells. Similarly, it is also encompassed that engineered to have knockout, knockdown, or increased expression of HHLA2 ligands (such as TMIGD2 and / or KIR3DL3) immune cells or other cells.

[0075] B cell receptors are present on B cells. The B cell antigen receptor is a complex between membrane immunoglobulins (mIgs) and other transmembrane polypeptides (e.g., Igα and Igβ). The signal transduction function of mIgs is triggered by cross-linking of receptor polypeptides with oligomeric or polymeric antigens. B cells can also be activated by anti-immunoglobulin antibodies. Upon BCR activation, various changes occur in B cells, including tyrosine phosphorylation.

[0076] Fc receptors involved in immune responses are found on a variety of cells. Fc receptors (FcRs) are cell surface receptors for the Fc portion of immunoglobulin polypeptides (Ig). To date, the human FcRs identified are those that recognize IgG (designated FcγR), IgE (FcεR1), IgA (Fcα), and polymeric IgM / A (FcμαR). FcRs are present in the following cell types: FcεR I (mast cells), FcεR.II (multiple leukocytes), FcαR (neutrophils), and FcμαR (glandular epithelium, hepatocytes) (Hogg, N. (1988) Immunol. Today 9: 185-86). Extensively studied FcγR is the center of cellular immune defense and is responsible for stimulating the release of inflammatory mediators and hydrolases involved in the pathogenesis of autoimmune diseases (Unkeless, JC et al. (1988) Annu. Rev. Immunol. 6: 251-81). FcγR provides a key connection between effector cells and Ig-secreting lymphocytes, because macrophages / monocytes, polymorphonuclear leukocytes and natural killer (NK) cell FcγRs confer elements of specific recognition mediated by IgG. Human leukocytes have at least three different receptors for IgG: hFcγRI (present on monocytes / macrophages), hFcγRII (on monocytes, neutrophils, eosinophils, platelets, B cells (possibly) and K562 cell lines) and FcγIII (on NK cells, neutrophils, eosinophils and macrophages).

[0077] With respect to T cells, the transmission of co-stimulatory signals to T cells involves signaling pathways that are not inhibited by cyclosporin A. Furthermore, co-stimulatory signals can induce cytokine secretion (e.g., IL-2 and / or IL-10) in T cells and / or can prevent the induction of unresponsiveness to antigen, the induction of anergy, or the induction of cell death (absence) in T cells.

[0078] When used with respect to polypeptides, such as HHLA2 and / or HHLA2 natural binding partners, such as TMIGD2 and / or KIR3DL3, the term "activity" includes activities inherent in the structure of the protein. For example, with respect to an HHLA2 ligand, the term "activity" includes the ability to modulate immune cell suppression by modulating inhibitory signals in immune cells (e.g., by engaging a natural receptor on the immune cell). Those skilled in the art will recognize that when the activated form of the HHLA2 ligand polypeptide binds to an inhibitory receptor, an inhibitory signal is generated in the immune cell.

[0079] The term "inhibitory signal" refers to a signal transmitted via inhibitory receptors (e.g., HHLA2, KLRB1, CTLA4, PD-1, etc.) of polypeptides on immune cells. Such signals antagonize signals generated via activating receptors (e.g., via TCR, CD3, BCR, TMIGD2, or Fc polypeptides) and can result in, for example, inhibition of second messenger generation; inhibition of proliferation; inhibition of effector function in immune cells, such as reduced phagocytosis, reduced antibody production, reduced cytotoxicity, inability of immune cells to produce mediators (such as cytokines (e.g., IL-2) and / or mediators of allergic reactions); or development of anergy.

[0080] The amount of a biomarker in a subject is "significantly" above or below the normal amount of a biomarker if the amount of the biomarker in the subject is greater or less than the normal or control level, respectively, by an amount that is the standard error of the assay used to assess the amount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% greater or less than that amount. Alternatively, if the amount of a biomarker in a subject is at least about two-fold higher or lower than the normal and / or control amount of the biomarker, respectively, and preferably at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 125%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 200%, 201%, 202%, 203%, 204%, 205%, 206%, 207%, 208%, 209%, 210%, 211%, 212%, 213%, 214%, 215%, 216%, 217%, 218%, 219%, 220%, 221 "Significantly" above or below a normal and / or control amount. An amount may be considered "significantly" above or below a normal and / or control amount if the amount is 15%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, two-fold, three-fold, four-fold, five-fold, or more, or any range therebetween, such as 5%-100%. Such significant modulation values ​​can be applied to any metric described herein, such as altered expression levels, altered activity, changes in cancer cell hyperproliferative growth, changes in cancer cell death, changes in biomarker inhibition, changes in test agent binding, etc.

[0081] If the "amount" of a marker in a subject, such as the expression or copy number of a marker or MCR or the protein level of a marker, is greater or less than a normal level, respectively, by the amount of the standard error of the assay used to assess the amount, and preferably at least two-fold greater or less than that amount, and more preferably three-fold, four-fold, five-fold, ten-fold or more, then the amount of the biomarker is "significantly" higher or lower than the normal amount of the marker. Alternatively, if the amount of a marker in a subject is at least about two-fold higher or lower, and preferably at least about three-fold, four-fold or five-fold higher or lower than the normal amount of the marker, then the amount can be considered to be "significantly" higher or lower than the normal amount.

[0082] " the expression level of the change " of term marker refers to test sample, the expression level of marker in the sample of the experimenter such as deriving from cancer or copy number, it is greater than or less than the standard error of the mensuration for assessing expression or copy number, and preferably than the expression level of marker or chromosomal region or copy number in control sample (for example, from the sample of the healthy subject not suffering from relevant disease), and preferably the average expression level of marker or chromosomal region or copy number in several control samples are greater or less than at least two times, and more preferably three times, four times, five times or ten times or more times.The expression level of change is greater or less than the standard error of the mensuration for assessing expression or copy number, and preferably than the expression level of marker in control sample (for example, from the sample of the healthy subject not suffering from relevant disease), and preferably the average expression level of marker or copy number in several control samples are greater or less than at least two times, and more preferably three times, four times, five times or ten times or more times.

[0083] The term "immunotherapy" refers to a form of targeted therapy that can include, for example, cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are viruses that can infect and lyse cancer cells without harming normal cells, making them potentially useful for immunomodulatory therapy. The replication of oncolytic viruses promotes tumor cell destruction and also produces dose amplification at the tumor site. They can also serve as carriers of anti-cancer genes, so that the anti-cancer genes are specifically delivered to the tumor site. Immunotherapy can be related to the passive immunity of the short-term protection of the host, by administering preformed antibodies for cancer antigens or disease antigens (for example, administering monoclonal antibodies optionally connected to chemotherapeutics or toxins, connected to tumor antigens). Immunotherapy can also focus on epitopes recognized by cytotoxic lymphocytes using cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc. can be used to selectively regulate and control biomolecules related to the initiation, progression, and / or pathology of tumors or cancers. As described above, immunotherapy for immune checkpoint targets (such as HHLA2, TMIGD2, KIR3DL3, etc.) is available.

[0084] Unless otherwise indicated herein, the terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric antibodies, humanized antibodies, and multispecific antibodies, as well as all fragments and derivatives thereof, which have at least one antigen binding site. Antibody derivatives may comprise a protein or chimeric portion conjugated to an antibody. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V H ) and heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as V L ) and the light chain constant region. The light chain constant region comprises one domain, CL. V H and V L The regions can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L They are 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 the binding domain that interacts with the antigen. The term "inactivated antibody" refers to an antibody that does not induce the complement system.

[0085] As used herein, the term "antibody" also includes the "antigen-binding portion" of an antibody (or simply "antibody portion"). As used herein, the term "antigen-binding portion" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., an HHLA2 polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be achieved 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 Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity-determining regions (CDRs). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, they can be joined using recombinant methods by synthetic linkers that enable them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide (referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proceedings of the National Academy of Sciences of the United States of America 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a specific scFv can be linked to a human immunoglobulin constant region cDNA or genomic sequence to generate an expression vector encoding a complete IgG polypeptide or other isotype. VH and VL can also be used to generate Fab, Fv or other fragments of immunoglobulins using protein chemistry or recombinant DNA technology. Other forms of single-chain antibodies are also contemplated, such as diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the 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, RJ et al. (1994) Structure 2:1121-1123).

[0086] In addition, antibody or its antigen-binding portion thereof can be the part of the larger immunoadhesion polypeptide formed by the covalent or non-covalent association of antibody or antibody portion with one or more other proteins or peptides.The example of such immunoadhesion polypeptide includes using streptavidin core region to prepare tetramer scFv polypeptide (Kipriyanov, SM et al. (1995) " human antibodies and hybridoma (Human Antibodies and Hybridomas) " 6:93-101) and using cysteine ​​residues, marker peptide and C-terminal polyhistidine tag to prepare divalent and biotinylated scFv polypeptide (Kipriyanov, SM et al. (1994) " molecular immunology (Mol.Immunol.) " 31:1047-1058).Antibody portion, such as Fab and F (ab ') 2 fragment, can use conventional techniques, such as using papain or pepsin digestion of full antibody respectively by full antibody preparation.In addition, antibody, antibody portion and immunoadhesion polypeptide can be obtained using standard recombinant DNA technology, as described herein.

[0087] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or isogenic; or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies can be fully human. In one embodiment, the antibodies of the present invention specifically or substantially specifically bind to an HHLA2 polypeptide or fragment thereof. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides from only one species that contain an antigen-binding site capable of immunoreacting with a specific epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides from multiple species that contain antigen-binding sites capable of interacting with a specific antigen. A monoclonal antibody composition typically exhibits a single binding affinity for the specific antigen with which it immunoreacts.

[0088] The term "body fluid" refers to fluids that are excreted or secreted from the body as well as fluids that are not normally excreted or secreted from the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, cerumen and earwax, Cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female semen, interstitial fluid, intracellular fluid, lymph, menstrual fluid, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomitus).

[0089] The term "cancer" or "tumor" or "hyperproliferative disorder" refers to the presence of cells that have characteristics characteristic of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological features. Cancer cells often take the form of tumors, but such cells may exist alone in an animal or may be non-tumorigenic cancer cells, such as leukemia cells. Cancers include, but are not limited to, B-cell cancers (e.g., multiple myeloma), Waldenstrom's macroglobulinemia ( macroglobulinemia), heavy chain disease (e.g., alpha chain disease, gamma chain disease, and mu chain disease), benign monoclonal gammopathy and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer or endometrial cancer, oral cancer or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematologic cancer, etc. Other non-limiting examples of cancer types suitable for the methods encompassed by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, tumor), leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma , medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma;Leukemia, such as acute lymphocytic leukemia and acute myeloid leukemia (myelocyte, promyelocytic, myelomonocytic, monocyte and erythroleukemia);Chronic leukemia (chronic myeloid (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 certain embodiments, cancer is epithelial in nature and includes but is not limited to bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer or skin cancer.In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer or colon cancer. In yet other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., ovarian serous cystadenocarcinoma), or breast cancer. Epithelial cancers can be characterized in various other ways, including but not limited to serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0090] The term "CDR" and its plural "CDRs" refer to complementary determining regions (CDRs), three of which constitute the binding characteristics of the light chain variable region (CDR-L1, CDR-L2 and CDR-L3), and three of which constitute the binding characteristics of the heavy chain variable region (CDR-H1, CDR-H2 and CDR-H3). CDR contributes to the functional activity of the antibody molecule and is separated by the amino acid sequence comprising the scaffold region or framework region. The precisely defined CDR boundaries and lengths depend on different classification and numbering systems. Therefore, CDRs can be quoted by Kabat, Chothia, contact (contact) or any other boundary definition method. Although the boundaries are different, each of these systems has a certain degree of overlap when constituting the so-called "hypervariable region" in the variable sequence. Therefore, the CDR limits according to these systems can be different in length and boundary regions about adjacent framework regions. See, e.g., Kabat, Chothia, and / or MacCallum et al. (Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., U.S. Department of Health and Human Services, 1992; Chothia et al. (1987) J. Mol. Biol. 196, 901; and MacCallum et al., J. Mol. Biol. (1996) 262, 732, each of which is incorporated herein by reference in its entirety).

[0091] As used herein, the term "classification" includes "associating" a sample with a disease state or "classifying" a sample as having a disease state. In some cases, "classification" is based on statistical evidence, empirical evidence, or both. In certain embodiments, methods and systems for classification use a so-called training set of samples with known disease states. Once established, the training data set serves as a basis, model, or template to which the characteristics of unknown samples are compared in order to classify the unknown disease state of the sample. In some cases, classifying a sample is analogous to diagnosing the disease state of the sample. In certain other cases, classifying a sample is analogous to distinguishing the disease state of the sample from other disease states.

[0092] As used herein, the term "coding region" refers to a region of nucleotide sequence containing codons translated into amino acid residues, while the term "non-coding region" refers to a region of nucleotide sequence not translated into amino acids (e.g., 5' and 3' untranslated regions).

[0093] "Complement of..." or "complementarity" refers to the broad concept of sequence complementarity between regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that adenine residues in a first nucleic acid region are capable of forming specific hydrogen bonds ("base pairing") with residues in a second nucleic acid region that are antiparallel to the first region (if the residues are thymine or uracil). Similarly, it is known that cytosine residues in a first nucleic acid chain are capable of base pairing with residues in a second nucleic acid chain that are antiparallel to the first chain (if the residues are guanine). If a first region of a nucleic acid and a second region of the same nucleic acid or a different nucleic acid are arranged in an antiparallel manner, at least one nucleotide residue in the first region is capable of base pairing with residues in the second region, then the first region is complementary to 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 portion and the second portion are arranged in an antiparallel manner, at least about 50%, and preferably at least about 75%, at least about 90% or at least about 95% of the nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion. In another embodiment, all of the nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion.

[0094] As used herein, the term "composite antibody" refers to an antibody having a variable region comprising germline or non-germline immunoglobulin sequences from two or more unrelated variable regions. Additionally, the term "composite human antibody" refers to an antibody having a constant region and a variable region, wherein the constant region is derived from a human germline or non-germline immunoglobulin sequence, and the variable region comprises a human germline or non-germline sequence from two or more unrelated human variable regions. Composite human antibodies can be used as an effective component in therapeutic agents according to the present invention because the antigenicity of composite human antibodies in humans is reduced.

[0095] The term "control" refers to any reference standard suitable for providing a comparison with the expression product in the test sample. In one embodiment, the control comprises obtaining a "control sample" from which the expression product level is detected and compared with the expression product level from the test sample. Such control samples can include any suitable sample, including but not limited to samples from control cancer patients with known results (which can be stored samples or previous sample measurements); normal tissues or cells separated from subjects (such as normal patients or cancer patients), cultured primary cells / tissues separated from subjects (such as normal patients or cancer patients), adjacent normal cells / tissues obtained from the same organ or body position of cancer patients, tissues or cell samples isolated from normal subjects, or primary cells / tissues obtained from a depository institution. In another preferred embodiment, the control can include reference standard expression product levels from any suitable source, including but not limited to housekeeping genes, expression product level ranges from normal tissues (or other previously analyzed control samples), expression product level ranges previously determined within a test sample from a group of patients, or a group of patients with a certain result (e.g., survival for one year, two years, three years, four years, etc.) or receiving a certain treatment (e.g., standard of care cancer therapy). Those skilled in the art will appreciate 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 can include normal or non-cancerous cells / tissue samples. In another preferred embodiment, the control can include a group of patients, such as a cancer patient or a group of cancer patients receiving certain treatment or a group of patients with a result relative to another result. In the former case, each patient's specific expression product level can be designated as the expression level of percentile, or expressed as the mean or average value higher or lower than the reference standard expression level. In another preferred embodiment, the control can include normal cells, cells from patients treated with combination chemotherapy, and cells from patients with benign cancers. In another embodiment, the control can also include measured values, such as the average expression level of a specific gene compared to the expression level of a housekeeping gene in the same group in a colony. Such colonies can include normal subjects, cancer patients who have not undergone any treatment (e.g., without treatment), cancer patients experiencing standard care therapy, or patients with benign cancers. In another preferred embodiment, the control comprises a ratiometric transformation of expression product levels, including but not limited to determining the ratio of the expression product levels of two genes in a test sample and comparing it to any suitable ratio of the same two genes in a reference standard; determining the expression product levels of two or more genes in a test sample and determining the difference in expression product levels in any suitable control; and determining the expression product levels of two or more genes in a test sample, normalizing their expression to the expression of a housekeeping gene in the test sample and comparing it to any suitable control.In a particularly preferred embodiment, the control comprises the control sample with identical pedigree and / or type as the test sample.In another embodiment, the control can be included in one group of patient samples (such as all patients suffer from cancer) or be grouped into the expression product level of percentile based on the patient sample of described group.In one embodiment, set up the control expression product level, wherein with respect to the higher or lower expression product level of for example specific percentile, be used as the basis of predicted result.In another preferred embodiment, use the expression product level from the cancer control patient with known results to set up the control expression product level, and the expression product level from the test sample is compared with the control expression product level as the basis of predicted result.As demonstrated by the following data, method of the present invention is not limited to using specific cut point when the expression product level of test sample is compared with the control.

[0096] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for use in antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0097] As used herein, "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, a preferred FcR is an FcR that binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors, FcγRII receptors including FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in: Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those to be identified in the future.

[0098] A molecule is "immobilized" or "attached" to a substrate if it is covalently or non-covalently associated with the substrate such that the substrate can be washed with a fluid (e.g., standard citrate water, pH 7.4) without a substantial portion of the molecule dissociating from the substrate.

[0099] As used herein, "framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0100] "Function conservative variants" are those in which a given amino acid residue in a protein or enzyme is changed without altering the overall conformation and function of the polypeptide, including but not limited to those in which an amino acid is replaced by an amino acid with similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, aromaticity, etc.). Amino acids other than those indicated as conservative may vary in a protein such that the percentage of protein or amino acid sequence similarity between any two proteins of similar function may be, for example, from 70% to 99%, as determined by an alignment scheme, such as by a clustering method, wherein the similarity is based on the MEGALIGN algorithm. "Function conservative variants" also include a polypeptide having at least 60% amino acid identity, preferably at least 75%, more preferably at least 85%, still preferably at least 90% and even more preferably at least 95% amino acid identity, as determined by the BLAST or FASTA algorithms, and having the same or substantially similar properties or functions as the native or parent protein to which it is compared.

[0101] As used herein, the term "heterologous antibody" is defined with respect to the transgenic non-human organism that produces such an antibody. The term refers to an antibody having an amino acid sequence or encoding nucleic acid sequence corresponding to that present in an organism that is not composed of the transgenic non-human animal, and typically from a species other than the species of the transgenic non-human animal.

[0102] As used herein, "homology" refers to the nucleotide sequence similarity between two regions of the same nucleic acid chain or between regions of two different nucleic acid chains. When the nucleotide residue positions in the two regions are occupied by the same nucleotide residues, the regions are homologous at the positions. If at least one nucleotide residue in each region is occupied by the same residue, the first district and the second district are homologous. The homology between the two regions is represented by the ratio of the nucleotide residue positions occupied by the same nucleotide residues in the two regions. By way of example, a region with the nucleotide sequence 5'-ATTGCC-3' and a region with the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first district comprises the first portion and the second district comprises the second portion, whereby at least about 50% of the nucleotide residue positions of each portion, and preferably at least about 75%, at least about 90% or at least about 95% are occupied by the same nucleotide residues. More preferably, all nucleotide residue positions in each portion are occupied by the same nucleotide residues.

[0103] 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, is introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to a particular subject's cell, but also to the progeny or potential progeny of such a cell. Because certain changes may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parental cell, but are still included within the scope of the term as used herein.

[0104] As used herein, the term "humanized antibody" is intended to include antibodies prepared by non-human cells with variable regions and constant regions, and the antibodies are changed and are more similar to antibodies prepared by human cells. For example, by changing the non-human antibody amino acid sequence to incorporate the amino acid present in the human germline immunoglobulin sequence. Humanized antibodies can, for example, include in CDR an amino acid residue that is not encoded by the human germline immunoglobulin sequence (for example, a mutation introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "humanized antibody" also includes antibodies wherein the CDR sequence derived from the germline of another mammalian species (such as a mouse) has been grafted onto a human framework sequence.

[0105] As used herein, humanized mice are mice that carry functional 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 therapeutic agents. Nude mice and severe combined immunodeficiency (SCID) mice can be used for this purpose. NCG mice, NOG mice, and NSG mice can be used to more effectively graft human cells and tissues than other models. Such humanized mouse models can be used to simulate the human immune system under healthy and pathological conditions, and can be used to evaluate therapeutic candidates in an in vivo environment associated with human pathology.

[0106] As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop. Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the most diversity of the six HVRs, and it is believed that H3 plays a unique role in conferring good specificity to antibodies. See, for example, Xu et 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 composed of heavy chains are functional and stable only in the absence of light chains (see, e.g., Hamers-Casterman et al. (1993) Nature 363:446-448 (1993) and Sheriff et al. (1996) Nature Struct. Biol. 3, 733-736).

[0107] As used herein, the term "immune cell" refers to a cell that plays a role in an 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.

[0108] As used herein, the term "immune disorder" includes immune diseases, conditions and predispositions including, but not limited to, cancer, chronic inflammatory diseases and disorders (including, for example, Crohn's disease, inflammatory bowel disease, reactive arthritis, and Lyme disease), insulin-dependent diabetes mellitus, organ-specific autoimmunity (including, for example, multiple sclerosis, Hashimoto's thyroiditis, autoimmune uveitis, and Graves' disease), contact dermatitis, psoriasis, transplant rejection, graft-versus-host disease, sarcoidosis, atopic conditions (including, for example, asthma and allergies, including, but not limited to, allergic rhinitis and gastrointestinal allergies such as food allergies), eosinophilia, conjunctivitis, glomerulonephritis, systemic lupus erythematosus, scleroderma, certain pathogen susceptibilities such as helminth susceptibility (including, for example, leishmaniasis), and certain viral infections (including, for example, HIV and bacterial infections such as tuberculosis and lepromatous leprosy), and malaria.

[0109] As used herein, the term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses indirectly achieved by activation of T cells (e.g., antibody production (humoral response)) and cytokine-responsive cells such as macrophages.

[0110] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate the host immune system to generate an immune response to a tumor or cancer in a subject. Various immunotherapeutic agents can be used in the compositions and methods described herein.

[0111] The term "immune checkpoints" refers to a group of molecules that fine-tune immune responses by lowering or suppressing anti-tumor immune responses on the cell surface of CD4+ and / or CD8+T cells. Immune checkpoint proteins are well known in the art and include but are not limited to CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP α (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin and A2aR (see, for example, WO 2012 / 177624). The term further encompasses biologically active protein fragments, and nucleic acids encoding full-length immune checkpoint proteins and biologically active protein fragments thereof. In some embodiments, the term further encompasses any fragment described according to the homology provided herein.

[0112] Immune checkpoints and their sequences are well known in the art, and representative embodiments are described below. For example, the term "PD-1" refers to a member of the immunoglobulin gene superfamily that acts as a co-inhibitory receptor with PD-L1 and PD-L2 as known ligands. PD-1 was previously identified using a method based on subtraction cloning to select genes that were upregulated during TCR-induced activated T cell death. Based on its ability to bind PD-L1, PD-1 is a member of the CD28 / CTLA-4 family of molecules. Like CTLA-4, PD-1 is rapidly induced on the surface of T cells in response to anti-CD3 (Agata et al. 25 (1996) "International Immunology (Int. Immunol.)" 8:765). However, compared to CTLA-4, PD-1 is also induced on the surface of B cells (in response to anti-IgM). PD-1 is also expressed on a subset of thymocytes and myeloid cells (Agata et al. (1996) ibid; Nishimura et al. (1996) "International Immunology" 8:773).

[0113] As used herein, the term "inhibit" and its grammatical equivalents refer to reducing, limiting and / or blocking a specific effect, function or interaction. In one embodiment, the term refers to reducing the level of a given output or parameter to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less than the amount in the corresponding control (e.g., background staining, HHLA2 signaling, HHLA2 immunosuppressive function, etc.). The reduced level of a given output or parameter does not necessarily (although possible) mean that the output or parameter is absolutely absent. The present invention does not require and is not limited to methods for completely eliminating an output or parameter. A given output or parameter can be determined using methods well known in the art, including but not limited to immunohistochemical assays, molecular biological assays, cell biological assays, clinical assays and biochemical assays, as discussed herein and in the Examples. Conversely, the terms "promote," "increase," and their grammatical equivalents refer to increasing the level of a given output or parameter, ie, as opposed to those stated with respect to inhibiting or decreasing.

[0114] 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 to each other (e.g., HHLA2 binds to TMIGD2 or HHLA2 binds to KIR3DL3). Typically, such an interaction results in an activity of one or both of the molecules (which produces a biological effect). The activity can be a direct activity of one or both of the molecules (e.g., signal transduction). Alternatively, one or both of the molecules in the interaction can be prevented from binding to their ligand and thereby remain inactive in terms of ligand binding activity (e.g., binding to their ligand and triggering or suppressing an immune response). Inhibiting such an interaction results in the destruction of the activity of one or more molecules involved in the interaction. Enhancing such an interaction is prolonging or increasing the probability of the physical contact and prolonging or increasing the probability of the activity.

[0115] The term "neoadjuvant therapy" refers to treatment given before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy.

[0116] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds HHLA2 and is substantially free of antibodies that do not bind HHLA2). However, an isolated antibody that specifically binds HHLA2 may have cross-reactivity with other B7 family proteins, each derived from a different species. For example, in some embodiments, the antibody maintains specific binding affinity for at least two species, such as humans and another animal, such as a non-rodent or other mammalian or non-mammalian species. However, in some embodiments, the antibody maintains a higher or substantially specific affinity and selectivity for human HHLA2. Furthermore, isolated antibodies are typically substantially free of other cellular material and / or chemicals. In one embodiment of the present invention, a combination of "isolated" monoclonal antibodies with different specificities for human HHLA2 are combined in a well-defined composition.

