KIR3DL3 as an HHLA2 receptor, anti-HHLA2 antibody, and uses thereof

Abstract

The present invention is based on the discovery of a monoclonal antibody that specifically binds partially to HHLA2 and its antigen-binding fragment, as well as immunoglobulins, polypeptides, their nucleic acids, and methods for using such antibodies for diagnostic, prognostic, and therapeutic purposes.

Description

Technology Field

[0001] 권리의 진술

[0002] The present invention was created with government support under authorization number P01 AI056299 issued by the National Institutes of Health. The government holds specific rights to the present invention. Background Technology

[0003] Many other immune checkpoints, such as CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, butyrophilin, and A2aR, negatively regulate the progression of the immune response based on complex and coupled interactions between numerous inputs. Although immune checkpoint inhibitors can modulate immune responses in some subjects, the expression of immune checkpoints and their interactions with their intrinsic binding partners vary among subjects and within their tissues. Therefore, it is highly necessary in the industry to identify novel immune checkpoints for use in interference. HHLA2 is a newly identified member of the B7 family that regulates T-cell function. HHLA2 has been identified as a specific ligand for TMIGD2, and the HHLA2 / TMIGD2 interaction selectively co-stimulates human T-cell growth and cytokine production via an AKT-dependent signaling cascade (Zhu et al. (2013) Nat. Comm. 4:2043; Janakiram et al. (2015) Clinical Cancer Research. 21:2359-2366). A second unspecified receptor for HHLA2 on activated T cells that exerts a co-inhibitory function has been proposed in several studies (Zhao et al. (2013) Proc. Natl. Acad. Sci. USA 110:9879-9884; Xiao and Freeman et al. (2015) Clinical Cancer Research 21:2201-2203; Wang et al. (2014) J. Immunol. 192:126.11). HHLA2 is expressed in various human cancers, and its co-inhibitory function makes HHLA2 a candidate for cancer immunotherapy.

[0004] The present invention is based, at least in part, on the finding that HHLA2, a member of the B7 gene family, is widely expressed in various tumors and antigen-presenting cells and acts as both an activating and inhibiting ligand for T cells. TMIGD2, expressed in naive T cells, is an activating receptor for HHLA2 and transmits a co-stimulating signal after binding to the T cell antigen receptor (TCR). TMIGD2 is downregulated after repeated TCR stimulation. It is possible for a putative inhibitory receptor for HHLA2 to be upregulated on activated T cells to regulate T cell activation. The present invention is based, at least in part, on the finding that HHLA2 binds to KIR3DL3, a receptor on T and NK cells, and that T cell activation is inhibited as a result of the HHLA2:KIR3DL3 interaction. Based on the observation that HHLA2 is highly expressed in tumors and can act as a checkpoint ligand, a panel of anti-HHLA2 human monoclonal antibodies (mAbs) was derived as candidate immune checkpoint inhibitors. Blocking and non-blocking anti-HHLA2 mAbs were identified by evaluating soluble human HHLA2-mIgG2a binding to TMIGD2-transfected 300.19 mouse progenitor-B leukemia cells or KIR3DL3-transfected 300.19 mouse progenitor-B leukemia cells. Anti-HHLA2 mAbs that block HHLA2 from binding to TMIGD2 and KIR3DL3, or that block KIR3DL3 more selectively but not TMIGD2, appeared to act as checkpoint inhibitor antibodies in T cell assays.

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

[0006] 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 having at least about 95% identity with a heavy chain CDR sequence selected from the group of sequences listed in Table 2; and / or b) a light chain CDR sequence having at least about 95% identity with a light chain CDR sequence selected from the group of 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 of sequences listed in Table 2; and / or b) a light chain sequence selected from the group of 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 of sequences listed in Table 2; and / or b) a light chain CDR sequence selected from the group of sequences listed in Table 2.

[0009] Additionally, a number of embodiments applicable to any aspect of the invention described herein are provided. For example, in one embodiment, the monoclonal antibody, or its antigen-binding fragment, is of chimeric, humanized, synthetic, rat, or human origin. In another embodiment, the monoclonal antibody, or its antigen-binding fragment, is selected from the group consisting of a detectable label, a reaction domain (effector domain), a Fc domain, and / or Fv, Fav, F(ab')2), Fab', dsFv, scFv, sc(Fv)2, and diabody fragment. In yet another embodiment, the monoclonal antibody, or its antigen-binding fragment, is obtained from the hybridoma ______ deposited under accession number ______. In another embodiment, a monoclonal antibody, or its antigen-binding fragment, 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. In a T cell activation assay, an HHLA2 mAb that blocks HHLA2 from binding to KIR3DL3 was found to be a checkpoint blocker. In another embodiment, a monoclonal antibody, or its antigen-binding fragment, specifically binds to HHLA2.

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

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

[0012] In another aspect, a vector comprising the isolated nucleic acid described in this specification is provided.

[0013] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid described herein, the vector described herein, the antibody described herein, or an antigen-binding fragment thereof, or the host cell deposited under accession number ______.

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

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

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

[0017] As described above, specific embodiments may be applied to any method described herein. For example, in one embodiment, at least one monoclonal antibody, or an antigen-binding fragment thereof, forms a complex with an HHLA2 polypeptide, and the complex is detected in the form of an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an immunochemical assay, a Western blot, or an intracellular flow assay.

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

[0019] As described above, specific embodiments may be applied to any method described herein. For example, in one embodiment, between the first time point and a subsequent time point, the subject receives treatment to improve the disability.

[0020] In another aspect, a method for predicting clinical outcomes of a subject suffering from a disorder associated with abnormal HHLA2 expression comprises: a) determining the level of HHLA2 in a sample of the subject using at least one monoclonal antibody or an antigen-binding fragment thereof as described herein; b) determining the level of HHLA2 in a sample of a control subject having good clinical outcomes using said at least one monoclonal antibody or an antigen-binding fragment thereof; and c) comparing the level of HHLA2 in a sample of the subject and in a sample of the control subject, wherein a significantly higher level of HHLA2 in a sample of the subject compared to the level in a sample of the control subject indicates that the subject has poor clinical outcomes.

[0021] In another aspect, a method for evaluating the efficacy of a treatment for a disorder associated with abnormal HHLA2 expression in a subject comprises: a) determining the level of HHLA2 in a first sample obtained from a subject before providing at least a portion of the treatment to the subject, using at least one monoclonal antibody or an antigen-binding fragment thereof as described herein; and b) determining the level of HHLA2 in a second sample obtained from the subject after providing a portion of the treatment, wherein a significantly lower level of HHLA2 in the second sample compared to the first sample is an indication that the treatment is effective for inhibiting the disorder in the subject.

[0022] In another aspect, a method for evaluating the efficacy of a test compound for inhibiting a disorder associated with abnormal HHLA2 expression in a subject comprises: a) determining the level of HHLA2 in a first sample obtained from a subject and exposed to the test compound using at least one monoclonal antibody or an antigen-binding fragment thereof as described herein; and b) determining the level of HHLA2 in a second sample obtained from a subject, wherein the second sample is not exposed to the test compound, and a significantly lower level of HHLA2 compared to the second sample is an indication that the test compound is effective for inhibiting the disorder in the subject.

[0023] As described above, specific embodiments may be applied to any method described herein. For example, in one embodiment, the first and second samples are part of a single sample obtained from the subject or part of a mixed sample obtained from the subject. In another embodiment, the disorder is cancer. In yet another embodiment, the cancer is selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancer infiltrated by immune cells expressing a receptor for HHLA2. In another embodiment, the sample comprises cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject. In another embodiment, a significantly higher level of HHLA2 comprises an increase of at least 20% between the level of HHLA2 in the subject sample and the normal level of HHLA2 in the sample of the control subject. In another embodiment, a significantly lower level of HHLA2 comprises a decrease of at least 20% in the level of HHLA2. In another embodiment, the subject is a human.

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

[0025] As described above, specific embodiments may be applied to any method described herein. For example, in one embodiment, the at least one monoclonal antibody, or its antigen-binding fragment, is conjugated to a cytotoxic agent. In another embodiment, the cytotoxic agent is selected from the group consisting of chemotherapy agents, biological agents, toxins, and radioisotopes. In yet another embodiment, the at least one monoclonal antibody, or its antigen-binding fragment, reduces the number of proliferating cells within the cancer and / or reduces the tumor volume or size of the cancer. In yet another embodiment, the at least one monoclonal antibody, or its antigen-binding fragment, is administered in a pharmaceutically acceptable formulation. In yet another embodiment, the method described herein also includes administering a therapeutic agent or therapy to a subject to treat cancer. In yet another embodiment, the method described herein also includes administering to a subject an additional therapy selected from the group consisting of immunotherapy, checkpoint inhibitors, cancer vaccines, chimeric antigen receptors, chemotherapy, radiation, targeted therapy, and surgery. In another embodiment, the cancer cells and / or tumor immune infiltrating cells of the subject express HHLA2. In yet another embodiment, the cancer is selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancer infiltrated by immune cells expressing a receptor for HHLA2. In yet another embodiment, the cancer is selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), head and neck cancer, liver cancer, ovarian cancer, prostate cancer, and uterine cancer. In yet another embodiment, the subject is an animal model of cancer. In yet another embodiment, the animal model is a mouse model, and optionally, the mouse model is a humanized mouse model.In another embodiment, the object 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 is provided to regulate an immune response by inhibiting the interaction between HHLA2 and its binding inhibitor receptor, KIRDL3.

[0027] In another aspect, a method is provided for regulating an immune response by selectively inhibiting the interaction between HHLA2 and its binding inhibitor receptor KIRDL3 without blocking or significantly inhibiting the interaction between HHLA2 and its binding stimulator receptor TMIGD2.

[0028] As described above, specific embodiments may be applied to any method described herein. For example, in one embodiment, the interaction between HHLA2 and KIRDL3 is blocked to use a checkpoint inhibitor cancer immunotherapy. In another embodiment, the interaction between HHLA2 and KIRDL3 is inhibited or blocked using an anti-HHLA2 antibody. In yet another embodiment, the anti-HHLA2 antibody is a checkpoint inhibitor for T cell activation for cancer immunotherapy.

[0029] In any drawing representing a bar histogram, curve, or other data associated with a legend, the said bar, curve, or other data displayed from left to right for each indication corresponds directly to the boxes from top to bottom or left to right of the legend. Brief explanation of the drawing

[0030] Figure 1a shows binding affinity data for anti-HHLA2 mAb on HHLA2-transfected 300.19 mouse precursor-B cell leukemia cell lines by flow cytometry. Figure 1b shows the anti-HHLA2 mAb blockade of TMIGD2-human IgG binding to HHLA2-transfused 300.19 mouse precursor-B cell leukemia cell lines by flow cytometry. Figure 2 shows Western blot data of proteins from nine human tumor cell lines probed with anti-HHLA2 mAb 8D2 (IHC mAb). Figure 3 shows HHLA2 mRNA expression compared to other checkpoint inhibitors in clear cell renal cell carcinoma (ccRCC) compared to normal kidney. Figure 4 shows HHLA2 expression in various cancers from the TCGA database. Figure 5a shows the results of HHLA2 immunohistochemistry (IHC) on negative control cells (300.19), HHLA-2 transfused 300.19 cells (positive control), OC1-Ly1 cells (negative tumor), and HDLM2 (positive Hodgkin lymphoma cell line) HHLA2 control cells. Figure 5b shows HHLA2 expression in a normal kidney. Figure 5c shows a representative image of HHLA2 expression in ccRCC from a microarray (TMA). Figure 5d shows a representative image of HHLA2 expression deficiency in other ccRCCs from a tumor microarray (TMA). Figure 6a shows the screening results of a representative set of ~300 doubly divided plasma membrane clones and a validation screen showing HHLA2 bound to KIR3DL3. A total of 5,682 cell surface receptor membrane clones were screened. Figure 6b shows the selective binding of HHLA2 to KIR3DL3. Figure 6c shows the gene ID and NCBI receipt information for the related biomarker. Figure 7 shows a schematic diagram of the Jurkat NFAT receptor gene analysis. Figure 8 shows a CAR-T cell model. Figure 9 shows inhibitory B7 family members that can be expressed by tumors. Figure 10 illustrates a model of HHLA2 interactions with two receptors (stimulatory and inhibitory HHLA2 receptors) for the regulation of 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 through a pathway involving AKT phosphorylation. With repeated T-cell activation, the expression of the stimulatory receptor TMIGD2 gradually disappears, and the expression of the inhibitory receptor KIR3DL3 becomes dominant. HHLA2 on APCs or tumor cells can interact with the second receptor and exert a co-inhibitory function. This figure is based on the literature of Xiao and Freeman et al. et al. (2015) Clinical Cancer Research It was adopted in 21:2201-2203). Figure 11 shows the stratification of HHLA2+ ve patients in non-small cell lung cancer (i.e., lung cancer tissues expressing HHLA2 based on immunohistochemistry (IHC) staining using HHLA2 mAbs). HHLA2 expression rates in PD-L1-positive and PD-L1-negative non-small cell lung cancer were calculated based on HHLA2 and PD-L1 immunohistochemistry studies (Cheng et al. (2018) Clinical Cancer Research 24:1954-1964). Figures 12a and 12b show the expression of TMIGD2 (Fig. 12a) or KIR3DL3 (Fig. 12b) on transfected 293T cells. TMIGD2 or KIR3DL3 cDNA in the pEF-Puro expression vector was transiently transfected into 293T cells, and after 48 to 72 hours, respectively, (1) goat-anti-mouse IgG F(ab) followed by TMIGD2 mAb (R&D systems catalog #MAB83162; clone #953743) 2-It was stained with PE (R&D Systems Catalog F0102B) or (2) KIR3DL3-PE conjugated mAb (R&D Systems Catalog # FAB8919R; clone #1136B) and detected by flow cytometry. Figures 12c and 12d show HHLA2-Fc bound to 293T cells transfected with TMIGD2 (Figure 12c) or KIR3DL3 (Figure 12d). HHLA2-mIgG2a transiently bound to 293T cells transfected with TMIGD2 or KIR3DL3 was detected by flow cytometry using PE-labeled Fab2 goat anti-mouse IgG2a antibody (absorbed for cross-reactivity with human Ig, Southern Biotech Catalog # 1082-09). Figures 13a and 13b show HHLA2 mAbs binding to HHLA2 in humans and Philippine monkeys. Different concentrations of HHLA2 mAbs were incubated with human or Philippine monkey HHLA2-transfected 300.19 progenitor-B cells at 4°C for 30 minutes. HHLA2 mAbs bound to the transfected 300.19 cells were detected by flow cytometry using PE-labeled goat anti-mouse IgG(H+L). Figure 14 shows a schematic diagram of the TMIGD2 / HHLA2 T cell co-stimulation assay. TMIGD2 Jurkat T cell NFAT-luciferase reporter gene cells were stimulated with CHO cells transfected with anti-CD3 scFV or anti-CD3 scFV + HHLA2. Figure 15 shows the expression of HHLA2 in CHO-anti-CD3 scFV cells. HHLA2 expression on CHO cells (clone #28) transfected with anti-CD3 scFV + HHLA2 was detected by flow cytometry using PE-conjugated 6F10 HHLA2 mAb. Figure 16 shows the expression of TMIGD2 in Jurkat NFAT reporter cells. The expression of TMIGD2 in TMIGD2-transfected Jurkat NFAT reporter cells (clone #62) is shown. Figure 17 shows the blockade of HHLA2 mAb by TMIGD2-mediated T cell co-stimulation. 2×10 HHLA2-TCR-CHO cells were placed in CHOK1 growth medium in a 96-well plate with a white, opaque bottom. 4 Cells were inoculated at a cell / well density. After incubating overnight at 37°C with 5% CO2, the cells were attached to the plate. The next day, the medium was carefully removed from each well, 50 µl of Jurkat cell medium containing the anti-HHLA2 antibody was added, and 4 to 5 × 10⁶ cells were added to 50 µl of Jurkat cell medium. 4 HHLA2-TCR-CHO cells were cultured for 1 hour before adding the MIGD2 NFAT Jurkat reporter cell line to the cell / well. The plate wells were mixed and incubated for approximately 3 to 6 hours. To develop the luciferase signal, 100 µl of ONE-Step was added according to the manufacturer's recommended protocol. TM A luciferase assay system (BPS Bioscience, Catalog #60690) was added to each well. Luminescence was read using a luminometer. Figure 18 shows a schematic diagram of the KIR3DL3 / HHLA2 checkpoint T cell assay. KIR3DL3 Jurkat T cell IL-2 promoter luciferase reporter gene cells were stimulated with CHO cells transfected with anti-CD3 scFV or anti-CD3 scFV + HHLA2. Figure 19 shows the expression of KIR3DL3 in Jurkat-IL-2 reporter clones. The expression of KIR3DL3 was detected in KIR3DL3-transfected Jurkat IL-2 reporter cell clones 1-6, 1-7, and 2-12. Figures 20a through 20c show HHLA2 mAb checkpoint blockade of KIR3DL3-mediated T cell inhibition. 2×10 HHLA2-TCR-CHO cells were placed in CHOK1 growth medium in a 96-well plate with a white, opaque bottom. 4 Cells were inoculated at a cell / well density. After incubating overnight at 37°C with 5% CO2, the cells were attached to the plate. The next day, the medium was carefully removed from each well, and 50 µl of Jurkat cell medium containing the anti-HHLA2 antibody was added. Additionally, 4 to 5 × 10⁶ cells were added to 50 µl of Jurkat cell medium containing 2 µg / ml of anti-CD28 antibody (BPS Bioscience #100186) (final concentration of 1 µg / ml in 100 µl assay mixture per well). 4 HHLA2-TCR-CHO cells were cultured for 1 hour before adding the KIR3DL3_IL2_Jurkat reporter cell line to the cell / well. The plate wells were mixed and incubated for approximately 5 hours. To develop the luciferase signal, 100 µl of ONE-Step was added according to the manufacturer's recommended protocol. TM A luciferase assay system (BPS Bioscience, Catalog #60690) was added to each well. Luminescence was read using a luminometer. Figures 21a to 21c show the titration of HHLA2 mAb in the Jurkat KIR3DL3 inhibition assay. Different concentrations of HHLA2 mAb 2C4 and 6F10 were evaluated in the Jurkat IL-2 reporter luciferase assay using Jurkat IL-2 luciferase clones 1-6, 1-7, and 2-12. Figure 22 shows that 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 maternal NFAT reporter cells. 2×10 HHLA2-TCR-CHO cells were placed in CHOK1 growth medium in white, opaque-bottomed 96-well plates.4 Cells were inoculated at a cell / well density. After incubating overnight at 37°C with 5% CO2, the cells were attached to the plate. The next day, the medium was carefully removed from each well, 50 µl of Jurkat cell medium containing the anti-HHLA2 antibody was added, and 4 to 5 × 10⁶ cells were added to 50 µl of Jurkat cell medium. 4 HHLA2-TCR-CHO cells were cultured for 1 hour before adding the Jurkat maternal_NFAT_Jurkat reporter cell line to the cell / well. The plate wells were mixed and incubated for approximately 3 to 6 hours. To develop the luciferase signal, 100 µl of ONE-Step was added according to the manufacturer's recommended protocol. TM A luciferase assay system (BPS Bioscience, Catalog #60690) was added to each well. Luminescence was read using a luminometer. Figures 23a to 23c illustrate a method for evaluating an HHLA2 mAb in a humanized SRG-15 mouse tumor model (containing both T and NK cells). An HHLA2 mAb is administered to humanized SRG-15 mice harboring HHLA2-expressing tumor cells, and tumor growth inhibition is evaluated. The figures are based on the literature of Herndler-Brandstetter D et al. [Herndler-Brandstetter D et al. It was adopted in

[2017] 114:E9626-E9634. Figures 24a and 24b show a method for evaluating HHLA2 mAb in a Philippine monkey T cell model. Philippine monkeys are administered HHLA2 mAb and immunized with KLH, and T cell-dependent antibodies and cell-mediated responses are evaluated. NK cytotoxicity is evaluated in vitro. Specific details for implementing the invention

[0031] The present invention is based, at least in part, on the finding that HHLA2, a member of the B7 gene family, is widely expressed in various tumors and antigen-presenting cells and acts as both an activating and inhibitory ligand to T cells. TMIGD2, expressed in naive T cells, is an activating receptor for HHLA2 and transmits a co-stimulating signal after binding to the T cell antigen receptor (TCR). TMIGD2 is downregulated after repeated TCR stimulation. Based on the observation that HHLA2 is highly expressed in tumors and can act as a checkpoint ligand, a panel of anti-HHLA2 human monoclonal antibodies (mAbs) was derived as candidate immune checkpoint inhibitor therapies. Considering that the same ligand binding domains of B7 family members B7-1 and B7-2 bind to both activating and inhibiting receptors (e.g., CD28 and CTLA-4), it is thought that anti-HHLA2 monoclonal antibodies that block TMIGD2 binding could be good candidates for blocking their binding to putative inhibiting receptors. By evaluating soluble hHHLA2-mIgG2a binding to TMIGD2-transfected 300.19 mouse progenitor-B leukemia cells, both blocking and non-blocking anti-HHLA2 mAbs were identified. 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 μg / 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 μg / mL, respectively. The variable region heavy and light chain gene sequences of these candidate therapeutic anti-HHLA2 antibodies are described in the present invention.

[0032] Anti-HHLA2 mAbs 1C8 and 6D10 were identified using good formalin-fixed, paraffin-embedded immunohistochemistry or Western blot reagents. In primary tumors from the TCGA database, HHLA2 was found to be highly expressed in lung, renal, pancreatic, and colorectal cancers and AML, and at intermediate levels in head and neck, liver, ovarian, prostate, and uterine cancers. The expression of HHLA2 mRNA in cancer is higher than in corresponding normal tissues.

[0033] By screening cell surfaces expressing a library of > 4,500 human plasma protein libraries containing over 3,500 different plasma membrane proteins with soluble human HHLA2-mIgG2a, KIR3DL3 (killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 3) was identified as a novel receptor for HHLA2. The cytoplasmic tail of KIR3DL3 contains an ITIM motif consisting of the sequence "VTYAQL," which represents an inhibitory receptor for HHLA2 that can act as a checkpoint receptor target for cancer immunotherapy. Since 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), the selectivity of HHLA2 to bind to KIR3DL3 was demonstrated.

[0034] Because HHLA2 is expressed at high levels in various types of tumors, anti-HHLA2 mAb checkpoint inhibitor therapies can increase the pool of patients who respond to checkpoint inhibitor treatment. Furthermore, patients who have developed resistance to PD-1 therapy may express HHLA2 as an alternative immune evasion strategy, and HHLA2 blockade may provide a way to overcome resistance to PD-1 immunotherapy.

[0035] Accordingly, the present invention provides a monoclonal antibody that specifically binds to HHLA2, and its antigen-binding fragment as well as immunoglobulin, polypeptide, nucleic acid thereof, and a method for using such antibody for diagnostic, prognostic, and therapeutic purposes.

[0036] I. 정의

[0037] Singular expressions are used herein to refer to one or more (i.e., at least one) of the grammatical objects of a singular expression. For example, "one element" means one element or more than one element.

[0038] The term "altered amount" of a marker refers to an increased or decreased copy number of the marker and / or increased or decreased nucleic acid levels of specific marker genes or genes in the sample compared to the marker in the control sample. Additionally, the term "altered amount" of a marker includes increased or decreased protein levels of the marker in the sample compared to the protein levels of the marker in normal and control samples.