[0117] As used herein, "isolated protein" refers to a protein that is substantially free of other proteins, cellular material, separation media, and culture media when isolated from a cell or produced by recombinant DNA technology, or substantially free of 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 of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of target polypeptides (e.g., immunoglobulins) or fragments thereof in which the protein is separated from cellular components of the cells from which it was isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of target protein or fragments thereof having less than about 30% (by dry weight) of non-target proteins (also referred to herein as "contaminating proteins"), more preferably less than about 20% of non-target proteins, still more preferably less than about 10% of non-target proteins, and most preferably less than about 5% of non-target proteins. When the antibody, polypeptide, peptide or fusion protein, or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it is preferably also substantially free of culture medium, i.e., culture medium comprises less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0118] As used herein, the term "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

[0119] As used herein, the term "K D" is intended to refer to the dissociation equilibrium constant for a particular antibody-antigen interaction. The binding affinity of the disclosed antibodies of the invention can be measured or determined by standard antibody-antigen assays, e.g., competition assays, saturation assays, or standard immunoassays such as ELISA or RIA.

[0120] As used herein, a "kit" is any article (e.g., package or container) comprising at least one reagent (e.g., probe) for specifically detecting or regulating the expression of a marker of the invention. The kit can be promoted, distributed, or sold as a unit for performing the methods of the invention.

[0121] As used herein, the term "monoclonal antibody" refers to an antibody that displays a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody that displays a single binding specificity and has variable and constant regions derived from human germline or non-germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas comprising B cells fused to immortalized cells obtained from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising human heavy and light chain transgenes.

[0122] A "marker" is a gene whose expression level in a tissue or cell, relative to its expression level in normal or healthy tissue or cells, 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 all or part of the sequence of any nucleic acid sequence listed in the sequence listing, or the complement of such a sequence. Marker nucleic acids also include RNA comprising all or part of the sequence of any nucleic acid sequence listed in the sequence listing, or the complement of such a sequence, in which all thymidine residues are replaced with uridine residues. A "marker protein" is a protein encoded by or corresponding to a marker of the present invention. A marker protein comprises all or part of the sequence of any sequence listed in the sequence listing. In some embodiments, HHLA2 as a whole is used as a marker. In other embodiments, fragments of HHLA2 are used as markers. The terms "protein" and "polypeptide" are used interchangeably.

[0123] As used herein, the term "modulate" includes upregulation and downregulation, eg, enhancing or inhibiting a response.

[0124] " normal " expression level of a marker is the expression level of the marker in the cell of the subject (for example, human patient) that is not affected by the disease relevant to abnormal marker level or illness." overexpression " or " significantly higher expression level " of a marker refers to the expression level in the test sample, which is greater than the standard error of the mensuration for evaluating expression, and preferably than the expression level of the marker in the control sample (for example, from the sample of the healthy subject not suffering from the marker related disease), and preferably the average expression level of the marker in several control samples is at least twice as large, and more preferably three times, four times, five times or ten times. " significantly lower expression level " of a marker refers to the expression level in the test sample, which is greater than the expression level of the marker in the control sample (for example, from the sample of the healthy subject not suffering from the marker related disease), and preferably the average expression level of the marker in several control samples is low at least two times, and more preferably three times, four times, five times or ten times.

[0125] Such "significant" levels can also apply to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, etc.

[0126] The term "predetermined" biomarker amount and / or activity measurement can be, by way of example only, a biomarker amount and / or activity measurement used to assess a subject for selection for a particular treatment, to assess response to treatment, such as one or more modulators of the HHLA2 pathway (such as modulators of HHLA2 and one or more natural binding partners (such as TMIGD2 and / or KIR3DL3)), alone or in combination with one or more immunotherapies, and / or to assess disease state. The predetermined biomarker amount and / or activity measurement can be determined in a population of patients with or without cancer. The predetermined biomarker amount and / or activity measurement can be a single number that applies equally to each patient, or the predetermined biomarker amount and / or activity measurement can vary according to a specific subpopulation of patients. The subject's age, weight, height, and other factors can affect the individual's predetermined biomarker amount and / or activity measurement. In addition, the predetermined biomarker amount and / or activity can be determined individually for each subject. In one embodiment, the amounts determined and / or compared in the methods described herein are based on absolute measurements. In another embodiment, the amount determined and / or compared in the methods described herein is based on relative measurements, such as ratios (e.g., cell ratios or serum biomarkers normalized to housekeeping or other generally constant biomarkers). Predetermined biomarker amounts and / or activity measurements can be any suitable standard. For example, predetermined biomarker amounts and / or activity measurements can be obtained from the same or different people for whom the patient selection is assessed. In one embodiment, predetermined biomarker amounts and / or activity measurements can be obtained from the previous assessment of the same patient. In this way, the process of patient selection can be monitored over time. In addition, if the subject is a person, the control can be obtained from the assessment of another one or more people, such as a selected group of people. In this way, the degree of selection of the person selected for its assessment can be compared with suitable other people, such as other people in a situation similar to the person of interest, such as those suffering from similar or identical symptom and / or those of the same ethnic group.

[0127] The term "predictive" includes the use of biomarker nucleic acid and / or protein status, e.g., over- or underactivity, emergence, expression, growth, remission, relapse, or resistance of a tumor, before, during, or after therapy, to determine the probability of a cancer's response to an immunomodulatory therapy, such as an HHLA2 pathway modulator therapy (e.g., a modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3, alone or in combination with an immunotherapy such as an immune checkpoint inhibition therapy). Such predictive utility of a biomarker can be confirmed by, for example, (1) increased or decreased copy number (e.g., by FISH, FISH plus SKY, single molecule sequencing, e.g., as described in the art at least in J. Biotechnol., 86:289-301, or qPCR), overexpression or underexpression (e.g., by ISH, Northern blot, or qPCR) of a biomarker nucleic acid in greater 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 the human cancer types or cancer samples assayed. Blot (or qPCR), increased or decreased biomarker protein (e.g., by IHC) and / or biomarker target or increased or decreased activity; (2) the presence or absence of its absolute or relative regulation in a biological sample from a subject (e.g., a human with cancer), such as a sample containing tissue, whole blood, serum, plasma, oral scrapings, saliva, cerebrospinal fluid, urine, stool, or bone marrow; (3) the presence or absence of its absolute or relative regulation in a clinical subset of patients with cancer (e.g., those who respond to or develop resistance to a particular immunomodulatory therapy (e.g., HHLA2 pathway modulator therapy (e.g., modulators of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3, alone or in combination with immunotherapy)).

[0128] The terms "prevent," "preventing," and "prevention," "prophylactic treatment," and the like refer to reducing the probability of a disease, disorder, or condition developing in a subject who does not have the disease, disorder, or condition but is at risk of or susceptible to developing the disease, disorder, or condition.

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

[0130] The term "response to therapy (e.g., HHLA2 pathway modulator therapy (e.g., modulators of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3, alone or in combination with immunotherapy, such as immune checkpoint inhibition therapy))" relates to any response to therapy (e.g., HHLA2 pathway modulator therapy (e.g., modulators of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3, alone or in combination with immunotherapy, such as immune checkpoint inhibition therapy)), and for cancer, preferably relates to changes in the number of cancer cells, tumor mass and / or volume after the start of neoadjuvant or adjuvant chemotherapy. Hyperproliferative disorder response can be assessed, for example, for efficacy or in a neoadjuvant or adjuvant setting, where the tumor size after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation. Response can also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Responses can also be recorded quantitatively, such as the percentage change in tumor volume, or qualitatively, such as "pathological complete response (pCR)", "clinical complete remission (cCR)", "clinical partial remission (cPR)", "clinically stable disease (cSD)", "clinically progressive disease (cPD)" or other qualitative criteria. The assessment of the response to the hyperproliferative disorder can be performed early after the start of neoadjuvant or adjuvant chemotherapy, for example, after a few hours, days, weeks, or preferably after a few months. A typical endpoint for the response assessment is at the end of neoadjuvant chemotherapy or when residual tumor cells and / or the tumor bed are surgically removed. This is typically three months after the start of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatments described herein can be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients in complete remission (CR), the number of patients in partial remission (PR), and the number of patients with stable disease (SD) at a time point at least 6 months after the end of therapy. A shorthand expression of this formula is CBR=CR+PR+SD over a 6-month period. In some embodiments, the CBR of a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more. Additional criteria for assessing response to cancer therapy are related to "survival," which includes all of the following: survival to death, also known as overall survival (wherein the death may be unrelated to the cause or associated tumor); "recurrence-free survival" (wherein the term recurrence shall include both local and distant recurrences); metastasis-free survival; disease-free survival (wherein the term disease shall include cancer and diseases associated therewith).The length of the survival period can be calculated by reference to the starting point (for example, the time of diagnosis or treatment start) and endpoint (for example, death, recurrence or metastasis) of the definition. In addition, the criteria of therapeutic efficacy can be extended to include the response to chemotherapy, the probability of survival, the probability of transfer within a given time period and the probability of tumor recurrence. For example, in order to determine an appropriate threshold value, a specific cancer treatment regimen can be applied to the subject population, and the result can be associated with the biomarker measurement value determined before applying any immunomodulatory therapy. The result measurement value can be the pathological response to the therapy given in a neoadjuvant environment. Alternatively, the result measurement value, such as overall survival and disease-free survival, can be monitored within a certain time period for the subject whose biomarker measurement value is known after immunomodulatory therapy. In certain embodiments, the dosage applied is a standard dose for cancer therapeutic agents known in the art. The time period of monitoring the subject can vary. For example, the subject can be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55 or 60 months.

[0131] The term "resistance" refers to an acquired or natural resistance of a cancer sample or mammal to an immunomodulatory therapy (i.e., no response to therapeutic treatment or a reduced or limited response), such as a response that is 5% or more, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, than that of a therapeutic treatment. The reduction in response can be measured by comparison with the same cancer sample or mammal before resistance was acquired or by comparison with a different cancer sample or mammal that is known to have no resistance to therapeutic treatment. Typical acquired resistance to chemotherapy is referred to as "multidrug resistance." 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 multidrug-resistant microorganism or a combination of microorganisms. The determination of resistance to therapeutic treatment is common in the art and within the skill of the skilled artisan, and can be measured as "sensitized" by, for example, cell proliferation assays and cell death assays as described herein. In some embodiments, the term "reversal of resistance" means that the use of a second agent in combination with a primary cancer therapy (e.g., chemotherapy or radiation therapy) is able to produce a statistically significant (e.g., p < 0.05) reduction in tumor volume when compared to the tumor volume of the untreated tumor when the primary cancer therapy (e.g., chemotherapy or radiation therapy) alone is unable to produce a statistically significant reduction in tumor volume compared to the tumor volume of the untreated tumor. This generally applies to tumor volume measurements made when the untreated tumor is growing regularly logarithmically.

[0132] The term "response" or "responsiveness" refers to a response to therapy. For example, an anticancer response includes a reduction in tumor size or inhibition of tumor growth. The term can also refer to an improved prognosis, such as an increase in the time before recurrence (this is the time period before the first recurrence in which a second primary cancer is considered a first event or until death without signs of recurrence), or an increase in overall survival (this is the time period from treatment to death due to any cause). Responding or having a response means that a beneficial endpoint is obtained when exposed to a stimulus. Alternatively, negative or harmful symptoms are minimized, alleviated or weakened when exposed to a stimulus. It should be understood that assessing the probability that a tumor or subject will show a good response is equivalent to assessing the probability that a tumor or subject will not show a good response (i.e., will show a lack of response or no response).

[0133] The term "tolerance" or "anergy" includes the refraction of cells (such as immune cells) to stimulation (for example, stimulation via activating receptors or cytokines). Anergy can occur, for example, due to exposure to immunosuppressants or exposure to high-dose antigens. Several independent methods can induce tolerance. A mechanism is called "anergy", which is defined as the state in which cells persist in vivo as anergy cells and do not differentiate into cells with effector functions. Such refraction is typically antigen-specific and persists after exposure to tolerance antigens stops. For example, T cell anergy is characterized by a lack of cytokine production, such as IL-2. When T cells are exposed to antigens and receive the first signal (T cell receptor or CD-3 mediated signal) in the absence of the second signal (co-stimulatory signal), T cell anergy occurs. Under these conditions, cells are exposed to the same antigen again (even if it occurs in the presence of co-stimulatory polypeptides) and cannot produce cytokines, and therefore cannot proliferate. However, if cultivated with cytokines (for example, IL-2), anergy T cells can proliferate. For example, T cell anergy can also be observed by lacking the IL-2 produced by T lymphocytes, as measured by ELISA or by using indicator cell lines to perform proliferation assays. Alternatively, a reporter gene construct can be used. For example, anergic T cells cannot start IL-2 gene transcription (Kang et al. (1992) Science 257:1134) induced by a heterologous promoter or by a polymer of the AP1 sequence that can exist in the enhancer under the control of the 5' IL-2 gene enhancer. Another mechanism is referred to as "exhaustion". T cell exhaustion is a state of T cell dysfunction that occurs during multiple chronic infections and cancers. This dysfunction is defined as poor effector function, the sustained expression of inhibitory receptors, and a transcriptional state different from the transcriptional state of functional effector T cells or memory T cells.

[0134] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. As used herein, reference is made to a nucleic acid molecule encoding an antibody or antibody portion (e.g., V or V) that binds to HHLA2. H 、V L , CDR3) (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 sequence encoding the antibody, or antibody portion, is free of other nucleotide sequences encoding antibodies, or antibody portions, that bind antigens other than HHLA2, which may naturally flank the nucleic acid in human genomic DNA.

[0135] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to a coding sequence. With respect to transcriptional regulatory sequences, operably linked means that the linked DNA sequences are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. With respect to switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.

[0136] By "overexpression" or "significantly higher expression level" of a marker is meant that the expression level in a test sample is greater than the standard error of the assay used to assess expression, and preferably is at least two-fold higher, and more preferably 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, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times higher, than the expression activity or level of the marker in a control sample (e.g., a sample from a healthy subject not suffering from a disease associated with the marker), and preferably the average expression level of the marker in several control samples. By "significantly lower expression level" of a marker is meant that the expression level in the test sample is at least two-fold lower, and more preferably 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, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times lower, than the expression level of the marker in a control sample (e.g., a sample from a healthy subject not suffering from the disease associated with the marker), and preferably the average expression level of the marker in several control samples.

[0137] Such antibodies as described herein can be used in any of the immunoassay formats known, including but not limited to radioimmunoassay, western blot assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay or slot blot assay. Other modifications of the general techniques and techniques for carrying out the various immunoassays mentioned above are known to those of ordinary skill in the art, such as in situ proximity ligation assay (PLA), fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), scattering immunoturbidimetric inhibition assay (NIA), enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), ELISA, etc., which can be used alone, or in combination with NMR, MALDI-TOF, LC-MS / MS, or in place of them.

[0138] Such reagents can also be used to monitor protein levels in cells or tissues (e.g., leukocytes or lymphocytes) as part of a clinical testing procedure, for example, to monitor the optimal dose of an inhibitor. 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, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, fluorescein, and aequorin; and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[0139] Such reagents can also be used with any number of biological samples. Biological samples can be collected from a variety of sources from the patient, including body fluid samples, cell samples, or tissue samples containing 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 sections, paraffin-embedded tissues, biopsies, whole blood, nipple aspirates, serum, plasma, oral scrapings, 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.

[0140] In some embodiments, the present invention provides the method for the biomarker amount of experimenter and / or the activity measurement value of experimenter.Sample can be collected from individual repeat (for example, every day, every week, every month, every year, every half a year or many times etc.) in a longitudinal time period.In a time period, obtain multiple samples from individual and can be used for verifying the result of earlier detection and / or the change of the biological pattern that for example disease progression, drug therapy etc. occur.For example, can obtain and monitor experimenter sample according to the combination of every month, every two months or January, two months or March intervals of the present invention.In addition, the biomarker amount and / or the activity measurement value of the experimenter that obtains in time can compare easily with each other and the biomarker amount and / or the activity measurement value of normal control during the monitoring time period, thereby provide as the value of the experimenter self of the interior or individual control for long-term monitoring.

[0141] The sample may contain living cells / tissue, fresh frozen cells, fresh tissue, a biopsy, fixed cells / tissue, cells / tissue embedded in a medium such as paraffin, a histological section, or any combination thereof.

[0142] Sample preparation and separation can involve any procedure depending on the type of sample collected and / or analysis of the biomarker measurements. By way of example only, such procedures include concentration, dilution, pH adjustment, removal of abundant polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, sample desalting, sample protein concentration, lipid extraction, and purification.

[0143] Sample preparation can also separate molecules that are bound to other proteins (e.g., carrier proteins) in non-covalent complexes. This process can separate those molecules that bind to a specific carrier protein (e.g., albumin), or use more general processes such as, for example, releasing bound molecules from all carrier proteins via protein denaturation using acid, followed by removal of the carrier protein.

[0144] Removing unwanted proteins (e.g., high abundance, no information or 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 can 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 based on size and molecular weight. Such filters can further include reverse osmosis, nanofiltration, ultrafiltration and microfiltration.

[0145] When used in relation to a reference polypeptide, the term "polypeptide fragment" or "fragment" refers to a polypeptide in which amino acid residues are deleted compared to the reference polypeptide itself, but the remaining amino acid sequence is generally identical to the polypeptide at the corresponding position in the reference polypeptide. Such deletions can occur at the amino terminus, internally, or at the carboxyl terminus of the reference polypeptide, or alternatively, at both. Fragments are typically at least 5, 6, 8, or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40, or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500, or more amino acids long. Their lengths may be, for example, at least and / or include 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 650, 660, 670, 680, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1020, 140 1260, 1280, 1300, 1320, 1340 or more, as long as their length is less than the length of the full-length polypeptide. Alternatively, they can be no longer than and / or exclude such ranges, as long as their length is less than the length of the full-length polypeptide.

[0146] The term "probe" refers to any molecule that is capable of selectively binding to a specific intended target molecule, such as a nucleotide transcript or protein encoded by or corresponding to a marker. Probes can be synthesized by one skilled in the art or derived from appropriate biological products. For the purpose of detecting a target molecule, probes can be specifically designed to be labeled, as described herein. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0147] As used herein, the term "rearranged" refers to a configuration of a heavy or light chain immunoglobulin locus in which the V segments encode primarily a complete V H and V L The domains are positioned in close proximity to the DJ segment or J segment in a conformational manner. Rearranged immunoglobulin loci can be identified by comparison to germline DNA; rearranged loci will have at least one recombined heptamer / nonamer homology element.

[0148] 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 also to the progeny of such a cell. Because certain changes may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0149] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from animals that are transgenic or transchromosomal for human immunoglobulin genes (e.g., mice) or hybridomas prepared therefrom (as further described below); (b) antibodies isolated from host cells transformed to express the antibodies, such as transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, generated or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, in vivo somatic mutagenesis) and thus the V H and V L The amino acid sequence of the region is as follows, in the human germline V H and VL While the sequences are not specifically identified, they may not naturally occur in the human antibody germline repertoire in vivo.

[0150] As used with reference to activated immune cells, the term "costimulation" includes the ability of a costimulatory polypeptide to provide a second non-activating receptor-mediated signal ("costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion in, for example, T cells that receive T cell receptor-mediated signals. Immune cells that receive cell receptor-mediated signals, such as via activating receptors, are referred to herein as "activated immune cells."

[0151] The term "co-stimulatory receptor" includes receptors, such as CD28, that transmit co-stimulatory signals to immune cells. As used herein, the term "inhibitory receptor" includes receptors (e.g., CTLA4, KIR3DL3 or PD-1) that transmit negative signals to immune cells. Even if co-stimulatory receptors (such as CD28) are not present on immune cells, inhibitory signals transduced by inhibitory receptors can also occur, and therefore, this is not merely a function of competition between inhibitory receptors and co-stimulatory receptors for binding co-stimulatory polypeptides (Fallarino et al. (1998) J. Exp. Med. 188: 205). The transmission of inhibitory signals to immune cells can result in the unresponsiveness or inability or programmed cell death of immune cells. Preferably, the transmission of inhibitory signals is operated by a mechanism that does not involve apoptosis. As used herein, the term "apoptosis" includes programmed cell death, which can be characterized using techniques known in the art. Apoptosis The characteristics of cell death can be, for example, cell shrinkage, membrane blebbing and chromatin condensation ending with cell fragmentation. Cells undergoing apoptosis also show a characteristic pattern of internucleosomal DNA cracking. Depending on the form of the polypeptide bound to the receptor, for example, by competing with the activated form of HHLA2 and / or KIR3DL3 to bind to one or more natural binding partners, the signal can be transmitted (for example, by the multivalent form of HHLA2 and / or KIR3DL3 polypeptides), or the signal can be suppressed (for example, by the soluble monovalent form of HHLA2 and / or KIR3DL3). However, there are cases where soluble polypeptides may be irritating. The effect of modulators can be easily demonstrated using conventional screening assays as described herein.

[0152] The terms "high," "low," "intermediate," and "negative," in connection with cellular biomarker expression, refer to the amount of a 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 but not limited to analysis of cellular levels, activity, structure, etc. of one or more biomarker genomic nucleic acids, RNAs, and / or polypeptides. In one embodiment, the terms refer to a defined percentage of a cell population that expresses a biomarker at the highest, intermediate, or lowest level, respectively. Such percentage can be defined as the top 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15% or more or any range (including end values) between the cell colonies of high expression or weak expression biomarkers. The term "low" does not include cells that cannot detectably express biomarkers because such cells are "negative" for biomarker expression. The term "intermediate" includes cells that express biomarkers but express at a level lower than that of a colony expressing biomarkers at a "high" level. In another embodiment, the term can also refer to or alternatively refer to a cell colony of biomarker expression identified by a qualitative or statistical plotting area. For example, cell populations sorted using flow cytometry can be differentiated based on biomarker expression levels by identifying different plots based on detectable moiety analysis, such as mean fluorescence intensity, according to methods well known in the art. Such plot areas can be refined for target biomarkers based on number, shape, overlap, etc., according to methods well known in the art. In yet another embodiment, the term can also be determined based on the presence or absence of expression of additional biomarkers.

[0153] As described above, the term "response" is generally associated with, for example, determining the effect of a clinical intervention on progression, efficacy, or outcome. In some embodiments, the response is directly related to changes in tumor mass and / or volume after the start of a clinical intervention (e.g., administration of anti-HHLA2 monoclonal antibodies, such as 2G2, 4D1, 8A12, 8D2, 1C8, 2C4, 6D10, 4E5, or 6F10 and polyclonal antibodies). For example, a hyperproliferative disorder can be assessed based on the size of the tumor after a systemic intervention compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound, or palpation. Response can also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Responses can also be recorded quantitatively, such as the percentage change in tumor volume, or qualitatively, such as "pathological complete response (pCR)," "clinical complete remission (cCR)," "clinical partial remission (cPR)," "clinically stable disease (cSD)," "clinically progressive disease (cPD)," or other qualitative criteria. Assessment may be performed after the start of clinical intervention, for example after hours, days, weeks or preferably months. Typical endpoints for response assessment are at the end of clinical intervention or when residual tumor cells and / or tumor bed are surgically removed.

[0154] As used herein, the term "specific binding" refers to the binding of an antibody to a predetermined antigen. Typically, in the case of human HHLA2 as an analyte and an antibody as a ligand, When determined by surface plasmon resonance (SPR) technology in an assay instrument, the antibody is approximately less than 10 -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower affinity (K D ), and binds to the predetermined antigen with an affinity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 times or greater than its affinity for binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The phrases "antibody that recognizes an antigen" and "antibody that is specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0155] As used herein, "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from a disease or condition associated with abnormal marker levels. The terms "subject" and "patient" are used interchangeably. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

[0156] The language "substantially free of chemical precursors or other chemicals" includes preparations of antibodies, polypeptides, peptides, or fusion proteins in which the protein is separated from chemical precursors or other chemicals involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of antibodies, polypeptides, peptides, or fusion proteins having less than about 30% (by dry weight) of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals, more preferably less than about 20% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals, still more preferably less than about 10% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals, and most preferably less than about 5% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals.

[0157] As used herein, the term "survival" includes all of the following: survival to death, also known as overall survival (wherein the death may be unrelated to the cause or associated tumor); "recurrence-free survival" (wherein the term recurrence should include both local and distant recurrences); metastasis-free survival; disease-free survival (wherein the term disease should include cancer and diseases associated therewith). The length of the survival period can be calculated by reference to a defined starting point (e.g., time of diagnosis or start of treatment) and an endpoint (e.g., death, recurrence, or metastasis). In addition, the criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0158] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or an analog of such RNA or cDNA) that is complementary to or homologous to all or a portion of a mature mRNA produced by transcription of a marker of the invention and normal post-transcriptional processing (e.g., splicing, if any) of the RNA transcript and reverse transcription of the RNA transcript.

[0159] As used herein, the term "T cell" includes CD4+T cells and CD8+T cells. The term T cell also includes type 1 T helper T cells and type 2 T helper T cells. The term "antigen presenting cell" includes professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) and other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).

[0160] Conventional T cells (also referred to as Tconv or Teff) have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) to increase immune responses by the expression of one or more T cell receptors thereof. Tconv or Teff are generally defined as any T cell population that is not Treg, and include, for example, primitive T cells, activated T cells, memory T cells, resting Tcon, or Tcon that has differentiated to, for example, Th1 or Th2 lineages. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is CD4+Teff or CD8+Teff, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17) and CD8+ cytotoxic T lymphocytes. As further described herein, cytotoxic T cells are CD8+T lymphocytes. "Original Tcon" is CD4 + T cell, it breaks up in bone marrow and successfully experiences the positive and negative process of center selection in thymus, but also not by being exposed to antigen activation.Original Tcon is characterized by the surface expression of L-selectin (CD62L) generally, the disappearance of activation markers such as CD25, CD44 or CD69, and the disappearance of memory markers such as CD45RO.Therefore it is believed that original Tcon is static and does not split, needs interleukin-7 (IL-7) and interleukin-15 (IL-15) for stable survival (at least referring to WO 2010 / 101870).The existence and activity of such cells are not needed when suppressing immune response. Unlike Tregs, Tcon are not anergic and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol. Sci. 356:625-637). In tumors, exhausted cells can exhibit hallmarks of anergy.