[0039] The term "altered activity" of a marker refers to increased or decreased activity of a marker in a disease state, e.g., a biological sample, compared to the activity of the marker in a normal, control sample. The altered activity of a marker may be the result of, for example, altered expression of the marker, altered protein levels of the marker, altered structure of the marker, or, for example, altered interactions with other proteins involved in the same or different pathways as the marker, or altered interactions with transcription activators or inhibitors.

[0040] The term "altered structure" of a marker relates to the presence of mutations or allelic variations in the marker gene or marker protein compared to the normal or wild-type gene or protein, for example, mutations that affect the expression or activity of the marker. For example, mutations include, but are not limited to, substitutions, deletions, or additions. Mutations may exist within the coding or non-coding regions of the marker.

[0041] The term "activating receptor" includes immune cell receptors that bind to antigens, complex with antigens (e.g., in the context of MHC polypeptides), or bind to antibodies. These activating receptors include T cell receptors (TCRs), B cell receptors (BCRs), cytokine receptors, LPS receptors, complement receptors, and Fc receptors.

[0042] T cell receptors appear on T cells and are associated with the CD3 polypeptide. T cell receptors are stimulated by antigens (as well as by polyclonal T cell activating agents) in the context of MHC polypeptides. T cell activation via TCRs results in numerous changes, such as protein phosphorylation, membrane lipid changes, ion flux, cyclic nucleotide changes, RNA transcription changes, protein synthesis changes, and cell volume changes.

[0043] The terms "Chimeric Antigen Receptor" or "CAR" relate to engineered T cell receptors (TCRs) possessing desired antigen specificity. T lymphocytes recognize specific antigens through the interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) type I or type II molecules. For initial activation and clonal expansion, naive T cells rely on specialized antigen-presenting cells (APCs) that provide additional co-stimulatory signals. Activation of the TCR in the absence of co-stimulation can result in non-response and clonal energies. To bypass immunization, alternative approaches have been developed for the induction of cytotoxic effector cells with transplanted recognition specificity. CARs have been constructed consisting of binding domains derived from natural ligands or antibodies specific to the cell-surface components of the TCR-associated CD3 complex. Upon antigen binding, these chimeric antigen receptors connect to the endogenous signaling pathways of effector cells and generate activation signals similar to those initiated by TCR complexes. Since the first report on chimeric antigen receptors, this concept has been steadily improved, the molecular design of chimeric receptors has been optimized, and many well-known binding domains, such as scFV, Fav, and other protein-binding fragments described herein, are routinely used.

[0044] Generally, CAR is a type of "cell therapy" (e.g., T cell therapy) considered for use according to the present invention. While there are representative embodiments of substances and methods for regulating immune cell activity by modulating HHLA2 pathways, such as regulating the interaction between HHLA2 and its natural binding partners like TMIGD2 and / or KIR3DL3, immune cell-based therapies and methods are also included. For example, T cells engineered to have knockout, knockdown, or increased expression of TMIGD2 and / or KIR3DL3 are considered. Similarly, immune cells or other cells engineered to have knockout, knockdown, or increased expression of HHLA2 ligands such as TMIGD2 and / or KIR3DL3 are also considered.

[0045] B cell receptors are presented on B cells. The B cell antigen receptor is a complex between membrane Ig (mIg) and other transmembrane polypeptides (e.g., Igα and Igβ). The signaling function of mIg is triggered by the cross-linking of the receptor polypeptide by oligomeric or multimeric antigens. Additionally, B cells can be activated by anti-immunoglobulin antibodies. Upon BCR activation, numerous changes in the B cell occur, including tyrosine phosphorylation.

[0046] Fc receptors are found in many cells involved in immune responses. Fc receptors (FcRs) are cell surface receptors for the Fc portion of immunoglobulin polypeptides (Ig). Among the human FcRs identified to date are those that recognize IgG (denoted as FcγR), IgE (Fcε R1), IgA (Fcα), and polymerized IgM / A (Fcμα R). FcRs are found in the following cell types: Fcε R1 (mast cells), Fcε R1 (polycythiocytes), Fcα R1 (neutrophils), and Fcμα R1 (glandular epithelium, hepatocytes) (Hogg, N. (1988)). Immunol. Today9:185-86). The extensively studied FcγR is central to cellular immune defense and plays a role in stimulating the release of inflammatory mediators and hydrolytic enzymes involved in the pathogenesis of autoimmune diseases (Unkeless, JC et al. (1988) Annu. Rev. Immunol. 6:251-81). Macrophages / monocytes, polymorphonuclear leukocytes, and natural killer (NK) cells. Because FcγR confers a specific recognition element mediated by IgG, FcγR provides an important link between effector cells and Ig-secreting lymphocytes. Human leukocytes possess at least three different receptors for IgG: h Fcγ RI (found on monocytes / macrophages), h Fcγ RII (on monocytes, neutrophils, eosinophils, platelets, possible B cells, and the K562 cell line), and Fcγ RIII (on NK cells, neutrophils, eosinophils, and macrophages).

[0047] With respect to T cells, the delivery of co-stimulatory signals to T cells involves signaling pathways that are not inhibited by cyclosporin A. Additionally, co-stimulatory signals can induce cytokine secretion (e.g., IL-2 and / or IL-10) in T cells and / or prevent the induction of non-response to antigens, anaphoria, or apoptosis (deletion) in T cells.

[0048] When used in relation to polypeptides, e.g., HHLA2 and / or HHLA2 natural binding counterparts, e.g., TMIGD2 and / or KIR3DL3, the term “activity” includes activity inherent in the structure of the protein. For example, in relation to HHLA2 ligands, the term “activity” includes the ability to regulate immune cell inhibition by modulating inhibitory signals in immune cells (e.g., by binding to natural receptors on immune cells). Those skilled in the art will recognize that an inhibitory signal is generated in immune cells when the active form of the HHLA2 ligand polypeptide binds to an inhibitory receptor.

[0049] The term “inhibitory signal” relates to signals transmitted via inhibitory receptors (e.g., HHLA2, KLRB1, CTLA4, PD-1, etc.) for polypeptides on immune cells. These signals antagonize signals via activating receptors (e.g., TCR, CD3, BCR, TMIGD2, or Fc polypeptides) and may result in, for example, inhibition of messenger II production; inhibition of proliferation; inhibition of effector function in immune cells, for example, reduced phagocytosis, reduced antibody production, reduced cytotoxicity, failure of mediator production in immune cells (cytokines (e.g., IL-2) and / or mediators of allergic reactions); or energization.

[0050] If the amount of a biomarker in a subject is greater or smaller than the normal or control level by an amount greater than the standard deviation of the analysis used to evaluate the amount, respectively, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the amount, the amount of the biomarker in the subject is "significantly" higher or lower than the normal amount of the biomarker. Alternatively, the amount of the biomarker in the subject is greater than the amount of the biomarker in the normal and / or control group, respectively, at least about 2, 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%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 2 times, 3 times, 4 times, 5 times, or more, or any range in between, e.g., 5% to 100%, higher or lower, the amount of the biomarker in the subject may be considered to be "significantly" higher or lower than the amount in normal and / or control subjects. Such significant control values ​​may be applied to any metrics described herein, e.g., altered levels of expression, altered activity, changes in hyperproliferative growth of cancer cells, changes in apoptosis of cancer cells, changes in biomarker inhibition, changes in test substance binding, etc.

[0051] If the amount of the marker in the subject is greater than or less than the normal level by an amount greater than the standard deviation of the analysis used to evaluate the amount, and preferably at least twice, and more preferably three, four, five, ten, or more times the amount, the “amount” of the marker in the subject, e.g., the expression or copy number of the marker or MCR, or the protein level of the marker is “significantly” higher or lower than the normal amount of the marker. Alternatively, if the amount of the marker in the subject is higher or lower than the normal amount of the marker, respectively, by at least about two times, and preferably at least about three, four, or five times, the amount of the marker in the subject may be considered “significantly” higher or lower than the normal amount.

[0052] The term “altered level of expression” of a marker relates to the expression level or copy number of a marker in a test sample, e.g., a sample derived from a subject with cancer, which is greater or smaller than the standard deviation of the analysis used to evaluate the expression or copy number, preferably at least twice the expression level or copy number of the marker or chromosomal region in a control sample (e.g., a sample from a healthy subject without associated disease), preferably at least twice the average expression level or copy number of the marker or chromosomal region in some control samples, and more preferably three times, four times, five times, or ten times or more than the standard deviation of the analysis used to evaluate the expression or copy number, preferably at least twice the expression level or copy number of the marker in a control sample (e.g., a sample from a healthy subject without associated disease), preferably at least three times, four times, five times, or ten times or more than the average expression level or copy number of the marker in some control samples.

[0053] The term "immunotherapy" relates to forms of targeted therapy that may include, for example, the use of cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are potentially useful in immunomodulatory therapy because they are viruses capable of infecting and lysing cancer cells while not damaging remaining living normal cells. Replication of oncolytic viruses promotes tumor cell destruction and induces dose amplification at the tumor site. Additionally, they can act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site. Immunotherapy may include passive immunity for short-term host protection achieved by the administration of pre-formed antibodies against cancer antigens or disease antigens (e.g., administration of monoclonal antibodies against tumor antigens, selectively linked to chemotherapy agents or toxins). Furthermore, immunotherapy may focus on the use of cytotoxic lymphocyte-recognizing epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc., can be used to selectively modulate biomolecules linked to the initiation, progression, and / or pathology of tumors or cancer. As described above, immunotherapy targeting immune checkpoint targets such as HHLA2, TMIGD2, KIR3DL3, etc., is useful.

[0054] Unless otherwise specified herein, the term “antibody” broadly includes naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, e.g., single-chain antibodies, chimeric and humanized antibodies and multispecific antibodies, as well as fragments and derivatives of all of the foregoing having at least one antigen-binding site. Antibody derivatives may comprise proteins or chemical moieties conjugated to antibodies. “Antibody” relates to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or the antigen-binding portions thereof. Each heavy chain is a heavy chain variable region (V in this specification). H It consists of a heavy chain invariant region (abbreviated as (V)) and a heavy chain invariant region. The heavy chain invariant region consists of three domains, CH1, CH2, and CH3. Each light chain is a light chain variable region (V in this specification). L It consists of a (abbreviated as ) and a light chain invariant region. The light chain invariant region consists of a single domain, CL. V H and V L The region can be further subdivided into a hyper-variability region called the Complementarity Determination Region (CDR), which is interspersed with a more conserved region called the Frame Region (FR). Each V H and V L It consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The term "inactivated antibody" relates to antibodies that do not induce the complement system.

[0055] As used herein, the term “antibody” also includes the “antigen-binding portion” (or simply “antibody portion”) of an antibody. As used herein, the term “antigen-binding portion” refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., HHLA2 polypeptide or a fragment thereof). It has been found that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the term “antigen-binding portion” of an antibody are: (i) a Fab fragment, which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab’)2 fragment, which is a divalent fragment comprising two Fab fragments connected by disulfide crosslinking at a hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of an antibody; and (v) a dAb fragment (Ward) consisting of a VH domain. et al. , (1989) Nature 341:544-546); and (vi) include an isolated complementarity determining region (CDR). Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be combined using a recombinant method by a synthetic linker capable of pairing the VL and VH regions to form a single protein chain that creates a monovalent polypeptide (known as single-stranded Fv (scFv); e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA(See 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). These single-strand antibodies are also intended to be included in the term "antigen-binding portion" of the antibody. Any VH and VL sequence of a specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences to generate an intact IgG polypeptide or an expression vector encoding another isotype. VH and VL can also be used to generate Fab, Fv, or other fragments of immunoglobulin using proteochemistry or recombinant DNA technology. Other forms of single-strand antibodies, such as diabodies, are also included. Diabodies are bivalent bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but because they use a linker that is too short to allow binding between the two domains on the same chain, the domains bind to complementary domains on different chains, creating two antigen-binding sites (e.g., Holliger, P. et al . (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al . (1994) Structure See 2:1121-1123).

[0056] Additionally, the antibody or its antigen-binding portion may be part of a larger immunoadhesive polypeptide formed by the covalent or non-covalent bonding of the antibody or antibody portion with one or more other proteins or peptides. An example of such an immunoadhesive polypeptide is the use of a streptavidin core region to form a tetrameric scFv polypeptide (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to make divalent and biotinylated scFv polypeptides (Kipriyanov, SM, et al.(1994) Mol. Immunol Includes 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques such as papain or pepsin digestion of whole antibodies. Furthermore, the antibody, antibody portions, and immunoadhesive polypeptide can be obtained using standard recombinant DNA techniques as described herein.

[0057] Antibodies may be polyclonal or monoclonal; heterogeneous, homologous, or homologous; or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies may also be fully human. In one embodiment, the antibody of the present invention binds specifically or substantially specifically to the HHLA2 polypeptide or a fragment thereof. As used herein, the terms “monoclonal antibody” and “monoclonal antibody composition” refer to a group of antibody polypeptides comprising only one type of antigen-binding site capable of immunoreacting with a specific epitope of an antigen, whereas the terms “polyclonal antibody” and “polyclonal antibody composition” refer to a group of antibody polypeptides comprising several types of antigen-binding sites capable of interacting with a specific antigen. Monoclonal antibody compositions generally exhibit a single-binding affinity for the specific antigen to which they immunoreact.

[0058] The term “body fluid” refers not only to fluids that are excreted or secreted from the body, but also to fluids that are generally not (e.g., amniotic fluid, aqueous fluid, bile, blood and plasma, cerebrospinal fluid, cholecysts and earwax, pre-ejaculatory fluid, chyle, chyme, feces, female ejaculatory fluid, interstitial fluid, intracellular fluid, lymph, menstruation, breast milk, mucus, pleural fluid, pus, saliva, semen, serum, sweat, joint fluid, tears, urine, vaginal fluid, vitreous fluid, vomit).

[0059] "Cancer," "tumor," or "hyperproliferative disorder" refers to the presence of cells possessing typical characteristics of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and specific characteristic morphological features. While cancer cells often take the form of tumors, these cells may exist alone in animals or be non-tumor cancer cells, such as leukemia cells. Cancer includes, but is not limited to, B-cell cancers, e.g., multiple myeloma, Waldenström macroglobulinemia, heavy chain diseases, e.g., alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal globulinemia, and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or cecum cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematological tissue cancer, etc.Other non-limiting examples of cancer types applicable to the methods included in the present invention are human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangiendothelial sarcoma, synovoma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystic carcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, biliary tract carcinoma, liver cancer, choriocarcinoma, seminoma, embryonic carcinoma, Wilms tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, pulmonary small cell carcinoma, bladder carcinoma, epithelial carcinoma, glioma. astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal adenoma, hemangioma, acoustic neuroma, oligodendron glioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemia, e.g., acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic, promyeloblastic, myelomonocytic, mononucleic, and erythroleukemia); chronic leukemia (chronic myeloid (granulocytic) leukemia and chronic lymphoblastic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is essentially epithelial and includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colorectal 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 another embodiment, the epithelial carcinoma is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial carcinoma may be characterized in various other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or unclassified.

[0060] The term "CDR" refers to the Complementarity Determining Region (CDR), where three components constitute the binding characteristics of the light chain variable region (CDR-L1, CDR-L2, and CDR-L3) and three components constitute the binding characteristics of the heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3). The CDR contributes to the functional activity of the antibody molecule and is distinguished by an amino acid sequence containing a scaffolding or framework region. The precise definitions of CDR boundaries and lengths vary depending on different classification and numbering systems. Therefore, the CDR may be referred to by Kabat, Chothia, related, or any other boundary definition. Despite the different boundaries, each of these systems has some degree of overlap in constituting the so-called "hypervariable region" within the variable sequence. Definitions of the CDR according to these systems may differ in length and boundary zones with respect to adjacent framework regions. Refer to the literature of Kabat, Chothia, and / or MacCallum et al. (Kabat et al. , in "Sequences of Proteins of Immunological Interest," 5 th Pan, US Department of Health and Human Services, 1992; Chothia et al. (1987) J. Mol. Biol. 196, 901; and MacCallum et al. , J. Mol. Biol. (1996) 262, 732, each of these is incorporated by reference in its entirety).

[0061] As used herein, the term “classification” includes “associating” or “categorizing” a sample with a disease state. In certain cases, “classification” is based on statistical evidence, empirical evidence, or both. In certain embodiments, the method and system of classification use a so-called training set of samples having known disease states. Once established, the training data set serves as a criterion, model, or template for comparing the features of unknown samples to classify the unknown disease state of the samples. In certain cases, the classification of a sample is similar to diagnosing the disease state of a sample. In certain other cases, the classification of a sample is similar to distinguishing the disease state of a sample from other disease states.

[0062] As used herein, the term “coding region” refers to a region of a nucleotide sequence containing a codon that translates into an amino acid residue, whereas the term “non-coding region” refers to a region of a nucleotide sequence that does not translate into an amino acid residue (e.g., 5’ and 3’ non-translating regions).

[0063] "Complementary" or "complementary" refers to a broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of the first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue of the second nucleic acid region that is antiparallel to the first region when the residue of the second nucleic acid region is thymine or uracil. Similarly, it is known that a cytosine residue of the first nucleic acid strand can base pair with a residue of the second nucleic acid strand that is antiparallel to the first strand when the residue of the second nucleic acid strand is guanine. When the first region of a nucleic acid and the second region of a nucleic acid are arranged in an antiparallel manner, if at least one nucleotide residue of the first region can base pair with a residue of the second region, the first region of the nucleic acid is complementary to the second region of the same or different nucleic acid. In one embodiment, the first region comprises a first portion and the second region comprises a second portion, thereby, when the first and second portions are arranged in an antiparallel manner, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion can be base paired with the nucleotide residues of the second portion. In another embodiment, all nucleotide residues of the first portion can be base paired with the nucleotide residues of the second portion.

[0064] As used herein, the term "complex antibody" refers to an antibody having a variable region comprising germline or non-germline immunoglobulin sequences derived from two or more unrelated variable regions. Additionally, the term "complex human antibody" refers to an antibody having a constant region derived from a human germline or non-germline immunoglobulin sequence and a variable region comprising human germline or non-germline sequences derived from two or more unrelated human variable regions. Because complex human antibodies have low antigenicity in the human body, they are useful as active ingredients of the therapeutic agent according to the present invention.

[0065] The term “control group” refers to any reference standard suitable for providing a comparison with the expression product in the test sample. In one embodiment, the control group comprises obtaining a “control sample” in which the level of the expression product is detected and compared to the level of the expression product from the test sample. Such control sample may include any suitable sample, including but not limited to a sample from a control cancer patient with known results (which may be a stored sample or a previous sample measurement); normal tissue or cells isolated from a subject such as a normal patient or cancer patient; cultured primary cells / tissues isolated from a subject such as a normal subject or cancer patient; adjacent normal cells / tissues obtained from the same organ or body location of the cancer patient; tissue or cells isolated from a normal subject; or primary cells / tissues obtained from a deposit institution. In another preferred embodiment, the control group may include housekeeping genes, a range of expression product levels from normal tissue (or other previously analyzed control samples), a range of expression product levels within test samples of a previously determined patient group, or reference standard expression product levels from any suitable source including, but not limited to, a set of patients having a specific outcome (e.g., survival over a period of 1, 2, 3, 4 years, etc.) or receiving a specific treatment (e.g., standard cancer therapy). Those skilled in the art will understand that such control samples and reference standard expression product levels may be used in combination as controls in the method of the present invention. In one embodiment, the control group may include normal or non-cancerous cell / tissue samples. In another preferred embodiment, the control group may include expression levels for a set of patients such as a set of cancer patients, or for a set of cancer patients receiving a specific treatment, or for a set of patients having one outcome versus another outcome.In the former case, each patient's specific expression product level may be assigned as a percentile level of expression, or expressed as higher or lower than the average of the reference standard expression level. In another preferred embodiment, the control group may include normal cells, cells of patients treated with combination chemotherapy, and cells of patients with benign cancer. In another embodiment, the control group may also include the average expression level of a specific gene in a population compared to a measured value, for example, the expression level of a housekeeping gene in the same population. Such a population may include normal subjects, cancer patients who have not received any treatment (i.e., no treatment experience), cancer patients receiving standard treatment regimens, or patients with benign cancer. In another preferred embodiment, the control group determines the ratio of expression product levels of two genes within a test sample and compares them to any suitable ratio of the same two genes within a reference standard; determines the expression product levels of two or more genes within a test sample and determines the difference in expression product levels within any suitable control group; and includes a ratio transformation of expression product levels, which includes but is not limited to determining the expression product levels of two or more genes within a test sample, normalizing their expression to the expression of a housekeeping gene within the test sample, and comparing them to any suitable control. In a particularly preferred embodiment, the control includes a control sample that is of the same lineage and / or type as the test sample. In another embodiment, the control may include expression product levels grouped into percentiles within a set of patient samples, such as all cancer patients, or based on this set. In one embodiment, a control expression product level is established, for example, that is higher or lower than a specific percentile, and is used as a basis for predicting results.In another preferred embodiment, the control expression product level is established using the expression product level of a cancer control patient with known results, and the expression product level of the test sample is compared to the control expression product level as a reference for predicting results. As demonstrated by the data below, the method of the present invention is not limited to the use of a specific cut-point when comparing the expression product level in the test sample to the control.

[0066] As used herein, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including natural-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 generally defined as the range from the amino acid residue at the Cys226 position, or from Pro230, to its carboxy-terminus. Natural-sequence Fc regions suitable for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0067] As used herein, "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a natural sequence human FcR. Furthermore, a preferred FcR binds to an IgG antibody (gamma receptor) and comprises receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors, wherein the FcγRII receptor comprises FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), and primarily their cytoplasmic domains have different similar amino acid sequences. The activating receptor FcγRIIA contains an immune receptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immune receptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (Reference [M. Da ron, Annu. Rev. Immunol. See 15:203-234 (1997)]). FcR is referenced in the literature [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)] was reviewed. Other FcRs, including those to be identified in the future, are included in the term "FcR" of this specification.

[0068] When a molecule is covalently or non-covalently associated with a substrate such that it can be rinsed with a liquid (e.g., standard saline citrate, pH 7.4) without any substantial fragment of the molecule dissociating from the substrate, the molecule is "fixed" or "attached" to the substrate.

[0069] As used herein, "framework" or "FR" residues are variable-domain residues other than HVR residues as defined herein.

[0070] "Function-conserving variants" are those in which amino acid residues given to a protein or enzyme are modified without altering the overall structure and function of the polypeptide, including but not limited to replacing amino acids with those having similar properties (e.g., polarity, hydrogen bond potential, acidity, basicity, hydrophobicity, aromaticity, etc.). Since amino acids other than those indicated as conserved may differ in the protein, the percentage of protein or amino acid sequence similarity between any two proteins of similar function may differ, and may be, for example, 70 to 99% as determined by an alignment method such as a cluster method based on the MEGALIGN algorithm. "Function-conserving variants" also include polypeptides having at least 60%, preferably at least 75%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% amino acid identity as determined by the BLAST or FASTA algorithms, and having properties or functions that are the same or essentially similar to the natural or maternal protein being compared.

[0071] As used herein, the term “heterogeneous antibody” is defined in relation to a transgenic non-human organism that produces such an antibody. This term refers to an antibody having an amino acid sequence or coding nucleic acid sequence corresponding to that found in an organism that is not composed of said transgenic non-human animal, generally from a species other than said transgenic non-human animal.

[0072] As used herein, "homologous" refers to nucleic acid sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. If nucleotide residue positions in two regions are occupied by the same nucleotide residue, then the regions are homologous at that position. If at least one nucleotide residue position in each of the first region and the second region is occupied by the same residue, the first region is homologous to the second region. Homologousness between two regions is expressed as the ratio of nucleotide residue positions in the two regions occupied by the same nucleotide residue. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first region comprises a first part and the second region comprises a second part, and at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each part are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each part are occupied by the same nucleotide residue.