[0161] As used herein, the term "unrearranged" or "germline configuration" with reference to a V segment refers to a configuration in which the V segment has not undergone recombination so as to be in close proximity to a D or J segment.

[0162] As used herein, the term "vector" refers to a nucleic acid capable of transporting another nucleic acid connected to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of the genes to which they are operatively connected. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Typically, the expression vectors used in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, the present 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 perform equivalent functions.

[0163] For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, typically 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, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions to the complement of the strand.

[0164] The percent identity between the two sequences is a function of the number of identical positions that the sequences share (i.e., % identity = # of identical positions / total position # x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The sequence comparison between the two sequences and the determination of percent identity can be accomplished using a mathematical algorithm, as described in the following non-limiting examples.

[0165] 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, Inc. website) using NWSgapdna. The 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 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 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, 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 (available on the World Wide Web at the GCG, Inc. website) using either the Blosum 62 matrix or the 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.

[0166] The nucleic acid and protein sequences of the present invention can further be used as "query sequences" to search against public databases, for example, to 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 = 100, word length = 12, 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, word length = 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 used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) (available on the World Wide Web at the NCBI website) can be used.

[0167] Nucleic acid can be present in full cell, cell lysate or in partially purified or substantially pure form. When separated by purification with other cellular components or other pollutants (for example, other cellular nucleic acids or proteins), nucleic acid is "isolated" or "becomes substantially pure" by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and other techniques well known in the art (see, F.Ausubel et al., eds. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)).

[0168] The term "determining an appropriate treatment regimen for a subject" means determining a treatment regimen to be initiated, modified, and / or terminated for a subject (i.e., a single therapy or a combination of different therapies for preventing and / or treating cancer in a subject) based on, substantially based on, or at least in part on the results of an analysis according to the present invention. One example is determining whether to provide targeted therapy for cancer to provide immunomodulatory therapy (e.g., HHLA2 pathway modulator therapy (e.g., a modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3)). Another example is initiating adjuvant therapy after surgery to reduce the risk of recurrence, and another is changing the dose of a specific chemotherapy. In addition to the results of an analysis according to the present invention, the determination may be based on the individual characteristics of the subject to be treated. In most cases, the actual determination of an appropriate treatment regimen for a subject will be made by the attending physician or doctor.

[0169] II. Monoclonal Antibodies, Immunoglobulins, and Peptides

[0170] The present invention relates in part to isolated monoclonal antibodies or fragments thereof (such as the monoclonal antibodies and polyclonal antibodies listed herein) directed against HHLA2. Such molecules are characterized in part by their ability to recognize HHLA2 proteins in diagnostic assays (such as immunohistochemistry (IHC), Western blotting, intracellular flow cytometry, ELISA, etc.). Such molecules are characterized in part by their ability to inhibit HHLA2 binding to receptors, such as receptors expressed on T cells (e.g., TMIGD2 and KIR3DL3).

[0171] The term "HHLA2", also known as human endogenous retrovirus-H long terminal repeat-associated protein 2, HERV-H LTR-associated 2, B7y, B7H7, B7-H5, B7-H7, refers to a member of the B7 family. The HHLA2 protein has limited expression in normal human tissues, but is widely expressed in human cancers. The HHLA2 protein is a membrane protein with three Ig-like domains (IgV-IgC-IgV), while other members of the B7 family typically have only two Ig domains (IgV-IgC). The HHLA2 protein in normal human tissues is expressed in epithelial cells of the kidney, intestine, gallbladder, and breast, as well as in placental trophoblasts. In the immune system, the HHLA2 protein is constitutively expressed on human monocytes / macrophages. HHLA2 regulates human T cell function, including, for example, HHLA2 inhibits T cell proliferation and cytokine production, and increases T cell production and cytokine production. HHLA2 is expressed at higher levels in a wide range of human cancers from the colorectum, kidney, lung, pancreas, ovary, and prostate. HHLA2 is also expressed in human cancers of the thyroid, melanoma, liver, bladder, colon, kidney, breast, and esophagus.

[0172] HHLA2 structure and function are well known in the art, as described above (see, e.g., Xiao et al. (2015) Clin Cancer Res 21:2201-2203, Janakiram et al. (2015) Clin Cancer Res 21:2359-2366, Mager et al. (1999) Genomics 21:2359-2366, Flajnik et al. (2012) Immunogenet. 64:571-590, Zhao et al. (2013) Proc. Nat. Acad. Sci. USA 110:9879-9884, and Zhu et al. (2013) Nat. Commun. 4:2043).

[0173] The term "HHLA2" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human HHLA2 cDNAs and human HHLA2 protein sequences are well known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Human HHLA2 variants include variant 1 (NM_007072.3 and NP_009003.1, which represent the longest transcript and encode the longest isoform a), variant 2 (NM_001282556.1 and NP_001269485.1, which represent the use of an alternative promoter and differ in the 5'UTR compared to variant 1), variant 3 (NM_001282557.1 and NP_001269486.1, which represent the use of an alternative promoter and differ in the 5'UTR compared to variant 1), variant 4 (NM_001282558.1 and NP_001282557.1), variant 5 (NM_001282558.1 and NP_001282558.1), variant 6 (NM_001282558.1 and NP_001282558.1), variant 7 (NM_001282558.1 and NP_001282558.1), variant 8 (NM_001282558.1 and NP_001282558.1), variant 9 (NM_001282558.1 and NP_001282558.1), variant 10 (NM_001282558.1 and NP_001282558.1), variant 11 (NM_001282558.1 and NP_001282558.1 1 ) and variant 5 (NM_001282559.1 and NP_001269488.1, encoding isoform c, representing the use of an alternative promoter and having multiple differences compared to variant 2, resulting in a different 5'UTR compared to variant 1 and leading to translation initiation at an alternative start codon, resulting in a different N-terminus and an isoform shorter 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_001128644.1 and NP_001122116.1). Representative sequences of HHLA2 orthologs are presented in Table 1 below.

[0174] Anti-HHLA2 antibodies suitable for detecting HHLA2 protein are well known in the art and include, for example, antibody catalog #: ab107119 and ab214327 (abcam), antibodies PA5-24146 and PA5-6313 (Thermo Fisher Scientific), antibodies MAB80841, AF8084, FAB80841R, FAB80841T and MAB8084 (Andy Biosciences), antibody AP52042PU-N (Origene), antibodies NBP2-49187, MAB80842, H00011148-B01P and NBP2-32420 (Novus Biologicals), antibody GTX51981 (GeneTex), antibody HPA055478 (Atlas Biosciences), antibody AKT50001 ... Antibodies), antibodies LS-C321945, LS-C308228, LS-C246742, LS-C246743, LS-C246744, LS-C236210, and LS-C249186 (LifeSpan Biosiences), etc.In addition, a number of siRNA, shRNA, and CRISPR constructs for reducing HHLA2 expression can be found in the commercial product lists of the above-mentioned companies, such as shRNA products #TL312462, TF312462, TR312462, TG312462, and TL312462V from Origene Technologies. Technologies) siRNA product #SR323358, SiRNA products #i009616, i009616a, i009616b, i009616c, i009616d, iV009616, iV009616a, iV009616b, iV009616c, iV009616d, iAAV00961600, iAAV00961601, iAAV00961602, iAAV00961603, iAAV00961604, iAAV00961605, iAAV00961606, iAAV00961607, iAAV00961608 8 and iAAV00961609, CRISPR products #K0950321, K0950301, K0950302, K0950303, K0950304, K0950305, K0950306, K0950307, ​​K0950308 and K0950311 (abm), siRNA product #sc-78498, shRNA products #sc-78498-V and sc-78498-SH, CRISPR products #sc-411576, sc-411576-HDR, sc-411576-NIC and sc-411576-NIC-2 (Santa Cruz Biotechnology), etc. It should be noted that the term can further be used to refer to any combination of the features described herein for the HHLA2 molecule. For example, any combination of sequence composition, percent identity, sequence length, domain structure, functional activity, etc. can be used to describe the HHLA2 molecules of the present invention.

[0175] The term "HHLA2 pathway" includes HHLA2 and the interaction of HHLA2 with one or more of its natural binding partners, such as TMIGD2 and KIR3DL3.

[0176] The term "TMIGD2" refers to a transmembrane and immunoglobulin domain containing 2, CD28H, IGPR1, and IGPR-1, which is a membrane protein with approximately 10% amino acid identity to CD28, CTLA-4, ICOS, and PD-1. TMIGD2 has an extracellular IgV-like domain, a transmembrane region, and a proline-rich cytoplasmic domain with two tyrosine signaling motifs. The TMIGD2 protein is constitutively expressed on all naive T cells and most natural killer (NK) cells, but not on T regulatory cells or B cells. TMIGD2 expression is slowly lost under repeated 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 to function to reduce cell migration and promote capillary formation during angiogenesis.

[0177] TMIGD2 structure and function 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) Nature Communications 4:2043, and Rahimi (2012) Cell 23:1646-1656).

[0178] 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_001162597.1; compared to variant 1, it uses an alternative in-frame splice site in the 3' coding region, thereby producing an isoform shorter than isoform 1) and isoform 3 (NM_001308232.1 and NP_001295161.1, compared to variant 1, it lacks an alternative in-frame exon in the 5' coding region, thereby producing an isoform shorter than isoform 1). The nucleic acid and polypeptide sequences of TMIGD2 orthologs in organisms other than humans are well known and include, for example, chimpanzee TMIGD2 (XM_009434393.2 and XP_009432668.2, and XM_001138228.4 and XP_001138228.3) and bovine TMIGD2 (XM_005208980.3 and XP_005209037.1, XM_005208979.3 and XP_005209036.1, and XM_002688933.5 and XP_002688979.1). Representative sequences of TMIGD2 orthologs are presented in Table 1 below.

[0179] Anti-TMIGD2 antibodies suitable for detecting TMIGD2 protein are well known in the art and include, for example, antibody catalog #MAB8316, MAB83162, FAB8316R, FAB83162R, FAB83162G, FAB83162N, FAB83162S, FAB83162T, FAB83162U and FAB83162V (Andy Biotech), antibody TA326695 (Orgene), antibodies PA5-52787 and PA5-38055 (Thermo Fisher Scientific), antibodies MAB83161 and NBP1-81164 (Rofos Biologics), etc. In addition, a variety of siRNA, shRNA, CRISPR constructs for reducing TMIGD2 expression can be found in the commercial product lists of the above-mentioned companies, such as shRNA products #TF317829, TG317829, TL317829, TR317829 and TL317829V, siRNA product #SR314913, and CRISPR products #KN204938, KN204938LP from OriGene Technologies. , KN204938RB and KN204938BN, siRNA products #i024914, i024914a, i024914b, i024914c, i024914d, iV024914, iV024914a, iV024914b, iV024914c, iV024914d, iAAV02491400, iAAV02491401, iAAV02491402, iAAV02491403 1403, iAAV02491404, iAAV02491405, iAAV02491406, iAAV02491407, iAAV02491408, and iAAV02491409, and CRISPR products #K2409321, K2409301, K2409302, K2409303, K2409304, K2409305, K2409306, K2409307, and K2409308 08 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.In addition, a plurality of CRISPR constructs for increasing TMIGD2 expression can be found in the commercial product lists of the above-mentioned 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 Biotechnology), etc. It should be noted that the term can further be used to refer to any combination of the features described herein for the TMIGD2 molecule. For example, any combination of sequence composition, percent identity, sequence length, domain structure, functional activity, etc. can be used to describe the TMIGD2 molecules of the present invention.

[0180] The interaction between TMIGD2 and HHLA2 and their functions are well known in the art, as described above (see, e.g., Xiao et al. (2015) Clin Cancer Res 21:2201-2203 and Janakiram et al. (2015) Clin Cancer Res 21:2359-2366).

[0181] 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 family of transmembrane glycoproteins expressed by a subset of natural killer cells and T cells. Killer cell immunoglobulin-like receptor (KIR) genes are polymorphic and highly homologous, and they are present in a cluster on chromosome 19q13.4 within the 1Mb leukocyte receptor complex (LRC). The gene content of the KIR gene cluster varies in haplotypes, although several "framework" genes are present in all haplotypes (KIR3DL3, KIR3DP1, KIR3DL4, KIR3DL2). KIR proteins are classified by the number of extracellular immunoglobulin domains (2D or 3D) and by whether they have long (L) or short (S) cytoplasmic domains. KIR proteins with long cytoplasmic domains transduce inhibitory signals via an immunotyrosine-based inhibitory motif (ITIM) upon ligand binding, while KIR proteins with short cytoplasmic domains lack ITIM motifs and instead associate with TYRO protein tyrosine kinase binding proteins to transduce activation signals. The ligands of several KIR proteins are subsets of class I HLA molecules; therefore, it is believed that KIR proteins play an important role in regulating immune responses. This gene is one of the "framework" loci present on all haplotypes. The KIR3DL3 protein has an N-terminal signal sequence, three Ig domains, a transmembrane region lacking positively charged residues, and a long cytoplasmic tail region containing an immunoreceptor tyrosine-based inhibitory motif (ITIM). KIR3DL3 lacks the stalk region present in other KIRs.

[0182] KIR3DL3 structure and function are well known in the art, as described above (see, e.g., Hsu et al. (2002) Immunol Rev. 190:40-52, Trompeter et al. (2005) J. Immunol. 174:4135-4143, Trundley et al. (2006) Immunogenetics 57:904-916, and Jones et al. (2006) Immunogenetics 58:614-627).

[0183] The term "KIR3DL3" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human KIR3DL3 cDNAs 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) can be encoded by transcript (NP_703144.3). The 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 macaque KIR3DL3 (NM_001104552.2 and NP_001098022.1), mouse KIR3DL3 (NM_001310690.1 and NP_001297619.1, NM_177749.4 and NP_808417.2, NM_177748.2 and NP_808416.1), and rat KIR3DL3 (NM_181479.2 and NP_852144.1). Representative sequences of KIR3DL3 orthologs are presented in Table 1 below.

[0184] Anti-KIR3DL3 antibodies suitable for detecting KIR3DL3 protein are well known in the art and include, for example, antibody catalog #: FAB8919R, MAB8919, FAB8919G, FAB8919N, FAB8919S, FAB8919T, FAB8919U and FAB8919V (Andy Biotechnology), antibody AP52374PU-N (Orgene), antibody PA5-26178 (Thermo Fisher Scientific), antibodies OAAB05761, OAAF08125, OAAN04122, OACA09134, OACA09135, OACD04988 and OASG01190 (Aviva Systems Biology), etc. In addition, a variety of siRNA, shRNA, and CRISPR constructs for reducing KIR3DL3 expression can be found in the commercial product lists of the above-mentioned companies, such as shRNA products #TF303684, TR303684, TG303684, TL303684, and TL303684V, siRNA product #SR314516, and CRISPR products #KN224383, KN224 from OriGene Technologies. 383BN, KN224383RB, and KN224383LP, siRNA products #i011627, i011627a, i011627b, i011627c, i011627d, iV011627, iV011627a, iV011627b, iV011627c, iV011627d, iAAV01162700, iAAV01162701, iAAV01162702, iAAV01162703, iAAV01162704, iAAV01162705, iAAV01162706, iAAV01162707, iAAV01162708, and iAAV01162709, and CRISPR products #K1151421, K1151401, K1151402, K1151403, K1151404, K1151405, K1151406, and K1151407 1407, K1151408 and K1151411 (Abm), siRNA product #sc-60892, shRNA product #sc-60892-SH and sc-60892-V, and CRISPR product #sc-406227, sc-406227-KO-2, sc-406227-HDR-2, sc-406227-NIC and sc-406227-NIC-2 (Santa Cruz Biotechnology), etc. It should be noted that the term can further be used to refer to any combination of the features described herein for the KIR3DL3 molecule.For example, any combination of sequence composition, percent identity, sequence length, domain structure, functional activity, etc. can be used to describe the KIR3DL3 molecules of the invention.

[0185] The term "peripheral blood cell subset" refers to cell types normally present in peripheral blood, including but not limited to eosinophils, neutrophils, T cells, monocytes, NK cells, granulocytes, and B cells.

[0186] The term "recombinant human antibody" includes all human antibodies prepared, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from animals that are transgenic or transchromosomal for human immunoglobulin genes (e.g., mice) or hybridomas prepared therefrom (as further described below); (b) antibodies isolated from host cells transformed to express the antibodies, such as transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, generated or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, in vivo somatic mutagenesis) and thus the V H and V L The amino acid sequence of the region is as follows, in the human germline V H and VL While the sequences are not specifically identified, they may not naturally occur in the human antibody germline repertoire in vivo.

[0187] The term "sample" 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 body fluid (e.g., as described above under the definition of "body fluid") or a tissue sample (e.g., a biopsy), such as a small intestine, colon sample, or surgical resection tissue. In some cases, the method of the present invention further comprises obtaining a sample from an individual before detecting or determining the presence or level of at least one marker in the sample.

[0188] As used herein, "RNA interference agent" is defined as any agent that interferes with or inhibits the expression of a target biomarker gene through RNA interference (RNAi). Such RNA interference agents include, but are not limited to, nucleic acid molecules, including RNA molecules or fragments thereof homologous to the target biomarker genes of the present invention, short interfering RNA (siRNA), and small molecules that interfere with or inhibit the expression of target biomarker nucleic acids through RNA interference (RNAi).

[0189] "RNA interference (RNAi)" is an evolutionarily conserved process whereby the expression or introduction of RNA of a sequence identical or highly similar to a target biomarker nucleic acid results in sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of the messenger RNA (mRNA) transcribed from the targeted gene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting the expression of the target biomarker nucleic acid. In one embodiment, the RNA is double-stranded RNA (dsRNA). The process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific nuclease Dicer, which promotes the cleavage of long dsRNA into double-stranded fragments called siRNA. siRNA is incorporated into a protein complex that recognizes and cleaves the target mRNA. RNAi can also be initiated by the introduction of nucleic acid molecules, such as synthetic siRNA, shRNA, or other RNA interfering agents, to inhibit or silence the expression of the target biomarker nucleic acid. As used herein, "inhibition of target biomarker nucleic acid expression" or "inhibition of marker gene expression" includes any reduction in the expression of a target biomarker nucleic acid or a protein encoded by a target biomarker nucleic acid, or the activity or level of a protein. The reduction can be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to the expression of a target biomarker nucleic acid or the activity or level of a protein encoded by a target biomarker nucleic acid that is not targeted by an RNA interfering agent.

[0190] In addition to RNAi, genome editing can be used to regulate the copy number or gene sequence of target biomarkers, such as constitutive or inducible knockout or mutation of target biomarkers (such as HHLA2 pathway components, 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 producing non-functional or null mutations). In such embodiments, CRISPR guide RNA and / or Cas enzymes can be expressed. For example, a vector containing only guide RNA can be administered to an animal or cell transgenic for the Cas9 enzyme. Similar strategies can be used (e.g., designing zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases). Such systems are well known in the art (see, e.g., U.S. Patent No. 8,697,359; Sander and Joung (2014) Nature Biotechnology 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Patent Publications 2014 / 0087426 and 2012 / 0178169; Boch et al. (2011) Nature Biotechnology 29:135-136; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al. (2011) PLoS One (2011) 6:e19722; Li et al. (2011) Nucl. Acids Res. 39:6315-6325; Zhang et al. (2011) Nature Biotechnology 29:149-153; Miller et al. (2011) Nature Biotechnology 29:143-148; Lin et al. (2014) Nucleic Acids Res. 42:e47). Such genetic strategies can use constitutive or inducible expression systems, according to methods well known in the art.

[0191] "Piwi-interacting RNAs (piRNAs)" are the largest class of small noncoding RNA molecules. piRNAs form RNA-protein complexes by interacting with piwi proteins. These piRNA complexes are associated with both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germline cells, particularly those involved in spermatogenesis. They differ from microRNAs (miRNAs) in size (26–31 nt 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, particularly of transposons. The majority of piRNAs are antisense to transposon sequences, suggesting that transposons are piRNA targets. In mammals, piRNA activity in transposon silencing appears to be most important during embryonic development, and in both C. elegans and humans, piRNAs are essential for spermatogenesis. piRNAs play a role in RNA silencing via the formation of the RNA-induced silencing complex (RISC).

[0192] "Aptamers" are oligonucleotides or peptide molecules that bind to specific target molecules. "Nucleic acid aptamers" are nucleic acid species that are engineered to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, through repeated rounds of in vitro selection or, equivalently, SELEX (systematic evolution of ligands by exponential enrichment). "Peptide aptamers" are artificial proteins that are selected or engineered to bind to specific target molecules. These proteins consist of one or more peptide loops of variable sequences displayed by a protein scaffold. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutagenesis or multiple rounds of variable region mutagenesis and selection. "Affinity multimeric proteins" (evolution of peptide aptamers) are small, highly stable proteins engineered to display peptide loops that provide a high-affinity binding surface for a specific target protein. It is a low molecular weight 12–14 kDa protein derived from the cystatin family of cysteine ​​protease inhibitors. Aptamers can be used in biotechnology and therapeutic applications because they provide molecular recognition properties comparable to commonly used biomolecules and antibodies. In addition to their discriminatory recognition, aptamers offer advantages over antibodies in that they can be fully engineered in a test tube, are easily produced by chemical synthesis, possess desirable storage properties, and induce little or no immunogenicity in therapeutic applications.

[0193] "Short interfering RNA" (siRNA), also referred to herein as "small interfering RNA", is defined as an agent used to inhibit the expression of a target biomarker nucleic acid, for example, by RNAi. siRNA can be chemically synthesized, can be produced by in vitro transcription, or can be produced in a host cell. In one embodiment, the siRNA is a double-stranded RNA (dsRNA) molecule having a length of about 15 to about 40 nucleotides, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides, and more preferably about 19, 20, 21, or 22 nucleotides, and can contain 3' and / or 5' overhangs of about 0, 1, 2, 3, 4, or 5 nucleotides in length on each strand. The length of the overhangs between the two strands is independent, 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 by degradation of the target messenger RNA (mRNA) or specific post-transcriptional gene silencing (PTGS).

[0194] In another embodiment, the siRNA is a small hairpin (also known as a 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 a similar sense strand. Alternatively, the sense strand can be before the nucleotide loop structure, and the antisense strand can be after it. These shRNAs can be contained in plasmids, retroviruses, and lentiviruses, and, for example, expressed by the pol III U6 promoter or another promoter (see, e.g., Stewart et al. (2003) RNA Apr; 9(4): 493-501, which is incorporated herein by reference).

[0195] RNA interfering agents, such as siRNA molecules, can be administered to a subject having or at risk of developing cancer to inhibit the expression of biomarker genes overexpressed in cancer and thereby treat, prevent, or inhibit cancer in the subject.

[0196] The term "small molecule" is a term of art and includes molecules less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively include peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. 1998. Science 282:63) and natural product extraction libraries. In another embodiment, the compound is a small organic non-peptide compound. In another embodiment, small molecules are not biosynthesized.

[0197] The term "selective modulator" or "selective modulation" as applied to a biologically active agent refers to the ability of the agent to modulate a target (such as a cell population, signaling activity, etc.) via direct or reciprocal interaction with the target as compared to an off-target cell population, signaling activity, etc. For example, an agent that selectively inhibits an interaction between HHLA2 and one or more natural binding partners (such as TMIGD2 and KIR3DL3) and / or such interaction on a target cell population as compared to another interaction between HHLA2 and another binding partner can have activity for an HHLA2 pathway modulator therapy (e.g., modulation of the interaction between HHLA2 and one or more natural binding partners (such as TMIGD2 and KIR3DL3)). For example, an inhibitory agent may be an inhibitory agent, wherein the interaction is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 2x (fold) or more (e.g., at least about 3x) of the activity of the agent toward at least another binding partner. x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 9 0x, 95x, 100x, 105x, 110x, 120x, 125x, 150x, 200x, 250x, 300x, 350x, 400x, 450x, 500x, 600x, 700x, 800x , 900x, 1000x, 1500x, 2000x, 2500x, 3000x, 3500x, 4000x, 4500x, 5000x, 5500x, 6000x, 6500x, 7000x, 7500x, 8000x, 8500x, 9000x, 9500x, 10000x or any range greater or therebetween, inclusive). Such metrics are typically expressed in terms of the relative amount of agent required to reduce the interaction / activity by half.

[0198] More generally, the term "selective" refers to a preferential effect or function. The term "selective" can be quantified with respect to a preferential effect in a particular target of interest relative to other targets. For example, a measured variable (e.g., regulation of Treg / Breg relative to other cells, such as other immune cells like Tcon) can be 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5.5-fold, 6.5-fold, 7.5-fold, 8.5-fold, 9.5-fold, 10. fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or any range (including end values, such as 50% to 16-fold) between them. The same multiple analysis can be used to confirm the amplitude of the effect in a given tissue or cell population, the variable measured, the effect of the measurement, etc., such as Treg:Tcon ratio, Breg:Tcon ratio, hyperproliferative cell growth rate or volume, Treg / Breg proliferation rate or number, etc.

[0199] In contrast, the term "specific" refers to an exclusive effect or function. For example, specific regulation of HHLA2-TMIGD2 and HHLA2-KIR3DL3 interactions refers to exclusive regulation of HHLA2 / TMIDG2 and HHLA2 / KIR3DL3 interactions, respectively, but does not include regulation of the interaction 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 target antigen without binding to other antigens. Typically, when using the target antigen as the analyte and the antibody as the ligand, When determined by surface plasmon resonance (SPR) technology in the assay instrument, the antibody is detected at a concentration of approximately less than 1 x 10 -7 M, such as approximately less than 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or even lower affinity (K D) and binds to the predetermined antigen with an affinity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 times or greater than its affinity for binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. In addition, K D It's K A The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0200] The term "sensitization" means changing a cell, such as a cancer cell or tumor cell, in a manner that allows for more effective treatment with a therapy (e.g., HHLA2 pathway modulator therapy (e.g., a modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and KIR3DL3), 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 excessively damaged by the therapy (e.g., HHLA2 pathway modulator therapy (e.g., a modulator of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and KIR3DL3), alone or in combination with an immunotherapy such as an immune checkpoint inhibition therapy). Increased or decreased sensitivity to therapeutic treatment is measured for the specific treatments and methods described herein below according to methods known in the art, including but not limited to cell proliferation assays (Tanigawa N, Kern DH, Kikasa Y, Morton DL, Cancer Res 1982;42:2159-2164), cell death assays (Weisenthal LM, Shoemaker RH, Marsden JA, Dill PL, Baker JA, Moran EM, Cancer Res 1984;94:161-173; Weisenthal LM, Lippman ME, Cancer Treat Rep 1985;69:615-632; Weisenthal LM, In: Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJ P, ed., Drug Resistance in Leukemia and Lymphoma., Langhorne, PA: Harwood Academic Publishers, 1993: 415-432; Weisenthal LM, Contrib Gynecol Obstet 1994; 19: 82-90). Sensitivity or resistance can also be measured in animals by measuring the reduction in tumor size over a period of time (e.g., 6 months for humans and 4-6 weeks for mice).If compared with the treatment sensitivity or resistance in the absence of such compositions or methods, the treatment sensitivity increases or resistance decreases 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 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times or more, then the compositions or methods sensitize the response to therapeutic treatment. The determination of sensitivity or resistance to therapeutic treatment is common in the art and within the skill range of ordinary technical clinicians. It should be understood that any method for enhancing the efficacy of immunomodulation as described herein can be equally applied to methods for sensitizing hyperproliferative or other cancerous cells (for example, resistant cells) to therapy.

[0201] The term "synergistic effect" means that the combined effect of two or more therapeutic agents (such as two or more HHLA2 pathway modulators, an HHLA2 pathway modulator and an immunotherapy, an HHLA2 pathway modulator alone or in combination with an immunotherapy such as an immune checkpoint inhibition therapy, etc.) can be greater than the sum of the individual effects of the individual anticancer agents.

[0202] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from a condition of interest (eg, cancer). The term "subject" is used interchangeably with "patient."

[0203] The term "survival" includes all of the following: survival to death, also known as overall survival (wherein the death may be unrelated to the cause or associated tumor); "recurrence-free survival" (wherein the term recurrence should include both local and distant recurrences); metastasis-free survival; disease-free survival (wherein the term disease should include cancer and diseases associated therewith). The length of the survival period can be calculated by reference to a defined starting point (e.g., time of diagnosis or start of treatment) and an endpoint (e.g., death, recurrence, or metastasis). In addition, the criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0204] The term "therapeutic effect" refers to a local or systemic effect caused by a pharmacologically active substance in an animal, particularly a mammal, and more particularly a human. Thus, the term refers to any substance intended to be used in the diagnosis, cure, alleviation, treatment or prevention of a disease or in the enhancement of a desired physical or psychological development and condition in a human or animal. The phrase "therapeutically effective amount" means the amount of such a substance that produces a certain desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. In certain embodiments, the therapeutically effective amount of a compound will depend on its therapeutic index, solubility, etc. For example, certain compounds discovered by the methods of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0205] As used herein, the terms "therapeutically effective amount" and "effective amount" mean an amount of a compound, material, or composition comprising a compound of the invention effective to produce a desired therapeutic effect in at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. The toxicity and therapeutic efficacy of the subject compounds can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, such as for determining LD50. 50 and ED 50 Compositions that exhibit a large therapeutic index are preferred. In some embodiments, the LD of the agent can be measured. 50 (lethal dose), and can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more relative to not administering the agent. Similarly, the ED of the agent can be measured. 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms), and can be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more relative to not administering the agent. Additionally, similarly, the IC of the agent can be measured. 50In some embodiments, the growth of cancer cells 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%, relative to the absence of the agent. Cancer cell growth can be promoted by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, a reduction of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% in the number of cancer cells and / or solid malignancies can be achieved.

[0206] The term "substantially free of chemical precursors or other chemicals" includes preparations of antibodies, polypeptides, peptides, or fusion proteins in which the protein is separated from chemical precursors or other chemicals involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of antibodies, polypeptides, peptides, or fusion proteins having less than about 30% (by dry weight) of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals, more preferably less than about 20% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals, still more preferably less than about 10% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals, and most preferably less than about 5% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein chemicals.

[0207] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA or miRNA binding site or variants thereof or analogs of such RNA or cDNA) that is complementary to or homologous to all or a portion of a mature mRNA produced by transcription of a marker of the invention and normal post-transcriptional processing (e.g., splicing, if any) of the RNA transcript and reverse transcription of the RNA transcript.

[0208] The term "vector" refers to a nucleic acid capable of transporting another nucleic acid connected to it. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of the genes to which they are operatively connected. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". Typically, the expression vectors used in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present 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 perform equivalent functions.

[0209] There is a known and established correspondence between the amino acid sequence of a particular protein and the nucleotide sequence that can encode that protein, as defined by the genetic code (shown below). Similarly, there is a known and established 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.

[0210] genetic code

[0211]

[0212]

[0213] The important and known feature of genetic code is its redundancy, thus for most of the amino acids for preparing proteins, more than one coding nucleotide triplet (shown above) can be adopted.Therefore, a plurality of different nucleotide sequences can encode a given amino acid sequence.This type of nucleotide sequence is considered to be functionally equivalent because they cause the production of identical amino acid sequences in all organisms (although some organisms can more effectively translate some sequences than others). In addition, occasionally, methylated variants of purine or pyrimidine can be present in a given nucleotide sequence.This type of methylation does not affect the coding relationship between trinucleotide codons and corresponding amino acids.

[0214] In view of the above, the nucleotide sequence of the DNA or RNA encoding the biomarker nucleic acid (or any portion thereof) can be used to obtain a polypeptide amino acid sequence using the genetic code that translates the DNA or RNA into an amino acid sequence. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence that can encode the polypeptide can be inferred by the genetic code (due to its redundancy, the genetic code will produce multiple nucleic acid sequences for any given amino acid sequence). Therefore, the description and / or disclosure of the nucleotide sequence encoding the polypeptide herein should be considered to also include the description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, the description and / or disclosure of the polypeptide amino acid sequence herein should be considered to also include the description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0215] Finally, nucleic acid and amino acid sequence information that can be used for nucleic acid and polypeptide molecules of the present invention are well known in the art and can be easily obtained from publicly available databases such as the U.S. National Center for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences that derive from publicly available sequence databases are provided in Table 1 below.

[0216] Table 1

[0217] SEQ ID NO: 1 Human HHLA2 variant 1 cDNA sequence (NM_007072.3, CDS region from positions 415-1659)

[0218]

[0219]

[0220]

[0221]

[0222] SEQ ID NO: 2 Human HHLA2 variant 1 amino acid sequence (NP_009003.1)

[0223]

[0224]

[0225] SEQ ID NO: 3 Human HHLA2 variant 2 cDNA sequence (NM_001282556.1, CDS from positions 224-1468) district)

[0226]

[0227]

[0228]

[0229]

[0230] SEQ ID NO:4 Human HHLA2 variant 2 amino acid sequence (NP_001269485.1)

[0231]

[0232] SEQ ID NO: 5 Human HHLA2 variant 3 cDNA sequence (NM_001282557.1, CDS from positions 155-1399) district)

[0233]

[0234]

[0235]

[0236]

[0237] SEQ ID NO:6 Human HHLA2 variant 3 amino acid sequence (NP_001269486.1)

[0238]

[0239] SEQ ID NO: 7 Human HHLA2 variant 4 cDNA sequence (NM_001282558.1, CDS from positions 302-1495) district)

[0240]

[0241]

[0242]

[0243]

[0244] SEQ ID NO: 8 Human HHLA2 variant 4 amino acid sequence (NP_001269487.1)

[0245]

[0246]

[0247] SEQ ID NO: 9 Human HHLA2 variant 5 cDNA sequence (NM_001282559.1, CDS from positions 232-1284) district)

[0248]

[0249]

[0250]

[0251] SEQ ID NO: 10 Human HHLA2 variant 5 amino acid sequence (NP_001269488.1)

[0252]

[0253]

[0254] SEQ ID NO: 11 Human TMIGD2 isoform 1 cDNA sequence (NM_144615.2, CDS from positions 47-895) district)

[0255]

[0256]

[0257] SEQ ID NO: 12 Human TMIGD2 isoform 1 amino acid sequence (NP_653216.2)

[0258]

[0259]

[0260] SEQ ID NO: 13 Human TMIGD2 isoform 2 cDNA sequence (NM_001169126.1, from positions 47-883 CDS area)

[0261]

[0262]

[0263] SEQ ID NO: 14 Human TMIGD2 isoform 2 amino acid sequence (NP_001162597.1)

[0264]

[0265]

[0266] SEQ ID NO: 15 Human TMIGD2 isoform 3 cDNA sequence (NM_001308232.1, from positions 47-535 CDS area)

[0267]

[0268]

[0269] SEQ ID NO: 16 Human TMIGD2 isoform 3 amino acid sequence (NP_001295161.1)

[0270]

[0271] SEQ ID NO: 17 Human KIR3DL3 cDNA sequence (NM_153443.4, CDS region from positions 51-1283)

[0272]

[0273]

[0274]

[0275] SEQ ID NO: 18 Human KIR3DL3 amino acid sequence (NP_703144.3)

[0276]

[0277] * Included in Table 1 are RNA nucleic acid molecules (e.g., thymine replaced by uridine), nucleic acid molecules encoding orthologs of the encoded proteins, and DNA or RNA nucleic acid sequences or portions thereof comprising a nucleic acid sequence that is 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 identical across its entire length to the nucleic acid sequence of any SEQ ID NO listed in Table 1. Such nucleic acid molecules can function as the full-length nucleic acids as further described herein. * Included in Table 1 are orthologs of the proteins, as well as polypeptide molecules comprising an amino acid sequence that is 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 identical across its entire length to the amino acid sequence of any SEQ ID NO listed in Table 1. Such polypeptides may have the functions of the full-length polypeptide as further described herein.

[0278] *Other known HHLA2, TMIGD2, and KIR3DL3 nucleic acid and amino acid sequences are included in Table 1.

[0279] In addition to being a stimulatory receptor (i.e., transmitting co-stimulatory signals to immune cells), HHLA2 polypeptides are also inhibitory receptors that can transmit inhibitory signals to immune cells, thereby inhibiting immune cell effector functions, or can, for example, promote co-stimulation of immune cells when binding to inhibitory or stimulatory receptors. HHLA2 binds to one or more receptors on T cells, such as TMIGD2, KIR3DL3, and / or other polypeptides.

[0280] The term "HHLA2 activity" encompasses the ability of an HHLA2 polypeptide to modulate inhibitory signaling in activated immune cells, for example, by engaging natural HHLA2 ligands on T cells. Modulation of inhibitory signaling in immune cells results in immune cell proliferation and / or modulation of cytokine secretion by the immune cells. Thus, the term "HHLA2 activity" encompasses the ability of an HHLA2 polypeptide to bind its natural ligand, modulate costimulatory or inhibitory signaling in immune cells, and modulate an immune response.

[0281] 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 has a more pronounced or synergistic responsiveness when treated with HHLA2 blockade in combination with other therapies. Various conditions that are responsive to HHLA2 blockade alone or in combination include, but are not limited to, melanoma (e.g., advanced or metastatic melanoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), breast cancer (e.g., HER-2 negative breast cancer, estrogen receptor + / HER-2- breast cancer, and triple negative breast cancer), pancreatic cancer (e.g., pancreatic adenocarcinoma), and Hodgkin's lymphoma, as well as bladder cancer, gastric cancer, head and neck cancer, kidney cancer, prostate cancer, gynecological cancer, colorectal cancer, ovarian cancer, and hematological cancers.

[0282] Preferred B7 polypeptides can provide co-stimulatory or inhibitory signals to immune cells, thereby promoting or inhibiting immune cell responses. For example, B7 family members that bind to co-stimulatory receptors increase T cell activation and proliferation, while B7 family members that bind to inhibitory receptors reduce co-stimulation. In addition, the same B7 family members can increase or decrease T cell co-stimulation. For example, when binding to co-stimulatory receptors, HHLA2 can induce co-stimulation of immune cells, or when binding to inhibitory receptors, HHLA2 can inhibit immune cells. When binding to inhibitory receptors, HHLA2 can transmit inhibitory signals to immune cells. Preferred B7 family members include HHLA2, B7-1, B7-2, B7h, PD-L1 or PD-L2 and soluble fragments or derivatives thereof. In one embodiment, B7 family members bind to one or more receptors on immune cells, such as TMIGD2, KIR3DL3, CTLA4, CD28, ICOS, PD-1 and / or other receptors, and according to the receptors, have the ability to transmit inhibitory or co-stimulatory signals to immune cells, preferably T cells.

[0283] Regulation of co-stimulatory signals leads to regulation of effector functions of immune cells. Thus, the term "HHLA2 ligand activity" includes the ability of an HHLA2 ligand polypeptide to bind to its natural receptor (e.g., HHLA2), to regulate immune cell co-stimulatory or inhibitory signaling, and to regulate immune responses.

[0284] It is demonstrated herein that the HHLA2 pathway is either a negative or 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. It is believed that HHLA2 binding to TMIGD2 is similar to HHLA2 binding to inhibitory receptors. Therefore, inhibiting HHLA2 binding to TMIGD2 is considered to inhibit HHLA2 binding to inhibitory receptors. Therefore, agents of the invention described herein that are HHLA2 pathway modulators (e.g., modulators of the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3) directly or indirectly modulate the interaction between HHLA2 and one or more natural binding partners, can upregulate or downregulate the immune system and thereby upregulate or downregulate the immune response. Agents that modulate such interactions can do so directly or indirectly.

[0285] The interaction between HHLA2 and one or more HHLA2 natural binding partners (such as TMIGD2 and / or KIR3DL3) results in the delivery of co-stimulatory or co-inhibitory immune signals. Thus, in one embodiment, agents that directly block such interactions (e.g., anti-HHLA2, anti-TMIGD2, and / or anti-KIR3DL3 blocking antibodies) can prevent inhibitory or stimulatory signaling and upregulate or downregulate the immune response. Alternatively, agents that indirectly block the interaction can prevent inhibitory signaling and upregulate the immune response. Exemplary agents for upregulating an immune response include antibodies directed against HHLA2 or KIR3DL3 that block the interaction between HHLA2 and KIR3DL3; inactive forms of HHLA2 or KIR3DL3 (e.g., dominant negative polypeptides) small molecules or peptides that block the interaction between HHLA2 and KIR3DL3; fusion proteins that bind to HHLA2 or KIR3DL3, respectively, and inhibit the interaction between HHLA2 and KIR3DL3 (e.g., the extracellular portion of HHLA2 or KIR3DL3 fused to the Fc portion of an antibody or immunoglobulin); nucleic acid molecules and / or genetic modifications that block transcription or translation of HHLA2 and / or KIR3DL3; inactive forms of native HHLA2 ligands and soluble forms of native KIR3DL3 ligands.

[0286] In other exemplary embodiments, agents that promote the binding of an HHLA2 polypeptide to one or more natural binding partners (such as a KIR3DL3 polypeptide) promote inhibitory signaling to immune cells. Agents that modulate such interactions can do so directly or indirectly. Thus, in one embodiment, agents that directly enhance the interaction between HHLA2 and KIR3DL3 (HHLA2 agonists and / or KIR3DL3 agonists) can promote inhibitory signaling and downregulate the immune response. Alternatively, agents that block the binding of KIR3DL3 to other targets increase the effective concentration of KIR3DL3 available for binding to HHLA2. Exemplary agents for downregulating the immune response include antibodies that activate HHLA2 or KIR3DL3 or promote the interaction between HHLA2 and KIR3DL3; small molecules or peptides that activate or promote the interaction between HHLA2 and KIR3DL3; and blocking antibodies that bind to natural binding partners of HHLA2 and KIR3DL3 other than HHLA2 and KIR3DL3, respectively. These relationships also apply to the interaction between HHLA2 and TMIGD2.

[0287] Additional reagents that can be used in the methods of the present invention include antibodies, small molecules, peptides, peptidomimetics, natural ligands and derivatives of natural ligands, which can bind to and / or activate or inhibit the protein biomarkers of the present invention (including the biomarkers listed in Table 1) or fragments thereof; RNA interference, antisense, nucleic acid aptamers, etc., which can downregulate the expression and / or activity of the biomarkers of the present invention (including the biomarkers listed in Table 1) or fragments thereof.

[0288] Isolated monoclonal antibodies or fragments thereof directed against HHLA2 are provided. In some embodiments, mAbs produced by hybridomas have been deposited with the American Type Culture Collection (ATCC) under the Budapest Treaty at ______ under accession number ______.

[0289] Because it is well known in the art that the antibody heavy chain and light chain CDR3 domains play a particularly important role in the binding specificity / affinity of the antibody for the antigen, the recombinant monoclonal antibodies of the present invention prepared as described above comprise the heavy chain and light chain CDR3 of the variable region of the present invention (e.g., comprising the sequence of Table 2 or a portion thereof). The antibody may further comprise the CDR2 of the variable region of the present invention (e.g., comprising the sequence of Table 2 or a portion thereof). The antibody may further comprise the CDR1 of the variable region of the present invention (e.g., comprising the sequence of Table 2 or a portion thereof). In other embodiments, the antibody may comprise any combination of CDRs.

[0290] The CDR1, CDR2, and / or CDR3 regions of the engineered antibodies described above may comprise an amino acid sequence that is identical to the amino acid sequence of the variable regions of the invention disclosed herein (e.g., comprising the sequences of Table 2 or portions thereof). However, one of ordinary skill will appreciate that some variations from identical CDR sequences may be possible while still retaining the ability of the antibody to effectively bind to HHLA2 (e.g., conservative sequence modifications). Thus, 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 invention (e.g., comprising the sequences of Table 2 or portions thereof).

[0291] Structural features of known non-human or human antibodies (e.g., mouse or non-rodent anti-human HHLA2 antibodies) can be used to generate structurally related human anti-human HHLA2 antibodies (such as mAb 8A12 and polyclonal antibodies 1.2 and 2.2) that retain at least one functional property of the antibodies of the invention (such as HHLA2 binding). Another functional property includes inhibition of binding of the original known non-human or human antibody in a competition ELISA assay.

[0292] In some embodiments, monoclonal antibodies capable of binding to human HHLA2 (such as mAb 8A12 and polyclonal antibodies 1.2 and 2.2) are provided, which comprise a heavy chain, wherein the variable domain comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the set of heavy chain variable domain CDRs presented in Table 2.

[0293] Similarly, monoclonal antibodies capable of binding to human HHLA2 (such as mAb 8A12 and polyclonal antibodies 1.2 and 2.2) are also provided, which comprise a light chain, wherein the variable domain comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the set of light chain variable domain CDRs presented in Table 2.

[0294] Also provided are monoclonal antibodies capable of binding to human HHLA2 (such as mAb 8A12 and polyclonal antibodies 1.2 and 2.2), comprising a heavy chain, wherein the variable domain comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the set of heavy chain variable domain CDRs presented in Table 2; and a light chain, wherein the variable domain comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the set of light chain variable domain CDRs presented in Table 2.

[0295] The 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.

[0296] The monoclonal antibodies of the present invention may comprise a heavy chain, wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of the heavy chain variable domain CDRs presented in Table 2; and a light chain, wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of the light chain variable domain CDRs presented in Table 2.

[0297] Such monoclonal antibodies may comprise a light chain, wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of CDR-L1, CDR-L2, and CDR-L3 as described herein; and / or a heavy chain, wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of CDR-H1, CDR-H2, and CDR-H3 as described herein. In some embodiments, the monoclonal antibody capable of binding to human HHLA2 comprises or consists of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as described herein.

[0298] The heavy chain variable domain of the monoclonal antibody of the present invention may comprise or consist of the vH amino acid sequence listed in Table 2, and / or the light chain variable domain of the monoclonal antibody of the present invention may comprise or consist of the vκ amino acid sequence listed in Table 2.

[0299] The monoclonal antibodies of the present invention can be produced and modified by any technology well known in the art. For example, such monoclonal antibodies can be mouse or non-rodent antibodies, such as those obtained from hybridomas deposited as a deposit at ATCC from ______. 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 domains comprise human acceptor framework regions and optionally human constant regions (if present) and non-human donor CDRs, such as mouse or non-rodent CDRs defined above.

[0300] The present invention further provides fragments of the monoclonal antibodies, including but not limited to Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies; as well as multispecific antibodies formed from antibody fragments. For example, members of immunosuppressive molecules such as HHLA2, PD-L2, PD-L1, CTLA-4, KIR3DL3, etc. can be detected in a bispecific or multispecific manner to effectively characterize the expression of such molecules.

[0301] 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 their variable domains comprise at least one CDR presented in Table 2. In one embodiment, the immunoglobulin heavy chain comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the set of heavy or light chain variable domain CDRs presented in Table 2. In another embodiment, the immunoglobulin light chain comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the set of light or heavy chain variable domain CDRs described herein (e.g., presented in Table 2).

[0302] In some embodiments, the immunoglobulin heavy chain 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 as described herein. Such an immunoglobulin heavy chain may comprise or consist of at least one of CDR-H1, CDR-H2, and CDR-H3. Such an immunoglobulin light chain may comprise or consist of at least one of CDR-L1, CDR-L2, and CDR-L3.

[0303] In other embodiments, the immunoglobulin heavy and / or light chains according to the invention comprise or consist of the vH or vκ variable domain sequences provided in Table 2, respectively.

[0304] The present invention further provides polypeptides having a sequence selected from the group consisting of a vH variable domain, a vκ variable domain, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences described herein.

[0305] The antibodies, immunoglobulins and polypeptides of the present invention can be used in isolated (eg, purified) form or contained in a carrier, such as a membrane or lipid vesicle (eg, liposome).

[0306] Table 2: Anti-HHLA2 monoclonal antibodies including mAbs 8A12, 6D10, 6F10, 8D2, 2G2, 2C4, 4D1, 1C8 and 4E5 Identification and sequencing of the leader sequence and variable regions of the

[0307] 8A12 heavy chain variable (vH) DNA and amino acid sequences*

[0308] MHC2554HC.1;M13 499.5.8A12.11.10.5

[0309] CDR analysis

[0310] GFTFNTNV....___IRTKTNNYAT___VGAMDY

[0311] Amino acid sequence in FASTA format (MHC2554HC.1\;M13F)

[0312] >MHC2554HC.1\;M13F

[0313]

[0314]

[0315] Nucleotide sequence in FASTA format (MHC2554HC.1\;M13F)

[0316] >MHC2554HC.1\;M13F.499.5.8A12.11.10.5

[0317] starting point

[0318]

[0319] Signal peptide (base pairs 1-57):

[0320] 1ATGCTGTTGG GGCTGAAGTG GATTTTCTTT GTTGTTTTTT ATCAAGGTGT GCATTGT

[0321] 57 (SEQ ID NO: 21)

[0322] / translation="MLLGLKWIFFVVFYQGVHC" (SEQ ID NO: 22)

[0323] Frame 1 (base pairs 58-132):

[0324] 58GAG GTGCAACTTG TTGAGACTGG TGGAGGATTG GTGCAGCCTA AAGGGTCATTGAAACTCTCA TGTGCAGCCT CT 132 (SEQ ID NO: 23)

[0325] / Translation = "EVQLVETGGGLVQPKGSLKLSCAAS" (SEQ ID NO: 24)

[0326] CDR-H1 (base pairs 133-153):

[0327] 133GGATTCACCTTTCAACACC AAT 153(SEQ ID NO:25)

[0328] / translation = "GFTFNTN" (SEQ ID NO: 26)

[0329] Frame 2 (base pairs 154-210):

[0330] 154GTCATGA ACTGGGTCCG CCAGGCTCCA GGAAAGGGTT TGGAATGGGT TGGTCGCATA

[0331] 210 (SEQ ID NO: 27)

[0332] / Translation = "VMNWVRQAPGKGLEWVGRI" (SEQ ID NO: 28)

[0333] CDR-H2 (base pairs 211-234):

[0334] 211AGAACTAAAA CTAATAATTA TGCA 234 (SEQ ID NO: 29)

[0335] / Translation = "RTKTNNYA" (SEQ ID NO: 30)

[0336] Frame 3 (base pairs 235-357):

[0337] 235ACATAT TATGCCGATT CAGTGAAAGG CAGGTTCACC ATCTCCAGAG ATGATTCACAAAGTATGCTC TATCTGCAAA TGAACAACTT GAAAACTGAG GACACAGCCA CGTATTTCTG TGTGGGA 357 (SEQ ID NO: 31)

[0338] / translation="TYYADSVKGRFTISRDDSQSMLYLQMNNLKTEDTATYFCVG" (SEQ ID NO:32)

[0339] CDR-H3 (base pairs 358-369):

[0340] 358GCT ATGGACTAC 369(SEQ ID NO:33)

[0341] / translation = "AMDY" (SEQ ID NO: 34)

[0342] Framework 4 (base pairs 370-402) :

[0343] 370T GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CA 402 (SEQ ID NO:35)

[0344] / translation = "WGQGTSVTVSS" (SEQ ID NO: 36)

[0345] MHC2554HC.1 499.5.8A12.11.10.5

[0346]

[0347] 8A12 light chain variable (vL) DNA and amino acid sequence*

[0348] MHC2554LC.2;M13.499.5.8A12.11.10.5

[0349] CDR analysis

[0350] ESVDNSGINF..___RAS.......___QQSYKDPPT

[0351] Amino acid sequence in FASTA format (MHC2554LC.2\;M13F)

[0352] >MHC2554LC.2\;M13F

[0353]

[0354]

[0355] Nucleotide sequence in FASTA format (MHC2554LC.2\;M13F)

[0356] >MHC2554LC.2\;M13F.499.5.8A12.11.10.5

[0357] starting point

[0358]

[0359]

[0360] Signal peptide (base pairs 1-60):

[0361] 1ATGGAGACAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG TTCCACAGGT

[0362] 60 (SEQ ID NO: 39)

[0363] / translation = "METDTLLLWVLLLWVPGSTG" (SEQ ID NO: 40)