[0073] As used herein, the term "host cell" is intended to refer to a cell into which the nucleic acid of the present invention, for example, the recombinant expression vector of the present invention, has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. These terms should be understood to refer not only to specific target cells but also to the offspring or potential offspring of such cells. Since specific modifications may occur in subsequent generations due to mutations or environmental influences, these offspring may not actually be identical to the parent cells, but are still included within the scope of the terms used herein.

[0074] As used herein, the term “humanized antibody” is intended to include antibodies produced by non-human cells having variable and constant regions modified to be more similar to antibodies produced by human cells. For example, by modifying the non-human antibody amino acid sequence to include amino acids found in human germline immunoglobulin sequences. Humanized antibodies may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., in vitro random or site-specific mutagenesis or in vivo somatic mutation), for example, CDRs. As used herein, the term “humanized antibody” also includes antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, is transplanted onto a human framework sequence.

[0075] As used herein, a humanized mouse is a mouse that carries a functioning human gene (e.g., HHLA2, TMIGD2, and / or KIR3DL3), cells, tissues, and / or organs. Humanized mice are generally used as small animal models in biological and medical research for human therapeutics. Nude mice and severe combined immunodeficiency (SCID) mice may be used for this purpose. NCG mice, NOG mice, and NSG mice may be used to transplant human cells and tissues more effectively than other models. These humanized mouse models may be used to model the human immune system in health and pathological scenarios and may enable the evaluation of therapeutic candidates in an in vivo environment related to human physiology.

[0076] As used herein, “hypervariable region,” “HVR,” or “HV” refers to a region of the antibody-variable domain that is hypervariable and / or forms a structurally defined loop in the sequence. Generally, antibodies contain six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the greatest variability among the six HVRs, and H3, in particular, is believed to play a unique role in conferring excellent specificity to the antibody. For example, Xu et al. See (2000) Immunity 13, 37-45; Johnson and Wu in Methods in Molecular Biology 248, 1-25 (Lo, ed., Human Press, Totowa, NJ, 2003)). In fact, naturally occurring cameloid antibodies consisting solely of heavy chains are functional and stable despite the absence of light chains (e.g., Hamers-Casterman et al. (1993) Nature 363:446-448 (1993) and Sheriff et al. (1996) See Nature Struct. Biol. 3, 733-736).

[0077] 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; and bone marrow cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0078] As used herein, the term “immune disorder” includes cancer, chronic inflammatory diseases and disorders (e.g., Crohn’s disease, inflammatory bowel disease, reactive arthritis, and Lyme disease), insulin-dependent diabetes mellitus, organ-specific autoimmunity (e.g., multiple sclerosis, Hashimoto’s thyroiditis, autoimmune uveitis, and Graves’ disease), contact dermatitis, psoriasis, transplant rejection, graft-versus-host disease, sarcoidosis, apotytic conditions (e.g., asthma and allergies, including but not limited to gastrointestinal allergies such as allergic rhinitis and food allergies), eosinophilia, conjunctivitis, glomerulonephritis, systemic lupus erythematosus, scleroderma, susceptibility to specific pathogens such as parasitic (e.g., leishmaniasis), and specific viral infections (e.g., HIV and bacterial infections such as Mycobacterium tuberculosis and Lycaenida), and malaria, including but not limited to immune diseases, conditions, and predispositions.

[0079] As used herein, the term “immune response” includes T cell-mediated and / or B cell-mediated immune responses. By example, an immune response includes T cell responses, e.g., cytokine production, and cytotoxicity. Additionally, the term immune response includes immune responses indirectly affected by T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages.

[0080] The term “immunotherapeutic agent” may include any molecule, peptide, antibody, or other substance capable of stimulating the host immune system to generate an immune response against a tumor or cancer of a subject. Various immunotherapeutic agents are useful for the compositions and methods described herein.

[0081] The term "immune checkpoint" refers to a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune immune responses, downregulating or inhibiting anti-tumor immune responses. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, and A2aR (see, e.g., WO 2012 / 177624). This term also includes biologically active protein fragments, as well as the full length of an immune checkpoint protein and nucleic acids encoding its biologically active protein fragments. In some embodiments, this term also includes any fragment according to the homology description provided herein.

[0082] Immune checkpoints and their sequences are well known in the art, and representative embodiments are described below. For example, the term "PD-1" refers to a member of the immunoglobulin gene superfamily that functions as a co-inhibitory receptor having PD-L1 and PD-L2 as known ligands. PD-1 was previously identified using a subtraction cloning-based approach to select genes upregulated in the TCR-induced activated T cell death process. PD-1 is a member of the CD28 / CTLA-4 family among molecules based on their ability to bind to PD-L1. Like CTLA-4, PD-1 is rapidly induced on the surface of T-cells in response to anti-CD3 (Agata et al. 25 (1996) Int. Immunol.8:765). However, unlike CTLA-4, PD-1 is also induced on the surface of B cells (in response to anti-IgM). PD-1 is also expressed in thymic and myeloid cell subpopulations (Agata et al. (1996) above; Nishimura et al. (1996) Int. Immunol. 8:773).

[0083] As used herein, the term “inhibition” and its grammatically equivalent terms mean reducing, limiting, and / or blocking a specific action, function, or interaction. In one embodiment, this term means reducing the level of a given yield or parameter (e.g., background staining, HHLA2 signaling, HHLA2 immunoinhibitory function, etc.) relative to the amount to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less compared to the amount of the corresponding control. A reduced level of a given yield or parameter may mean the complete absence of this yield or parameter, but is not required. The present invention does not require, but is not limited to, a method of completely eliminating this yield or parameter. A given yield or parameter may be determined using methods well known in the art, including but not limited to immunohistochemical, molecular biological, cellular biological, clinical, and biochemical analyses as discussed in this specification and examples. The opposite terms "promote," "increase," and grammatically equivalent terms mean increasing the level of a given yield or parameter, as opposed to what is described for inhibition or reduction.

[0084] As used herein, the term "interaction" refers to physical contact (e.g., binding) between a molecule and another molecule when referring to an interaction between two molecules (e.g., binding between HHLA2 and TMIGD2 or binding between HHLA2 and KIR3DL3). Generally, such an interaction results in the activity (producing a biological effect) of one or both of the molecules. This activity may be a direct activity of one or both of the molecules (e.g., signal transduction). Alternatively, in this interaction, one or both molecules may have their ligand binding blocked and thus remain inactive with respect to ligand binding activity (e.g., binding to their ligand and inducing or inhibiting an immune response). Inhibiting this interaction results in the disruption of the activity of one or more molecules involved in this interaction. Enhancing this interaction is to extend or increase the possibility of physical contact and to extend or increase the possibility of activity.

[0085] The term "neoadjuvant therapy" refers to treatment administered prior to major treatment. Examples of neoadjuvant therapy may include chemotherapy, radiation therapy, and hormone therapy.

[0086] As used herein, the term “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to HHLA2 is substantially free of antibodies that do not bind to HHLA2). However, an isolated antibody that specifically binds to HHLA2 may have cross-reactivity to other B7 family proteins from different species. For example, in some embodiments, the antibody maintains specific binding affinity for at least two species, e.g., humans and other animals, e.g. rodents, or other mammalian or non-mammalian species. However, in some embodiments, the antibody maintains a higher or actual specific affinity and selectivity for human HHLA2. Additionally, the isolated antibody is generally substantially free of other cellular material and / or chemicals. In one embodiment of the invention, a combination of “isolated” monoclonal antibodies having different specificities for human HHLA2 is combined into a well-defined composition.

[0087] As used herein, “isolated protein” refers to a protein that, when isolated from a cell or produced by recombinant DNA technology, is substantially free of other proteins, cellular material, isolation medium, and culture medium, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. The “isolated” or “purified” protein or its biologically active portion is free of cellular material or other contaminating proteins derived from the cell or tissue source from which the antibody, polypeptide, peptide, or fusion protein is released, or, if chemically synthesized, is substantially free of chemical precursors or other chemicals. The term “substantially free of cellular material” includes a protein isolated from a cellular component, isolated or produced by recombinant DNA, comprising a target polypeptide (e.g., immunoglobulin) or a fragment thereof. In one embodiment, the term “substantially free of cellular material” comprises a preparation of a target protein or a fragment thereof having less than about 30% (dry weight) of non-target protein (also referred to herein as “contamination protein”), more preferably less than about 20% of non-target protein, more preferably less than about 10% of non-target protein, and most preferably less than about 5% of non-target protein. When an antibody, polypeptide, peptide, or fusion protein or a fragment thereof, for example, a biologically active fragment thereof, is produced by recombination, preferably the culture medium is also substantially free, i.e., the culture medium is less than about 20% of the volume of the protein preparation material, more preferably less than about 10%, and most preferably less than about 5%.

[0088] As used herein, the term "isotype" refers to a class of antibodies (e.g., IgM or IgG1) encoded by a heavy chain constant region gene.

[0089] As used in this specification, the term "K" D" is intended to refer to the dissociation equilibrium constant of a specific antibody-antigen interaction. The binding affinity of the antibody of the disclosed invention can be measured or determined by standard antibody-antigen assays, e.g., competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA.

[0090] As used herein, "kit" is any product (e.g., package or container) comprising at least one reagent, e.g., a probe for specifically detecting or controlling the expression of a marker of the present invention. The kit may be promoted, distributed, or sold as a constituent unit to carry out the method of the present invention.

[0091] As used herein, the term “monoclonal antibody” refers to an antibody that exhibits single-bind specificity and affinity for a specific epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody that exhibits single-bind specificity and has variable and constant regions derived from human germline or non-germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising a fusion of B cells and immortalized cells obtained from a genetically modified non-human animal, e.g., a genetically modified mouse, having a genome containing human heavy chain transgenes and light chain transgenes.

[0092] A “marker” is a gene whose expression level is altered from the expression level of normal or healthy tissues or cells and is associated with a disease state, e.g., cancer. A “marker nucleic acid” is a nucleic acid (e.g., mRNA, cDNA) encoded by or corresponding to the marker of the present invention. Such marker nucleic acids comprise DNA (e.g., cDNA) comprising the whole or partial sequence of any nucleic acid sequence listed in the sequence list or a complement thereof thereof. Marker nucleic acids also comprise RNA comprising the whole or partial sequence of any nucleic acid sequence listed in the sequence list or a complement thereof thereof, wherein all thymidine residues are substituted with uridine residues. A “marker protein” is a protein encoded by or corresponding to the marker of the present invention. The marker protein comprises the whole or partial sequence of any sequence listed in the sequence list. In some embodiments, the whole HHLA2 is used as a marker. In other embodiments, a fragment of HHLA2 is used as a marker. The terms “protein” and “polypeptide” are used interchangeably.

[0093] As used herein, the term "regulation" includes up-regulation and down-regulation, e.g., increasing or inhibiting a response.

[0094] The "normal" level of marker expression is the level of marker expression in the cells of a subject, e.g., a human patient, who does not suffer from a disease or disorder associated with abnormal marker levels. The "over-expression" or "significantly high expression level" of the marker refers to an expression level of the test sample that is greater than the standard error of the analysis used to evaluate the expression, preferably at least twice, more preferably three, four, five, or ten times the marker expression level of the control sample (e.g., a sample from a healthy subject not having a marker-associated disease), preferably the average marker expression level of several control samples. The "significantly low expression level" of the marker refers to an expression level of the test sample that is at least twice, more preferably three, four, five, or ten times lower than the marker expression level of the control sample (e.g., a sample from a healthy subject not having a marker-associated disease), preferably the average marker expression level of several control samples.

[0095] This level of "significance" may also be applied to any other measured parameters described herein, e.g., expression, inhibition, cytotoxicity, cell growth, etc.

[0096] The term "predetermined" biomarker amount and / or activity measure may be a biomarker amount and / or activity measure used to evaluate a subject who may be selected for a specific treatment, merely by example, to evaluate the response to a therapeutic agent alone or in combination with one or more immunotherapies, such as one or more modulators of the HHLA2 pathway, e.g., HHLA2, and modulators of one or more natural binding partners, e.g., TMIGD2 and / or KIR3DL3, and / or to evaluate a disease state. The predetermined biomarker amount and / or activity measure may be determined in a population of patients with or without cancer. The predetermined biomarker amount and / or activity measure may be a single number applicable equally to all patients, or the predetermined biomarker amount and / or activity measure may vary depending on a specific subgroup of patients. A subject's age, weight, height, and other factors may influence an individual's predetermined biomarker amount and / or activity measure. Furthermore, predetermined biomarker amounts and / or activities may be determined individually for each subject. In one embodiment, the amounts determined and / or compared in the method described herein are based on absolute measurements. In another embodiment, the amounts determined and / or compared in the method described herein are based on relative measurements, e.g., ratios (e.g., cell ratios or serum biomarkers normalized to housekeeping or other generally constant biomarker expression). The predetermined biomarker amounts and / or activity measurements may be any suitable standard. For example, the predetermined biomarker amounts and / or activity measurements may be obtained from the same or different humans being evaluated in the patient selection. In one embodiment, the predetermined biomarker amounts and / or activity measurements may be obtained from previous evaluations of the same patient. In this manner, the course of the patient selection can be monitored over time.Additionally, if the subject is human, the control group may be obtained from the evaluation of other humans or multiple humans, for example, a selected group of humans. In this manner, the degree of selection of the human being evaluated may be compared with other suitable humans, for example, other humans in a similar position to the human of interest, for example, people experiencing similar or the same condition and / or the same racial group.

[0097] The term “predictive” includes the use of biomarker nucleic acid and / or protein status, e.g., hyper- or hypo-activity, development, expression, growth, reduction, recurrence, or resistance of the tumor prior, during, or after therapy, to determine the likelihood of a cancer’s response to an immunomodulatory therapy, such as HHLA2 pathway modulator therapy (e.g., a modulator of the interaction between HHLA2 and one or more natural binding partners, e.g., TMIGD2 and / or KIR3DL3, alone or in combination with immunotherapy, e.g., immune checkpoint inhibitor therapy). The predictive use of such biomarkers is e.g., (1) increased or decreased copy number (e.g., FISH, FISH + SKY, single-molecule sequencing, e.g., at least J. Biotechnol.(by single-molecule sequencing as described in , 86:289-301, or by qPCR), overexpression or underexpression of biomarker nucleic acids (e.g., by ISH, Northern blot, or qPCR), increased or decreased biomarker proteins (e.g., by IHC) and / or biomarker targets, or increased or decreased activity of the analyzed human cancer type or cancer sample, e.g., 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; (2) its complete or relatively controlled presence or absence in biological samples from subjects suffering from cancer, e.g., human, e.g., tissue, whole blood, serum, plasma, oral scrape, saliva, cerebrospinal fluid, urine, feces, or bone marrow samples; (3) its complete or relatively controlled presence or absence in a clinical subgroup of patients with cancer (e.g., responding to or resistant to specific immunomodulatory therapies (e.g., HHLA2 pathway modulator therapy (e.g., HHLA2 and one or more natural binding partners, e.g., TMIGD2 and / or KIR3DL3, interaction modulators alone or in combination with immunotherapy)).

[0098] The terms "prevention," "preventive treatment," and similar terms mean reducing the likelihood of occurrence of a disease, disability, or condition in a subject who does not have the disease, disability, or condition but is at risk of or conducive to its occurrence.

[0099] The term “prognosis” includes the prediction of possible progression and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides an individual’s cancer prognosis. For example, the prognosis may be surgery, the occurrence of a clinical subtype of cancer (e.g., solid tumors such as lung cancer, melanoma, and renal cell carcinoma), the occurrence of one or more clinical factors, the occurrence of bowel cancer, or recovery from the disease.

[0100] The term "response to therapy" (e.g., HHLA2 pathway modulator therapy (e.g., a modulator of the interaction between HHLA2 and one or more natural binding partners, e.g., TMIGD2 and / or KIR3DL3, alone or in combination with immunotherapy, e.g., immune checkpoint inhibitor therapy)) relates to any response to therapy (e.g., HHLA2 pathway modulator therapy (e.g., a modulator of the interaction between HHLA2 and one or more natural binding partners, e.g., TMIGD2 and / or KIR3DL3, alone or in combination with immunotherapy, e.g., immune checkpoint inhibitor therapy)), and in the case of cancer, preferably to changes in cancer cell number, tumor mass, and / or volume following the initiation of neoadjuvant or adjuvant chemotherapy. The hyperproliferative response may be evaluated, for example, for efficacy or in neoadjuvant or adjuvant situations, wherein the tumor size following systemic intervention may be measured by CT, PET, mammography, ultrasound, or palpation and compared to the initial size and volume. Response may also be evaluated by caliper measurements of the tumor or pathological examination following biopsy or surgical resection. Response may be recorded in a quantitative manner, such as a percentage change in tumor volume, or in a qualitative manner, such as "pathological complete response (pCR)," "clinical complete remission (cCR)," "clinical partial remission (cPR)," "clinical stable disease (cSD)," "clinical progressive disease (cPD)," or other qualitative criteria. Evaluation of the response to hyperproliferative disorder may be performed early after the initiation of neoadjuvant or adjuvant therapy, for example, after a few hours, days, weeks, or preferably after a few months. Typical endpoints for response evaluation are the completion of neoadjuvant chemotherapy or the time of surgical removal of residual tumor cells and / or tumor site. This is typically 3 months after the initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the treatment described herein may be determined by measuring the clinical benefit rate (CBR).The clinical benefit rate can be measured by determining the sum of the percentage of patients with complete remission (CR), the number of patients with partial remission (PR), and the number of patients with stable disease (SD) at least 6 months after the end of therapy. The formula for this is CBR over 6 months = CR + PR + SD. In some embodiments, the CBR for a specific cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or higher. Additional criteria for evaluating the response to cancer therapy relate to "survival," which includes all of the following: survival to death, also known as overall survival (where said death may be unrelated to cause or related to the tumor); "recurrence-free survival" (where the term recurrence includes both local and primary recurrence); metastasis-free survival; and disease-free survival (where the term disease includes cancer and associated diseases). The above survival period may be calculated by referring to a defined start time (e.g., time of diagnosis or initiation of treatment) and end time (e.g., death, recurrence, or metastasis). Additionally, criteria for therapeutic efficacy may be extended to include response to chemotherapy, survival probability, probability of metastasis within a given time, and probability of tumor recurrence. For example, to determine an appropriate threshold, a specific cancer treatment regimen may be administered to a target population, and the outcome may be correlated with biomarker measurements determined prior to the administration of any immunomodulatory therapy. The outcome measurement may be the pathological response to the given regimen in a neoadjuvant setting. Alternatively, outcome degrees such as overall survival and disease-free survival may be monitored over time for subjects following an immunomodulatory therapy with known biomarker measurements. In certain embodiments, the dosage is a standard dosage known in the art for cancer treatments. The monitoring time of the subjects may vary.For example, the subject may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.

[0101] The term "resistance" (or "tolerance") means acquired or natural resistance of a cancer sample or mammal to immunomodulatory therapy (i.e., becoming unresponsive to treatment or having a reduced or limited response), e.g., 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, or a reduced response of 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more. The reduction in response may be measured by comparison with the same cancer sample or mammal before resistance was acquired, or by comparison with another cancer sample or mammal known to be non-resistant to treatment. Typical acquired resistance to chemotherapy is referred to as "multidrug resistance." Multidrug resistance may be mediated by P-glycoproteins or other mechanisms, or may occur when mammals are infected with multidrug-resistant microorganisms or combinations of microorganisms. Determining resistance to treatment is routine in the art and can be measured, for example, through cell proliferation assays and apoptosis assays, as described herein as "sensitization." In some embodiments, the term "reverse resistance" means that in situations where the primary cancer therapy (e.g., chemotherapy or radiation therapy) alone does not produce a statistically significant reduction in tumor volume compared to the volume of the untreated tumor, the use of a second agent in combination with the primary cancer therapy (e.g., chemotherapy or radiation therapy) can significantly reduce the tumor volume to a statistically significant level (e.g., p<0.05) compared to the volume of the untreated tumor. This generally applies to tumor volume measurements when the untreated tumor is growing logarithmically.

[0102] The terms "response" or "responsiveness" refer to a response to therapy. For example, an anti-cancer response includes a reduction in tumor size or inhibition of tumor growth. The term may also refer to an improved prognosis, such as an increased time to recurrence—that is, the time from the first screening for a second primary cancer to death without evidence of recurrence—or an increased overall survival—that is, the time from treatment to death from any cause. To respond or to have a response means that there is a beneficial endpoint achieved upon exposure to a stimulus. Alternatively, negative or harmful symptoms are minimized, alleviated, or attenuated upon exposure to the stimulus. It will be recognized that assessing the likelihood of a tumor or subject exhibiting a favorable response is equivalent to assessing the likelihood that the tumor or subject will not exhibit a favorable response (i.e., exhibit a lack of response or become non-responsive).

[0103] The terms "tolerance" or "non-responsiveness" refer to the refractiveness of cells, such as immune cells, to stimuli, for example, through activating receptors or cytokines. Non-responsiveness can occur, for example, due to exposure to immunosuppressants or high doses of antigens. Several independent mechanisms can induce tolerance. One mechanism is referred to as "anergy," defined as a state in which cells persist in vivo as non-responsive cells without differentiating into cells with effector functions. This refractiveness is generally antigen-specific and persists even after exposure to the tolerance antigen has ceased. For example, T cell anergy is characterized by a lack of cytokine production, such as IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, if cells are re-exposed to the same antigen (even if re-exposure occurs in the presence of a co-stimulating polypeptide), cytokine production fails, and consequently, proliferation fails. However, energizing T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell energization may be observed through a deficiency in IL-2 production by T lymphocytes, measured by ELISA or proliferation assays using indicator cell lines. Alternatively, reporter gene constructs may be used. For example, energizing T cells fail to initiate IL-2 gene transcription induced by a promoter of heterologous origin or by a multimer of the AP1 sequence found within the enhancer under the control of a 5' IL-2 gene enhancer (Kang et al. (1992) Science257:1134). Another mechanism is referred to as "exhaustion." T cell exhaustion is a state of T cell dysfunction that occurs in the course of many chronic infections and cancers. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from functional effector or memory T cells.

[0104] As used herein, the term "nucleic acid molecule" means including DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA. As used herein, antibodies or antibody portions that bind to HHLA2 (e.g., V H, V L The term “isolated nucleic acid molecule” in relation to nucleic acids encoding HHLA2 (e.g., mAbs 2G2, 4D1, 8A12, 8D2, 1C8, 2C4, 6D10, 4E5, and 6F10 and polyclonal antibodies) means a nucleic acid molecule in which the nucleotide sequence encoding the antibody or antibody portion is free from other nucleotide sequences encoding the antibody or antibody portion that bind to HHLA2 and other antigens, wherein the other sequence may naturally be present next to the nucleic acid within human genomic DNA.

[0105] Nucleic acids are "operably linked" when positioned in a functional relationship with other nucleic acid sequences. For example, if a promoter or enhancer influences the transcription of a coding sequence, this promoter or enhancer is operably linked to the coding sequence. In relation to transcriptional regulatory sequences, being operably linked means that the linked DNA sequences are continuous, adjacent, and within a reading frame where necessary to link two protein-coding regions. In the case of switch sequences, being operably linked indicates that the sequence can influence switch recombination.

[0106] "Over-expression" or "significantly high level of expression" of a marker refers to an expression level of a test sample that is greater than the standard error of the analysis used to evaluate expression, preferably at least twice the marker expression activity or level of a control sample (e.g., a sample of a healthy subject not having a marker-related disease), and preferably at least twice the average marker expression level of several control samples, 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 times or more higher. "Significantly lower expression levels" of the marker refer to expression levels of test samples that are at least twice, 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 times or more lower than the marker expression levels of control samples (e.g., samples from healthy subjects without marker-related diseases).

[0107] The antibodies described in the present invention may be used in any one of the well-known forms of immunoassay, including but not limited to radioimmunoassay, Western blot analysis, immunofluorescence analysis, enzyme immunoassay, immunoprecipitation analysis, chemiluminescence analysis, immunohistochemical analysis, dot blot analysis, or slot blot analysis. General techniques used to perform other variations of the various immunoassays and techniques mentioned above, such as in situ proximity ligation (PLA), fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), turbidity inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA), ELISA, etc., either alone or in combination with NMR, MALDI-TOF, LC-MS / MS, or in alternative applications, are known to those skilled in the art.

[0108] Reagents may also be used to monitor protein levels in cells or tissues, e.g., leukocytes or lymphocytes, for example, as part of a clinical trial procedure to monitor the optimal dosage of an inhibitor. Detection may be facilitated by the binding (e.g., physical linkage) of an antibody and a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include mustard hydrogen peroxide, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, danyl chloride, or phycoeridrine; Examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and equorin, and examples of suitable radioactive materials include 125 I, 131 I, 35S or 3 H is included.

[0109] These reagents may be used with any number of biological samples. Biological samples may be collected from various sources of the patient, including body fluid samples containing nucleic acids and / or proteins, cell samples, or tissue samples. In a preferred embodiment, the target and / or control samples are selected from the group consisting of cells, cell lines, tissue slides, paraffin-embedded tissue, biopsies, whole blood, nipple aspirates, serum, plasma, oral scrapes, saliva, cerebrospinal fluid, urine, feces, and bone marrow. In one embodiment, the sample is serum, plasma, or urine. In another embodiment, the sample is serum.

[0110] Samples may be collected repeatedly from an individual over a long period (e.g., at least once in the order of days, weeks, months, years, etc.). Obtaining multiple samples from an individual over time may be used to verify initial detection results and / or to identify changes in biological patterns as a result of, for example, disease progression or drug treatment. For example, subject samples may be taken and monitored according to the present invention at monthly, bi-monthly, or in combinations of one-month, two-month, or three-month intervals. Additionally, the biomarker quantity and / or activity measurements of the subject obtained over time can be conveniently compared with each other as well as with a general control group during the monitoring period, thereby providing the subject's own values ​​as an internal or individual control group for long-term monitoring.

[0111] The sample may include living cells / tissues, fresh frozen cells, fresh tissues, biopsies, fixed cells / tissues, cells / tissues embedded in a medium such as paraffin, tissue slides, or any combination thereof.

[0112] Sample preparation and separation may include any procedures depending on the type of collected sample and / or biomarker measurement analysis. Such procedures include, by example, concentration, dilution, pH titration, removal of high-concentration polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalination of the sample, concentration of sample proteins, extraction and purification of lipids.

[0113] Sample preparation may involve isolating molecules bound to non-covalent complexes with other proteins (e.g., transport proteins). This process may involve isolating molecules bound to specific transport proteins (e.g., albumin), or using more general processes such as protein denaturation—e.g., acid denaturation—to release molecules bound to all transport proteins and remove the transport proteins.

[0114] Removal of unwanted proteins from a sample (e.g., high concentrations, information-free, or undetectable proteins) 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 concentrations of proteins. Sample preparation may include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isopotential focusing, and related techniques. Molecular-weight filters include membranes that separate molecules based on size and molecular weight. Reverse osmosis, nanofiltration, ultrafiltration, and microfiltration may be additionally used for these filters.

[0115] When used with respect to a reference polypeptide, the term “polypeptide fragment” or “fragment” refers to a polypeptide from which amino acid residues have been deleted compared to the reference polypeptide itself, wherein the remaining amino acid sequence is generally identical to the corresponding position of the reference polypeptide. Such deletions may occur at the amino-terminus, internally, or at the carboxy-terminus of the reference polypeptide, or alternatively, both. The fragment is typically at least 5, 6, 8, or 10 amino acid lengths, at least 14 amino acid lengths, at least 20, 30, 40, or 50 amino acid lengths, at least 75 amino acid lengths, or at least 100, 150, 200, 300, 500, or more amino acid lengths. These are, for example, within the limit of being shorter than the length of the full-length polypeptide, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1020, 1040, 1060, 1080, 1100, 1120, 1140, 1160, 1180, 1200, 1220, 1240, 1260, 1280, 1300, 1320, 1340, or longer and / or including this length. Alternatively, they may not be longer than the above range within the limit of being shorter than the length of the full-length polypeptide and / or the above range may be excluded.

[0116] The term “probe” refers to any molecule capable of selectively binding to a specifically intended target molecule, e.g., a nucleotide transcript or a protein encoded by or corresponding to a marker. The probe may be synthesized by a person skilled in the art or derived from a suitable biological agent. For the purpose of detecting the target molecule, the probe may be specifically designed to be labeled as described herein. Examples of molecules that may be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0117] As used herein, the term "rearrangement" refers to V-segments that are essentially each complete V H and V L It refers to the arrangement of a heavy or light chain immunoglobulin locus located immediately adjacent to the DJ or J segment in the form encoding the domain. The rearranged immunoglobulin locus can be identified by comparison with germline DNA; the rearranged locus will have at least one recombinant heptamer / nonamer homolog.

[0118] As used herein, the term "recombinant host cell" (or simplified to "host cell") refers to a cell into which a recombinant expression vector has been introduced. The term should be understood to refer not only to specific target cells but also to the offspring of such cells. Because specific modifications may occur in subsequent generations due to mutations or environmental influences, the offspring may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0119] As used herein, the term “recombinant human antibody” comprises all human antibodies produced, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomic of the human immunoglobulin gene or hybridomas produced therefrom (as further described below); (b) antibodies isolated from host cells transformed to express antibodies, e.g., transfectomas; (c) antibodies isolated from a library of recombinant, recombinant human antibodies; and (d) antibodies produced, expressed, generated, or isolated by any other means including splicing a human immunoglobulin gene sequence into another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, these recombinant human antibodies may be affected by in vitro mutagenesis (or, in vivo mutagenesis if transgenic animals of human Ig sequences are used), and accordingly, the V of the recombinant antibody H and V L The amino acid sequence of the region is human germline V H and V L It is a sequence derived from and related to this sequence, but which may not naturally exist within the in vivo human antibody germline repertoire.

[0120] The term “co-stimulation” as used in relation to activated immune cells includes the ability of a co-stimulation polypeptide to provide a second, non-activating receptor-mediated signal (“co-stimulation signal”) that includes proliferative or effector functions. For example, the co-stimulation signal may induce cytokine secretion, e.g., cytokine secretion from T cells that have received a T cell-receptor-mediated signal. Immune cells that have received a cell-receptor-mediated signal, e.g., a cell-receptor-mediated signal via an activating receptor, are referred to herein as “activated immune cells.”

[0121] The term “co-stimulatory receptor” includes receptors that transmit co-stimulatory signals to immune cells, e.g., CD28. As used herein, the term “inhibitor receptor” includes receptors that transmit negative signals to immune cells (e.g., CTLA4, KIR3DL3, or PD-1). Even if a co-stimulatory receptor (e.g., CD28) is not present on the immune cell, an inhibitory signal converted by an inhibitory receptor may occur, and accordingly, the inhibitory signal is not merely a function of competition between the inhibitory receptor and the co-stimulatory receptor for the binding of the co-stimulatory polypeptide (Fallarino et al. (1998) J. Exp. Med.188:205). Inhibitory signaling to immune cells can result in non-reactive, energized, or programmed cell death within the immune cells. Preferably, the transmission of inhibitory signals operates through mechanisms not associated with apoptosis. As used herein, the term "apoptosis" includes programmed cell death that can be characterized using techniques known in the art. Apoptotic cell death may be characterized, for example, by cell shrinkage reaching the peak of cell fragmentation, membrane blistering, and chromatin condensation. Additionally, cells undergoing apoptosis exhibit a characteristic pattern of DNA cleavage between nucleosomes. Depending on the form of the polypeptide that binds to the receptor, for example, through competition with the active forms of HHLA2 and / or KIR3DL3 to bind to one or more natural binding partners, a signal may be transmitted (e.g., by the multivalent form of the HHLA2 and / or KIR3DL3 polypeptide) or inhibited (e.g., by the soluble, monovalent form of HHLA2 and / or KIR3DL3). However, there are instances where the soluble polypeptide can stimulate. The effect of the modulator can be easily demonstrated using the general screening assays described herein.

[0122] The terms “high,” “low,” “medium,” and “negative” relating to 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. The expression of the biomarker may be determined according to any method described herein, including but not limited to the analysis of the cellular level, activity, structure, etc., of one or more biomarker genomic nucleic acids, ribonucleic acids, and / or polypeptides. In one embodiment, these terms refer to a defined percentage of a cell population expressing the biomarker at the highest, medium, or lowest level, respectively. These percentages may be defined as any range including, or between, the top 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15% or more of the cell populations that express the biomarker highly or weakly. Since cells that do not detectably express the biomarker have "negative" biomarker expression, the term "low" excludes cells that do not detectably express the biomarker. The term "intermediate" includes cells that express the biomarker but at a lower level than the population expressing it at a "high" level. In other embodiments, this term may also refer to, or alternatively refer to, a population of cells expressing the biomarker identified by qualitative or statistical plot regions. For example, cell populations classified using flow cytometry may be distinguished based on biomarker expression levels by identifying distinct plots based on detectable moiety analysis, such as methods based on average fluorescence intensity, according to methods well known in the art. These plot regions may be refined according to numerical values, shapes, overlapping, etc., based on methods well known in the art for the biomarker of interest.In another embodiment, this term may also be determined by the presence or absence of additional biomarker expression.

[0123] As described above, the term “response” generally relates to determining, for example, the effect, efficacy, or outcome of the progression of a clinical intervention. In some embodiments, the response is directly related to changes in the mass and / or volume of the tumor following the initiation of the clinical intervention (e.g., administration of anti-HHLA2 monoclonal and polyclonal antibodies such as 2G2, 4D1, 8A12, 8D2, 1C8, 2C4, 6D10, 4E5, or 6F10). For example, a hyperproliferative response may be evaluated by measuring the size of the tumor after the systemic intervention compared to the starting size and volume using CT, PET, mammography, ultrasound, or palpation. Additionally, the response may be evaluated by caliper measurements or pathological examinations of the tumor following a biopsy or surgical resection. Response may be recorded in a quantitative manner, such as the percentage change in tumor volume, or in a qualitative manner, such as "pathological complete response (pCR)," "clinical complete remission (cCR)," "clinical partial remission (cPR)," "clinical stable disease (cSD)," "clinical progressive disease (cPD)," or other qualitative criteria. Evaluation may be performed early after the initiation of clinical intervention, for example, after a few hours, days, weeks, or preferably months. Response evaluation is typically concluded at the end of the clinical intervention or at the time of surgical removal of residual tumor cells and / or the tumor site.

[0124] As used herein, the term “specific binding” relates to an antibody that binds to a predetermined antigen. Typically, the antibody is approximately 10 when determined by surface plasmon resonance (SPR) technology on a BIACORE® analyzer using human HHLA2 as the analyte and the antibody as the ligand. -7 Less than M, for example, about 10 -8 M, 10-9 M or 10 -10 It binds with an affinity of less than or lower than M, and binds to the predetermined antigen with an affinity 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-fold or greater than the binding affinity to other non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigen. The phrases "antibody recognizing an antigen" and "antibody specific to an antigen" are used interchangeably with the term "antibody specifically binding to an antigen" in this specification.

[0125] As used herein, “Subject” refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from a disease or disorder associated with abnormal marker levels. The term “Subject” is interchangeable with “Patient.” The term “Non-human Animal” includes all vertebrates, e.g., non-human primates, mammals such as sheep, dogs, cattle, chickens, amphibians, reptiles, etc., and non-mammals.

[0126] The term “substantially free of chemical precursors or other compounds” includes preparations of antibodies, polypeptides, peptides, or fusion proteins separated from chemical precursors or other compounds associated with the synthesis of proteins. In one embodiment, the term “substantially free of chemical precursors or other compounds” 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 compounds, more preferably less than about 20% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein compounds, more preferably less than about 10% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein compounds, and most preferably less than about 5% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein compounds.

[0127] As used herein, the term "survival" includes all of the following: survival to death, also known as total survival (wherein death may be unrelated to the cause or related to the tumor); "recurrence-free survival" (wherein the term recurrence includes both local and primary recurrence); metastasis-free survival; and disease-free survival (wherein the term disease includes cancer and related diseases). The duration of said survival may be calculated with reference to a defined starting point (e.g., time of diagnosis or start of treatment) and an endpoint (e.g., death, recurrence, or metastasis). Additionally, the criteria for therapeutic efficacy may be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time, and probability of tumor recurrence.

[0128] "Transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or an analog of RNA or cDNA) that is complementary or homologous to all or part of the completed mRNA produced by the transcription of the marker of the present invention and, in any case, the general post-transcriptional process of the RNA transcript (e.g., splicing) and the reverse transcription of the RNA transcript.

[0129] As used herein, the term "T cell" includes CD4+ T cells and CD8+ T cells. Additionally, the term "T cell" includes both T helper type 1 T cells and T helper type 2 T cells. The term "antigen-presenting cell" includes specialized antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).

[0130] Traditional T cells, also known as Tconv or Teff, possess effector functions (e.g., cytokine secretion, cytotoxicity, anti-self-recognition, etc.) that enhance the immune response due to the ability to express one or more T cell receptors. Tconv or Teff is generally defined as any population of T cells that are not Tregs, including, for example, naive T cells, activated T cells, memory T cells, resting Tcons, or Tcons differentiated into Th1 or Th2 lineages. In some embodiments, Teff is a subpopulation of non-Treg T cells. In some embodiments, Teff is a 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. "Naive Tcons" are CD4 cells that have differentiated in the bone marrow and successfully undergone positive and negative central screening in the thymus but have not yet been activated by antigen exposure. + They are T cells. Naive Tcons are generally characterized by surface expression of L-selectin (CD62L), the absence of activating markers such as CD25, CD44, or CD69, and the absence of memory markers such as CD45RO. Therefore, naive Tcons are considered to be quiescent and non-dividing, requiring interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see at least WO 2010 / 101870). The presence and activity of these cells are undesirable in the context of suppressing the immune response. Unlike Tregs, Tcons are not energetic 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, depleted cells may exhibit characteristics of anergy.

[0131] The terms “unrearranged” or “germline arrangement” as used herein in relation to V segments refer to an arrangement in which the V segment is not rearranged so as to be immediately adjacent to the D or J segment.

[0132] As used herein, the term "vector" refers to a nucleic acid capable of transporting other connected nucleic acids. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA fragments can be ligated. Another type of vector is a viral vector to which additional DNA fragments can be ligated within the viral genome. Certain vectors are capable of autonomous replication in the introduced host cell (e.g., bacterial vectors of bacterial replication origin and episomal mammalian vectors). Other vectors (e.g., non-episosomal mammalian vectors) are incorporated into the host cell's genome upon introduction into the host cell and are consequently replicated along with the host genome. Additionally, certain vectors can regulate the expression of operably linked genes. In this specification, such vectors are referred to as "recombinant expression vectors" or, for simplicity, "expression vectors." Generally, expression vectors useful for recombinant DNA technology are in the form of plasmids. In this specification, "plasmid" and "vector" may be interchangeably used for plasmids, which are the most commonly used form of a vector. However, the present invention is intended to include other forms of expression vectors, such as viral vectors that provide equivalent functions (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0133] In the case of nucleic acids, the term "substantial homology" indicates that when two nucleic acids, or when optimally aligned and compared, are identical in their specified sequences, including appropriate nucleotide insertions or deletions, in at least about 80% of nucleotides, generally in at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more nucleotides, and more preferably in at least about 97%, 98%, 99%, or more nucleotides. Alternatively, substantial homology exists when a fragment hybridizes to the strand's complement under selective hybridization conditions.

[0134] The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that must be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % identity = number of identical positions / total number of positions × 100). Comparing sequences and determining the percentage identity between two sequences can be achieved using a mathematical algorithm as described in the non-limiting examples below.

[0135] The percentage identity between two nucleotide sequences can be determined using the NWSgapdna.CMP matrix and the GAP program of the GCG software package (available on the GCG website) using gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Additionally, the percentage 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)) integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, percentage identity between two amino acid sequences can be determined using the Blosum 62 matrix or PAM250 matrix, and the Needleman and Wunsch (J. Mol. Biol. (48):444 453 (1970)) algorithm integrated into the GAP program of the GCG software package, using gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6.

[0136] The nucleic acid and protein sequences of the present invention may also be used as "query sequences" to perform a search in a public database, for example, to identify related sequences. Such a search is [Altschul, et al.This can be performed using the NBLAST and XBLAST programs (version 2.0) of [(1990) J. Mol. Biol. 215:403 10]. To obtain a nucleotide sequence homologous to the nucleic acid molecule of the present invention, a BLAST nucleotide search can be performed using the NBLAST program, score=100, wordlength=12. To obtain an amino acid sequence homologous to the protein molecule of the present invention, a BLAST protein search can be performed using the XBLAST program, score=50, wordlength=3. To obtain a gap alignment for comparison purposes, [Altschul et al. Gap BLAST as described in

[1997] Nucleic Acids Res. 25(17):3389 3402] may be used. When using BLAST and Gap BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used (available on the NCBI website).

[0137] Nucleic acids may exist in intact cells, cell lysates, or in partially purified or substantially pure forms. Alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other standard techniques well known in this field (F. Ausubel, et al. When purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by means of ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987), the nucleic acid is “isolated” or “substantially purified.”

[0138] The term “determining an appropriate therapeutic regimen for a subject” is considered to mean determining the initiation, modification, and / or termination of a therapeutic regimen for a subject (i.e., a monotherapy or combination with other therapies used to prevent and / or treat the subject’s cancer) based on the results of the analysis according to the present invention, or essentially, or at least partially. One embodiment is determining whether to provide a targeted therapy for cancer to provide immunomodulatory therapy (e.g., HHLA2 pathway modulator therapy (e.g., modulators of interactions between HHLA2 and one or more natural binding partners such as TMIGD2 and / or KIR3DL3)). Another embodiment is initiating adjuvant therapy after surgery for the purpose of reducing the risk of recurrence, and another embodiment is changing the dosage of a specific chemotherapy. The decision may be based on the individual characteristics of the subject being treated, in addition to the results of the analysis according to the present invention. In most cases, the determination of a therapeutic regimen actually suitable for the subject is performed by a physician.

[0139] II. 단일클론성 항체, 면역글로불린 및 폴리펩타이드

[0140] The present invention relates in part to a monoclonal antibody or a fragment thereof isolated for HHLA2 (e.g., the monoclonal antibody and polyclonal antibody listed herein). These molecules are characterized in part by exhibiting the ability to recognize the HHLA2 protein in diagnostic assays such as immunohistochemistry (IHC), Western blot, intercellular flow, ELISA, etc. These molecules are characterized in part by exhibiting the ability to inhibit HHLA2 by binding to receptors such as receptors expressed on T-cells (e.g., TMIGD2 and KIR3DL3).

[0141] 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, and B7-H7, refers to a member of the B7 family. The expression of the HHLA2 protein is limited in normal human tissues but is broadly expressed in cancer. The HHLA2 protein is a protein member possessing three Ig-like domains (IgV-IgC-IgV), whereas other members of the B7 family generally possess only two Ig domains (IgV-IgC). In normal human tissues, the HHLA2 protein is expressed in the epithelium of the kidney, intestines, gallbladder, and breast, as well as in placental trophoblast cells. In the immune system, the HHLA2 protein is constitutively expressed in human monocytes / macrophages. HHLA2 regulates human T-cell function, including inhibiting T-cell proliferation and cytokine production, and increasing T-cell and cytokine production. HHLA2 is expressed at high levels in a wide range of human cancers, including the colorectal, kidney, lung, pancreas, ovary, and prostate. Additionally, HHLA2 is expressed in human cancers of the thyroid, melanoma, liver, bladder, colon, kidney, breast, and esophagus.

[0142] The structure and function of HHLA2 are well known in this field as described above (e.g., literature [Xiao et al . (2015) Clinical Cancer Research 21:2201-2203, Janakiram et al . (2015) Clinical Cancer Research 21:2359-2366, Mager et al. (1999) Genomics 21:2359-2366, Flajnik et al. (2012) Immunogenet. 64:571-590, Zhao et al. (2013) Proc. Natl. Acad. Sci. USA 110:9879-9884 and Zhu et al. (2013) Nat. Commun. [Refer to 4:2043]).

[0143] The term "HHLA2" is intended to include its fragments, variants (e.g., allelic variants), and derivatives. Representative human HHLA2 cDNA and human HHLA2 protein sequences are well known in the field and are publicly available from the National Center for Biotechnology Information (NCBI). Human HHLA2 variants include Variant 1 (NM_007072.3 and NP_009003.1, representing the longest transcript and encoding the longest isomorph a), Variant 2 (NM_001282556.1 and NP_001269485.1, indicating the use of an alternative promoter and differing in the 5' UTR compared to Variant 1), Variant 3 (NM_001282557.1 and NP_001269486.1, indicating the use of an alternative promoter and differing in the 5' UTR compared to Variant 1), and Variant 4 (NM_001282558.1 and NP_001269487.1, encoding isomorph b, indicating the use of an alternative promoter, differing in the 5' UTR compared to Variant 1, and an alternative within the 3' coding region). Includes variants with a deleted in-frame exon, producing a shorter isoform than isoform a), and variant 5 (NM_001282559.1 and NP_001269488.1, encoding isoform c, indicating the use of an alternative promoter, having several differences compared to variant 2, with a unique 5' UTR, translation initiated at an alternative start codon compared to variant 1, a unique N-terminus, and producing a shorter isoform than isoform a). Nucleic acid and polypeptide sequences of HHLA2 orthologs in non-human organisms are well known, including, for example, frog HHLA2 (NM_001128644.1 and NP_001122116.1). Representative sequences of HHLA2 orthologs are shown in Table 1 below.

[0144] Anti-HHLA2 antibodies suitable for HHLA2 protein detection are well known in this field, for example: Cat #: ab107119 and ab214327 (abcam), antibodies PA5-24146 and PA5-6313 (ThermoFisher Scientific), antibodies MAB80841, AF8084, FAB80841R, FAB80841T and MAB8084 (R&D Systems), antibody AP52042PU-N (Origene), antibodies NBP2-49187, MAB80842, H00011148-B01P and NBP2-32420 (Novus Biologicals), antibody GTX51981 (GeneTex), antibody HPA055478 (Atlas Antibodies), antibodies LS-C321945, LS-C308228, Includes LS-C246742, LS-C246743, LS-C246744, LS-C236210 and LS-C249186 (LifeSpan Biosciences), etc.In addition, various siRNA, shRNA, and CRISPR constructs for reducing HHLA2 expression include Origene Technologies' shRNA products # TL312462, TF312462, TR312462, TG312462, and TL312462V, siRNA product # SR323358, and abm's siRNA products # i009616, i009616a, i009616b, i009616c, i009616d, iV009616, iV009616a, iV009616b, iV009616c, iV009616d, iAAV00961600, iAAV00961601, iAAV00961602, iAAV00961603, iAAV00961604, iAAV00961605, iAAV00961606, iAAV00961607, iAAV00961608 and iAAV00961609, CRISPR products # K0950321, K0950301, K0950302, K0950303, K0950304, K0950305, K0950306, K0950307, ​​K0950308 and K0950311, Santa Cruz Biotechnology's siRNA product # sc-78498, shRNA products # sc-78498-V and sc-78498-SH, CRISPR products # sc-411576, sc-411576-HDR, sc-411576-NIC and It can be found in the product list of the aforementioned company, such as c-411576-NIC-2. It should be noted that this term may also be used to refer to any combination of features described herein in relation to the HHLA2 molecule. For example, any combination of sequence composition, percentage identity, sequence length, domain structure, functional activity, etc., may be used to describe the HHLA2 molecule of the present invention.