[0364] Frame 1 (base pairs 61-129):

[0365] 61GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTGGGGCA GAGGGCCACCGTCTCCTGC 129 (SEQ ID NO:41)

[0366] / Translation = "DIVLTQSPASLAVSLGQRATVSC" (SEQ ID NO: 42)

[0367] CDR-L1 (base pairs 130-174):

[0368] 130A GAGCCAGCGAAAGTGTTGAT AATTCTGGCA TAAATTTTACAC 174(SEQ ID NO:43)

[0369] / Translation = "RASESVDNSGINFIH" (SEQ ID NO: 44)

[0370] Frame 2 (base pairs 175-219):

[0371] 175TGGTAC CAGCAGAAAC CAGGACAGTC ACCCAAACTC CTCCTCTAT 219 (SEQ ID NO:45)

[0372] / Translation = "WYQQKPGQSPKLLLY" (SEQ ID NO: 46)

[0373] CDR-L2 (base pairs 220-240):

[0374] 220C GTGCATCCAA CCTAAAATCT 240(SEQ ID NO:47)

[0375] / translation = "RASNLKS" (SEQ ID NO: 48)

[0376] Frame 3 (base pairs 241-336):

[0377] 241GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAATCCTGTGGAGA CTGGTGATGT TGCAACCTAT TACTGT 336 (SEQ ID NO:49)

[0378] / Translation = "GIPARFSGSGSRTDFTLTINPVETGDVATYYC" (SEQ ID NO: 50)

[0379] CDR-L3 (base pairs 337-363):

[0380] 337CAGC AAAGTTATAA GGATCCTCCT ACG 363 (SEQ ID NO:51)

[0381] / translation="QQSYKDPPT" (SEQ ID NO: 52)

[0382] Framework 4 (base pairs 364-393) :

[0383] 364TTCGGTA CTGGGACCAA GCTGGAGCTG AAG 393 (SEQ ID NO:53)

[0384] / translation = "FGTGTKLELK" (SEQ ID NO: 54)

[0385] MHC2554LC.2.499.5.8A12.11.10.5

[0386] area Sequence fragments residue length leader sequence METDTLLLWVLLLWVPGSTG 1-20 20 LFR1 DIVLTQSPASLAVSLGQRATVSC 21-43 23 CDR-L1 RASESVDNSGINFIH 44-58 15 LFR2 WYQQKPGQSPKLLLY 59-73 15 CDR-L2 RASNLKS 74-80 7 LFR3 GIPARFSGSGSRTDFTLTINPVETGDVATYYC 81-112 32 CDR-L3 QQSYKDPPT 113-121 9 LFR4 FGTGTKLELK 122-131 10

[0387] 6D10 heavy chain variable (vH) DNA and amino acid sequences*

[0388] MHC2555HC.1;M13.499.5.6D10.11.11.7

[0389] CDR analysis

[0390]

[0391]

[0392]

[0393]

[0394] Signal peptide (base pairs 1-57):

[0395] 1ATGCTGTTGG GGCTGAAGTG GATTTTCTTT GTTGTTTTTT ATCAAGGTGT GCATTGT

[0396] 57 (SEQ ID NO: 57)

[0397] / translation="MLLGLKWIFFVVFYQGVHC" (SEQ ID NO: 58)

[0398] Frame 1 (base pairs 58-132):

[0399] 58GAG GTGCAGCTTG TTGAGACTGG TGGAGGATTG GTGCAGCCTA AAGGGTCATTGAAACTCTCA TGTGCAGCCT CT 132 (SEQ ID NO:59)

[0400] / Translation = "EVQLVETGGGLVQPKGSLKLSCAAS" (SEQ ID NO: 60)

[0401] CDR-H1 (base pairs 133-153):

[0402] 133GGATTCACCTTCAATACC AAT 153(SEQ ID NO:61)

[0403] / translation = "GFTFNTN" (SEQ ID NO: 62)

[0404] Frame 2 (base pairs 154-210):

[0405] 154GTCATGA ACTGGGTCCG CCAGGCTCCA GGAAAGGGTT TGGAATGGGT TGCTCGCATA

[0406] 210(SEQ ID NO:63)

[0407] SYNONYMS “VMNWVRQAPGKGLEWVARI”(SEQ ID NO:64)

[0408] CDR-H2 (base pairs 211-234):

[0409] 211TGCA 234(SEQ ID NO:65)

[0410] SYNONYMS “RTKTNNYA”(SEQ ID NO:66)

[0411] Frame 3 (base pairs 235-357):

[0412] 235ACATAT TATGCCGATT CAGTGAAAGA CAGGTTCACC ATCTTCAGAG ATGATTCACAAAGCATTCTC TATCTGCAAA TGAACAACTT GAAAACTGAG GACACAGCCA TGTATTACTG TGTGGGA 357(SEQ ID NO:67) .

[0413] SPECIAL: “TYYADSVKDRFTIFRDDSQSILYLQMNNLKTEDTAMYYCVG”(SEQ ID NO:68)

[0414] CDR-H3 (base pairs 358-369):

[0415] 358GCT ATGGACTAC 369(SEQ ID NO:69)

[0416] / spelling “AMDY”(SEQ ID NO:70)

[0417] Framework 4 (base pairs 370-402) :

[0418] 370T GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CA 402(SEQ ID NO:71)

[0419] / DESCRIPTION “WGQGTSVTVSS”(SEQ ID NO:72)

[0420] MHC2555HC.1;M13.499.5.6D10.11.11.7

[0421]

[0422]

[0423] 6D10 light chain variable (vL) DNA and amino acid sequences*

[0424] MHC2555LC.1;M13.499.5.6D10.11.11.7

[0425] CDR analysis

[0426] ESVDNYGISF..___RAS.......___QQSSKDPPT

[0427] Amino acid sequence in FASTA format (MHC2555LC.1\;M13F)

[0428] >MHC2555LC.1\;M13F.499.5.6D10.11.11.7

[0429]

[0430]

[0431] Nucleotide sequence in FASTA format (MHC2555LC.1\;M13F)

[0432] >MHC2555LC.1\;M13F.499.5.6D10.11.11.7

[0433] starting point

[0434]

[0435] Signal peptide (base pairs 1-60):

[0436] 1ATGGAGACAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG TTCCACAGGT

[0437] 60 (SEQ ID NO: 75)

[0438] / translation = "METDTLLLWVLLLWVPGSTG" (SEQ ID NO: 76)

[0439] Frame 1 (base pairs 61-129):

[0440] 61GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTAGGGCA GAGGGCCACCATCTCCTGC 129 (SEQ ID NO:77)

[0441] / Translation = "DIVLTQSPASLAVSLGQRATISC" (SEQ ID NO: 78)

[0442] CDR-L1 (base pairs 130-174):

[0443] 130A GAGCCAGCGAGAGTGTTGAT AATTATGGCA TTAGTTTTAT GCAC 174 (SEQ ID NO:79)

[0444] / translation = "RASESVDNYGISFMH" (SEQ ID NO: 80)

[0445] Frame 2 (base pairs 175-219):

[0446] 175TGGTAC CAGCAGAAAC CAGGACAGCC ACCCAAACTC CTCATCTAT 219 (SEQ ID NO:81)

[0447] / Translation = "WYQQKPGQPPKLLIY" (SEQ ID NO: 82)

[0448] CDR-L2 (base pairs 220-240):

[0449] 220C GTGCATCCAA CCTAAAATCT 240(SEQ ID NO:83)

[0450] / translation = "RASNLKS" (SEQ ID NO: 84)

[0451] Frame 3 (base pairs 241-336):

[0452] 241GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAATCCTGTGGAGA CTGGTGATGT TGCTACCTAT TACTGT 336 (SEQ ID NO:85)

[0453] / Translation = "GIPARFSGSGSRTDFTLTINPVETGDVATYYC" (SEQ ID NO: 86)

[0454] CDR-L3 (base pairs 337-363):

[0455] 337CAGCAAAGTAGTAAGGATCCTCCTACG 363(SEQ ID NO:87)

[0456] / translation = "QQSSKDPPT" (SEQ ID NO: 88)

[0457] Framework 4 (base pairs 364-393) :

[0458] 364TTCGGTACTGGGACCAAGCTAGAGCTGAAA 393(SEQ ID NO:89)

[0459] / translation = "FGTGTKLELK" (SEQ ID NO: 90)

[0460] MHC2555LC.1 499.5.6D10.11.11.7

[0461] area Sequence fragments residue length leader sequence METDTLLLWVLLLWVPGSTG 1-20 20 LFR1 DIVLTQSPASLAVSLGQRATISC 21-43 23 CDR-L1 RASESVDNYGISFMH 44-58 15 LFR2 WYQQKPGQPPKLLIY 59-73 15 CDR-L2 RASNLKS 74-80 7 LFR3 GIPARFSGSGSRTDFTLTINPVETGDVATYYC 81-112 32 CDR-L3 QQSSKDPPT 113-121 9 LFR4 FGTGTKLELK 122-131 10

[0462] 6F10 heavy chain variable (vH) DNA and amino acid sequences*

[0463] MHC2557HCO.1;M13.499.5.6F10.A4.10

[0464] CDR analysis

[0465] GYTFTTYT....___INPSSGYT..___ARHPWDSDY

[0466] Amino acid sequence in FASTA format (MHC2557HCO.1\;M13F)

[0467] >MHC2557HCO.1\;M13F.499.5.6F10.A4.10

[0468]

[0469]

[0470] Nucleotide sequence in FASTA format (MHC2557HCO.1\;M13F)

[0471] >MHC2557HCO.1\;M13F.499.5.6F10.A4.10

[0472] starting point

[0473]

[0474]

[0475] Signal peptide (base pairs 1-57):

[0476] 1ATGGAAAGGC ACTGGATCTT TCTCTTCCTG TTGTCAGTAA CTGCAGGTGT CCACTCC

[0477] 57(SEQ ID NO:93)

[0478] / translation = "MERHWIFLFLLSVTAGVHS" (SEQ ID NO:94)

[0479] Frame 1 (base pairs 58-132):

[0480] 58CAG GTCCACCTGC AGCAGTCTGC AGCTGAACTG GCAAGACCTG GGGCCTCAGTGAAGATGTCC TGCAAGGCTT CT 132(SEQ ID NO:95)

[0481] / translation = "QVHLQQSAAELARPGASVKMSCKAS" (SEQ ID NO:96)

[0482] CDR-H1 (base pairs 133-153):

[0483] 133GGCTACAC CTTTACTACC TAC 153(SEQ ID NO:97)

[0484] / translation = "GYTFTTY" (SEQ ID NO:98)

[0485] Frame 2 (base pairs 154-210):

[0486] 154ACGATGC ACTGGGTAAA ACAGAGGCCT

[0487] 181GGACAGGGTC TGGAATGGAT TGGACACATT 210(SEQ ID NO:99)

[0488] / translation = "TMHWVKQRPGQGLEWIGHI" (SEQ ID NO:100)

[0489] CDR-H2 (base pairs 211-228):

[0490] 211AATCCTAGCAGTGGATAT 228(SEQ ID NO:101)

[0491] / translation = "NPSSGY" (SEQ ID NO: 102)

[0492] Frame 3 (base pairs 229-351):

[0493] 229AC TGATTACAAT CAGAAATTCA AGGACAAGAC CACATTGACT GCAGACAAATCCTCCAGTAC AGCCTACATG CAACTGAACA GCCTGACATC TGAGGACTCT GCGGTCTATT ACTGTGCAAGA 351 (SEQ ID NO: 103)

[0494] / translation="TDYNQKFKDKTTLTADKSSSTAYMQLNSLTSEDSAVYYCAR" (SEQ ID NO:104)

[0495] CDR-H3 (base pairs 352-372):

[0496] 352CACCCCTGG GACTCGGACT AC 372(SEQ ID NO:105)

[0497] / translation = "HPWDSDY" (SEQ ID NO: 106)

[0498] Framework 4 (base pairs 373-405) :

[0499] 373TGGGGCCA AGGCACCACT CTCACAGTCT CCTCA 405 (SEQ ID NO:107)

[0500] / translation = "WGQGTTLTVSS" (SEQ ID NO: 108)

[0501] MHC2557HCO.1 499.5.6F10.A4.10

[0502]

[0503] 6F10 light chain variable (vL) DNA and amino acid sequences*

[0504] MHC2557LC.1;M13.

[0505] CDR analysis

[0506] ENIDSY......___AAT..........___QHYYITPFT

[0507] Amino acid sequence in FASTA format (MHC2557LC.1\;M13F)

[0508] >MHC2557LC.1\;M13F.499.5.6F10.A4.10

[0509]

[0510]

[0511] Nucleotide sequence in FASTA format (MHC2557LC.1\;M13F)

[0512] >MHC2557LC.1\;M13F.499.5.6F10.A4.10

[0513] starting point

[0514]

[0515] Signal peptide (base pairs 1-60):

[0516] 1ATGAGTGTGC CCACTCAGCT CCTGGGGTTG CTGCTGCTGT GGCTTACAGA TGCCAGATGT

[0517] 60 (SEQ ID NO: 111)

[0518] / translation="MSVPTQLLGLLLLWLTDARC" (SEQ ID NO: 112)

[0519] Frame 1 (base pairs 61-129):

[0520] 61GACATCCAGATGACTCAGTCTCCAGCTTCCCTGTCTGCATCTGTGGGAGAAACTGTCACCATCACATGT 129(SEQ ID NO:113)

[0521] / Translation = "DIQMTQSPASLSASVGETVTITC" (SEQ ID NO: 114)

[0522] CDR-L1 (base pairs 130-162):

[0523] 130C GAGCAAGTGA GAATATTGAC AGTTATTTAG CA 162 (SEQ ID NO: 115)

[0524] / Translation = "RASENIDSYLA" (SEQ ID NO: 116)

[0525] Frame 2 (base pairs 163-207):

[0526] 163TGGTATCAGCAGAAACAGGGAAGATCTCCTCAGCTCCTGGTCTAT 207(SEQ ID NO:117)

[0527] / translation="WYQQKQGRSPQLLVY" (SEQ ID NO: 118)

[0528] CDR-L2 (base pairs 208-228):

[0529] 208GCT GCAACAAACT TAGCAGAT 228(SEQ ID NO:119)

[0530] / translation = "AATNLAD" (SEQ ID NO: 120)

[0531] Frame 3 (base pairs 229-324):

[0532] 229GG TGTGCCATCA AGGTTCAGTG GCAGTGGATC AGGCACACAG TTTTCTCTCCAGATCAACCG CCTGCAGTCT GAAGATGTTG CGAGATATTA CTGT 324 (SEQ ID NO: 121)

[0533] / translation="GVPSRFSGSGSGTQFSLQINRLQSEDVARYYC" (SEQ ID NO:122)

[0534] CDR-L3 (base pairs 325-351):

[0535] 325CAACAT TATTATATTA CTCCATTCAC G 351(SEQ ID NO:123)

[0536] / translation = "QHYYITPFT" (SEQ ID NO: 124)

[0537] Framework 4 (base pairs 352-381) :

[0538] 352TTCGGCTCG GGGACAAATTGGAAATAGC A 381 (SEQ ID NO: 125)

[0539] / translation = "FGSGTKLEIA" (SEQ ID NO: 126)

[0540] MHC2557LC.1 499.5.6F10.A4.10

[0541] area Sequence fragments residue length leader sequence MSVPTQLLGLLLLWLTDARC 1-20 20 LFR1 DIQMTQSPASLSASVGETVTITC 21-43 23 CDR-L1 RASENIDSYLA 44-54 11 LFR2 WYQQKQGRSPQLLVY 55-69 15 CDR-L2 AATNLAD 70-76 7 LFR3 GVPSRFSGSGSGTQFSLQINRLQSEDVARYYC 77-108 32 CDR-L3 QHYYITPFT 109-117 9 LFR4 FGSGTKLEIA 118-127 10

[0542] 8D2 heavy chain variable (vH) DNA and amino acid sequences*

[0543] MHC2558HC.1;M13.499.8D2.6.8

[0544] CDR analysis

[0545] GFTFNTNV....___IRTKTNNYAT___VGAMDY

[0546] Amino acid sequence in FASTA format (MHC2558HC.1\;M13F)

[0547] >MHC2558HC.1\;M13F.499.8D2.6.8

[0548]

[0549]

[0550] Nucleotide sequence in FASTA format (MHC2558HC.1\;M13F)

[0551] >MHC2558HC.1\;M13F.499.8D2.6.8

[0552] starting point

[0553]

[0554]

[0555] Signal peptide (base pairs 1-57):

[0556] 1ATGCTGTTGG GGCTGAAGTG GATTTTCTTT GTTGTTTTTT ATCAAGGTGT GCATTGT

[0557] 57 (SEQ ID NO: 129)

[0558] / translation = "MLLGLKWIFFVVFYQGVHC" (SEQ ID NO: 130)

[0559] Frame 1 (base pairs 58-132):

[0560] 58GAG GTGCAACTTG TTGAGACTGG TGGAGGATTG GTGCAGCCTA AAGGGTCATTGAAACTCTCA TGTGCAGCCT CT 132 (SEQ ID NO: 131)

[0561] / Translation = "EVQLVETGGGLVQPKGSLKLSCAAS" (SEQ ID NO: 132)

[0562] CDR-H1 (base pairs 133-153):

[0563] 133GGATTCACCTTTCAACACC AAT 153(SEQ ID NO:133)

[0564] / translation = "GFTFNTN" (SEQ ID NO: 134)

[0565] Frame 2 (base pairs 154-210):

[0566] 154GTCATGA ACTGGGTCCG CCAGGCTCCA GGAAAGGGTT TGGAATGGGT TGGTCGCATA

[0567] 210 (SEQ ID NO: 135)

[0568] / Translation = "VMNWVRQAPGKGLEWVGRI" (SEQ ID NO: 136)

[0569] CDR-H2 (base pairs 211-234):

[0570] 211AGAACTAAAA CTAATAATTA TGCA 234 (SEQ ID NO:137)

[0571] / Translation = "RTKTNNYA" (SEQ ID NO: 138)

[0572] Frame 3 (base pairs 235-357):

[0573] 235ACATAT TATGCCGATT CAGTGAAAGG CAGGTTCACC ATCTCCAGAG ATGATTCACAAAGTATGCTC TATCTGCAAA TGAACAACTT GAAAACTGAG GACACAGCCA CGTATTTCTG TGTGGGA 357 (SEQ ID NO: 139)

[0574] / translation="TYYADSVKGRFTISRDDSQSMLYLQMNNLKTEDTATYFCVG" (SEQ ID NO:140)

[0575] CDR-H3 (base pairs 358-369):

[0576] 358GCT ATGGACTAC 369(SEQ ID NO:141)

[0577] / translation = "AMDY" (SEQ ID NO: 142)

[0578] Framework 4 (base pairs 370-402) :

[0579] 370T GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CA 402 (SEQ ID NO:143)

[0580] / translation = "WGQGTSVTVSS" (SEQ ID NO: 144)

[0581] MHC2558HC.1 499.8D2.6.8

[0582]

[0583] 8D2 light chain variable (vL) DNA and amino acid sequence*

[0584] MHC2558LC.2;M13.499.8D2.6.8

[0585] CDR analysis

[0586] ESVDNSGINF..___RAS.......___QQSYKDPPT

[0587] Amino acid sequence in FASTA format (MHC2558LC.2\;M13F)

[0588] >MHC2558LC.2\;M13F.499.8D2.6.8

[0589]

[0590] Nucleotide sequence in FASTA format (MHC2558LC.2\;M13F)

[0591] >MHC2558LC.2\;M13F.499.8D2.6.8

[0592] starting point

[0593]

[0594] Signal peptide (base pairs 1-60):

[0595] 1ATGGAGACAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG TTCCACAGGT

[0596] 60 (SEQ ID NO: 147)

[0597] / translation = "METDTLLLWVLLLWVPGSTG" (SEQ ID NO: 148)

[0598] Frame 1 (base pairs 61-129):

[0599] 61GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTGGGGCA GAGGGCCACCGTCTCCTGC 129 (SEQ ID NO:149)

[0600] / Translation = "DIVLTQSPASLAVSLGQRATVSC" (SEQ ID NO: 150)

[0601] CDR-L1 (base pairs 130-174):

[0602] 130A GAGCCAGCGAAAGTGTTGAT AATTCTGGCA TAAATTTTACAC 174(SEQ ID NO:151)

[0603] / translation = "RASESVDNSGINFIH" (SEQ ID NO: 152)

[0604] Frame 2 (base pairs 175-219):

[0605] 175CTGGTAC CAGCAGAAAC CAGGACAGTC ACCCAAACTC CTCCTCTAT 219 (SEQ ID NO:153)

[0606] / Translation = "WYQQKPGQSPKLLLY" (SEQ ID NO: 154)

[0607] CDR-L2 (base pairs 220-240):

[0608] 220C GTGCATCCAA CCTAAAATCT 240(SEQ ID NO:155)

[0609] / translation = "RASNLKS" (SEQ ID NO: 156)

[0610] Frame 3 (base pairs 241-336):

[0611] 241GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAATCCTGTGGAGA CTGGTGATGT TGCAACCTAT TACTGT 336 (SEQ ID NO: 157)

[0612] / Translation = "GIPARFSGSGSRTDFTLTINPVETGDVATYYC" (SEQ ID NO: 158)

[0613] CDR-L3 (base pairs 337-363):

[0614] 337CAGCAAAGTTATAAGGATCCTCCTACG 363(SEQ ID NO:159)

[0615] / translation = "QQSYKDPPT" (SEQ ID NO: 160)

[0616] Framework 4 (base pairs 364-393) :

[0617] 364TTCGGTACTGGGACCAAGCTGGAGCTG AAG 393(SEQ ID NO:161)

[0618] / Translation = "FGTGTKLELK" (SEQ ID NO: 162)

[0619] MHC2558LC.2 499.8D2.6.8

[0620]

[0621]

[0622] 2G2 heavy chain variable (vH) DNA and amino acid sequences*

[0623] MHC2561HC.1;M13.499.5.2G2.13.3.10

[0624] CDR analysis

[0625] GLTFSSYA....___ISSGGSHT..___TRLGRAFDY

[0626] Amino acid sequence in FASTA format (MHC2561HC.1\;M13F)

[0627] >MHC2561HC.1\;M13F.499.5.2G2.13.3.10

[0628]

[0629]

[0630] Nucleotide sequence in FASTA format (MHC2561HC.1\;M13F)

[0631] >MHC2561HC.1\;M13F.499.5.2G2.13.3.10

[0632] starting point

[0633]

[0634] Signal peptide (base pairs 1-57):

[0635] 1ATGAACTTCGGGCTCAGCTT GATTTTCCTT GTCCTTGTTT TAAAAGGTGT CCAGTGT

[0636] 57 (SEQ ID NO: 165)

[0637] / Translation = "MNFGLSLIFLVLVLKGVQC" (SEQ ID NO: 166)

[0638] Frame 1 (base pairs 58-132):

[0639] 58GAA GTGATGCTGG TGGAGTCAGG GGGAGGCTTA GTGAAGCCTG GAGGATCCCTGAAACTCTCC TGTGCAGTCT CT 132 (SEQ ID NO: 167)

[0640] / Translation = "EVMLVESGGGLVKPGGSLKLSCAVS" (SEQ ID NO:168)

[0641] CDR-H1 (base pairs 133-153):

[0642] 133GGATTAAC TTTTAGTAGT TAT 153(SEQ ID NO:169)

[0643] / Translation = "GLTFSSY" (SEQ ID NO:170)

[0644] Frame 2 (base pairs 154-210):

[0645] 154GCCATGT CTTGGGTTCG CCAGACTCCG GAGAAGAGGC TGGAGTGGGT CGCAACCATT

[0646] 210(SEQ ID NO:171)

[0647] / Translation = "AMSWVRQTPEKRLEWVATI" (SEQ ID NO:172)

[0648] CDR-H2 (base pairs 211-228):

[0649] 211AGTAGTGGTG GTAGTCAC 228(SEQ ID NO:173)

[0650] / Translation = "SSGGSH" (SEQ ID NO:174)

[0651] Frame 3 (base pairs 229-351):

[0652] 229AC CTACTATCCA GACAGTGTGA AGGGGCGATT CATCATTTCT AGAGACAATGCCAAGAACAC CCTGTACCTG CAAATGAACA GTCTGAGGTC TGAGGACACG GCCATGTATT ACTGTACAAGA 351(SEQ ID NO:175)

[0653] / Translation = "TYYPDSVKGRFIISRDNAKNTLYLQMNSLRSEDTAMYYCTR" (SEQ ID NO:176)

[0654] CDR-H3 (base pairs 352-372):

[0655] 352CTGGGACGG GCCTTTGACT AC 372 (SEQ ID NO:177)

[0656] / translation = "LGRAFDY" (SEQ ID NO: 178)

[0657] Framework 4 (base pairs 373-405) :

[0658] 373TGGGGCCA AGGCACCACT CTCACAGTCT CCTCA 405 (SEQ ID NO: 179)

[0659] / translation = "WGQGTTLTVSS" (SEQ ID NO: 180)

[0660] MHC2561HC.1 499.5.2G2.13.3.10

[0661]

[0662] 2G2 light chain variable (vL) DNA and amino acid sequence*

[0663] MHC2561LC.1;M13.499.5.2G2.13.3.10

[0664] CDR analysis

[0665] QDVSTA......___WAS.....___QQDYSTPWT

[0666] Amino acid sequence in FASTA format (MHC2561LC.1\;M13F)

[0667] >MHC2561LC.1\;M13F.499.5.2G2.13.3.10

[0668]

[0669]

[0670] Nucleotide sequence in FASTA format (MHC2561LC.1\;M13F)

[0671] >MHC2561LC.1\;M13F.499.5.2G2.13.3.10

[0672] starting point

[0673]

[0674]

[0675] Signal peptide (base pairs 1-60):

[0676] 1ATGGAGTCAC AGATTCAGGT CTTTGTATTC GTGTTTCTCT GGTTGTCTGG TGTTGACGGA

[0677] 60(SEQ ID NO:183)

[0678] / translation = "MESQIQVFVFVFLWLSGVDG" (SEQ ID NO:184)