[0145] The term "HHLA2 pathway" includes the interaction of HHLA2 with one or more natural binding partners, such as TMIGD2 and KIR3DL3.

[0146] The term "TMIGD2" refers to a membrane protein sharing approximately 10% amino acid identity with CD28, CTLA-4, ICOS, and PD-1, specifically transmembrane and immunoglobulin domain containing 2, CD28H, IGPR1, and IGPR-1. TMIGD2 possesses one extracellular IgV-like domain, a transmembrane region, and a proline-rich cytoplasmic domain containing two tyrosine signaling motifs. The TMIGD2 protein is constitutively expressed on all naive T cells and most natural killer (NK) cells, but not on regulatory T cells or B cells. TMIGD2 expression is gradually lost upon repeated stimulation of T cells. Consistently, TMIGD2 is expressed in only about half of memory T cells, and TMIGD2-negative T cells exhibit terminal differentiation and senescence phenotypes. In addition, TMIGD2 was found to be expressed in endothelial and epithelial cells and to function in reducing cell migration and promoting capillary formation during the angiogenesis process.

[0147] The structure and function of TMIGD2 are well known in this field as described above (e.g., literature [Xiao et al . (2015) Clinical Cancer Research 21:2201-2203, Janakiram et al . (2015) Clinical Cancer Research 21:2359-2366, Zhu et al. (2013) Nat. Commun. 4:2043 and Rahimi (2012) Cell [See 23:1646-1656]).

[0148] The term "TMIGD2" is intended to include its fragments, variants (e.g., allelic variants), and derivatives. Representative human TMIGD2 cDNA and human TMIGD2 protein sequences are well known in the field and are publicly available from the National Center for Biotechnology Information (NCBI). Human TMIGD2 isomorphs include isomorph 1 (NM_144615.2 and NP_653216.2), isomorph 2 (NM_001169126.1 and NP_001162597.1; using alternative in-frame splice sites in the 3' coding region compared to isomorph 1, generating a shorter isomorph compared to isomorph 1), and isomorph 3 (NM_001308232.1 and NP_001295161.1; having a missing alternative in-frame exon in the 5' coding region compared to isomorph 1, generating a shorter isomorph compared to isomorph 1). The nucleic acid and polypeptide sequences of the TMIGD2 ortholog of organisms other than humans are well known, 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 the TMIGD2 ortholog are shown in Table 1 below.

[0149] Anti-TMIGD2 antibodies suitable for detecting TMIGD2 protein are well known in this field, including, for example, antibodies Cat # MAB8316, MAB83162, FAB8316R, FAB83162R, FAB83162G, FAB83162N, FAB83162S, FAB83162T, FAB83162U and FAB83162V (R&D systems), antibody TA326695 (Origene), antibodies PA5-52787 and PA5-38055 (ThermoFisher Scientific), antibodies MAB83161 and NBP1-81164 (Novus Biologicals), etc.In addition, various siRNA, shRNA, and CRISPR constructs for reducing TMIGD2 expression include Origene Technologies' shRNA products # TF317829, TG317829, TL317829, TR317829, and TL317829V, siRNA product # SR314913, and CRISPR products # KN204938, KN204938LP, KN204938RB, and KN204938BN, and Abm's siRNA products # i024914, i024914a, i024914b, i024914c, i024914d, iV024914, iV024914a, iV024914b, iV024914c, iV024914d, iAAV02491400, iAAV02491401, iAAV02491402, iAAV02491403, iAAV02491404, iAAV02491405, iAAV02491406, iAAV02491407, iAAV02491408 and iAAV02491409 and CRISPR products # K2409321, K2409301, K2409302, K2409303, K2409304, K2409305, K2409306, K2409307, K2409308 and K2409311, Santa Cruz Biotechnology's siRNA product # sc-97757, shRNA product # sc-97757-SH and can be found in the product list of the aforementioned companies, such as sc-97757-V and CRISPR products # sc-414261, sc-414261-HDR, sc-414261-NIC, and sc-414261-NIC-2, and Vigene Biosciences' shRNA products # SH888208 and SH874720.Additionally, various CRISPR constructs for increasing the expression of TMIGD2 can be found in the product list of the aforementioned companies, such as Abm’s CRISPR products # K2409378, K2409377, K2409376, K2409375, K2409374, K2409373, K2409372, and K2409371, and Santa Cruz Biotechnology’s CRISPR products # sc-414261-ACT, sc-414261-ACT-2, sc-414261-LAC, and sc-414261-LAC-2. It should be noted that this term may also be used to refer to any combination of features described herein in relation to the TMIGD2 molecule. For example, any combination of sequence composition, percentage identity, sequence length, domain structure, functional activity, etc., may be used to describe the TMIGD2 molecule of the present invention.

[0150] The interaction between TMIGD2 and HHLA2, as well as their functions, are well known in this field as described above (e.g., Xiao et al . (2015) Clinical Cancer Research 21:2201-2203 and Janakiram et al . (2015) Clinical Cancer Research See 21:2359-2366).

[0151] The term "KIR3DL3" refers to a member of a transmembrane glycoprotein family expressed by natural killer cells and T cell subpopulations, also known as Killer cell immunoglobulin-like receptor 3DL3, CD158Z, KIR3DL7, KIR44, KIRC1, KIR2DS2, and three Ig domains and long cytoplasmic tail 3. Killer cell immunoglobulin-like receptor (KIR) genes are polymorphic, highly homologous, and found in a cluster on chromosome 19q13.4 within the 1 Mb leukocyte receptor complex (LRC). Although several "framework" genes are found in all haplotypes (KIR3DL3, KIR3DP1, KIR3DL4, KIR3DL2), the genetic content of the KIR gene cluster varies by haplotype. KIR proteins are classified according to the number of extracellular immunoglobulin domains (2D or 3D) and whether they possess a long cytoplasmic domain (L) or a short cytoplasmic domain (S). KIR proteins with a long cytoplasmic domain transmit inhibitory signals upon ligand binding via the immunotyrosine-based inhibitory motif (ITIM), whereas KIR proteins with a short cytoplasmic domain are associated with TYRO proteins that bind to tyrosine kinases and lack the ITIM motif, instead transmitting an activation signal. The ligands of some KIR proteins are subgroups of HLA class I molecules; therefore, KIR proteins are thought to play an important role in regulating immune responses. This gene is one of the "framework" loci present in all haplotypes.The KIR3DL3 protein has an N-terminal signal sequence, three Ig domains, a transmembrane region lacking positively charged residues, and a long cytoplasmic tail containing an immune receptor tyrosine-based inhibition motif (ITIM). KIR3DL3 lacks the stalk region found in other KIRs.

[0152] The structure and function of KIR3DL3 are well known in this field as described above (e.g., literature [Hsu et al . (2002) Immunol Rev. 190:40-52, Trompeter et al . (2005) J. Immunol. 174:4135-4143, Trundley et al . (2006) Immunogenet. 57:904-916 and Jones et al . (2006) Immunogenet. [See 58:614-627]).

[0153] The term "KIR3DL3" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human KIR3DL3 cDNA and human KIR3DL3 protein sequences are well known in the field 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 the transcript (NP_703144.3). The nucleic acid and polypeptide sequences of KIR3DL3 ossolologs in non-human organisms are well known, for example, chimpanzee KIR3DL3 (XM_003316679.3 and XP_003316727.3), rhesus 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 KIR3DL3 ossololog sequences are shown in Table 1 below.

[0154] Anti-KIR3DL3 antibodies suitable for detecting KIR3DL3 protein are well known in this field, including, for example, antibodies Cat #: FAB8919R, MAB8919, FAB8919G, FAB8919N, FAB8919S, FAB8919T, FAB8919U and FAB8919V (R&D systems), antibody AP52374PU-N (Origene), antibody PA5-26178 (ThermoFisher Scientific), antibodies OAAB05761, OAAF08125, OAAN04122, OACA09134, OACA09135, OACD04988 and OASG01190 (Aviva Systems Biology).In addition, various siRNA, shRNA, and CRISPR constructs for reducing KIR3DL3 expression include Origene Technologies' shRNA products # TF303684, TR303684, TG303684, TL303684, TL303684V, siRNA product # SR314516 and CRISPR products # KN224383, KN224383BN, KN224383RB and KN224383LP, and Abm's 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, K1151407, K1151408 and K1151411, Santa Cruz Biotechnology's siRNA product # sc-60892, shRNA product # sc-60892-SH and sc-60892-V and CRISPR products # sc-406227, sc-406227-KO-2, sc-406227-HDR-2, sc-406227-NIC, and sc-406227-NIC-2, etc., can be found in the product list of the company referenced above. It should be noted that this term may also be used to refer to any combination of features described herein in relation to the KIR3DL3 molecule. For example, any combination of sequence composition, percentage identity, sequence length, domain structure, functional activity, etc., may be used to describe the KIR3DL3 molecule of the present invention.

[0155] The term "peripheral blood cell subtype" refers to cell types commonly found in peripheral blood, including but not limited to eosinophils, neutrophils, T cells, monocytes, NK cells, granulocytes, and B cells.

[0156] The term “recombinant human antibody” includes all human antibodies produced, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomic animals of human immunoglobulin genes or hybridomas produced therefrom (as further described below), (b) antibodies isolated from host cells transformed for antibody expression, e.g., from transfactomas, (c) antibodies isolated from a library of recombinant, recombinant human antibodies, and (d) antibodies produced, expressed, generated, or isolated by any other means including splicing a human immunoglobulin gene sequence into another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, if transgenic animals of human Ig sequences are used, in vivo mutagenesis), and accordingly, the V of the recombinant antibody H and V L The amino acid sequence of the region is human germline V H and V L It is a sequence derived from and related to this sequence, but which may not naturally exist within the in vivo human antibody germline repertoire.

[0157] The term “sample” used to detect or determine the presence or level of at least one biomarker is generally whole blood, plasma, serum, saliva, urine, stool, tears, and any other body fluid (e.g., as described above according to the definition of “body fluid”), or tissue samples such as small intestine, colon samples, or surgically resected tissue (e.g., biopsy). In certain examples, 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.

[0158] As used herein, "RNA interference substance" is defined as any substance that interferes with or inhibits the expression of a target biomarker through RNA interference (RNAi). Such RNA interference substances include, but are not limited to, nucleic acid molecules comprising RNA molecules or fragments thereof that are homologous to the target biomarker gene of the present invention, short interfering RNA (siRNA), and small molecules that interfere with or inhibit the expression of the target biomarker nucleic acid through RNA interference (RNAi).

[0159] RNA interference (RNAi) is an evolutionarily conserved process in which the expression or introduction of RNA with a sequence identical or very similar to that of a target biomarker nucleic acid results in sequence-specific degradation of messenger RNA (mRNA) transcribed from a target gene or sequence-specific post-transcriptional gene silencing (PTGS) (Coburn, G. and Cullen, B. (2002)). J. of Virology(See 76(18):9225), and this results in the inhibition of the expression of the target biomarker nucleic acid. In one embodiment, this RNA is double-stranded RNA (dsRNA). This process has been described in plant, invertebrate, and mammalian cells. Originally, RNAi is initiated by a dsRNA-specific endonuclease Dicer that facilitates the cleavage of long dsRNA into a double-stranded fragment called siRNA. The siRNA is incorporated into a protein complex that recognizes and cleaves the target mRNA. Additionally, RNAi may be initiated by the introduction of nucleic acid molecules, such as synthetic siRNA, shRNA, or other RNA interfering substances, to inhibit or silence the expression of the target biomarker nucleic acid. As used herein, "inhibition of target biomarker nucleic acid expression" or "inhibition of marker gene expression" includes any reduction in the expression, activity, or level of the target biomarker nucleic acid or the protein encoded by the target biomarker nucleic acid. This reduction may be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to the expression of the target biomarker nucleic acid not targeted by the RNA interference substance or the activity or level of the protein encoded by this target biomarker nucleic acid.

[0160] In addition to RNAi, genome editing may be used to regulate the copy number or gene sequence of a biomarker of interest, such as constitutive or inductive knockout or mutation of biomarkers of interest like HHLA2 pathway components, e.g., HHLA2, TMIGD2, and / or KIR3DL3. For example, a CRISPR-Cas system may be used for the precise editing of genomic nucleic acids (e.g., to generate non-functional or null mutations). In such embodiments, CRISPR guide RNA and / or Cas enzymes may be expressed. For example, a vector containing only guide RNA may be administered to animals or cells transgenic of the Cas9 enzyme. Similar strategies may be used (e.g., designer zinc fingers, transcriptional activator-like effectors (TALEs), or homing meganucleases). Such systems are well known in the field (e.g., U.S. Patent No. 8,697,359; literature [Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; US Pat. Publ. 2014 / 0087426 and 2012 / 0178169; Boch et al. (2011) Nat. Biotech. 29:135-136; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al. (2011) PLoS One 6:e19722; Li et al. (2011) Nucl. Acids Res. 39:6315-6325; Zhang et al. (2011) Nat. Biotech. 29:149-153; Miller et al. (2011) Nat. Biotech. 29:143-148; Lin et al. (2014) Nucl. Acids Res. [See 42:e47]). In these genetic strategies, constitutive expression systems or inductive expression systems can be used according to methods well known in this field.

[0161] "Peewe-interacting RNA (piRNA) is the largest class of small non-coding RNA molecules. PiRNA forms RNA-protein complexes through interactions with peewe proteins. These piRNA complexes are associated with both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germline cells, particularly in spermatogenesis. They are distinguished from microRNA (miRNA) by their size (26–31 nt rather than 21–24 nt), loss of sequence conservation, and increased complexity. However, like other small RNAs, piRNA is thought to be associated with gene silencing, particularly the silencing of transposons. Most piRNAs are antisense to transposon sequences, suggesting that transposons are piRNA targets. PiRNA activity of transposons is most important in mammalian embryonic development, and C. elegans piRNA is required for sperm formation in humans. piRNA plays a role in silencing RNA through the formation of the RNA-induced silencing complex (RISC).

[0162] An "aptamer" is an oligonucleotide or peptide molecule that binds to a specific target molecule. "Nucleic acid aptamers" are nucleic acid species produced through intra-test selection or SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets, such as small molecules, proteins, nucleic acids, as well as cells, tissues, and organisms. "Peptide aptamers" are artificial proteins selected or produced to bind to a specific target molecule. These proteins consist of one or more peptide loops of variable sequences marked by a protein scaffold. They are typically isolated from combinatorial libraries and are often subsequently improved through direct mutation or variable region mutagenesis and selection processes. "Affimer proteins," which are an evolution of peptide aptamers, are small, highly stable proteins produced to exhibit peptide loops that provide a high-affinity binding surface for specific target proteins. This is a low molecular weight protein of 12 to 14 kDa derived from the cystatin cysteine ​​protease inhibitor family. Aptamers are useful in biotechnological and therapeutic applications because they provide molecular recognition properties comparable to antibodies, which are commonly used biomolecules. In addition to differential recognition, aptamers offer advantages over antibodies because they can be produced entirely in vitro, can be easily produced by chemical synthesis, possess desirable storage properties, and induce little to no immunogenicity in therapeutic applications.

[0163] "Short interfering RNA" (siRNA), also referred to herein as "small interfering RNA," is defined as a substance that acts to inhibit the expression of target biomarker nucleic acids, for example, by RNAi. siRNA may be chemically synthesized, produced by transcription in vitro, or 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, more preferably about 19, 20, 21, or 22 nucleotides, and may include a 3' and / or 5' overhang having a length of about 0, 1, 2, 3, 4, or 5 nucleotides on each strand. The lengths of these overhangs are independent between the two strands, that is, the overhang length of one strand does not depend on the overhang length of the other strand. Preferably, siRNA can promote RNA interference through the degradation of target messenger RNA (mRNA) or specific post-transcriptional gene silencing (PTGS).

[0164] In another embodiment, the siRNA is a small hairpin (or stem loop) RNA (shRNA). In one embodiment, this shRNA consists of a short (e.g., 19 to 25 nucleotides) antisense strand, followed by a 5 to 9 nucleotide loop and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop and the antisense strand may follow. This shRNA may be included in plasmids, retroviruses, and lentiviruses and, for example, the pol III U6 promoter or other promoters (e.g., the literature [Stewart, et al. (2003) RNA [See Apr;9(4):493-501] (incorporated by reference in this specification) may be expressed.

[0165] RNA interference substances, such as siRNA molecules, may be administered to patients who have cancer or are at risk of having cancer in order to treat, prevent, or inhibit cancer in subjects by inhibiting the expression of biomarker genes that are overexpressed in cancer.

[0166] The term "small molecule" is a term in the art and includes molecules with a molecular weight of less than about 1,000 or less than about 500. In one embodiment, the small molecule does not include only peptide bonds. In another embodiment, the small molecule is not an oligomer. Exemplarily, small molecule compounds for which activity can be screened include peptides, peptidomimetic compounds, nucleic acids, carbohydrates, and small organic molecules (e.g., polyketides) (Cane et al. 1998. Science 282:63) and a library of natural extracts are included, but not limited thereto. In other embodiments, this compound is a small organic non-peptide compound. In further embodiments, the small molecule is not a biosynthetic material.

[0167] The terms "selective modulator" or "selective modulation," applied to biologically active substances, refer to the ability of a substance to regulate targets, such as cell populations or signaling activities, in comparison to non-target cell populations or signaling activities through direct or indirect interactions with the target. For example, a substance that selectively inhibits interactions between HHLA2 and one or more natural binding partners such as TMIGD2 and KIR3DL3, interactions between HHLA2 and other binding partners, and / or these interactions on a cell population of interest may be active for HHLA2 pathway modulator therapy (e.g., as a modulator of interactions between HHLA2 and one or more natural binding partners such as TMIGD2 and KIR3DL3, 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%, 2× (times) or greater of a modifier (e.g., at least about 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, 50×, 55×, 60×, 65×, 70×, 75×, 80×, 85×, 90×, 95×, 100×, 105×, 110×, 120×, 125×, 150×, 200×, 250×, 300×, 350×, 400×, 450×, 500×, 600×, 700×, 800×, 900×, 1000×, 1500×, 2000×, 2500×, 3000×, 3500×, 4000×, 4500×, 5000×, 5500×, 6000×, 6500×, 7000×, 7500×, 8000×, 8500×, 9000×, 9500×, 10000× or more, or any range between, including)).These indicators are generally expressed in terms of the relative amount of substance required to reduce the interaction / activity by half.

[0168] More generally, the term "selective" refers to a preferential action or function. The term "selective" can be quantified in terms of the preferential effect on a specific target of interest relative to other targets. For example, the measured variable (e.g., regulation of Treg / Breg versus other cells, e.g., other immune cells such as Tcon) is 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 11x, 12x, 13x, 14x, It may vary by 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 times or more, or any range between (e.g., 50% to 16 times). The magnitude of the effect can be determined in a given tissue, cell population, measurement variable, measurement effect, etc., such as Treg:Tcon ratio, Breg:Tcon ratio, hyperproliferative cell growth rate or volume, Treg / Breg proliferation rate or number, etc., using the same multiplication analysis.

[0169] In contrast, the term "specific" refers to an exclusive action or function. For example, the specific regulation of the HHLA2-TMIGD2 and HHLA2-KIR3DL3 interaction refers to the exclusive regulation of each of the HHLA2-TMIGD2 and HHLA2-KIR3DL3 interactions, rather than the regulation of HHLA2 by other ligands. In another example, the specific binding of an antibody to a predetermined antigen refers to the ability of an antibody to bind to the antigen of interest without binding to other antigens. Generally, when determined by Surface Plasmon Resonance (SPR) technology on a BIACORE® analyzer using the antigen of interest as the analyte and the antibody as the ligand, the antibody is approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or a value less than or equal to approximately 1×10 -7 Affinity less than M (K D It binds with ), and binds to the predetermined antigen with an affinity of 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-fold or greater compared to the binding affinity for non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or closely associated antigen. Additionally, K D is K A It is the opposite of. The phrases "antibody recognizing an antigen" and "antibody specific to an antigen" are used interchangeably with the term "antibody specifically binding to an antigen" in this specification.

[0170] The term "sensitize" refers to a therapy (e.g., It means altering cells, such as cancer cells or tumor cells, in a manner that allows for more effective treatment with HHLA2 pathway modulator therapy (e.g., modulators of interactions between HHLA2 and one or more natural binding partners such as TMIGD2 and KIR3DL3) alone or in combination with immunotherapy such as immune checkpoint inhibitor therapy). In some embodiments, normal cells are altered by therapy (e.g., It is not affected to the extent of being excessively impaired by HHLA2 pathway modulator therapy (e.g., modulators of interactions between HHLA2 and one or more natural binding partners such as TMIGD2 and KIR3DL3) alone or in combination with immunotherapy such as immune checkpoint inhibitor therapy. Increased or decreased sensitivity depending on therapeutic treatment is particularly related to cell proliferation assays (Tanigawa N, Kern DH, Kikasa Y, Morton DL, Cancer Res 1982; 42: 2159-2164), apoptosis 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 AJP, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, PA: Harwood Academic Publishers, 1993: 415-432; The treatments and methods described in the present specification below, including but not limited to Weisenthal LM, Contrib Gynecol Obstet 1994; 19: 82-90), are measured according to methods known in the art. Additionally, sensitivity or resistance may be measured in animals by measuring tumor size reduction over a period of time, e.g., 6 months in humans and 4 to 6 weeks in mice.If the increase in therapeutic sensitivity or the decrease in resistance is 5% or more, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, or up to 2, 3, 4, 5, 10, 15, 20 times or more, compared to the therapeutic sensitivity or resistance in the absence of the composition or method, the composition or method sensitizes the response to therapeutic treatment. Determining sensitivity or resistance to therapeutic treatment is common practice in this field and within the skill of those skilled in the art. Any method described herein for enhancing the efficacy of immunomodulation can be equally applied to a method for sensitizing hyperproliferative or cancerous cells (e.g., resistant cells) to a therapy.

[0171] The term "synergy effect" refers to the combined effect of two or more therapeutic substances, such as two or more HHLA2 pathway modulators that may be greater than the sum of the independent effects of the anticancer drugs alone, HHLA2 pathway modulators and immunotherapy, or combinations with HHLA2 pathway modulators alone or immunotherapy such as immune checkpoint inhibitor therapy.

[0172] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from the condition of interest (e.g., cancer). The term "subject" is used interchangeably with "patient."

[0173] The term "survival" includes all of the following: survival to death, also referred to as total survival (where death may be unrelated to the cause or related to the tumor); "recurrence-free survival" (where the term recurrence includes both local and primary recurrence); metastasis-free survival; and disease-free survival (where the term disease includes cancer and related diseases). The duration of said survival may be calculated with reference to a defined starting point (e.g., time of diagnosis or start of treatment) and an endpoint (e.g., death, recurrence, or metastasis). Additionally, the criteria for therapeutic efficacy may be extended to include response to chemotherapy, survival probability, probability of metastasis within a given time, and probability of tumor recurrence.

[0174] The term "therapeutic effect" refers to a local or systemic effect caused by a pharmacologically active substance in animals, particularly mammals, and particularly humans. Therefore, this term refers to any substance intended for use in the diagnosis, healing, alleviation, treatment, or prevention of a disease in animals or humans, or for the improvement of desirable physical or mental development and condition. The phrase "therapeutic effective dose" refers to an amount of such substance that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. In certain embodiments, the therapeutic effective dose of a compound will vary depending on the therapeutic index, solubility, etc. For example, a specific compound developed by the method of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0175] As used herein, the terms “therapeutic effective dose” and “effective dose” refer to an amount of a compound, material, or composition comprising a compound of the present invention that is effective in producing some desired therapeutic effect in at least a subpopulation of animal cells at a reasonable benefit / risk ratio applicable to any medical treatment. The toxicity and therapeutic efficacy of the target compound are, for example, LD50 50 and ED50 It can be determined through standard pharmaceutical procedures for cell culture or experimental animals. A composition exhibiting a large therapeutic index is preferred. In some embodiments, LD50 50 (Lethal dose) is measurable and may be reduced by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more relative to the substance compared to the case where the substance is not administered. Similarly, ED 50 (i.e., the concentration that achieves maximum-half inhibition of symptoms) may also be measured and may be increased by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more for the substance compared to the case where the substance is not administered. Also, similarly, IC 50(i.e., the concentration that achieves maximum-half cytotoxic or cytotoxic effects on cancer cells) can also be measured and, for example, compared to the case where the substance is not administered, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more for the substance. In some embodiments, cancer cell growth in the analysis may 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 100%. Cancer cell death may be promoted by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In other embodiments, the number of cancer cells and / or solid malignancy may be achieved at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0176] The term “substantially free of chemical precursors or other compounds” comprises preparations of antibodies, polypeptides, peptides, or fusion proteins separated from chemical precursors or other compounds associated with the synthesis of proteins. In some embodiments, the term “substantially free of chemical precursors or other compounds” comprises 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 compounds, more preferably less than about 20% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein compounds, more preferably less than about 10% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein compounds, and most preferably less than about 5% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein compounds.

[0177] "Transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, complete miRNA, precursor miRNA, primary 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 part of the complete mRNA produced by transcription of the marker of the present invention and, in any case, general post-transcriptional processes of RNA transcripts (e.g., splicing) and reverse transcription of RNA transcripts.

[0178] The term "vector" refers to a nucleic acid capable of transporting other connected nucleic acids. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA fragments can be ligated. Another type of vector is a viral vector to which additional DNA fragments can be ligated within the viral genome. Certain vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors of bacterial replication origin and episomal mammalian vectors). Other vectors (e.g., non-episosomal mammalian vectors) are incorporated into the host cell's genome upon introduction into the host cell and are consequently replicated along with the host genome. Additionally, certain vectors can regulate the expression of operably linked genes. In this specification, such vectors are referred to as "recombinant expression vectors" or, for simplicity, "expression vectors." Generally, expression vectors useful for recombinant DNA technology are in the form of plasmids. In this specification, "plasmid" and "vector" may be interchangeably used for plasmids, which are the most commonly used form of a vector. However, the present invention is intended to include other forms of expression vectors, such as viral vectors that provide equivalent functions (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0179] As defined by the genetic code (see below), there is a known clear correlation between the amino acid sequence of a specific protein and the nucleotide sequence that can encode this protein. Likewise, as defined by the genetic code, there is a known clear correlation between the nucleotide sequence of a specific nucleic acid and the amino acid sequence encoded by the nucleic acid.

[0180] genetic code

[0181] Alanine (Ala, A) GCA, GCC, GCG, GCT

[0182] Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT

[0183] Asparagine (Asn, N) AAC, AAT

[0184] Aspartic acid (Asp, D) GAC, GAT

[0185] Cysteine ​​(Cys, C) TGC, TGT

[0186] Glutamic acid (Glu, E) GAA, GAG

[0187] Glutamine (Gln, Q) CAA, CAG

[0188] Glycine (Gly, G) GGA, GGC, GGG, GGT

[0189] Histidine (His, H) CAC, CAT

[0190] Isoleucine (Ile, I) ATA, ATC, ATT

[0191] Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG

[0192] Lysine (Lys, K) AAA, AAG

[0193] Methionine (Met, M) ATG

[0194] Phenylalanine (Phe, F ) TTC, TTT

[0195] Proline (Pro, P) CCA, CCC, CCG, CCT

[0196] Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT

[0197] Threonine (Thr, T) ACA, ACC, ACG, ACT

[0198] Tryptophan (Trp, W) TGG

[0199] Tyrosine (Tyr, Y) TAC, TAT

[0200] Valin (Val, V) GTA, GTC, GTG, GTT

[0201] Termination signal (end) TAA, TAG, TGA

[0202] An important and well-known feature of the genetic code is redundancy, where one or more coding nucleotide triplets can be applied for most amino acids required to make a protein (see above). Therefore, multiple different nucleotide sequences can encode a given amino acid sequence. These nucleotide sequences are considered functionally equivalent because they produce the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than others). Additionally, methylated variants of purines or pyrimidines may occasionally be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0203] In the foregoing view, a nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any part thereof) can be used to derive a polypeptide amino acid sequence by using a genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for a polypeptide amino acid sequence, a nucleotide sequence capable of encoding this polypeptide can be inferred from the genetic code (due to its redundancy, multiple nucleic acid sequences will be generated for any given amino acid sequence). Accordingly, the description and / or disclosure of a nucleotide sequence encoding a polypeptide in this specification should be deemed to include the description and / or disclosure of an amino acid sequence encoded by the nucleotide sequence. Likewise, the description and / or disclosure of a polypeptide amino acid sequence in this specification should be deemed to include the description and / or disclosure of all possible nucleotide sequences capable of encoding this amino acid sequence.

[0204] Finally, nucleic acid and amino acid sequences for nucleic acid and polypeptide molecules useful for the present invention are well known in the art and are readily available in publicly available databases such as the National Center for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences derived from publicly available databases are provided in Table 1 below.

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] * Table 1 includes RNA nucleic acid molecules (e.g., thymine substituted with uridine), nucleic acid molecules encoding the ortholog of an coding protein, as well as DNA or RNA nucleic acid sequences or parts thereof comprising nucleic acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity over the entire length of any sequence number listed in Table 1. Such nucleic acid molecules may have the function of a full-length nucleic acid as further described herein.

[0224] * Table 1 includes not only the ortholog of a protein, but also polypeptide molecules or parts thereof comprising amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity over the entire length of the amino acid sequence of any sequence number listed in Table 1. Such polypeptides may have the function of full-length polypeptides as further described herein.

[0225] * Table 1 includes other known HHLA2, TMIGD2, and KIR3DL3 nucleic acid sequences and amino acid sequences.

[0226] In addition to acting as a stimulatory receptor (i.e., transmitting co-stimulatory signals to immune cells), the HHLA2 polypeptide may be an inhibitory receptor capable of inhibiting immune cell effector function by transmitting an inhibitory signal to immune cells when, for example, when binding to an inhibitory receptor or a stimulatory receptor, or it may promote co-stimulation of immune cells. HHLA2 binds to one or more receptors, for example, TMIGD2, KIR3DL3 and / or other polypeptides on T-cells.

[0227] The term "HHLA2 activity" includes the ability of the HHLA2 polypeptide to regulate inhibitory signals in activated immune cells, for example, by binding to natural HHLA2 ligands on T-cells. The regulation of inhibitory signals in immune cells leads to the regulation of immune cell proliferation and / or cytokine secretion. Therefore, the term "HHLA2 activity" includes the ability of the HHLA2 polypeptide to bind to natural ligands, the ability to regulate immune cell co-stimulation or inhibitory signals, and the ability to regulate immune responses.

[0228] In some embodiments, cancer-like conditions respond to HHLA2 blockade alone. In other embodiments, cancer-like conditions respond to HHLA2 blockade alone but respond significantly or synergistically better when treated with a combination of HHLA2 blockade and other therapies. Conditions that respond to HHLA2 blockade alone or in combination include melanoma (e.g., advanced or metastatic melanoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), and breast cancer (e.g. , HER-2 negative breast cancer, estrogen-receptor+ / HER-2- breast cancer and triple-negative breast cancer), pancreatic cancer (e.g., pancreatic adenocarcinoma) and Hodgkin lymphoma, as well as bladder cancer, gastric cancer, head and neck cancer, kidney cancer, prostate cancer, gynecologic cancer, colorectal cancer, ovarian cancer and hematological cancer, are included but not limited thereto.

[0229] A preferred B7 polypeptide can promote or inhibit immune cell responses by providing co-stimulatory or inhibitory signals to immune cells. For example, a B7 family member that binds to a co-stimulatory receptor increases T cell activation and proliferation, whereas a B7 family member that binds to an inhibitory receptor decreases co-stimulation. Additionally, the same B7 family member can increase or decrease T cell co-stimulation. For example, when bound to a co-stimulatory receptor, HHLA2 can induce co-stimulation of immune cells, and when bound to an inhibitory receptor, HHLA2 can inhibit immune cells. When bound to an inhibitory receptor, HHLA2 can deliver an inhibitory signal to immune cells. Preferred B7 family members include HHLA2, B7-1, B7-2, B7h, PD-L1, or PD-L2 and their soluble fragments or derivatives. In one embodiment, a B7 family member binds to one or more receptors on an immune cell, e.g., TMIGD2, KIR3DL3, CTLA4, CD28, ICOS, PD-1 and / or other receptors, and, depending on the receptor, has the ability to transmit an inhibitory signal or a co-stimulatory signal to an immune cell, preferably a T cell.

[0230] The modulation of co-stimulatory signals leads to the modulation of effector functions in immune cells. Therefore, the term "HHLA2 ligand activity" includes the ability of HHLA2 ligand polypeptides to bind to natural receptors (e.g., HHLA2), the ability to modulate immune cell co-stimulatory or inhibitory signals, and the ability to modulate immune responses.

[0231] In this specification, it has been demonstrated that the HHLA2 pathway is a negative or positive regulator of immune function and can regulate immune function by modulating the interaction between HHLA2 and one or more natural binding partners, such as TMIGD2 and / or KIR3DL3. HHLA2 binding to TMIGD2 can be a positive regulator of immune function. HHLA2 binding to inhibitory receptors is a negative regulator of immune function. HHLA2 binding to TMIGD2 is considered to be similar to HHLA2 binding to inhibitory receptors. Therefore, inhibiting HHLA2 binding to TMIGD2 is considered to inhibit HHLA2 binding to inhibitory receptors. Therefore, the material of the present invention described herein as an HHLA2 pathway modulator (e.g., a modulator of the interaction between HHLA2 and one or more natural binding partners such as TMIGD2 and / or KIR3DL3) modulates the interaction between HHLA2 and one or more natural binding partners and can upregulate or downregulate the immune response by upregulating or downregulating the immune system, either directly or indirectly. The material modulating this interaction can do so directly or indirectly.

[0232] Interactions between HHLA2 and one or more natural binding partners of HHLA2, such as TMIGD2 and / or KIR3DL3, result in the transmission of immune signaling co-stimulation or co-inhibition. Accordingly, in one embodiment, a substance that directly blocks these interactions (e.g., anti-HHLA2, anti-TMIGD2 and / or anti-KIR3DL3 blocking antibodies) may block inhibiting or stimulating signaling and may upregulate or downregulate the immune response. Alternatively, a substance that indirectly blocks these interactions may block inhibiting signaling and may upregulate the immune response. Examples of substances for upregulating the immune response include antibodies against HHLA2 or KIR3DL3 that block the interaction between HHLA2 and KIR3DL3; non-activated 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 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 variants that block the transcription or translation of HHLA2 and / or KIR3DL3; and non-activated forms of natural HHLA2 ligands and soluble forms of natural KIR3DL3 ligands are included.

[0233] In another exemplary embodiment, a substance that promotes the binding of an HHLA2 polypeptide to one or more natural binding partners, such as a KIR3DL3 polypeptide, promotes an inhibitory signal to immune cells. A substance that modulates this interaction may do so directly or indirectly. Thus, in one embodiment, a substance that directly enhances the interaction between HHLA2 and KIR3DL3 (an HHLA2 agonist and / or a KIR3DL3 agonist) may promote inhibitory signaling and downregulation of the immune response. Alternatively, a substance that blocks the binding of KIR3DL3 to other targets increases the effective concentration of KIR3DL3 available to bind to HHLA2. Exemplarily, a substance that downregulates the immune response is an antibody against HHLA2 or KIR3DL3 that activates or promotes the interaction between HHLA2 and KIR3DL3; a small molecule or peptide that activates or promotes the interaction between HHLA2 and KIR3DL3; It includes blocking antibodies that bind to natural binding partners of HHLA2 and KIR3DL3 other than HHLA2 and KIR3DL3. Their relationship also applies to the interaction between HHLA2 and TMIGD2.

[0234] Additional materials useful for the method of the present invention include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives thereof that can bind to and / or activate or inhibit the protein biomarker of the present invention, comprising the biomarker or fragment thereof listed in Table 1; RNA interference, antisense, nucleic acid aptamers, etc., that can downregulate the expression and / or activity of the biomarker of the present invention, comprising the biomarker or fragment thereof listed in Table 1.

[0235] A monoclonal antibody directly isolated against HHLA2 or a fragment thereof is provided. In some embodiments, an mAb produced by a hybridoma is deposited at accession number ______, ______, with the American Type Culture Collection (ATCC), a U.S. strain depositary, under the terms of the Treaty of Budapest.

[0236] Since it is well known in the art that the heavy chain and light chain CDR3 domains of an antibody play a particularly important role in the binding specificity / affinity of the antibody to an antigen, the recombinant monoclonal antibody of the present invention prepared as above preferably comprises the heavy chain and light chain CDR3 of the variable region of the present invention (e.g., the sequence of Table 2 or a portion thereof). The antibody may also comprise the CDR2 of the variable region of the present invention (e.g., the sequence of Table 2 or a portion thereof). The antibody may also comprise the CDR1 of the variable region of the present invention (e.g., the sequence of Table 2 or a portion thereof). In other embodiments, the antibody may comprise any combination of CDRs.

[0237] The CDR1, 2 and / or 3 regions of the engineered antibody described above may include an amino acid sequence (e.g., including the sequence of Table 2 or a portion thereof) that is the same as that of the variable region of the present invention described herein. However, a person skilled in the art will recognize that some deviation from the said CDR sequence may be possible while maintaining the ability of the antibody to effectively bind to HHLA2 (e.g., conservative sequence modification). Accordingly, in other embodiments, the engineered antibody may consist of one or more CDRs that are, for example, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to one or more CDRs of the present invention (e.g., including the sequence of Table 2 or a portion thereof).

[0238] Structural properties of known non-human or human antibodies (e.g., mouse, non-rodent anti-human HHLA2 antibodies) may be used to generate structurally related human anti-human HHLA2 antibodies (e.g., mAb 8A12 and polyclonal antibodies 1.2 and 2.2) that retain at least one functional property of the antibody of the present invention, such as binding to HHLA2. Other functional properties include inhibiting the binding of the original known non-human or human antibody in a competitive ELISA assay.

[0239] In some embodiments, a monoclonal antibody capable of binding to human HHLA2 (e.g., mAb 8A12 and polyclonal antibodies 1.2 and 2.2) is provided, comprising a heavy chain of a variable domain having at least one CDR having a sequence identical to the group of heavy chain variable domain CDRs shown in Table 2 by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%.

[0240] Similarly, a monoclonal antibody capable of binding to human HHLA2 (e.g., mAb 8A12 and polyclonal antibodies 1.2 and 2.2) is provided, comprising a light chain of a variable domain having at least one CDR having a sequence identical to the group of light chain variable domain CDRs shown in Table 2 by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%.

[0241] A heavy chain of a variable domain comprising at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the group of heavy chain variable domain CDRs shown in Table 2; Monoclonal antibodies capable of binding to human HHLA2 (e.g., mAb 8A12 and polyclonal antibodies 1.2 and 2.2) are also provided, comprising a light chain of a variable domain having at least one CDR having a sequence identical to the group of light chain variable domain CDRs shown in Table 2, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%.

[0242] Those skilled in the art may achieve this by introducing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions within a given CDR that is equivalent to the given CDR.

[0243] The monoclonal antibody of the present invention may comprise a heavy chain of a variable domain comprising at least one CDR having a sequence selected from the group of heavy chain variable domain CDRs shown in Table 2, and a light chain of a variable domain comprising at least one CDR having a sequence selected from the group of light chain variable domain CDRs shown in Table 2.

[0244] Such monoclonal antibodies may comprise a light chain of a variable domain comprising 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 of a variable domain comprising 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, a monoclonal antibody capable of binding to human HHLA2 may comprise or be composed of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as described herein.

[0245] The heavy chain variable domain of the monoclonal antibody of the present invention may include or be composed of the vH amino acid sequence presented in Table 2, and / or the light chain variable domain of the monoclonal antibody of the present invention may include or be composed of the vκ amino acid sequence presented in Table 2.

[0246] The monoclonal antibodies of the present invention may be produced and modified by any technique well known in the art. For example, such monoclonal antibodies may be rodent or non-rodent antibodies, such as those obtained from a hybridoma deposited at ATCC under accession number ______. Similarly, such monoclonal antibodies may be chimeric, preferably mouse / human chimeric antibodies. In some embodiments, the monoclonal antibodies are humanized antibodies, such as those comprising a variable domain of a human acceptor framework region and, if present, optionally a human invariant domain, and a non-human donor CDR such as a mouse or non-rodent CDR as defined above.

[0247] The present invention also provides fragments of said monoclonal antibodies comprising, but not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody; and multispecific antibodies formed from the antibody fragments. For example, many immunosuppressive molecules such as HHLA2, PD-L2, PD-L1, CTLA-4, KIR3DL3, etc. can be detected in a bispecific or multispecific manner to effectively characterize the expression of these molecules.

[0248] Other fragments of the monoclonal antibody of the present invention are also considered. For example, individual immunoglobulin heavy chains and / or light chains are provided in which the variable domain comprises at least one CDR shown 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 group of heavy or light chain variable domain CDRs shown 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 group of light chain or heavy chain variable domain CDRs described herein (e.g., shown in Table 2).

[0249] 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. The immunoglobulin heavy chain may comprise or be composed of at least one of CDR-H1, CDR-H2, and CDR-H3. The immunoglobulin light chain may comprise or be composed of at least one of CDR-L1, CDR-L2, and CDR-L3.

[0250] In another embodiment, the immunoglobulin heavy chain and / or light chain according to the present invention may comprise or be composed of each of the vH or vκ variable domain sequences presented in Table 2.

[0251] The present invention also provides a polypeptide having a sequence selected from the group consisting of the vH variable domain, vκ variable domain, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences described herein.

[0252] The antibodies, immunoglobulins, and polypeptides of the present invention may be used in an isolated (e.g., purified) or contained in a vector such as a membrane or lipid vesicle (e.g., liposomes).

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] * CDR definition and protein sequence numbering according to Kabat. CDR amino acid sequences are indicated by underscores in the order of CDR1, CDR2, and CDR3.

[0296]

[0297] III. Nucleic Acids, Vectors, and Recombinant Host Cells

[0298] A further object of the present invention is to the nucleic acid sequence encoding the monoclonal antibody, its fragment, immunoglobulin, and polypeptide of the present invention.

[0299] 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 the vL domain of at least one effective anti-HHLA2 mAb isolated from polyclonal antibodies 1.2 and / or 2.2.

[0300] Typically, the nucleic acid is a DNA or RNA molecule that can be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0301] The terms “vector,” “cloning vector,” and “expression vector” refer to a means of transport into which a DNA or RNA sequence (e.g., a foreign gene) is introduced into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Accordingly, a further object of the present invention is to a vector comprising the nucleic acid of the present invention.

[0302] These vectors may include regulatory elements such as promoters, enhancers, and terminators to induce or control the expression of the polypeptide when administered to a subject. Examples of promoters and enhancers used in expression vectors for animals include the initial promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), and the promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of the immunoglobulin H chain.

[0303] Any expression vector for animal cells may be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), and pSG1 beta d2-4- (Miyaji H et al. 1990). Other representative examples of plasmids include replication plasmids containing a replication origin or integration plasmids such as pUC, pcDNA, pBR, etc. Representative examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. These recombinant viruses can be produced using techniques known in the field, such as transfection of packaging cells or transient transfection of helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Specific protocols for producing these replication-deficient recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056 and WO 94 / 19478.

[0304] A further object of the present invention relates to cells transfected, infected, or transformed with nucleic acids and / or vectors according to the present invention. The term “transformation” means “introducing a foreign (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell so 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 “transformed.”

[0305] The nucleic acid of the present invention can be used to produce the recombinant polypeptide of the present invention in a suitable expression system. The term "expression system" means, for example, a host cell under suitable conditions for the expression of a protein encoded by a foreign gene carried by a vector and introduced into a host cell, and a suitable vector.

[0306] General expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces Yeast and mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) are included, as well as primary or identified mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neurons, adipocytes, etc.). Examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells deficient in dihydrofolate reductase (hereinafter referred to as the "DHFR" gene) (Urlaub G et al; 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"). YB2 / 0 cells are preferred because the ADCC activity of chimeric or humanized antibodies increases when expressed in cells.

[0307] The present invention also relates to a method for producing recombinant host cells expressing the antibody or polypeptide of the present invention 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 a cell that expresses and / or secretes the antibody or polypeptide. Such recombinant host cells may be used for the production of the antibody and polypeptide of the present invention.

[0308] In another aspect, the present invention provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Accordingly, the polynucleotides of this embodiment may be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides of the present invention may be used to identify, isolate, or amplify partial or full-length clones of a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated from, or complementary to, human or mammalian nucleic acid library cDNA. Preferably, the cDNA library comprises at least 80% of the full-length sequence, preferably at least 85% or 90% of the full-length sequence, and more preferably at least 95% of the full-length sequence. The cDNA library may be normalized to increase the expression of rare sequences. Hybridization conditions of low or moderate severity are typically, but not exclusively, applied to sequences having reduced sequence identity relative to the complementary sequence. Conditions of moderate and high severity may be selectively applied to sequences of high identity. Conditions of low severity may be applied to enable selective hybridization of sequences having about 70% sequence identity and to identify orthologous or paralogous sequences. Optionally, the polynucleotide of the present invention will encode at least a portion of the antibody encoded by the polynucleotide described herein. The polynucleotide of the present invention comprises a nucleic acid sequence that can be used for selective hybridization to the polynucleotide encoding the antibody of the present invention. For example, see Ausubel; Colligan, each incorporated herein by reference in its entirety.

[0309] IV. Antibody Production Methods

[0310] The antibodies and fragments thereof, immunoglobulins and polypeptides of the present invention may be produced by any technology known in the art, such as, but not limited to, any chemical, biological, genetic, or enzymatic technology, or any combination thereof.

[0311] If the amino acid sequence of the desired sequence is known, a person skilled in the art can easily produce the said antibody or polypeptide using standard polypeptide production techniques. For example, using a well-known solid-phase method, preferably using a commercially available peptide synthesis apparatus (e.g., apparatus from Applied Biosystems (Foster City, California)) and synthesizing according to the manufacturer's instructions. Alternatively, the antibody and other polypeptides of the present invention can be synthesized using recombinant DNA techniques well known in the art. For example, these fragments can be obtained as DNA expression products after incorporating a DNA sequence encoding the desired (poly)peptide into an expression vector and introducing this vector into a suitable eukaryotic or prokaryotic host to express the desired polypeptide, and these can subsequently be isolated using well-known techniques.