[0679] Frame 1 (base pairs 61-129):

[0680] 61GACATTGTGA TGACCCAGTC TCACAAATTC ATGTCCACAT CAGTAGGAGA CAGGGTCAGCATCACCTGC 129(SEQ ID NO:185)

[0681] / translation = "DIVMTQSHKFMSTSVGDRVSITC" (SEQ ID NO:186)

[0682] CDR-L1 (base pairs 130-162):

[0683] 130A AGGCCAGTCA GGATGTGAGT ACTGCTGTAG CC 162(SEQ ID NO:187)

[0684] / translation = "KASQDVSTAVA" (SEQ ID NO:188)

[0685] Frame 2 (base pairs 163-207):

[0686] 163TGGTATCA ACAAAAGCCA GGGCAATCTC CTAAAGTTCT GATTTAC 207(SEQ ID NO:189)

[0687] / translation = "WYQQKPGQSPKVLIY" (SEQ ID NO:190)

[0688] CDR-L2 (base pairs 208-228):

[0689] 208TGG GCATCCACCC GGCACACT 228(SEQ ID NO:191)

[0690] / translation = "WASTRHT" (SEQ ID NO: 192)

[0691] Frame 3 (base pairs 229-324):

[0692] 229GG AGTCCCTGAT CGCTTCACAG GCAGTGGATC TGGGACAGAT TTTACTCTCACCATCAGCAG TGTGCAGGCT GAAGACCTGG CACTTTATTA CTGT 324 (SEQ ID NO: 193)

[0693] / translation="GVPDRFTGSGSGTDFTLTISSVQAEDLALYYC" (SEQ ID NO: 194)

[0694] CDR-L3 (base pairs 325-351):

[0695] 325CAGCAA GATTATAGCA CTCCGTGGAC G 351(SEQ ID NO:195)

[0696] / translation = "QQDYSTPWT" (SEQ ID NO: 196)

[0697] Framework 4 (base pairs 352-381) :

[0698] 352TTCGGTGGA GGCACCAAGC TGGAAATCAA A 381 (SEQ ID NO: 197)

[0699] / translation = "FGGGTKLEIK" (SEQ ID NO: 198)

[0700] MHC2561LC.1 499.5.2G2.13.3.10

[0701] area Sequence fragments residue length leader sequence MESQIQVFVFVFLWLSGVDG 1-20 20 LFR1 DIVMTQSHKFMSTSVGDRVSITC 21-43 23 CDR-L1 KASQDVSTAVA 44-54 11 LFR2 WYQQKPGQSPKVLIY 55-69 15 CDR-L2 WASTRHT 70-76 7 LFR3 GVPDRFTGSGSGTDFTLTISSVQAEDLALYYC 77-108 32 CDR-L3 QQDYSTPWT 109-117 9 LFR4 FGGGTKLEIK 118-127 10

[0702] 2C4 heavy chain variable (vH) DNA and amino acid sequences*

[0703] MHC2562HC.1;M13.499.5.2C4.E6.6.8.1

[0704] CDR analysis

[0705] GFSFTDYN....___IDPYYGRI..___ATGAYTSGYSWFAY

[0706] Amino acid sequence in FASTA format (MHC2562HC.1\;M13F)

[0707] >MHC2562HC.1\;M13F.499.5.2C4.E6.6.8.1

[0708]

[0709]

[0710] Nucleotide sequence in FASTA format (MHC2562HC.1\;M13F)

[0711] >MHC2562HC.1\;M13F.499.5.2C4.E6.6.8.1

[0712] starting point

[0713]

[0714] Signal peptide (base pairs 1-57):

[0715] 1ATGGGATGGA CCTGGATCTT TATTTTAATC CTGTCAGTAA CTACAGGTGT CCACTCT

[0716] 57 (SEQ ID NO: 201)

[0717] / Translation = "MGWTWIFILILSVTTGVHS" (SEQ ID NO: 202)

[0718] Frame 1 (base pairs 58-132):

[0719] 58GAG GTCCACCTGC AGCAGTCTGG ACCTGAGCTG GAGAAGCCTG GCGTTTCAGTGAAGATATCC TGCAAGGCTT CT 132 (SEQ ID NO: 203)

[0720] / Translation = "EVHLQQSGPELEKPGVSVKISCKAS" (SEQ ID NO: 204)

[0721] CDR-H1 (base pairs 133-153):

[0722] 133GGTTTCTCATTCACTGACTAC 153(SEQ ID NO:205)

[0723] / translation = "GFSFTDY" (SEQ ID NO: 206)

[0724] Frame 2 (base pairs 154-210):

[0725] 154AACATGA ACTGGGTGAA ACAGAGCAGT GGAAAGAGCC TTGAGTGGAT TGGAAATATT

[0726] 210(SEQ ID NO:207)

[0727] / Translation = "NMNWVKQSSGKSLEWIGNI" (SEQ ID NO:208)

[0728] CDR-H2 (base pairs 211-228):

[0729] 211GATCCTTACT ATGGACGT 228(SEQ ID NO:209)

[0730] / Translation = "DPYYGR" (SEQ ID NO:210)

[0731] Frame 3 (base pairs 229-351):

[0732] 229AT TAACTATAAC CAGAAATTCA AGGGCAAGGC CACATTGAGT GTAGACAAATCCTCCAGCAC AGCCTACATG CACCTCAAGA GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAACT 351(SEQ ID NO:211)

[0733] / Translation = "INYNQKFKGKATLSVDKSSSTAYMHLKSLTSEDSAVYYCAT" (SEQ ID NO:212)

[0734] CDR-H3 (base pairs 352-387):

[0735] 352GGGGCCTAC ACCTCGGGCT ACTCCTGGTT TGCTTAC 387(SEQ ID NO:213)

[0736] / Translation = "GAYTSGYSWFAY" (SEQ ID NO:214)

[0737] Framework 4 (base pairs 388-420) :

[0738] 388TGG GGCCAAGGGA CTCTGGTCAC TGTCTCTGCA 420(SEQ ID NO:215)

[0739] / translation = "WGQGTLVTVSA" (SEQ ID NO: 216)

[0740] MHC2562HC.1 499.5.2C4.E6.6.8.1

[0741]

[0742]

[0743] 2C4 light chain variable (vL) DNA and amino acid sequences*

[0744] MHC2562LC.2;M13.499.5.2C4.E6.6.8.1

[0745] CDR analysis

[0746] SSVSSSY.....___RTS.....___QQWSGYPFT

[0747] Amino acid sequence in FASTA format (MHC2562LC.2\;M13F)

[0748] >MHC2562LC.2\;M13F.499.5.2C4.E6.6.8.1

[0749]

[0750]

[0751] Nucleotide sequence in FASTA format (MHC2562LC.2\;M13F)

[0752] >MHC2562LC.2\;M13F.499.5.2C4.E6.6.8.1

[0753] starting point

[0754]

[0755]

[0756] Signal peptide (base pairs 1-66):

[0757] 1ATGGGTTTAC AGGTGCAGGT TATCAGCTTC CTGTTAATCA GTGTCACAGT CATAATGTCCAGAGGA 66 (SEQ ID NO: 219)

[0758] / translation="MGLQVQVISFLLISVTVIMSRG" (SEQ ID NO: 220)

[0759] Frame 1 (base pairs 67-135):

[0760] 67GAAA ATGTGCTCACCCAGTCTCCA GGAATAATGG CTGCCTCTCT GGGGGAGAAGGTCACCATGA CCTGC 135 (SEQ ID NO: 221)

[0761] / translation="ENVLTQSPGIMAASLGEKVTMTC" (SEQ ID NO: 222)

[0762] CDR-L1 (base pairs 136-171):

[0763] 136AGTGC CAGCTCAAGTGTAAGTTCCA GTTACTTGCA C 171 (SEQ ID NO: 223)

[0764] / Translation = "SASSSVSSSYLH" (SEQ ID NO: 224)

[0765] Frame 2 (base pairs 172-216):

[0766] 172TGGTACCAG CAGAGGTCAG GCGCTTCCCC CAAACCCTTG ATTCAT 216 (SEQ ID NO: 225)

[0767] / translation="WYQQRSGASPKPLIH" (SEQ ID NO: 226)

[0768] CDR-L2 (base pairs 217-237):

[0769] 217AGGA CATCCAACCT GGCTTCT 237 (SEQ ID NO: 227)

[0770] / translation = "RTSNLAS" (SEQ ID NO: 228)

[0771] Frame 3 (base pairs 238-333):

[0772] 238GGT GTCCCAGCTC GCTTCAGTGG CAGTGGGTCT GGGACCTCTT ACTCTCTCACAATCAGCAGC GTGGAGGCTG AAGATGATGC AACTTATTAC TGC 333 (SEQ ID NO: 229)

[0773] / translation="GVPARFSGSGSGTSYSLTISSVEAEDDATYYC" (SEQ ID NO: 230)

[0774] CDR-L3 (base pairs 334-360):

[0775] 334CAGCAGTGGAGTGGTTACCATTCACG 360(SEQ ID NO:231)

[0776] / translation="QQWSGYPFT" (SEQ ID NO: 232)

[0777] Framework 4 (base pairs 361-390) :

[0778] 361TTCGGCTCGG GGACAAAGTT GGAAAATAAAA 390 (SEQ ID NO: 233)

[0779] / translation = "FGSGTKLEIK" (SEQ ID NO: 234)

[0780] MHC2562LC.2 499.5.2C4.E6.6.8.1

[0781] area Sequence fragments residue length leader sequence MGLQVQVISFLLISVTVIMSRG 1-22 22 LFR1 ENVLTQSPGIMAASLGEKVTMTC 23-45 23 CDR-L1 SASSSVSSSYLH 46-57 12 LFR2 WYQQRSGASPKPLIH 58-72 15 CDR-L2 RTSNLAS 73-79 7 LFR3 GVPARFSGSGSGTSYSLTISSVEAEDDATYYC 80-111 32 CDR-L3 QQWSGYPFT 112-120 9 LFR4 FGSGTKLEIK 121-130 10

[0782] 4D1 heavy chain variable (vH) DNA and amino acid sequences*

[0783] MHC2563HC.1;M13.499.5.4D1.5.16

[0784] CDR analysis

[0785] GYTFTDYA....___ISTYYGDT..___ARGNYDDWYFNV

[0786] Amino acid sequence in FASTA format (MHC2563HC.1\;M13F)

[0787] >MHC2563HC.1\;M13F.499.5.4D1.5.16

[0788]

[0789]

[0790] Nucleotide sequence in FASTA format (MHC2563HC.1\;M13F)

[0791] >MHC2563HC.1\;M13F.499.5.4D1.5.16

[0792] starting point

[0793]

[0794]

[0795] Signal peptide (base pairs 1-57):

[0796] 1ATGGATTGGA GCTGTATCAT CTTCTTTCTG GTAGCAACAG CTACAGGTGT GCACTCC57(SEQID NO:237)

[0797] / translation = "MDWSCIIFFLVATATGVHS" (SEQ ID NO: 238)

[0798] Frame 1 (base pairs 58-132):

[0799] 58CAG GTCCAGCTGC AGCAGTCTGG GGCTGAACTG GTGAGGCCTG GGGTCTCAGTGAAGATTTCC TGCAAGGGTT CT 132 (SEQ ID NO: 239)

[0800] / Translation = "QVQLQQSGAELVRPGVSVKISCKGS" (SEQ ID NO: 240)

[0801] CDR-H1 (base pairs 133-153):

[0802] 133GGCTACACATTCACTGATTAT 153(SEQ ID NO:241)

[0803] / translation = "GYTFTDY" (SEQ ID NO: 242)

[0804] Frame 2 (base pairs 154-210):

[0805] 154GCTATGC ACTGGGTGAA GCAGAGTCAT GCAAAGAGTC TAGAGTGGAT TGGAAGTATT

[0806] 210(SEQ ID NO:243)

[0807] / translation = "AMHWVKQSHAKSLEWIGSI"(SEQ ID NO:244)

[0808] CDR-H2 (base pairs 211-228):

[0809] 211AGTACTTACT ATGGTGAT 228(SEQ ID NO:245)

[0810] / translation = "STYYGD"(SEQ ID NO:246)

[0811] Frame 3 (base pairs 229-351):

[0812] 229AC TAATTACAAC CAGAAATTCA AGGGCAAGGC CACAATGACT GTAGACAAATCCTCCAGCAC AGCCTATATG GAACTTGCCA GACTGACATC TGAGGATTCT GCCATCTATT ACTGTGCAAGA 351(SEQ ID NO:247)

[0813] / translation = "TNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCAR"(SEQ ID NO:248)

[0814] CDR-H3 (base pairs 352-381):

[0815] 352GGTAATTAC GACGACTGGT ACTTCAATGT C 381(SEQ ID NO:249)

[0816] / translation = "GNYDDWYFNV"(SEQ ID NO:250)

[0817] Framework 4 (base pairs 382-414) :

[0818] 382TGGGGCGCA GGGACCACGG TCACCGTCTC CTCA 414(SEQ ID NO:251)

[0819] / Translation = "WGAGTTVTVSS" (SEQ ID NO: 252)

[0820] MHC2563HC.1 499.5.4D1.5.16

[0821]

[0822] 4D1 light chain variable (vL) DNA and amino acid sequence*

[0823] MHC2563LC.1;M13.499.5.4D1.5.16

[0824] CDR analysis

[0825] QDISGY......___STS.......___LQYASSPYT

[0826] Amino acid sequence in FASTA format (MHC2563LC.1\;M13F)

[0827] >MHC2563LC.1\;M13F.499.5.4D1.5.16

[0828]

[0829]

[0830] Nucleotide sequence in FASTA format (MHC2563LC.1\;M13F)

[0831] >MHC2563LC.1\;M13F.499.5.4D1.5.16

[0832] starting point

[0833]

[0834] Signal peptide (base pairs 1-60):

[0835] 1ATGAGGATTC CTGCTCACGT TTTTGGCTTC TTGTTGCTCT GGTTTCCAGG TGCCAGATGT

[0836] 60 (SEQ ID NO: 255)

[0837] / Translation = "MRIPAHVFGFLLLWFPGARC" (SEQ ID NO: 256)

[0838] Frame 1 (base pairs 61-129):

[0839] 61(SEQ ID NO:257) 129(SEQ ID NO:257) 129(SEQ ID NO:257)

[0840] SPECIFIER “DIQMTQSPSSLSASLGERVSLTC”(SEQ ID NO:258)

[0841] CDR-L1 (base pairs 130-162):

[0842] 130C GGGCAAGTCA GGTACTTAG GGTTACTTAA GC 162(SEQ ID NO:259)

[0843] / Article “RASQDISGYLS”(SEQ ID NO:260)

[0844] Frame 2 (base pairs 163-207):

[0845] 163TGGCTTCA GCAGAAACCA GATGGAACTA TTAAACGTCT GATTTAT 207(SEQ ID NO:261)

[0846] SUBJECT TO “WLQQKPDGTIKRLIY”(SEQ ID NO:262)

[0847] CDR-L2 (base pairs 208-228):

[0848] 208AGC ACATCCACTT TAGTTCT 228(SEQ ID NO:263)

[0849] SYNONYMS “STSTLDS”(SEQ ID NO:264)

[0850] Frame 3 (base pairs 229-324):

[0851] 229GG TGTCCCAAAA AGGTTCAGTG GCAGTAGGTC TGGGTCAGAT TATTCTCTCACCATCAGCAG CCTAGAGTCT GAAGATTTTG CAGACTATTA CTGT 324(SEQ ID NO:265)

[0852] / DEFINITION“GVPKRFSGSRSGSDYSLTISSLESEDFADYYC”(SEQ ID NO:266)

[0853] CDR-L3 (base pairs 325-351):

[0854] 325CTACAATATGCTAGTT CTCCGTACAC G 351(SEQ ID NO:267)

[0855] / Translation = "LQYASSPYT" (SEQ ID NO: 268)

[0856] Framework 4 (base pairs 352-381) :

[0857] 352TTCGGAGGG GGGGCCAAGC TGGAAATAAA A 381 (SEQ ID NO: 269)

[0858] / Translation = "FGGGAKLEIK" (SEQ ID NO: 270)

[0859] MHC2563LC.1 499.5.4D1.5.16

[0860] area Sequence fragments residue length leader sequence MRIPAHVFGFLLLWFPGARC 1-20 20 LFR1 DIQMTQSPSSLSASLGERVSLTC 21-43 23 CDR-L1 RASQDISGYLS 44-54 11 LFR2 WLQQKPDGTIKRLIY 55-69 15 CDR-L2 STSTLDS 70-76 7 LFR3 GVPKRFSGSRSGSDYSLTISSLESEDFADYYC 77-108 32 CDR-L3 LQYASSPYT 109-117 9 LFR4 FGGGAKLEIK 118-127 10

[0861] 1C8 heavy chain variable (vH) DNA and amino acid sequence*

[0862] MHC2564HC.1;M13.499.5.1C8.9.12.2

[0863] CDR analysis

[0864] GFNIKDTY....___IDPANGKT..___AWLLPYYFDY

[0865] Amino acid sequence in FASTA format (MHC2564HC.1\;M13F)

[0866] >MHC2564HC.1\;M13F.499.5.1C8.9.12.2

[0867]

[0868]

[0869] Nucleotide sequence in FASTA format (MHC2564HC.1\;M13F)

[0870] >MHC2564HC.1\;M13F.499.5.1C8.9.12.2

[0871] starting point

[0872]

[0873]

[0874] Signal peptide (base pairs 1-57):

[0875] 1ATGAAATGCA GCTGGATTAT CTTCTTCCTG ATGGCTGTGG TTACAGGGGT CAATTCA57(SEQID NO:273)

[0876] SPECIFIER “MKCSWIIFFLMAVVTGVNS”(SEQ ID NO:274)

[0877] Frame 1 (base pairs 58-132):

[0878] 58GAG GTTCAGCTGC AGCAGTCTGG GGCAGAACTT GTGCAGCCAG GGGCCTCAGTCAAGTTGTCC TGTACAGCTT CT 132(SEQ ID NO:275)

[0879] / designation “EVQLQQSGAELVQPGASVKLSCTAS”(SEQ ID NO:276)

[0880] CDR-H1 (base pairs 133-153):

[0881] 133GGCTTCAA TATTAAAGAC ACC 153(SEQ ID NO:277)

[0882] / designation “GFNIKDT”(SEQ ID NO:278)

[0883] Frame 2 (base pairs 154-210):

[0884] 154TATATGC ACTGGGTAAA ACAGAGGCCT GAACAGGGCC TGGAGTGGAT TGGAAGGATT

[0885] 210(SEQ ID NO:279)

[0886] SUBJECT TO “YMHWVKQRPEQGLEWIGRI”(SEQ ID NO:280)

[0887] CDR-H2 (base pairs 211-228):

[0888] 211GATCCTGCGA ATGGTAAA 228(SEQ ID NO:281)

[0889] SYNONYMS “DPANGK”(SEQ ID NO:282)

[0890] Frame 3 (base pairs 229-351):

[0891] 229AC TATTTTTGAC CCGAAGTTCC AGGTCAAGGC CACTATAACT GCGACACATCCTCCAACAC AGTCTACCTG CATCTCAGCA GCCTGACATC TGAGGACACT GCCATCTATT ACTGTGCTTGG 351(SEQ ID NO:283);

[0892] SPECIAL: “TIFDPKFQVKATITADTSSNTVYLHLSSLTSEDTAIYYCAW”(SEQ ID NO:284)

[0893] CDR-H3 (base pairs 352-375):

[0894] 352TTACTTCCT TACTACTTTG ACTAC 375(SEQ ID NO:285)

[0895] SYNONYMS “LLPYYFDY”(SEQ ID NO:286)

[0896] Framework 4 (base pairs 376-408) :

[0897] 376TGGGG CCAAGGCACC ACTCTCACAG TCTCCTCA 408(SEQ ID NO:287)

[0898] SUBJECT TO “WGQGTTLTVSS”(SEQ ID NO:288)

[0899] MHC2564HC.1 499.5.1C8.9.12.2

[0900]

[0901] 1C8 light chain variable (vL) DNA and amino acid sequence*

[0902] MHC2564LCB.4;M13.499.5.1C8.9.12.2

[0903] CDR switch

[0904] SSISSSN.....___GTS.....___QKWSHYPLT

[0905] Amino acid sequence in FASTA format (MHC2564LCB.4\;M13F)

[0906] >MHC2564LCB.4\;M13F.499.5.1C8.9.12.2

[0907]

[0908] Nucleotide sequence in FASTA format (MHC2564LCB.4\;M13F)

[0909] >MHC2564LCB.4\;M13F.499.5.1C8.9.12.2

[0910] starting point

[0911]

[0912] Signal peptide (base pairs 1-66):

[0913] 1ATGGATTTTC ATGTGCAGAT TTTCAGCTTC ATGCTAATCA GTGTCACAGT CATGTTGTCCAGTGGG 66 (SEQ ID NO: 291)

[0914] / Translation = "MDFHVQIFSFMLISVTVMLSSG" (SEQ ID NO: 292)

[0915] Frame 1 (base pairs 67-135):

[0916] 67GAAA TTGTACTCAC CCAGTCTCCA GCAGTCATGG CTGCATCTCC AGGGGAGAAGGTCACCATCA CCTGC 135 (SEQ ID NO: 293)

[0917] / Translation = "EIVLTQSPAVMAASPGEKVTITC" (SEQ ID NO: 294)

[0918] CDR-L1 (base pairs 136-171):

[0919] 136AGCGT CAGTTCAAGT ATAAGTTCCA GCAACTTGCA C 171 (SEQ ID NO: 295)

[0920] / Translation = "SVSSSISSSNLH" (SEQ ID NO: 296)

[0921] Frame 2 (base pairs 172-216):

[0922] 172TGGTACCAG CAGAAGTCAG GAACCTCGCC CAAACTCTGG ATTTAT 216 (SEQ ID NO: 297)

[0923] / Translation = "WYQQKSGTSPKLWIY" (SEQ ID NO: 298)

[0924] CDR-L2 (base pairs 217-237):

[0925] 217GGCA CATCCAACCT GGCTTCT 237 (SEQ ID NO: 299)

[0926] / translation = "GTSNLAS" (SEQ ID NO: 300)

[0927] Frame 3 (base pairs 238-333):

[0928] 238GGA GTCCCTGTTC GCTTCAGTGG CAGTGGATCT GGGACCTCTT ATTCTCTCACAATCAGCAGC ATGGAGGCTG AAGATGCTGC CACTTATTAC TGT 333 (SEQ ID NO: 301)

[0929] / translation="GVPVRFSGSGSGTSYSLTISSMEAEDAATYYC" (SEQ ID NO:302)

[0930] CDR-L3 (base pairs 334-360):

[0931] 334CAAAAGT GGAGTCATTA CCCGCTCACG 360 (SEQ ID NO:303)

[0932] / translation = "QKWSHYPLT" (SEQ ID NO: 304)

[0933] Framework 4 (base pairs 361-390) :

[0934] 361TTCGGTGCTGGGACCAAGCT GGAGCTGAAA 390(SEQ ID NO:305)

[0935] / translation = "FGAGTKLELK" (SEQ ID NO: 306)

[0936] MHC2564LCB.4 499.5.1C8.9.12.2

[0937]

[0938]

[0939] 4E5 heavy chain variable (vH) DNA and amino acid sequences*

[0940] MHC2556HCN.1;M13.499.5.4E5.20.22

[0941] CDR analysis

[0942] GYTFTTYT....___INPSSGYT..___ARHPWDSNY

[0943] Amino acid sequence in FASTA format (MHC2556HCN.1\;M13F)

[0944] >MHC2556HCN.1\;M13F

[0945]

[0946]

[0947] Nucleotide sequence in FASTA format (MHC2556HCN.1\;M13F)

[0948] >MHC2556HCN.1\;M13F

[0949] starting point

[0950]

[0951] Signal peptide (base pairs 1-57):

[0952] 1ATGGAAAGGC ACTGGATCTT TCTCTTCCTG TTGTCAGTAA CTGCAGGTGT CCACTCC

[0953] 57 (SEQ ID NO: 309)

[0954] / translation = "MERHWIFLFLLSVTAGVHS" (SEQ ID NO: 310)

[0955] Frame 1 (base pairs 58-132):

[0956] 58CAG GTCCAGCTGC AGCAGTCTGC AGCTGAACTG GCAAGACCTG GGGCCTCAGTGAAGATGTCC TGCAAGGCTT CT 132(SEQ ID NO:311)

[0957] / Translation = "QVQLQQSAAELARPGASVKMSCKAS" (SEQ ID NO:312)

[0958] CDR-H1 (base pairs 133-153):

[0959] 133GGCTACAC CTTTACTACC TAC 153(SEQ ID NO:313)

[0960] / Translation = "GYTFTTY" (SEQ ID NO:314)

[0961] Frame 2 (base pairs 154-210):

[0962] 154ACGATGC ACTGGGTAAA ACAGAGGCCT GGACAGGGTC TGGAGTGGAT TGGACACATT

[0963] 210(SEQ ID NO:315)

[0964] / Translation = "TMHWVKQRPGQGLEWIGHI" (SEQ ID NO:316)

[0965] CDR-H2 (base pairs 211-228):

[0966] 211AATCCTAGCA GTGGATAT 228(SEQ ID NO:317)

[0967] / Translation = "NPSSGY" (SEQ ID NO:318)

[0968] Frame 3 (base pairs 229-351):

[0969] 229AC TGAGTACAAT CAGAAATTCA AGGACAAGAC CACACTGACT GCAGACAAATCCTCCAGCAC AGCCCACATG CAACTGAGCA GCCTAACATC TGAGGACTCT GCGGTCTATT ACTGTGCAAGA 351(SEQ ID NO:319)

[0970] / translation="TEYNQKFKDKTTLTADKSSSTAHMQLSSLTSEDSAVYYCAR" (SEQ ID NO:320)

[0971] CDR-H3 (base pairs 352-372):

[0972] 352CACCCCTGG GACTCGAACT AC 372 (SEQ ID NO:321)

[0973] / translation = "HPWDSNY" (SEQ ID NO: 322)

[0974] Framework 4 (base pairs 373-405) :

[0975] 373TGGGGCCA AGGCACCACT CTCACAGTCT CCTCA 405 (SEQ ID NO:323)

[0976] / translation = "WGQGTTLTVSS" (SEQ ID NO: 324)

[0977] MHC2556HCN.1 499.5.4E5.20.22

[0978]

[0979] 4E5 light chain variable (vL) DNA and amino acid sequence*

[0980] MHC2556LCN.2;M13.499.5.4E5.20.22

[0981] CDR analysis

[0982] ENIDSY......___AAT..........___QHYYITPFT

[0983] Amino acid sequence in FASTA format (MHC2556LCN.2\;M13F)

[0984] >MHC2556LCN.2\;M13F

[0985]

[0986]

[0987] Nucleotide sequence in FASTA format (MHC2556LCN.2\;M13F)

[0988] >MHC2556LCN.2\;M13F

[0989] starting point

[0990]

[0991]

[0992] Signal peptide (base pairs 1-60):

[0993] 1ATGAGTGTGC CCACTCAGCT CCTGGGGTTG CTGCTGCTGT GGCTTACAGA TGCCAGATGT

[0994] 60 (SEQ ID NO: 327)

[0995] / translation = "MSVPTQLLGLLLLWLTDARC" (SEQ ID NO: 328)

[0996] Frame 1 (base pairs 61-129):

[0997] 61GACATCCAGA TGACTCAGTC TCCAGCTTCC CTGTCTGCAT CTGTGGGAGA AACTGTCACCATCACATGT 129 (SEQ ID NO:329)

[0998] / translation="DIQMTQSPASLSASVGETVTITC" (SEQ ID NO: 330)

[0999] CDR-L1 (base pairs 130-162):

[1000] 130C GAGCAAGTGA GAATATTGAC AGTTATTTAG CA 162 (SEQ ID NO: 331)

[1001] / Translation = "RASENIDSYLA" (SEQ ID NO: 332)

[1002] Frame 2 (base pairs 163-207):

[1003] 163TGGTATCAGCAGAAACAGGGAAGATCTCCTCAGCTCCTGGTCTAT 207(SEQ ID NO:333)

[1004] / translation="WYQQKQGRSPQLLVY" (SEQ ID NO: 334)

[1005] CDR-L2 (base pairs 208-228):

[1006] 208GCT GCAACAAACT TAGCAGAT 228 (SEQ ID NO:335)

[1007] / translation = "AATNLAD" (SEQ ID NO: 336)

[1008] Frame 3 (base pairs 229-324):

[1009] 229GG TGTGCCATCA AGGTTCAGTG GCAGTGGATC AGGCACACAG TATTCTCTCAAGATCAACAG CCTGCAGTCT GAAGATGTTG CGAGATATTA CTGT 324 (SEQ ID NO: 337)

[1010] / translation="GVPSRFSGSGSGTQYSLKINSLQSEDVARYYC" (SEQ ID NO:338)

[1011] CDR-L3 (base pairs 325-351):

[1012] 325CAACAT TATTATATTA CTCCATTCAC G 351(SEQ ID NO:339)

[1013] / Translation = "QHYYITPFT" (SEQ ID NO: 340)

[1014] Framework 4 (base pairs 352-381) :

[1015] 352TTCGGCTCG GGGACAAAGT TGGAAATAAA A 381 (SEQ ID NO: 341)

[1016] / translation = "FGSGTKLEIK" (SEQ ID NO: 342)

[1017] MHC2556LCN.2 499.5.4E5.20.22

[1018] area Sequence fragments residue length leader sequence MSVPTQLLGLLLLWLTDARC 1-20 20 LFR1 DIQMTQSPASLSASVGETVTITC 21-43 23 CDR-L1 RASENIDSYLA 44-54 11 LFR2 WYQQKQGRSPQLLVY 55-69 15 CDR-L2 AATNLAD 70-76 7 LFR3 GVPSRFSGSGSGTQYSLKINSLQSEDVARYYC 77-108 32 CDR-L3 QHYYITPFT 109-117 9 LFR4 FGSGTKLEIK 118-127 10

[1019] * Numbering is based on Kabat's CDR definition and protein sequence. The CDR amino acid sequences are underlined in the order of CDR1, CDR2, and CDR3, respectively.

[1020] Table 3: Anti-HHLA2 Summary of mAb binding and ligand blocking characteristics

[1021]

[1022]

[1023] 1 : Binding to HHLA2-transfected 300.19 mouse pre-B leukemia cells determined by flow cytometry

[1024] 2 : Blockade of HHLA2-mIgG2a binding to TMIGD2-transfected 300.19 mouse pre-B leukemia cells determined by flow cytometry

[1025] 3： Undetermined: 8A12 - not measurable due to low binding; 4E5 - culture supernatant

[1026] III. Nucleic Acids, Vectors, and Recombinant Host Cells

[1027] Another object of the present invention relates to nucleic acid sequences encoding the monoclonal antibodies and fragments thereof, immunoglobulins and polypeptides of the present invention.

[1028] For example, in a specific 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 specific embodiment, the present invention relates in part to a nucleic acid sequence encoding the vH domain or vL domain of at least one effective anti-HHLA2 mAb isolated from polyclonal antibodies 1.2 and / or 2.2.

[1029] Typically, the nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[1030] The terms "vector", "cloning vector" and "expression vector" mean a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell in order to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Therefore, another object of the present invention relates to a vector comprising a nucleic acid of the present invention.

[1031] Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to initiate or direct expression of the polypeptide when administered to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the early promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney murine leukemia virus (Kuwana Y et al. 1987), the promoter of immunoglobulin H chain (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983), etc.

[1032] Any expression vector of animal cells can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1βd2-4- (Miyaji H et al. 1990), etc. Other representative examples of plasmids include replication plasmids containing a replication origin, or integration plasmids, such as pUC, pcDNA, pBR, etc. Representative examples of viral vectors include adenoviral vectors, retroviral vectors, herpes virus vectors, and AAV vectors. Such recombinant viruses can 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 viral packaging cells include PA317 cells, PsiCRIP cells, GPenv positive cells, 293 cells, etc. Detailed protocols for generating such replication-defective recombinant viruses can be found in, for example, WO 95 / 14785, WO 96 / 22378, US Pat. No. 5,882,877, US Pat. No. 6,013,516, US Pat. No. 4,861,719, US Pat. No. 5,278,056, and WO 94 / 19478.

[1033] Another object of the present invention relates to a cell transfected, infected or transformed by a nucleic acid and / or vector according to the present invention. The term "transformation" means the introduction of an "exogenous" (i.e., external or extracellular) gene, DNA or RNA sequence into a host cell such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is "transfected."

[1034] The nucleic acids of the present invention can be used to produce the recombinant polypeptides of the present invention in a suitable expression system. The term "expression system" means a host cell and a compatible vector for expressing a protein encoded by an exogenous DNA carried by a vector and introduced into the host cell under suitable conditions.

[1035] Common expression systems include Escherichia coli (E.coli) host cells and plasmid vectors, insect host cells and baculovirus vectors and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces (Kluyveromyces) or Saccharomyces (Saccharomyces) yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) and primary or established mammalian cell cultures (e.g., produced by lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neurons, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells in which the dihydrofolate reductase gene (hereinafter referred to as "DHFR" gene) is deficient (Urlaub G et al.; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"), etc. YB2 / 0 cells are preferred because the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in these cells.

[1036] The present invention also relates to a method for producing a recombinant host cell expressing an antibody or polypeptide according to the present invention, the method comprising the steps of: (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vitro or in vitro, (ii) culturing the obtained recombinant host cell in vitro or in vitro, and (iii) optionally selecting cells that express and / or secrete the antibody or polypeptide. Such recombinant host cells can be used to produce the antibodies and polypeptides of the present invention.

[1037] On the other hand, the present invention provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Therefore, the polynucleotides of the present embodiment can be used to separate, detect and / or quantify nucleic acids comprising such polynucleotides. For example, the polynucleotides of the present invention can be used to identify, separate or amplify partial or full-length clones in a preserved library. In certain embodiments, the polynucleotides are genomic sequences or cDNA sequences that are isolated from a human or mammalian nucleic acid library or are complementary to cDNAs from a human or mammalian nucleic acid library. Preferably, the cDNA library contains at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and most preferably at least 95% full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low or medium stringency hybridization conditions are generally, but not exclusively, used with sequences that have reduced sequence identity relative to the complementary sequence. Medium and high stringency conditions can optionally be used for sequences with greater identity. Low stringency conditions allow selective hybridization of sequences with approximately 70% sequence identity and can be used to identify orthologous or paralogous sequences. Optionally, the polynucleotides of the present invention encode at least a portion of an antibody encoded by a polynucleotide described herein. The polynucleotides of the present invention comprise nucleic acid sequences that can be used to selectively hybridize with a polynucleotide encoding an antibody of the present invention. See, e.g., Ausubel, supra; Colligan, supra, each of which is incorporated herein by reference in its entirety.

[1038] IV. Methods for Producing Antibodies

[1039] The antibodies and fragments thereof, immunoglobulins and polypeptides of the present invention can be produced by any technique known in the art, such as, but not limited to, any chemical, biological, genetic or enzymatic technique, alone or in combination.

[1040] The amino acid sequence of known desired sequence, those skilled in the art can easily produce described antibody or polypeptide by the standard technique for producing polypeptide.For example, they can use the solid phase method of knowing, preferably use the peptide synthesis device (such as by Applied Biosystems, Inc. (Applied Biosystems), Foster City, California manufacture) that can be commercially available and synthesize according to the instructions of manufacturer.Alternately, antibody of the present invention and other polypeptide can be synthesized by recombinant DNA technology as known in the art.For example, these fragments can be incorporated into the dna sequence dna of required (many) peptide of encoding and this type of carrier is incorporated into and expresses required polypeptide, can use the technology of knowing subsequently by them from its suitable eukaryotic or prokaryotic host after being separated as DNA expression product.

[1041] In particular, the present invention further relates to a method for producing an antibody or polypeptide of the present invention, said method comprising the steps of: (i) culturing a transformed host cell according to the present invention under conditions suitable for allowing expression of said antibody or polypeptide; and (ii) recovering the expressed antibody or polypeptide.

[1042] The antibodies and other polypeptides of the present invention are suitable for separation from culture medium by conventional immunoglobulin purification procedures, such as agarose protein A, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, New York, New York (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each incorporated herein by reference in its entirety.

[1043] Chimeric antibodies of the present invention (e.g., mouse-human chimeras or non-rodent-human chimeras) can be produced by obtaining nucleic acid sequences encoding VL and VH domains as previously described; constructing human chimeric antibody expression vectors by inserting them into expression vectors of genes encoding human antibody CH and human antibody CL in animal cells; and expressing the coding sequence by introducing the expression vector into animal cells. The CH domain of human chimeric antibodies can be any region belonging to human immunoglobulins, such as IgG classes or subclasses thereof, such as IgG1, IgG2, IgG3, and IgG4. Similarly, the CL of human chimeric antibodies can be any region belonging to Ig, such as κ or λ classes. Chimeric and humanized monoclonal antibodies comprising human and non-human parts (which can be prepared using standard recombinant DNA techniques) are within the scope of the present invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using the methods described in Robinson et al. International Patent Publication No. PCT / US86 / 02269; Akira et al. European Patent Application No. 184,187; Taniguchi, M. European Patent Application No. 171,496; Morrison et al. European Patent Application No. 173,494; Neuberger et al. PCT Application No. WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application No. 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proceedings of the National Academy of Sciences of the United States of America 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proceedings of the National Academy of Sciences of the United States of America 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, SL (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) J. Immunol. 141:4053-4060.

[1044] In addition, humanized antibodies can be prepared according to standard protocols, such as those disclosed in U.S. Patent No. 5,565,332. In another embodiment, antibody chains or specific binding pairs members can be recombined between a carrier of a nucleic acid molecule comprising a fusion of a polypeptide chain encoding a specific binding pair member and a component of a replicable generic display package and a carrier containing a nucleic acid molecule encoding a second polypeptide chain of a single binding pair member, using techniques known in the art (e.g., as described in U.S. Patent Nos. 5,565,332, 5,871,907 or 5,733,743). Humanized antibodies of the present invention can be produced by: obtaining a nucleic acid sequence encoding CDR domains as previously described; constructing a humanized antibody expression vector by inserting them into an expression vector of a gene encoding (i) the same heavy chain constant region as that of a human antibody and (ii) the same light chain constant region as that of a human antibody in an animal cell; and expressing a gene by introducing an expression vector into an animal cell. The humanized antibody expression vector may be of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain are present on separate vectors or of a type in which both genes are present on the same vector (tandem type).

[1045] 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 MS. et al. 1985). Antibodies can be humanized using various techniques known in the art, including, for example, CDR-grafting (EP 239,400; PCT Publication WO 91 / 09967; U.S. Patents 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA et al. (1994)) and chain shuffling (U.S. Patent 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application EP 125023 and International Patent Application Publication No. WO 96 / 02576).

[1046] Similarly, bispecific or multispecific antibodies as described herein can be prepared according to standard procedures. For example, trioma and hybridoma are two examples of cell lines that can secrete bispecific or multispecific antibodies. The example of bispecific and multispecific antibodies produced by hybridoma or trioma is disclosed in U.S. Patent No. 4,474,893. Such antibodies can also be constructed by chemical means (Staerz et al. (1985) "Nature" 314: 628, and Perez et al. (1985) "Nature" 316: 354) and hybridoma technology (Staerz and Bevan (1986) "Proceedings of the National Academy of Sciences of the United States of America", 83: 1453, and Staerz and Bevan (1986) "Modern Immunology (Immunol.Today)" 7: 241). Alternatively, such antibodies can also be generated by: preparing heterogeneous hybridomas by fusing hybridomas or other cells preparing different antibodies, then identifying clones that produce and assemble 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 fragment thereof of one or more biomarkers of the invention, including one or more immunosuppressive biomarkers described herein.

[1047] In addition, methods for producing antibody fragments are well known. For example, the Fab fragments of the present invention can be obtained by treating an antibody that specifically reacts with human HHLA2 (such as mAb 8A12 and polyclonal antibodies 1.2 and 2.2) with a protease such as papain. Alternatively, Fab can be produced by inserting DNA encoding the Fab of the antibody into a vector in a prokaryotic or eukaryotic expression system, and introducing the vector into a prokaryotic or eukaryotic organism (where appropriate) to express the Fab.

[1048] Similarly, the F(ab')2 fragment of the present invention can be obtained by treating an antibody that specifically reacts with HHLA2 with the protease, pepsin. Alternatively, the F(ab')2 fragment can be produced by binding the Fab' described below via a thioether bond or a disulfide bond.

[1049] The Fab' fragment of the present invention can be obtained by treating F(ab')2 that specifically reacts with HHLA2 with the reducing agent, dithiothreitol. Alternatively, the Fab' fragment can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic or eukaryotic expression vector, and introducing the vector into the prokaryotic or eukaryotic organism (where appropriate) for expression.

[1050] In addition, the scFv 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 a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into a prokaryotic or eukaryotic organism (where appropriate) to express the scFv. To generate a humanized scFv fragment, a well-known technique known as CDR transplantation can be used, which involves selecting complementary determining regions (CDRs) from a donor scFv fragment and transplanting them onto a human scFv fragment framework of a known three-dimensional structure (see, e.g., WO98 / 45322; WO 87 / 02671; U.S. Patent No. 5,859,205; U.S. Patent No. 5,585,089; U.S. Patent No. 4,816,567; EP0173494).

[1051] V. Modification of Antibodies, Immunoglobulins, and Peptides

[1052] It is envisioned that the amino acid sequence of the antibodies described herein is modified. 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 transplanting the CDRs in only the VH and VL of an antibody derived from a non-human animal into the FRs of the VH and VL of a human antibody, the antigen-binding activity is reduced compared to the antigen-binding activity of the original antibody derived from the non-human animal. It is believed that several amino acid residues in not only the CDRs but also the FRs of the VH and VL of the non-human antibody are directly or indirectly associated with the antigen-binding activity. Therefore, replacing these amino acid residues with different amino acid residues in the FRs of the VH and VL of a human antibody will reduce the binding activity, and can be corrected by replacing amino acids with amino acid residues of the original antibody derived from the non-human animal.

[1053] Modifications and changes can be made in the structure of the antibodies of the present invention and in the DNA sequences encoding them, and still obtain functional molecules encoding antibodies and polypeptides with desired characteristics. For example, certain amino acids can be replaced by other amino acids in the protein structure without a large amount of activity loss. Because the interactive ability and properties of proteins define the biological functional activity of proteins, certain amino acid substitutions can be made in the protein sequence and of course in its DNA coding sequence, while still obtaining proteins with similar properties. It is therefore envisioned that various changes can be made in the antibody sequences of the present invention or the corresponding DNA sequences encoding the polypeptides without causing a large amount of loss of their biological activity.

[1054] In one embodiment, amino acid changes can be achieved by altering codons in the DNA sequence to encode conservative substitutions based on the conservation of the genetic code. Specifically, there is a known and defined correspondence between the amino acid sequence of a particular protein and the nucleotide sequence that can encode said protein, as defined by the genetic code (shown below). Similarly, there is a known and defined 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 the genetic code chart above).

[1055] As noted above, an important and known feature of the genetic code is its redundancy, whereby for most amino acids used to make proteins, more than one coding nucleotide triplet (shown above) can be employed. Thus, multiple different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although some organisms may translate some sequences more efficiently than others). Additionally, occasionally, methylated variants of purines or pyrimidines may be present in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[1056] When making changes to the amino sequence of a polypeptide, the hydrophilicity index of the amino acids can be considered. The importance of the hydrophilic amino acid index in conferring interactive biological functions to proteins is generally understood in the art. It is believed that the relative hydrophilic character of the amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Based on their hydrophobicity and charge characteristics, each amino acid is assigned a hydrophilicity index, which are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (<RTI 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[1057] It is known in the art that certain amino acids can be replaced by other amino acids having similar hydrophilicity indices or scores and still produce a protein having similar biological activity, i.e., still obtain a biologically functionally equivalent protein.

[1058] As indicated above, amino acid substitutions are therefore generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various aforementioned characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[1059] Another type of amino acid modification of the antibody of the present invention can be used to change the original glycosylation pattern of the antibody to, for example, increase stability. "Alteration" means deleting one or more carbohydrate moieties present in the antibody and / or adding one or more glycosylation sites that do not exist in the antibody. The glycosylation of the antibody is usually N-connected. "N-connected" refers to the connection of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, in which X is any amino acid except proline, are recognition sequences for the enzymatic connection of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in the polypeptide produces potential glycosylation sites. Adding glycosylation sites to the antibody is conveniently achieved (for N-connected glycosylation sites) by changing the amino acid sequence so that it contains one or more of the tripeptide sequences described above. Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These programs are advantageous because they do not require the production of antibodies with glycosylation capabilities for N-connected or O-connected glycosylation in host cells. Depending on the coupling mode used, the sugar can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. Such methods are described, for example, in WO 87 / 05330.

[1060] Similarly, removal of any carbohydrate moieties present on the antibody can be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine) while keeping the antibody intact. Chemical deglycosylation is described by Sojahr H. et al. (1987) and Edge, A S. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by using various endoglycosidases and exoglycosidases, as described by Thotakura, N R. et al. (1987).

[1061] Other modifications may involve the formation of immunoconjugates. For example, in one type of covalent modification, an antibody or protein is covalently linked to one of various nonprotein polymers (e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes) as listed in U.S. Patents 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

[1062] Conjugation of an antibody or other protein of the invention to a xenologous agent can be prepared using a variety of bifunctional protein coupling agents, including but not limited to N-succinimidyl (2-pyridyldithiol) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl diimidoadipate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido 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 1,5-difluoro-2,4-dinitrobenzene). For example, carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies (WO 94 / 11026).

[1063] In another aspect, the present invention features antibodies that specifically bind to HHLA2 that are conjugated to a therapeutic moiety, such as a cytotoxin, a drug, and / or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." Cytotoxins or cytotoxic agents include any agent that is harmful to cells (e.g., kills cells). Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, 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 dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin D (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine). The antibodies of the present invention can be conjugated to radioactive isotopes, such as radioiodine, to generate cytotoxic radiopharmaceuticals for the treatment of related disorders, such as cancer.

[1064] Conjugated anti-HHLA2 antibodies can be used diagnostically or prognostically to monitor polypeptide levels in tissues as part of a clinical testing program, for example, to determine the efficacy of a given treatment regimen or to select patients most likely to respond to immunotherapy. For example, cells can be permeabilized in a flow cytometry assay to allow antibodies that bind to HHLA2 (such as mAb 8A12 and polyclonal antibodies 1.2 and 2.2) to target the intracellular epitope they recognize and to detect binding by analyzing the signal emitted by the conjugated molecule. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin (PE); examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H. As used herein, the term "labeled" with respect to an antibody is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, such as a radioactive agent or fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indole (Cy5)), as well as indirect labeling of the antibody through reactivity with a detectable substance.

[1065] The antibody conjugates of the present invention can be used to modify a given biological response. The therapeutic moiety is not considered to be limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having a desired biological activity. Such proteins can include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other cytokines or growth factors.

[1066] Techniques for conjugating such therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological and Clinical Applications. Antibodies '84: Biological And Clinical Applications," Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62: 119-58 (1982).

[1067] In some embodiments, conjugation can be performed using a "cleavable linker" that is conducive to the release of the cytotoxic agent or growth inhibitor in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-bonded linker can be used (see, for example, U.S. Patent No. 5,208,020). Alternatively, a fusion protein comprising an antibody and a cytotoxic agent or growth inhibitor can be prepared by recombinant technology or peptide synthesis. The length of DNA can include corresponding regions of two parts that are adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[1068] VI . Uses and methods of the present invention

[1069] The anti-HHLA2 antibodies, immunoglobulins, polypeptides, and nucleic acids of the invention described herein can be used in a variety of predictive medical assays (e.g., diagnostic assays, prognostic assays, and monitoring clinical trials) based on the detection of HHLA2 levels, and in some embodiments, can be used for therapeutic purposes (e.g., therapeutic and prophylactic) alone or when conjugated to toxic compounds or other therapeutic agents. As used herein, the term "detection" includes qualitative and / or quantitative detection (measuring levels) with or without reference to a control. As used herein, the HHLA2 polypeptides of the invention or fragments thereof have one or more of the following activities: 1) binding to one or more of their natural binding partners (such as TMIGD2 and / or KIR3DL3) and / or modulating the activity of such natural binding partners; 2) modulating intracellular or intercellular signaling, such as co-immunosuppressive signaling; 3) modulating lymphocyte activation and / or proliferation; 4) modulating the immune response of an organism (e.g., a mammalian organism such as a mouse, non-rodent, or human); and 5) modulating immune cell anergy.

[1070] Thus, one aspect of the present invention relates to diagnostic assays for determining the level of an HHLA2 polypeptide in the context of a biological sample (e.g., blood, serum, cells, or tissue), thereby determining the level of an HHLA2 polypeptide in the sample, determining whether an individual has a disease, and / or determining the status of such a disease as indicated by the level of HHLA2. For example, the antibodies of the present invention can be used to stage cancer diseases associated with HHLA2.

[1071] The invention also provides prognostic (or predictive) assays for determining whether an individual is at risk for developing such a condition.Another aspect of the invention relates to monitoring the effect of an agent (eg, drug, compound) on the expression or activity of HHLA2 in a clinical trial.

[1072] The present invention also provides for the detection of HHLA2 as a means of identifying agents that transduce HHLA2 signaling.Agents that transduce HHLA2 signaling attenuate immune responses and can be used in autoimmune diseases, asthma, and for establishing tolerance.

[1073] In any of the methods described herein, HHLA2 can be detected alone or in combination with the expression of other molecules (such as other immune checkpoints and / or co-stimulatory molecules). The combined detection of several molecules (e.g., sequentially or simultaneously) can provide useful information about the synergy of therapeutic intervention and / or personalized higher resolution diagnosis of disease subtypes. In some embodiments, HHLA2 is detected with a combination of multiple markers.

[1074] 1 . Diagnostic tests

[1075] The present invention provides, in part, methods, systems, and codes for accurately classifying whether a biological sample expresses cell-restricted HHLA2 and / or whether the level of cell-restricted HHLA2 is modulated (e.g., upregulated or downregulated), thereby indicating the status of a target condition (such as cancer). In some embodiments, the present invention can be used to classify a sample (e.g., from a subject) as associated with or at risk for a cancer or subtype mediated by HHLA2 using statistical algorithms and / or empirical data (e.g., the presence, absence, or level of HHLA2).

[1076] An exemplary method for detecting the level of HHLA2 or its fragments, and therefore for determining whether a sample is associated with a disease or condition mediated by aberrant expression (e.g., upregulation or downregulation) of HHLA2 or its clinical isoforms, involves obtaining a biological sample from a test subject and contacting the biological sample with an antibody or antigen-binding fragment thereof capable of detecting HHLA2 of the present invention, such that the level of HHLA2 is detected in the biological sample. In some embodiments, at least one antibody or antigen-binding fragment thereof is used, wherein two, three, four, five, six, seven, eight, nine, ten, or more such antibodies or antibody fragments may be used in combination (e.g., in a sandwich ELISA) or in tandem. In some cases, the statistical algorithm is a single learning statistical classifier system. For example, the single learning statistical classifier system can be used to classify a sample as an HHLA2 sample based on both the predicted or probability value and the presence or level of HHLA2. Use of a single learning statistical classifier system typically classifies a sample as an HHLA2 sample with a sensitivity, specificity, positive predictive value, negative predictive value, and / or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[1077] Other suitable statistical algorithms are well known to those skilled in the art.For example, learning statistical classifier systems include machine learning algorithms that can adapt to complex data sets (for example, the group of target markers) and make decisions based on such data sets. In certain embodiments, a single learning statistical classifier system is used, such as a classification tree (for example, random forest). In other embodiments, the combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more learning statistical classifier systems is preferably used in series. Examples of learning statistical classifier systems include, but are not limited to, those using: inductive learning (e.g., decision / classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), probably approximately correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), fuzzy neural networks (NFN), network structures, perceptrons such as multilayer perceptrons, multilayer feedforward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in known environments such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in unknown environments, active learning in unknown environments, learning-action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g., kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithm, Gauss-Newton algorithm, a mixture of Gaussian algorithm, gradient descent algorithm, and learning vector quantization (LVQ). In certain embodiments, the methods of the present invention further comprise sending the HLA2 sample typing results to a clinician, such as a histopathologist or oncologist.