[0312] In particular, the present invention further relates to a method for producing an antibody or polypeptide of the present invention, the method comprising steps of: (i) culturing a transformed host cell according to the present invention under suitable conditions that enable the expression of said antibody or polypeptide; and (ii) recovering the expressed antibody or polypeptide.

[0313] The antibodies and other polypeptides of the present invention are appropriately separated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-cephalosporose, 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”) may also be applied for purification. For example, see Colligan’s literature [Current Protocols in Immunology] or [Current Protocols in Protein Science] (John Wiley & Sons, NY, NY, 1997–2001), for example, Chapters 1, 4, 6, 8, 9, and 10, each incorporated herein by reference in their entirety.

[0314] The chimeric antibody of the present invention (e.g., a mouse-human chimera or a non-rodent-human chimera) can be produced by obtaining a nucleic acid sequence encoding the VL and VH domains as described above, constructing a human chimeric antibody expression vector by inserting it into an animal cell expression vector having genes encoding human antibody CH and human antibody CL, and introducing this expression vector into animal cells to express the encoding sequence. The CH domain of the human chimeric antibody may be any region belonging to human immunoglobulins, such as IgG or its subgroups, e.g., IgG1, IgG2, IgG3, and IgG4. Similarly, the CL of the human chimeric antibody may be any region belonging to Ig, such as kappa or lambda. Chimeric and humanized monoclonal antibodies containing both human and non-human portions and capable of being produced using standard recombinant DNA technology are within the scope of the present invention. These chimeric and humanized monoclonal antibodies utilize recombinant DNA technology known in this field, for example, in the literature [Robinson et al. International Patent Publication 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 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) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Patent No. 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. It can be produced using the method described in [141:4053-4060].

[0315] Additionally, humanized antibodies may be produced according to standard protocols as disclosed in U.S. Patent No. 5,565,332. In other embodiments, antibody chains or specific binding pair members may be produced by recombination between a vector containing a nucleic acid molecule encoding a fusion of a polypeptide chain of a specific binding pair member and a component of a replicatable genetic display package using techniques known in the art, e.g., as described in U.S. Patents No. 5,565,332, No. 5,871,907, or No. 5,733,743, and a vector containing a nucleic acid molecule encoding a second polypeptide chain of a single binding pair member. The humanized antibody of the present invention can be produced by obtaining a nucleic acid sequence encoding a CDR domain as described above, constructing a humanized antibody expression vector by inserting it into an expression vector for animal cells having a gene encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and introducing this expression vector into animal cells to express this gene. The humanized antibody expression vector may be of a type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain exist in separate vectors, or of a type in which the two genes exist in the same vector (tandem type).

[0316] Methods for producing humanized antibodies based on existing recombinant DNA and gene transfusion technologies are well known in the field (see, for example, the literature [iechmann L. et al. 1988; Neuberger M S. et al. 1985]). Antibodies can be humanized using various techniques known in the field, including, for example, CDR-grafting (EP No. 239,400; PCT Publication WO91 / 09967; U.S. Patents No. 5,225,539; No. 5,530,101; and No. 5,585,089), veneering or surface treatment (EP No. 592,106; EP No. 519,596; the literature [Padlan EA (1991); Studnicka GM et al. (1994); Roguska M A. et al. (1994)]), and chain shuffling (U.S. Patent No. 5,565,332). General recombinant DNA technology for producing such antibodies is also known (see European patent application EP 125023 and international patent application WO 96 / 02576).

[0317] Similarly, the bispecific or multispecific antibodies described herein may be produced according to standard procedures. For example, triomas and hybrid hybridomas are two examples of cell lines capable of secreting bispecific or multispecific antibodies. Examples of bispecific or multispecific antibodies produced by hybrid hybridomas or triomas are disclosed in U.S. Patent No. 4,474,893. These antibodies are produced by chemical means (Staerz et al. (1985) Nature 314:628 and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA , 83:1453 and Staerz and Bevan (1986) Immunol. TodayIt may also be constructed by 7:241). Alternatively, these antibodies may be generated by creating a heterohybridoma through the fusion of hybridomas or other cells that produce different antibodies, and by identifying a clone that produces and co-assembles the desired antibody. They may also be generated by chemical or genetic splicing of complete immunoglobulin chains or parts thereof, such as Fab and Fv sequences. The antibody component may bind to a polypeptide or fragment thereof of one or more biomarkers of the present invention, comprising one or more immunoglobulin biomarkers described herein.

[0318] Additionally, methods for producing antibody fragments are well known. For example, the Fab fragment of the present invention can be obtained by treating antibodies that act specifically on human HHLA2 (such as mAb 8A12 and polyclonal antibodies 1.2 and 2.2) with a protease such as papain. Additionally, Fab can be produced by inserting DNA encoding the Fab of the antibody into a vector for a prokaryotic expression system or a eukaryotic expression system, and introducing this vector into a prokaryote or eukaryote (appropriately) to express the Fab.

[0319] Similarly, the F(ab')2 fragment of the present invention can be obtained by treating an antibody that acts specifically on HHLA2 with the protease pepsin. Additionally, the F(ab')2 fragment can be produced by the binding of Fab' as described below through thioether bonds or disulfide bonds.

[0320] The Fab' fragment of the present invention can be obtained by treating F(ab')2, which acts specifically on HHLA2, with the reducing agent dithiothreitol. Additionally, the Fab' fragment can be produced by inserting DNA encoding the Fab' fragment of the antibody into an expression vector for prokaryotes or eukaryotes, and introducing this vector into a prokaryote or eukaryote (appropriately) to perform expression thereof.

[0321] Additionally, the scFv of the present invention may be produced by obtaining cDNA encoding the VH and VL domains as described above, constructing DNA encoding the scFv, inserting this DNA into an expression vector for prokaryotes or eukaryotes, and introducing this expression vector into prokaryotes or eukaryotes (appropriately) to express the scFv. To generate humanized scFv fragments, a well-known technique of CDR grafting may be used, in which a complementation determining region (CDR) is selected from a donor scFv fragment and grafted onto a human scFv fragment framework of a known three-dimensional structure (see, for example, 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).

[0322] V. Modification of Antibodies, Immunoglobulins, and Polypeptides

[0323] Amino acid sequence modifications of antibodies described herein are considered. For example, this may be necessary to improve the binding affinity and / or other biological properties of the antibody. It is known that when humanized antibodies are produced by simply grafting only the CDRs of VH and VL of non-human animal-derived antibodies onto the FRs of VH and VL of human antibodies, the antigen-binding activity is reduced compared to the original non-human animal-derived antibody. It is thought that some amino acid residues of VH and VL of FR, as well as the CDRs of non-human antibodies, are directly or indirectly related to antigen-binding activity. Therefore, substituting these amino acid residues with different amino acid residues derived from the FRs of VH and VL of human antibodies will reduce binding activity, and this can be corrected by substituting these amino acids with amino acid residues of the original non-human animal-derived antibody.

[0324] Modifications and changes may be made in the structure of the antibody of the present invention, or in the DNA sequence encoding it, thereby enabling the acquisition of functional molecules encoding antibodies and polypeptides that still possess desired characteristics. For example, specific amino acids may be substituted with other amino acids in the protein structure without significant loss of activity. Since the interaction ability and characteristics of a protein define its biological functional activity, specific amino acid substitutions may be made in the protein sequence and, of course, its DNA encoding sequence, yet proteins with similar characteristics can still be obtained. Therefore, it is considered that various changes may be made in the antibody sequence of the present invention or the corresponding DNA sequence encoding the polypeptide without significant loss of biological activity.

[0325] In one embodiment, a change in amino acids can be achieved by altering the codons of the DNA sequence to encode a conserved substitution based on the conservation of the genetic code. Specifically, as defined by the genetic code (shown below), there is a known clear correlation between the amino acid sequence of a specific protein and the nucleotide sequence capable of encoding this protein. Likewise, as defined by the genetic code (see the list of genetic codes above), there is a known clear correlation between the nucleotide sequence of a specific nucleic acid and the amino acid sequence encoded by this nucleic acid.

[0326] As described above, an important and well-known characteristic of the genetic code is redundancy; therefore, for most amino acids used to make proteins, one or more coding nucleotide triplets may apply (as described above). Accordingly, multiple different nucleotide sequences can encode a given amino acid sequence. These nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than others). Additionally, methylated variants of purines or pyrimidines may occasionally be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0327] When making changes to the amino acid sequence of polypeptides, the hydropathic index of amino acids may be considered. It is generally understood in this field that the hydropathic amino acid index is important in conferring interactive biological functions upon proteins. It is accepted that the relative hydropathic properties of amino acids contribute to the secondary structure of proteins, which defines interactions between proteins and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens. 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); 아스파라긴(-3.5); 라이신(-3.9); 및 아르기닌(-4.5)과 같이, 각각의 아미노산은 이들의 소수성 및 전하 특성에 따라 하이드로패스 지수가 지정된다.

[0328] It is known in this field that a specific amino acid can be substituted with another amino acid having a similar hydropass index or score and a protein with similar biological activity can still be produced, that is, a protein that is still biologically functionally equivalent can be obtained.

[0329] As explained above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Examples of substitutions considering 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.

[0330] For example, other types of amino acid modifications to the antibody of the present invention may be useful when altering the original glycosylation pattern of the antibody to increase stability. "Modification" means removing one or more carbohydrate moietys found in the antibody or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of the antibody is typically N-linking. "N-linking" refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid excluding proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Therefore, the presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Adding glycosylation sites to antibodies is easily achieved by modifying the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Another type of covalent modification involves chemically or enzymatically binding glycosides to antibodies. This procedure is advantageous in that it does not require the production of antibodies in host cells capable of N- or O-linked glycosylation. Depending on the binding method used, the sugar may be attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group such as that of cysteine, (d) a free hydroxyl group such as that of serine, threonine, or hydroxyproline, (e) an aromatic residue such as that of phenylalanine, tyrosine, or tryptophan, or (f) an amide group of glutamine. For example, such a method is described in WO87 / 05330.

[0331] Similarly, the removal of any carbohydrate moiety present in antibodies can be achieved chemically or enzymatically. Chemical deglycosylation requires exposing the antibody to a trifluoromethanesulfonic acid compound or an equivalent compound. This treatment results in the cleavage of most or all sugars, excluding the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while the antibody remains intact. Chemical deglycosylation is described in the literature [Sojahr H. et al. (1987)] and [Edge, A. S. et al. (1981)]. Enzymatic cleavage of antibody carbohydrate moiety can be achieved using various endo- and exo-glycosidases, as described in the literature [Thotakura, N. R. et al. (1987)].

[0332] Other modifications may include forming an immunoconjugate. For example, in one type of covalent modification, the antibody or protein is covalently linked to one of various non-protein polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner specified in U.S. Patents No. 4,640,835; No. 4,496,689; No. 4,301,144; No. 4,670,417; No. 4,791,192, or No. 4,179,337.

[0333] The conjugation of the antibody or other protein of the present invention to a heterogeneous substance comprises N-succinimidyl(2-pyridyldite)propionic acid (SPDP), succinimidyl(N-maleimidomethyl)cyclohexane-1-carboxylic acid, iminothiolane (IT), dual-functional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), activated esters (e.g., disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-activated fluorine. It can be made using various dual-functional protein binding materials including, but not limited to, a compound (1,5-difluoro-2,4-dinitrobenzene). For example, carbon-labeled 1-isothiocyanatobenzyl methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating material for conjugating radionucleotides to antibodies (WO 94 / 11026).

[0334] In another aspect, the present invention relates to an antibody that specifically binds to HHLA2 conjugated to a therapeutic moiety, such as a cytotoxin, drug, and / or radioisotope. When conjugated to a cytotoxin, the antibody conjugate is referred to as an "immunotoxin." The cytotoxin or cytotoxic substance comprises any substance that is harmful to cells (e.g., kills cells). Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, and tetracaine. Lidocaine, propranolol, and puromycin, and their analogs or homologues are included.Therapeutic substances include metabolic antagonists (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thioepahlorambucil, 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 (formerly actinomycin), bleomycin, mitramycin, and Antibodies of the present invention include, but are not limited to, antramycin (AMC) and anti-mitotic substances (e.g., vincristine and vinblastine). Antibodies of the present invention may be conjugated to a radioisotope, e.g., radioactive iodine, to produce a cytotoxic radiodrug for the treatment of related disorders such as cancer.

[0335] Conjugated anti-HHLA2 antibodies may be used diagnostically or prognostically as part of clinical trial procedures to monitor polypeptide levels in tissues, for example, to determine the efficacy of a given therapeutic regimen or to screen patients most likely to respond to immunotherapy. For example, cells may be permeated in flow cytometry to allow antibodies binding to HHLA2 (such as mAb 8A12 and polyclonal antibodies 1.2 and 2.2) to target recognized intracellular epitopes and to detect binding by analyzing signals emitted from the conjugated molecules. Detection can be facilitated by conjugating (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include mustard bean peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; Examples of suitable prosthetic 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 equorin; and examples of suitable radioactive materials include 125 I, 131 I, 35 S or 3 H is included. The term “label” as used in this specification in relation to antibodies is intended to include not only directly labeling the antibody by binding (i.e., physically linking) it with a detectable substance such as a radioactive substance or fluorescent dye (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)) to the antibody, but also indirectly labeling the antibody through reactivity with the detectable substance.

[0336] The antibody conjugate of the present invention may be used to modify a given biological response. The therapeutic moiety should not be interpreted as being limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, enzymatically active toxins or their active fragments such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-gamma; 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.

[0337] The technique of conjugating these therapeutic moieties to antibodies is, for example, in the literature [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 담체s Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475 506 (1985)]; ["Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303 16 (Academic Press 1985)] 및 [Thorpe et al. , "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119 58 (1982)]과 같이 잘 알려져 있다.

[0338] In some embodiments, conjugation may be performed using a "cleavable linker" that facilitates the release of a cytotoxic substance or growth inhibitor to the cell. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (see, for example, U.S. Patent No. 5,208,020) may be used. Alternatively, a fusion protein containing an antibody and a cytotoxic substance or growth inhibitor may be created through recombinant technology or peptide synthesis. The DNA may include regions separated by regions encoding two adjacent parts of the conjugate or regions encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0339] VI. Uses and Methods of the Present Invention

[0340] The anti-HHLA2 antibodies, immunoglobulins, polypeptides, and nucleic acids of the present invention described herein may be used in numerous predictive medical analyses based on the detection of HHLA2 levels (e.g., diagnostic analyses, prognostic analyses, and clinical trial monitoring), and in some embodiments may be used for therapeutic purposes (e.g., therapeutic and prophylactic) either alone or when conjugated to toxin 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 group. As described herein, the HHLA2 polypeptide of the present invention or a fragment thereof has one or more of the following activities: 1) binding to and / or binding to natural binding partners such as TMIGD2 and / or KIR3DL3 or modulating the activity of such binding partners; 2) modulating intracellular or intercellular signaling, such as co-immunoinhibitory signaling; 3) modulating the activation and / or proliferation of lymphocytes; 4) Regulation of immune responses in organisms, e.g., mice, non-rodent animals, or mammals such as humans; and 5) Regulation of immune cell energy.

[0341] Accordingly, one aspect of the present invention relates to a diagnostic analysis for determining HHLA2 polypeptide levels associated with a biological sample (e.g., blood, serum, cell, or tissue), thereby determining the levels of HHLA2 polypeptide in the sample, determining whether an individual is suffering from a disorder, and / or determining the state of such disorder manifested by such HHLA2 levels. For example, the antibody of the present invention is useful for demonstrating cancerous diseases associated with HHLA2.

[0342] The present invention also provides a prognostic (or predictive) analysis to determine whether an individual is at risk of developing such disorders. Another aspect of the present invention relates to monitoring the effects of HHLA2 expression or active substances (e.g., drugs, compounds) in clinical trials.

[0343] The present invention also provides HHLA2 detection as a means for identifying substances that transmit HHLA2 signals. Substances that transmit HHLA2 signals will attenuate the immune response and will be useful for autoimmune diseases and asthma, as well as for establishing tolerance.

[0344] In any method described herein, HHLA2 may 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 multiple molecules (e.g., sequentially or simultaneously) may provide useful information regarding the synergistic effects and / or personalization of therapeutic interventions and the high-resolution diagnosis of subtypes of disorders. In some embodiments, HHLA2 is detected in combination with one or more markers.

[0345] 1. Diagnostic analysis

[0346] The present invention provides, in part, a method, system, and code for accurately classifying whether a biological sample expresses cell-limiting HHLA2 and / or whether the level of cell-limiting HHLA2 is regulated (e.g., upregulated or downregulated), thereby indicating the state of a disorder of interest, such as cancer. In some embodiments, the present invention is useful for classifying samples (e.g., samples from subjects) associated with or at risk of HHLA2-mediated cancer or a subtype thereof using statistical algorithms and / or empirical data (e.g., the presence, absence, or level of HHLA2).

[0347] An exemplary method useful for detecting levels of HHLA2 or fragments thereof, and thereby classifying whether a sample is associated with a disease or disorder mediated by abnormal (e.g., upregulation or downregulation) expression of HHLA2 or a clinical subtype thereof, comprises obtaining a biological sample from a test subject and contacting this biological sample with an antibody of the present invention or an antigen-binding fragment thereof capable of detecting HHLA2, such as detecting levels of HHLA2 in the biological sample. In some embodiments, at least one antibody or an antigen-binding fragment thereof is used, wherein two, three, four, five, six, seven, eight, nine, ten, or more of these antibodies or antibody fragments may be used in combination (e.g., in a sandwich ELISA) or sequentially. In certain examples, the statistical algorithm is a single-learned statistical classification system. For example, a single-learned statistical classification system may be used to classify a sample as an HHLA2 sample based on predictions or probabilities and the presence or level of HHLA2. Using a single learning statistical classification system, samples are typically classified as HHLA2 samples with sensitivity, specificity, positive predictive values, negative predictive values, and / or 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% overall accuracy.

[0348] Other suitable statistical algorithms are well known to those skilled in the art. For example, a learning statistical classification system includes machine learning algorithm techniques capable of adapting to a complex dataset (e.g., a panel of markers of interest) and making decisions based on such dataset. In some embodiments, a single learning statistical classification system, such as a classification tree (e.g., a random forest), is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more learning statistical classification systems is used, preferably in tandem. Examples of learning statistical classification systems include, but are not limited to, inductive learning (e.g., decision / classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), PAC learning (Probably Approximately Correct learning), connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro-fuzzy networks (NFN), network structures, perceptrons such as multilayer perceptrons, multilayer feed-forward networks, applications of neural networks, Bayesian networks of belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment, passive learning in an unknown environment, active learning in an unknown environment, learning behavior-value functions, applications of reinforcement learning, such as inexperienced learning, adaptive dynamic learning, and temporal difference learning), and the use of genetic algorithms and evolutionary programming. Other learning statistical classification systems include support vector machines (e.g., kernel methods), multivariate adaptive regression splines (MARS), Lievenberg-Markworth algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, the method of the present invention further comprises sending HHLA2 sample classification results to a clinician, e.g., a histopathologist or oncologist.

[0349] In another embodiment, the method of the present invention further provides a diagnosis in the form of a probability that an individual will have a condition or disorder associated with HHLA2. For example, an individual may have a probability of about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher of having this condition or disorder. In another embodiment, the method of the present invention further provides a prognosis of the condition or disorder of the individual. In some examples, the method of classifying the sample as an HHLA2 sample is based on the symptoms of the individual (e.g., clinical factors) obtained from the sample. Symptoms or groups of symptoms may be, 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 diagnosing an individual with a condition or disorder related to HHLA2, a drug (e.g., a chemotherapy agent) useful for treating one or more symptoms related to this condition or condition is administered to the individual in a therapeutically effective amount.

[0350] In one embodiment, the method further comprises obtaining a control biological sample (e.g., a biological sample from a subject not having an HHLA2-mediated condition or disorder), a biological sample from a subject during a period of remission of an HHLA2-mediated condition or disorder or prior to the onset of such condition or disorder, or a biological sample from a subject during treatment for the onset of an HHLA2-mediated condition or disorder.

[0351] An example of a method for detecting the presence or absence of an HHLA2 polypeptide or a fragment thereof is an antibody of the present invention capable of binding to the HHLA2 polypeptide, preferably a detectably labeled antibody. The antibody may be polyclonal, and more preferably monoclonal. These may be labeled. The term “label” in relation to antibodies is intended to include not only directly labeling a probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, but also indirectly labeling the probe or antibody through reactivity with another directly labeled reagent. An example of indirect labeling includes detecting a primary antibody using a fluorescently labeled secondary antibody. The term “biological sample” is intended to include tissues, cells, and biological fluids isolated from a subject, such as serum, as well as tissues, cells, and fluids present within the subject. That is, the detection method of the present invention may be used to detect HHLA2 or a fragment thereof in biological samples in vivo as well as in vitro. In vitro techniques for the detection of HHLA2 polypeptides include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, immunohistochemistry (IHC), intracellular flow cytometry and related techniques, and immunofluorescence. Additionally, in vivo techniques for the detection of HHLA2 polypeptides or fragments thereof include introducing labeled anti-HHLA2 antibodies into a target. For example, these antibodies may be labeled alone or in combination with imaging for other molecules, such as cell type markers (e.g., CD8+ T cell markers), with radioactivity, luminescence, fluorescence, or other similar markers whose presence and location within the target can be detected by standard imaging techniques.

[0352] In one embodiment, the biological sample contains polypeptide molecules from the test subject. A preferred biological sample is serum, a tumor microenvironment sample, a peritumoral sample, or an intratumoral sample isolated from the subject by conventional means.

[0353] In another embodiment, the method further comprises obtaining a control biological sample from a control subject, contacting the control sample with a compound or substance capable of detecting the HHLA2 polypeptide or a fragment thereof so that the presence of the HHLA2 polypeptide or a fragment thereof is detected in the biological sample, and comparing the presence of the HHLA2 polypeptide or a fragment thereof in the control sample with the presence of the HHLA2 polypeptide or a fragment thereof in the test sample.

[0354] In another embodiment, the antibody may be associated with a component or device for use in an ELISA or RIA. Non-limiting examples include antibodies immobilized on a solid surface for use in these analyses (e.g., linked to and / or conjugated to a light or radiation-emitting detectable label as described above). In other embodiments, the antibody is associated with a device or strip for detecting HHLA2 using an immunochromatographic or immunochemical assay, such as a "sandwich" or competitive assay, immunohistochemistry, or immunofluorescence microscopy. Additional examples of such devices or strips are designed for home testing or rapid point-of-care testing. Further examples include those designed for the simultaneous analysis of multiple analytes within a single sample. For example, the unlabeled antibody of the present invention may be applied to the "capture" of an HHLA2 polypeptide in a biological sample and may conjugate the captured (or immobilized) HHLA2 polypeptide to a labeled form of the anti-HHLA2 antibody of the present invention for detection. For example, other standard embodiments of immunoassay, including immunodiffusion, immunoelectrophoresis, immunohistopathology, immunohistochemistry, and histopathology-based analysis methods, are well known to those skilled in the art.