[1078] In another embodiment, the methods of the present invention further provide a diagnosis in the form of a probability that an individual has a condition or disorder associated with HHLA2. For example, an individual may have a probability of approximately 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of having the condition or disorder. In yet another embodiment, the methods of the present invention further provide a prognosis for an individual's condition or disorder. In some cases, the method for classifying a sample as an HHLA2 sample is further based on symptoms (e.g., clinical factors) of the individual from whom the sample was obtained. Symptoms or groups of symptoms can include, for example, lymphocyte count, white blood cell count, erythrocyte sedimentation rate, diarrhea, abdominal pain, cramps, fever, anemia, weight loss, anxiety, depression, and combinations thereof. In some embodiments, after an individual is diagnosed with a condition or disorder associated with HHLA2, the individual is administered a therapeutically effective amount of a drug (eg, a chemotherapeutic agent) useful for treating one or more symptoms associated with the condition or disorder.

[1079] In one embodiment, the method further involves obtaining a control biological sample (e.g., a biological sample from a subject not suffering from a condition or disorder mediated by HHLA2), a biological sample obtained from a subject during remission or prior to developing a condition or disorder mediated by HHLA2, or a biological sample obtained from a subject during treatment for the development of a condition or disorder mediated by HHLA2.

[1080] An exemplary method for detecting the presence or absence of an HHLA2 polypeptide or fragment thereof is an antibody or fragment thereof of the present invention capable of binding to an HHLA2 polypeptide, preferably an antibody with a detectable label. Antibodies can be polyclonal or, more preferably, monoclonal. Such agents can be labeled. The term "labeled" with respect to antibodies is intended to encompass both direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling through reactivity with another directly labeled agent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody. The term "biological sample" is intended to include tissues, cells, and biological fluids, such as serum, isolated from a subject, as well as tissues, cells, and fluids present in a subject. That is, the detection methods of the present invention can be used to detect HHLA2 or fragments thereof in biological samples both in vitro and in vivo. In vitro techniques for detecting HHLA2 polypeptides include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, immunohistochemistry (IHC), intracellular flow cytometry and related techniques, and immunofluorescence. In addition, in vivo techniques for detecting HHLA2 polypeptides or fragments thereof include introducing a labeled anti-HHLA2 antibody into a subject. For example, the antibody can be labeled with radioactivity, luminescence, fluorescence, or other similar markers whose presence and location in the subject can be detected by standard imaging techniques, which can be used alone or in combination with imaging of other molecules, such as cell type markers (e.g., CD8+ T cell markers).

[1081] In one embodiment, the biological sample contains polypeptide molecules from a test subject.Preferred biological samples are serum, tumor microenvironment, peritumoral material, or intratumoral material isolated from a subject by conventional means.

[1082] In another embodiment, the method further involves obtaining a control biological sample from a control subject; contacting the control sample with a compound or agent capable of detecting an HHLA2 polypeptide or fragment thereof, such that the presence of the HHLA2 polypeptide or fragment thereof is detected in the biological sample; and comparing the presence of the HHLA2 polypeptide or fragment thereof in the control sample to the presence of the HHLA2 polypeptide or fragment thereof in the test sample.

[1083] In yet other embodiments, the antibodies can be associated with components or devices used for antibodies in ELISA or RIA. Non-limiting examples include immobilization of the antibodies on a solid surface for use in these assays (e.g., linked and / or conjugated to a detectable label based on light or radiation emission as described above). In other embodiments, the antibodies are associated with devices or strips for detecting HHLA2 using immunochromatographic or immunochemical assays, such as in "sandwich" or competitive assays, immunohistochemistry, immunofluorescence microscopy, and the like. Additional examples of such devices or strips include those designed for home testing or rapid point-of-care testing. Further examples include those designed for simultaneous analysis of multiple analytes in a single sample. For example, unlabeled antibodies of the present invention can be applied to "capture" HHLA2 polypeptides in a biological sample, and the captured (or immobilized) HHLA2 polypeptides can be bound to a labeled form of the anti-HHLA2 antibody of the present invention for detection. Other standard examples of immunoassays are well known to those skilled in the art and include assays based on, for example, immunodiffusion, immunoelectrophoresis, immunohistopathology, immunohistochemistry, and histopathology.

[1084] 2 . Prognostic assays

[1085] The diagnostic methods described herein can further be used to identify subjects suffering from or at risk for developing a condition associated with HHLA2. As used herein, the term "abnormal" encompasses upregulation or downregulation of HHLA2 that deviates from normal HHLA2 levels. Abnormal expression or activity encompasses increased or decreased expression or activity, as well as expression or activity that does not follow a normal developmental or subcellular expression pattern. For example, abnormal HHLA2 levels are intended to encompass situations in which mutations in the HHLA2 gene or regulatory sequences, or amplification of the chromosomal HHLA2 gene, lead to upregulation or downregulation of HHLA2. As used herein, the term "undesirable" encompasses undesirable phenomena involved in biological responses, such as immune cell activation. For example, the term "undesirable" encompasses undesirable HHLA2 in a subject.

[1086] A variety of conditions associated with HHLA2 are known to those skilled in the art, as further explained in the Examples. HHLA2 is expressed by a variety of tumor types, including selected lymphoid malignancies, virally induced cancers, and a variety of solid tumors. Generally, HHLA2 is a poor prognostic marker because it activates immune checkpoint regulators that inhibit strong immune responses against the condition in need. However, immunosuppression is required to downregulate the immune response in the treatment of a variety of conditions (such as autoimmune diseases, inflammatory diseases, etc.).

[1087] The assays described herein (such as the preceding diagnostic assays or the following assays) can be used to identify subjects suffering from, or at risk for developing, a condition associated with dysregulated HHLA2 activation. Thus, the present invention provides a method for identifying a condition associated with aberrant or unwanted HHLA2 activation, wherein a test sample is obtained from a subject and assayed for HHLA2, wherein the presence of an HHLA2 polypeptide is diagnostic for a subject suffering from, or at risk for developing, a condition associated with aberrant or unwanted HHLA2 activity. As used herein, a "test sample" refers to a biological sample obtained from a subject. For example, the test sample can be a biological fluid (e.g., cerebrospinal fluid or serum), a cell sample, or a tissue, such as a histopathological section of a tumor microenvironment, peritumoral region, and / or intratumoral region. In preferred embodiments, the sample comprises cells expressing mature, membrane-bound HHLA2 and / or HHLA2 fragments.

[1088] Furthermore, the prognostic assays described herein can be used to determine whether an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) can be administered to a subject to treat such a condition associated with aberrant or unwanted HHLA2 activity. For example, such methods can be used to determine whether a subject can be effectively treated with one or a combination of agents. Thus, the present invention provides methods for determining whether a subject can be effectively treated with one or more agents for treating a condition associated with aberrant or unwanted HHLA2 activation, wherein a test sample is obtained and HHLA2 is detected (e.g., wherein the abundance of the HHLA2 polypeptide is diagnostic for a subject to which an agent can be administered to treat a condition associated with aberrant or unwanted HHLA2 activation).

[1089] The methods described herein can be performed, for example, by utilizing a prepackaged diagnostic kit comprising at least one antibody reagent described herein, which can be conveniently used, for example, in a clinical setting to diagnose a patient exhibiting symptoms or a family history of a disease or condition involving HHLA2.

[1090] Furthermore, any cell type or tissue in which HHLA2 is expressed can be used in the diagnostic assays described herein.

[1091] Another aspect of the present invention includes the use of the compositions and methods described herein for association and / or stratification analysis, wherein HHLA2 is analyzed in biological samples from individuals with a condition associated with aberrant HHLA2 activation, and the information is compared with information from controls, preferably of similar age and ethnicity (e.g., individuals who do not have the condition; controls may also be referred to as "healthy" or "normal" individuals or at an earlier time point in a given time-lapse study). Appropriate selection of patients and controls is important for the success of association and / or stratification studies. Therefore, pools of individuals with well-characterized phenotypes are highly desirable. Guidelines for disease diagnosis, disease susceptibility screening, disease prognosis, determining drug responsiveness (pharmacogenomics), drug toxicity screening, and the like are described herein.

[1092] Different research designs can be used for genetic association and / or stratification studies (" Modern Epidemiology ", Wilkins Publishing Company (Lippincott Williams & Wilkins) (1998), 609-622). Observational studies are the most commonly performed, wherein the patient's response is not disturbed. The first type of observational study identifies a sample of a person in which the suspected cause of disease exists and another sample of a person in which the suspected cause of disease does not exist, and then compares the frequency of disease progression in the two samples. These sampling populations are referred to as cohorts, and the research is a prospective study. Another type of observational study is a case-control or retrospective study. In a typical case-control study, samples are collected from individuals with a target phenotype (case) (such as some manifestations of the disease) and from the population (target population) drawn therefrom without the individual (control) of the phenotype. The possible cause of disease is then studied retrospectively. Because the time and cost of collecting samples in case-control studies are significantly less than those of prospective studies, case-control studies are the most commonly used research design in genetic association studies, at least during the exploration and discovery phase.

[1093] After obtaining all relevant phenotypes and / or genotype information, carry out statistical analysis to determine whether there is any significant association between the existence of allele or genotype and individual phenotypic characteristics.Preferably, before carrying out the statistical test of genetic correlation, first carry out data inspection and cleaning.The epidemiology and the clinical data of sample can be summarized by the descriptive statistics of table well known in the art and figure.Preferably carry out data verification to check data completion, inconsistent entries and outliers.Then chi-square test and t-test (if distribution is abnormal, then use Wilcoxon rank sum test) can be used to check the significant difference between the case of discrete and continuous variable and contrast respectively.

[1094] The important decision of carrying out genetic association test is to determine significant level, under described level, when the p value of test reaches this level, can claim significant association.In the exploratory analysis that tracks positive hit in subsequent confirmatory test, unadjusted p value<0.2 (significant level on loose side) can be used for example to generate the hypothesis of significant association of HHLA2 level with some phenotypic characteristics of disease.Preferably realize p value<0.05 (significant level traditionally used in this area) so that described level is considered to have association with disease.When tracking hit in confirmatory test in more samples of same origin or in different samples of different origin, multiple tests are adjusted to avoid excessive number of hits, while experimental error rate is maintained at 0.05.Although there are different methods to adjust multiple tests to control different kinds of error rate, commonly used and conservative method is Bonferroni correction to control experiment or overall error rate (《Multiple comparisons and multiple tests (Multiple comparisons and multiple tests)》, Westfall et al., SAS Institute (SAS Institute) (1999)). Permutation tests that control the false discovery rate (FDR) may be more powerful (Benjamini and Hochberg, Journal of the Royal Statistical Society, Series B 57, 1289-1300, 1995, in Resampling-based Multiple Testing, Westfall and Young, Wiley (1993). Controlling multiplicity with such methods is preferred when the tests are dependent and controlling the false discovery rate is sufficient relative to controlling the experimental error rate.

[1095] Once it is found that the individual risk factors (hereditary or non-hereditary) for disease susceptibility, it is possible to set a classification / prediction scheme to predict the category (for example, disease or non-disease) that the individual is in according to its phenotype and / or genotype and other non-hereditary risk factors. The logistic regression of discrete traits and the linear regression of continuous traits are standard techniques for such tasks (" Applied Regression Analysis (Applied Regression Analysis) ", Draper and Smith, Willy (1998)). In addition, other techniques can also be used to set classification. Such techniques include but are not limited to MART, CART, neural networks and the discriminant analysis (" Statistical Learning Essentials (The Elements of Statistical Learning) ", Hastie, Tibshirani & Friedman, Springer (2002)) that are suitable for comparing the performance of different methods.

[1096] 3 . Monitoring effects during clinical trials

[1097] Monitoring the effects of an agent (e.g., a compound, drug, or small molecule) on an HHLA2 polypeptide or fragment thereof (e.g., regulation of cell proliferation and / or migration) can be applied not only to basic drug screening but also to clinical trials. For example, the effectiveness of an agent in reducing HHLA2 gene expression, polypeptide levels, or downregulating HHLA2 activity, as determined by a screening assay as described herein, can be monitored in clinical trials of subjects who exhibit reduced HHLA2 gene expression, polypeptide levels, or downregulated HHLA2 activity, or in clinical trials of subjects who exhibit reduced HHLA2 expression detectable by an anti-HHLA2 antibody or fragment as described herein. In such clinical trials, the expression or activity of an HHLA2 gene and / or symptom or marker of a condition of interest can be used as a "readout" or marker of the phenotype of a particular cell, tissue, or system. Similarly, the effectiveness of an agent determined by a screening assay as described herein to increase HHLA2 gene expression, polypeptide levels, or increase HHLA2 activity can be monitored in clinical trials of subjects who exhibit increased HHLA2 gene expression, polypeptide levels, or increased HHLA2 activity, or can be monitored in clinical trials of subjects who exhibit increased HHLA2 detectable by an anti-HHLA2 antibody or fragment as described herein. In such clinical trials, the expression or activity of an HHLA2 gene and / or symptom or marker of a disorder of interest can be used as a "readout" or marker of the phenotype of a particular cell, tissue, or system, such as for an autoimmune disorder.

[1098] For example, and without limitation, genes, including HHLA2, that are modulated in cells by treatment with an agent (e.g., a compound, drug, or small molecule) that modulates HHLA2 activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of an agent on a condition associated with aberrant HHLA2 activation, for example in a clinical trial, cells can be isolated, nucleic acids and / or proteins prepared, and the levels of HHLA2 and / or other genes implicated in the condition associated with aberrant HHLA2 activation analyzed. Gene expression levels (e.g., gene expression patterns) can be analyzed by measuring the amount of polypeptide produced using one of the methods described herein or by measuring the levels of HHLA2 or other genes. In this manner, gene expression patterns can serve as markers indicative of a cell's physiological response to the agent. Thus, this response state can be determined before treating an individual with the agent and at various points during treatment.

[1099] In preferred embodiments, the present invention provides a method for monitoring the effectiveness of treating a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate identified by a screening assay described herein), comprising the following steps: (i) obtaining a pre-administration sample from the subject prior to administration of the agent; (ii) detecting the level of an HHLA2 polypeptide or fragment thereof in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of an HHLA2 polypeptide or fragment thereof in the post-administration sample; (v) comparing the level of the HHLA2 polypeptide or fragment thereof in the pre-administration sample to the level of the HHLA2 polypeptide in the one or more post-administration samples; and (vi) altering administration of the agent to the subject accordingly. For example, it may be desirable to increase administration of the agent to reduce HHLA2 below the detected level, i.e., to increase the effectiveness of the agent. According to such embodiments, HHLA2 can be used as an indicator of the effectiveness of the agent, even in the absence of an observable phenotypic response. Similarly, HHLA2 analysis, such as by immunohistochemistry (IHC), can also be used to select patients for HHLA2 immunotherapy to inhibit one or more immune checkpoints. As described herein, patients with tumors that have HHLA2 activation are more likely to respond to HHLA2 mAb immunotherapy. Immunotherapy initially leads to immune activation, and activated T cells will express IFN-γ, which in turn upregulates HHLA2 activation. Typically, this will lead to a downregulation of HHLA2 engagement and immune response, but because HHLA2 can be blocked by anti-HHLA2 mAbs as described herein, the immune response continues until the desired condition, such as the tumor, is eliminated. In contrast, mAbs that actively signal through HHLA2 directly downregulate the immune response.

[1100] 4 . Therapeutic methods and uses

[1101] In some embodiments, the antibodies, fragments, or immunoconjugates of the invention (e.g., anti-HHLA2 antibodies alone or conjugated to a therapeutic moiety) can be used to treat any condition associated with abnormal or undesirable HHLA2 activation (e.g., cancer). In certain embodiments, the treatment is directed to a mammal, such as a human. Such antibodies of the invention can be used alone or in combination with any suitable agent or appropriate therapy for the target condition. For example, it is believed that therapeutic synergy is demonstrated when treating cells containing HHLA2 or another immune checkpoint or co-stimulatory molecule.

[1102] It is well known that therapeutic monoclonal antibodies can lead to the depletion of cells carrying extracellular antigens specifically recognized by the antibody. This depletion can be mediated by at least three mechanisms: antibody-mediated cytotoxicity (ADCC), complement-dependent lysis, and direct anti-tumor inhibition of tumor growth through signals given by the antigen targeted by the antibody.

[1103] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to an antibody that binds to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described by Gazzano-Santoro et al. (1997).

[1104] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to and subsequently kill antigen-bearing target cells. To assess the ADCC activity of a target molecule, an in vitro ADCC assay, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337, can be performed. As is well known in the art, the Fc portion can be engineered to achieve a desired interaction with an Fc receptor, or its lack thereof.

[1105] For antibody-mediated binding, neutralization and / or modulation of intracellular targets, certain modifications should be made.As described herein, certain antibody formats (such as sFv and Fab) are suitable for intracellular expression of antibody-like molecules. Methods for making and using such adaptive antibody-like molecules for targeted expression in different compartments of cells (including the nucleus, ER, cytoplasm, Golgi apparatus, plasma membrane, mitochondria) where they act in specific ways against antigens or molecules are well known (see, at least U.S. Patent Publication Nos. 2008-0233110 and 2003-0104402; Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893; Chen et al. (1994) Human Gene Therapy 5:595-601; Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:5932-5936; Mhashilkar et al. (1995) EMBO J. 14:1542-1551; Marasco et al. (1997) Gene Therapy Therapy 4:11-15; Richardson et al. (1995) Proceedings of the National Academy of Sciences of the United States of America 92:3137-3141; and Duan et al. (1994) Human Gene Therapy 5:1315-1324).

[1106] As used herein, the term "intracellular immunoglobulin molecule" is a complete immunoglobulin, which is identical to naturally occurring secretory immunoglobulin, but is retained in the cell after synthesis."intracellular immunoglobulin fragment" refers to any fragment, including single-chain fragments of intracellular immunoglobulin molecules. Therefore, intracellular immunoglobulin molecules or their fragments are not secreted or expressed on the outer surface of the cell. Single-chain intracellular immunoglobulin fragments are referred to as "single-chain immunoglobulins" in this article. As used herein, the term "intracellular immunoglobulin molecule or its fragment" is understood to encompass "intracellular immunoglobulin," "single-chain intracellular immunoglobulin" (or its fragment), "intracellular immunoglobulin fragment," "intracellular antibody" (or its fragment) and "intracellular antibody" (or its fragment). Thus, the terms "intracellular immunoglobulin," "intracellular Ig," "intracellular antibody" and "intracellular antibody" can be used interchangeably herein, and are all encompassed within the gene definition of "intracellular immunoglobulin molecule or its fragment." In some embodiments, the intracellular immunoglobulin molecule of the present invention or its fragment can comprise two or more subunit polypepti...

Claims

1. An anti-HHLA2 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises: a) a heavy chain variable region (VH), wherein the VH comprises a VH CDR1 as shown in SEQ ID NO: 206, a VH CDR2 as shown in SEQ ID NO: 210, and a VH CDR3 as shown in SEQ ID NO: 214; and b) a light chain variable region (VL), wherein the VL comprises a VL CDR1 as shown in SEQ ID NO: 224, a VL CDR2 as shown in SEQ ID NO: 228, and a VL CDR3 as shown in SEQ ID NO:

232.

2. The anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: a) VH comprising the amino acid sequence shown in SEQ ID NO: 199; and b) VL, said VL comprising the amino acid sequence shown in SEQ ID NO:

217.

3. The anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof: i) is a chimeric antibody, a humanized antibody, a composite antibody or a murine antibody; ii) is detectably labeled; iii) contains an effector domain; iv) selected from the group consisting of: Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 and diabody fragments; v) inhibiting: 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; or vi) Specifically binds to HHLA2.

4. An isolated nucleic acid molecule that hybridizes under stringent conditions to the complement of a nucleic acid encoding the anti-HHLA2 monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or to a nucleic acid encoding the anti-HHLA2 monoclonal antibody or an antigen-binding fragment thereof according to claim 1.

5. A vector comprising the isolated nucleic acid according to claim 4.

6. A host cell comprising the isolated nucleic acid according to claim 4.

7. A device or kit comprising at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

8. The device or kit according to claim 7, comprising a label for detecting the at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof, or comprising a label for detecting a complex comprising the anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof.

9. A method for producing at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, the method comprising the steps of: (i) culturing a transformed host cell that has been transformed with a nucleic acid comprising a sequence encoding the anti-HHLA2 monoclonal antibody under conditions suitable for allowing expression of at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2; and (ii) recovering the expressed anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof.

10. Use of the anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a device or kit for detecting the presence or level of an HHLA2 polypeptide, wherein the detection comprises obtaining a sample and detecting the polypeptide in the sample.

11. The use according to claim 10, wherein the at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof forms a complex with an HHLA2 polypeptide, and the complex is detected in the form of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blotting or using intracellular flow cytometry.

12. Use of at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a device or kit for monitoring the progression of a condition associated with abnormal HHLA2 expression in a subject, wherein the monitoring comprises: a) detecting the level of HHLA2 in a sample from a subject at a first time point using at least one of the devices or kits described; b) repeating step a) at a subsequent time point; and c) comparing the levels of HHLA2 detected in steps a) and b) to monitor the progression of said condition in said subject.

13. The use of claim 12, wherein between the first time point and the subsequent time point, the subject undergoes treatment to improve the condition.

14. Use of at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a device or kit for predicting clinical outcome of a subject suffering from a disorder associated with aberrant HHLA2 expression, wherein the prediction comprises: a) using the device or kit to determine the level of HHLA2 in a sample from a subject; b) determining the level of HHLA2 in a sample from a control subject with a good clinical outcome using the at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof; and c) comparing the level of HHLA2 in the sample from the subject with the level in the sample from the control subject; wherein a significantly higher level of HHLA2 in the subject sample compared to the level in the sample from the control subject indicates that the subject has a worse clinical outcome.

15. The use according to claim 14, wherein the significantly higher level of HHLA2 comprises at least a 20% increase in the level of HHLA2 in the sample from the subject relative to the normal level of HHLA2 in the sample from the control subject.

16. Use of at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a device or kit for assessing the efficacy of a therapy for a disorder associated with aberrant HHLA2 expression in a subject, wherein the assessment comprises: a) determining the level of HHLA2 in a first sample obtained from the subject using the device or kit 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 said subject after providing said portion of said therapy, wherein a significantly lower level of HHLA2 in the second sample relative to the first sample indicates that the therapy is effective in inhibiting the disorder in the subject.

17. The use according to claim 16, wherein the significantly lower level of HHLA2 comprises at least a 20% reduction in the level of HHLA2.

18. Use of at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a device or kit for evaluating the efficacy of a test compound for inhibiting a disorder associated with abnormal HHLA2 expression in a subject, wherein the evaluation comprises: a) using the device or kit to determine the level of HHLA2 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 said subject, in (i) the second sample was not exposed to the test compound, and a significantly lower level of HHLA2 relative to the second sample indicates that the test compound is effective in inhibiting the condition in the subject; and / or (ii) the first sample and the second sample are parts of a single sample obtained from the subject or parts of a pooled sample obtained from the subject.

19. Use according to any one of claims 12 to 18, wherein the disorder is cancer.

20. The use according to claim 19, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, leukemia, B-cell lymphoma and cancer infiltrating immune cells expressing receptors for HHLA2.

21. The use according to claim 20, wherein the leukemia is B-CLL.

22. The use according to any one of claims 10-21, wherein the sample, the subject sample, the control subject's sample, the first sample and / or the second sample comprises cells, serum, peritumoral tissue and / or intratumoral tissue.

23. The use according to claim 21, wherein the subject is a human.

24. Use of at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a pharmaceutical formulation for treating a subject suffering from cancer.

25. The use according to claim 24, wherein a) the at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent; b) the cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, a biological agent, a toxin, and a radioisotope; c) the at least one anti-HHLA2 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; d) administering the at least one monoclonal antibody or antigen-binding fragment thereof in a pharmaceutically acceptable dosage form; e) cancer cells and / or tumor immune-infiltrating cells in the subject express HHLA2; f) the cancer is selected from the group consisting of lung cancer, kidney cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, leukemia, B-cell lymphoma, and cancer infiltrating immune cells expressing receptors for HHLA2; g) administering to the subject a further therapeutic agent or regimen for treating cancer; h) administering to the subject an additional therapy selected from the group consisting of: immunotherapy, checkpoint blockade, cancer vaccines, chimeric antigen receptors, chemotherapy, radiation, targeted therapy, and surgery; and / or i) The subject is an animal model of cancer.

26. The use according to claim 25, wherein the leukemia is B-CLL.

27. The use according to claim 25 or 26, wherein the animal model is a mouse model.

28. The use according to claim 27, wherein the mouse model is a humanized mouse model.

29. The use according to claim 28, wherein the subject is a mammal.

30. The use according to claim 29, wherein the subject is a humanized mouse or a human.

31. Use of at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a pharmaceutical preparation for modulating an immune response in a subject, wherein the modulation comprises administering the anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof inhibits the interaction between HHLA2 and KIRDL3.

32. The use according to claim 31, wherein a) Blocking the interaction between HHLA2 and KIRDL3 for checkpoint blockade cancer immunotherapy; b) using anti-HHLA2 antibodies to inhibit or block the interaction between HHLA2 and KIRDL3; and / or c) The anti-HHLA2 antibody is a checkpoint inhibitor of T cell activation for cancer immunotherapy.

33. Use of at least one anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a pharmaceutical preparation for modulating an immune response in a subject, wherein the modulation comprises administering the anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-HHLA2 monoclonal antibody or antigen-binding fragment thereof does not block or significantly inhibit the interaction between HHLA2 and its binding stimulatory receptor TMIGD2.

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