[0355] 2. Prognosis analysis

[0356] The diagnostic method described herein may also be used to identify subjects having HHLA2-associated disorders or at risk of developing such disorders. As used herein, the term "abnormal" includes upregulation or downregulation of HHLA2 that deviates from normal HHLA2 levels. Abnormal expression or activity includes increased or decreased expression or activity, as well as expression or activity that does not follow normal expression patterns or intracellular expression patterns. For example, abnormal HHLA2 levels are intended to include cases where HHLA2 is upregulated or downregulated due to mutations in the HHLA2 gene or regulatory sequences, or amplification of the chromosomal HHLA2 gene. As used herein, the term "unwanted" includes unwanted phenomena associated with biological responses, such as immune cell activation. For example, the term "unwanted" includes undesirable HHLA2 in a subject.

[0357] Many disorders associated with HHLA2, as further described in the examples, are known to those skilled in the art. HHLA2 is expressed by various tumor types, including malignant lymphomas, virus-induced cancers, and many solid tumors. HHLA2 is generally an adverse prognostic marker because it activates immune checkpoint regulators that inhibit a strong immune response to necessary conditions. However, immunosuppression is necessary to downregulate the immune response in treating many disorders, such as autoimmune diseases and inflammatory diseases.

[0358] The analyses described herein, such as the diagnostic analyses described above or the analyses described below, may be used to identify subjects having disorders or at risk of developing disorders associated with regulatory errors of HHLA2 activation. Accordingly, the present invention provides a method for obtaining a test sample from a subject and detecting HHLA2 to identify disorders associated with abnormal or unwanted HHLA2 activation, wherein if an HHLA2 polypeptide is present, the subject is diagnosed as having a disorder associated with abnormal or unwanted HHLA2 activation or at risk of developing such disorder. As used herein, "test sample" refers to a biological sample obtained from a subject of interest. For example, the test sample may be a biological fluid (e.g., cerebrospinal fluid or serum), a cell sample or tissue, e.g., a histopathological slide of the tumor microenvironment, a peritumoral region and / or an intratumoral region. In a preferred embodiment, the sample comprises cells expressing mature membrane-bound HHLA2 and / or HHLA2 fragments.

[0359] Additionally, the prognostic analysis described herein may be used to determine whether a substance (e.g., agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) for treating disorders associated with abnormal or unwanted HHLA2 activity can be administered to a subject. For example, such a method may be used to determine whether a subject can be effectively treated with a single substance or a combination of substances. Accordingly, the present invention provides a method for obtaining a test sample and detecting HHLA2 to determine whether a subject can be effectively treated with one or more substances for treating disorders associated with abnormal or unwanted HHLA2 activity (e.g., whereby if HHLA2 polypeptide is abundant, it is diagnosed that a substance for treating disorders associated with abnormal or unwanted HHLA2 activity can be administered to the subject).

[0360] The method described herein may be performed using a pre-packaged diagnostic kit that includes, for example, at least one antibody reagent described herein and can be conveniently used in a clinical setting to diagnose, for example, a patient exhibiting HHLA2-related symptoms or related diseases or a family history of disease.

[0361] Additionally, any cell type or tissue expressing HHLA2 may be used for the prognostic analysis described herein.

[0362] Another aspect of the present invention involves the use of the compositions and methods described herein for analyzing HHLA2 in biological samples from individuals with disorders associated with abnormal HHLA2 activation and analyzing associations and / or stratification by comparing the information with information from a control group (e.g., individuals without the disorder; the control group may also refer to "healthy" or "normal" individuals or an early point in time in a given time-course study), preferably a control group of similar age and race. Appropriate selection of patients and controls is important for successful association and / or stratification studies. Therefore, a pool of individuals with well-characterized phenotypes is highly desirable. Criteria for disease diagnosis, disease predisposition screening, disease prognosis, determination of drug responsiveness (pharmacogenomics), drug toxicity screening, etc., are described herein.

[0363] Other research designs may be used for genetic association and / or stratification studies (Modern Epidemiology, Lippincott Williams & Wilkins (1998), 609-622). Observational studies that do not affect patient response are conducted most frequently. The first type of observational study involves identifying samples from individuals with a suspected cause of the disease and those without, and comparing the incidence of the disease in the two samples. This sampled population is called a cohort, and such studies are prospective. Other types of observational studies are case-control or retrospective studies. In a typical case-control study, samples are collected from individuals possessing the phenotype of interest, such as specific signs of the disease (case group), or from individuals without the phenotype of the population from which conclusions are to be drawn (target population) (control group). The possible causes of the disease are then investigated retrospectively. Because the time and cost of sample collection in case-control studies are significantly lower than those of prospective studies, case-control studies are more commonly used as a research design for genetic association studies, at least in the exploratory and discovery phases.

[0364] After obtaining relevant phenotypic and / or genotypic information, statistical analysis is performed to determine whether there is any significant correlation between the presence of an individual's allele or genotype and phenotypic characteristics. Preferably, data checking and cleaning are performed first before conducting statistical tests for genetic association. The epidemiological and clinical data of the samples may be summarized in descriptive statistics, along with tables and graphs well known in the field. Data verification is preferably performed to identify data completeness, discrepancies, and outliers. Subsequently, the chi-square test and t-test (or Wilcoxon rank-sum test in the case of non-normal distribution) may be used, respectively, to identify significant differences between the case group and the control group for discrete and continuous variables.

[0365] A critical decision in conducting genetic association tests is determining the significance level at which a significant association can be established when the test's p-value reaches that level. In exploratory analyses where positive hits are tracked in subsequent confirmatory tests, an unadjusted p-value <0.2 (the significance level in terms of tolerance) can be used, for example, to formulate a hypothesis regarding a significant association between HHLA2 levels and specific phenotypic traits of a disease. It is desirable to achieve a p-value <0.05 (the significance level traditionally used in this field) for the level to be considered as having an association with the disease. When hits are tracked in confirmatory analyses of more samples from the same source or different samples from different sources, adjustments for multiple tests will be made to avoid excess hits while maintaining the per-experiment error rate at 0.05. While there are other methods for adjusting multiple tests to control different types of error rates, a generally used but somewhat conservative method for controlling per-experiment or per-family error rates is the Bonferroni correction (Multiple comparisons and multiple tests, Westfall et al, SAS Institute (1999)). Permutation tests can be more powerful for controlling the false detection rate (FDR) (Benjamini and Hochberg, Journal of the Royal Statistical Society, Series B 57, 1289-1300, 1995, Resampling-based Multiple Testing, Westfall and Young, Wiley (1993)). These methods for controlling multipleity are preferred when the test is dependent and controlling the false detection rate is sufficient, in contrast to controlling the error rate per experiment.

[0366] When individual genetic or non-genetic risk factors are identified as predisposing factors for a disease, classification / prediction schemes may be established to predict the category (e.g., disease or non-disease) to which an individual belongs based on their phenotype and / or genotype and other non-genetic risk factors. Logistic regression for discrete characteristics and linear regression for continuous characteristics are standard techniques for this task (Applied Regression Analysis, Draper and Smith, Wiley (1998)). Additionally, other techniques may be used to establish classifications. These techniques include, but are not limited to, MART, CART, neural networks, and discriminant analysis, which are suitable for comparing the performance of different methods (The Elements of Statistical Learning, Hastie, Tibshirani & Friedman, Springer (2002)).

[0367] 3. Monitoring of efficacy during clinical trials

[0368] Monitoring the effects of substances (e.g., compounds, drugs, or small molecules) on the HHLA2 polypeptide or fragments thereof (e.g., regulation of cell proliferation and / or migration) may be applied to clinical trials as well as to basic drug screening. For example, the efficacy of a substance determined to reduce HHLA2 gene expression and polypeptide levels or downregulate HHLA2 activity through screening analysis as described herein may be monitored in clinical trials of subjects by whether reduced HHLA2 gene expression and polypeptide levels or downregulated HHLA2 activity are observed, or by whether reduced HHLA2 expression detectable by anti-HHLA2 antibodies or fragments described herein is observed in clinical trials of subjects. In such clinical trials, the expression or activity of the HHLA2 gene and / or symptoms or markers of the disorder of interest may be used as "readouts" or markers of the phenotype of specific cells, tissues, or bodies. Similarly, the efficacy of a substance determined to increase HHLA2 gene expression and polypeptide levels or increase HHLA2 activity through screening analysis as described herein may be monitored in clinical trials of subjects by whether it exhibits increased HHLA2 and polypeptide levels or increased HHLA2 activity, or by whether it exhibits increased HHLA2 detectable by an anti-HHLA2 antibody or a fragment described herein in clinical trials of subjects. In such clinical trials, the expression or activity of the HHLA2 gene and / or symptoms or markers of the disorder of interest may be used as "readouts" or markers of the phenotype of specific cells, tissues, or bodies for, for example, autoimmune disorders.

[0369] For example, without limitation, genes (including HHLA2) regulated within cells by treatment with a substance (e.g., a compound, drug, or small molecule) that regulates HHLA2 activity (e.g., identified in screening analyses as described herein) may be identified. Thus, for example, in clinical trials, to study the effect of a substance on disorders associated with abnormal HHLA2 activity, cells may be isolated and nucleic acids and / or proteins may be prepared and analyzed for the levels of HHLA2 and / or other genes associated with disorders associated with abnormal HHLA2 activity. Gene expression levels (e.g., gene expression patterns) may be analyzed by measuring the amount of polypeptide produced or by measuring the levels of HHLA2 or other genes using one of the methods described herein. In this method, the gene expression pattern may serve as a marker indicating the physiological response of the cell to the substance. Thus, this response state may be determined before treating the individual with the substance and at various points in the course of treatment.

[0370] In a preferred embodiment, the present invention provides a method for monitoring the therapeutic efficacy of a substance (e.g., an agonist, an antagonist, a peptidomimetic, a polypeptide, a peptide, a nucleic acid, a small molecule, or other drug candidate identified by the screening assay described herein) on a subject, comprising the steps of: (i) obtaining a pre-administration sample from a subject before administering the substance; (ii) detecting a level of an HHLA2 polypeptide or a fragment thereof in the pre-administration sample; (iii) obtaining one or more post-administration samples from a subject; (iv) detecting a level of an HHLA2 polypeptide or a fragment thereof in the post-administration sample; (v) comparing the level of an HHLA2 polypeptide or a fragment thereof in the pre-administration sample with the HHLA2 polypeptide in the post-administration sample or the samples; and (vi) changing the administration of the substance to the subject accordingly. For example, it may be desirable to increase the dosage of the substance to lower HHLA2 to a level lower than detected, that is, to increase the efficacy of the substance. According to these embodiments, HHLA2 can be used as an indicator of the efficacy of the substance even in the absence of an observable phenotypic response. Similarly, HHLA2 analysis by methods such as immunohistochemistry (IHC) can also be used to select patients to receive HHLA2 immunotherapy that inhibits one or more immune checkpoints. Tumor patients with HHLA2 activity are more likely to respond to the HHLA2 mAb immunotherapy described herein. This immunotherapy will initially result in immune activation, and activated T cells will express IFN-gamma, which will consequently upregulate HHLA2. Generally, this results in the engagement of HHLA2 and downregulation of the immune response, but since HHLA2 can be blocked by the anti-HHLA2 mAb described herein, the immune response continues until the desired state, such as the tumor, is eliminated.In contrast, mAbs that actively transmit signals via HHLA2 directly downregulate the immune response.

[0371] 4. Treatment methods and uses

[0372] In some embodiments, the antibodies, fragments, or immunoconjugates of the present invention (e.g., anti-HHLA2 antibodies alone or conjugated with a therapeutic moiety) are useful for treating any disorder (e.g., cancer) associated with abnormal or unwanted HHLA2 activity. In certain embodiments, the treatment is for mammals such as humans. These antibodies of the present invention may be used alone or in combination with any substance or therapy suitable for treating the disorder of interest. For example, therapeutic synergy is thought to occur when treating cells containing HHLA2 and other immune checkpoint or co-stimulatory molecules.

[0373] It is well known that therapeutic monoclonal antibodies can deplete cells that express antigens specifically recognized by the antibody extracellularly. This depletion can be mediated through 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.

[0374] "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 when the first component of the complement system binds to an antibody that binds to a cognate antigen. To evaluate complement activation, CDC assays, for example, the assay described in the literature [Gazzano-Santoro et al. (1997)], may be performed.

[0375] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which a secreted antibody bound to an Fc receptor (FcR) present on specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) causes these cytotoxic effector cells to specifically bind to target cells bearing an antigen, thereby killing the target cells. To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay as disclosed in U.S. Patent No. 5,500,362 or No. 5,821,337 may be performed. As is well known in the art, the Fc portion may be manipulated to result in a desired interaction with the Fc receptor or a deficiency thereof.

[0376] For antibody-mediated binding, neutralization, and / or modulation of intracellular targets, specific modifications must be made. As described herein, specific antibody forms, such as sFv and Fab, are suitable for the intracellular expression of antibody-like molecules. Methods for constructing and using such suitable antibody-like molecules for targeted expression in other compartments of the cell, including the nucleus, ER, cytoplasm, Golgi, plasma membrane, mitochondria, and sites corresponding to antigens or molecules in specific pathways, are well known (at least U.S. Patent Publications No. 2008-0233110 and No. 2003-0104402; literature [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 4:11-15; Richardson et al. (1995) Proc. Natl. Acad. Sci. USA 92:3137-3141; and Duan et al.(1994) Human Gene Therapy [See 5:1315-1324]).

[0377] As used herein, the term "intracellular immunoglobulin molecule" refers to a complete immunoglobulin that is naturally secreted but remains inside the cell after synthesis. "Intracellular immunoglobulin fragment" refers to any fragment comprising a single-chain fragment of an intracellular immunoglobulin molecule. Accordingly, intracellular immunoglobulin molecules or fragments thereof are not secreted or expressed on the external surface of the cell. A single-chain intracellular immunoglobulin fragment refers to "single-chain immunoglobulin" in this specification. As used herein, the term "intracellular immunoglobulin molecule or fragment thereof" is understood to include "intracellular immunoglobulin," "single-chain intracellular immunoglobulin (or fragment thereof)," "intracellular immunoglobulin fragment," "intracellular antibody (or fragment thereof)," and "intrabody (or fragment thereof)." As such, the terms “intracellular immunoglobulin,” “intracellular Ig,” “intracellular antibody,” and “intrabody” may be used interchangeably herein and are all included in the general definition of “intracellular immunoglobulin molecule or fragment thereof.” The intracellular immunoglobulin molecule or fragment thereof of the present invention may, in some embodiments, comprise two or more subunits, for example, “a first intracellular immunoglobulin subunit polypeptide” and “a second intracellular immunoglobulin subunit polypeptide.” However, in other embodiments, the intracellular immunoglobulin may be a “single-chain intracellular immunoglobulin” comprising only one polypeptide. As used herein, “single-chain intracellular immunoglobulin” is defined as any single fragment having a desired activity, for example, intracellular binding activity to an antigen.Accordingly, single-chain intracellular immunoglobulins include not only those comprising both heavy and light chain variable regions that work together to bind to an antigen, but also single-chain intracellular immunoglobulins having only a single variable region that binds to an antigen, e.g., a "camelized" heavy chain variable region as described herein. Intracellular immunoglobulins or Ig fragments may be expressed substantially anywhere within the cell, such as within the cytoplasm, on the inner surface of the cell membrane, or within subcellular compartments (also referred to as cell subcompartments or cell compartments) (e.g., nucleus, Golgi, endoplasmic reticulum, endosomes, mitochondria, etc.). Additional cell subcompartments include those described herein and those well known in the art.

[0378] These intracellular immunoglobulins are expressed within a recipient cell or host cell containing an antigen to be targeted. The host cell of the present invention is preferably a eukaryotic cell or cell line, and preferably a plant, animal, vertebrate, mammalian, rodent, mouse, primate, or human cell or cell line.

[0379] Without being bound by theory, it is thought that the intracellular expression of the immunoglobulin polypeptide described in this specification allows for intracellular targeting and binding to HHLA2, thereby modulating the ability to propagate signaling when activated by binding to an inhibitory receptor and / or the ability to regulate signaling when increasing the local efficacy concentration of multiple HHLA2 molecules, and thus stereochemically modulates the ability of the signaling molecule.

[0380] In some embodiments, the antibody of the present invention may be conjugated to a therapeutic moiety such as a growth inhibitor, a cytotoxic substance, or a prodrug-activating enzyme as described above. The antibody of the present invention may be useful for targeting said growth inhibitor, cytotoxic substance, or prodrug to cells that express less or overexpress HHLA2 than the desired amount.

[0381] Accordingly, the object of the present invention relates to a method for treating disorders associated with abnormal HHLA2 activity, comprising administering the antibody, fragment, or immunoconjugate of the present invention to a subject requiring treatment in a therapeutically effective amount.

[0382] Alternatively, in some embodiments, the antibody or antigen-binding fragment of the present invention is useful for therapeutic applications in addition to diagnostic, prognostic, and prophylactic applications regarding the upregulation of immune responses. The upregulation of immune responses may take the form of enhancing existing immune responses or inducing early immune responses. For example, enhancing immune responses using the target composition and method is useful in cases where immunological defense against cancer and microbial (e.g., bacteria, viruses, or parasites) infections is improved. For example, as described herein, the upregulation or enhancement of immune response functions is useful for inducing tumor immunity.

[0383] In another embodiment, the immune response may be stimulated by the method described herein so that existing resistance, clonal deletions, and / or depletion (e.g., T cell depletion) is overcome. For example, an immune response to an antigen for which the subject cannot exhibit a significant immune response, such as an autoantigen like a tumor-specific antigen, may be induced by administering a suitable substance described herein that upregulates the immune response. In one embodiment, an autoantigen like a tumor-specific antigen may be co-administered. In another embodiment, the immune response may be stimulated with an antigen (e.g., an autoantigen) to treat a neurological disorder. In another embodiment, the subject substance may be used as an adjuvant to enhance the response to an exogenous antigen during the active immunization process.

[0384] In certain examples, to further enhance the immune response, it may be desirable to additionally administer other substances that upregulate the immune response, such as forms of other B7 family members that signal via co-stimulatory receptors. Additionally, substances that upregulate the immune response may be used prophylactically in vaccines against various polypeptides (e.g., polypeptides derived from pathogens). Immunity to pathogens (e.g., viruses) can be induced by vaccinating viral proteins with substances that upregulate the immune response in appropriate adjuvants.

[0385] Alternatively, in some embodiments, the antibody and antigen-binding fragment of the present invention are for diseases that upregulate immune action, e.g., asthma, autoimmune diseases (glomerulonephritis, arthritis, dilated cardiomyopathy-like disease, ulcerative colitis, Sjögren's syndrome, Crohn's disease, systemic lupus erythematosus, chronic rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, scleroderma, periarteritis nodosum, rheumatic fever, vitiligo, insulin-dependent diabetes mellitus, Behcet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, pernicious anemia, Goodpasture syndrome, infertility, chronic active hepatitis, pemphigus, autoimmune thrombocytopenic purpura, and autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, anti-phospholipid syndrome, atopic allergy, autoimmune atrophic Gastritis, achlorogenic autoimmunity, celiac disease, Cushing's syndrome, dermatomyositis, discoid lupus, erythematosis, Goodpasture syndrome, Hashimoto's thyroiditis, idiopathic renal myofascial atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes mellitus, Lambert-Eaton syndrome, lupoid hepatitis, some cases of lymphopenia, mixed connective tissue disease, pemphigus, pemphigus vulgaris, pernicious anemia, lenticular uveitis, nodular pericarritis, polylinear autosyndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's disease, recurrent polychondritis, Schmidt syndrome, restricted scleroderma (or Crest syndrome), sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, insulin resistance type B, ulcerative colitis and It is useful for therapeutic applications in addition to diagnostic, prognostic, and preventive applications (such as treatment and delaying the onset or progression of the disease) to inhibit Wegener's granulomatosis.

[0386] Similarly, the antibodies and antigen-binding fragments of the present invention are useful for therapeutic applications in addition to diagnostic, prognostic, and prophylactic applications (such as treatment and delaying the onset or progression of the disease) for persistent infectious diseases (e.g., viral infectious diseases including retroviruses such as HPV, HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV-1 and HIV-2), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpesviruses such as HSV-1 and HSV-2, and influenza viruses). Other pathogen-related antigens that may be used as described herein are antigens of various parasites, including malaria, preferably NANP repeat-based malaria peptides. Additionally, Aspergillus ( Aspergillus ), Brugia( Brugia ), Candida( Candida ), Chlamydia( Chlamydia ), Coccidias ( Coccidia ), Cryptococcus( Cryptococcus ), Dirofilaria( Dirofilaria ), Gonococcus( Gonococcus ), histoplasma( Histoplasma ), Leishmania( Leishmania ), Mycobacterium( Mycobacterium ), Mycoplasma( Mycoplasma ), Paramecium( Paramecium ), Pertussis( Pertussis ), Plasmodium( Plasmodium ), Pneumococcus( Pneumococcus ), Pneumocystis( Pneumocystis ), Rickettsia( Rickettsia ), Salmonella( Salmonella ), Shigela( Shigella ), Staphylococcus ( Staphylococcus ), Streptococcus( Streptococcus ), Toxoplasma( Toxoplasma ) and Vibrio cholerae ( Vibrio cholerae This includes bacteria, fungi, and other parasitic diseases such as ). An example of a species is Neisseria gonorrhea ( Neisseria gonorrhoeae ), Mycobacterium tuberculosis( Mycobacterium tuberculosis ), Candida albicans( Candida albicans ), Candida tropicalis( Candida tropicalis ), Trichomonas vaginalis( Trichomonas vaginalis ), Haemophilus vaginalis ( Haemophilus vaginalis ), Group B Streptococcus sp., Microplasma hominis( Human microplasma ), Haemophilus Ducray( Hemophilus ducreyi ), Granuloma Inguinal ( Inguinal granuloma ), Lymphopathia Venereum( Venereal lymphopathy ), Treponema pallidium ( Treponema pallidum ), Brucella avibotus ( Brucella abortion Brucella melitensis, Brucella suis ( Brucella his ), Brucella canis ( Brucella canis ), Campylobacter fetus ( Campylobacter fetus ), Campylobacter fetus intestinalis( Campylobacter fetus intestinalis ), Leptospira Pomona ( Leptospira pomona ), Listeria monocytogenes( Listeria monocytogenes ), Brucella obis ( Brucella ovine ), Chlamydia sitasi( Chlamydia psittaci ), Trichomonas poetus ( Trichomonas foetus ), Toxoplasma gondii ( Toxoplasma gondii ), Isquerichia coli ( Escherichia coli ), Actinobacillus equili ( Actinobacillus equinus ), Salmonella abotus obis ( Salmonella abortion of eggs ), Salmonella avocados equity ( Salmonella abortion in horses ), Pseudomonas aeruginosa( Pseudomonas aeruginosa ), Corynebacterium equi( Corynebacterium equi ), Corynebacterium piogenes ( Corynebacterium pyogenes ), Actiobacillus seminis( Actinobacillus seminis ), Mycoplasma bovigenitalium ( Mycoplasma bovigenitalium ), Aspergillus fumigatus ( Aspergillus fumigatus ), Absidia Ramosa( Branched absidium ), Trypanosoma Equiperdum( Trypanosoma equiperdum ), Babesia Cavalli( Babesia equina ), Clostridium tetani( Clostridium tetani), Clostridium botulinum ( Clostridium botulinum ); or for example, Paracoccidioides brasiliensis( Paracoccidioides brasiliensis Fungi such as ); or other pathogens, for example, Plasmodium falciparum ( Plasmodium falciparum ) is included. It also includes priority pathogens of the U.S. National Institute of Allergy and Infectious Diseases (NIAID). These include variola major (smallpox) and Bacillus anthracis ( Bacillus anthracis )(Anthracnose), Yersinia pestis( Yersinia pestis )(Black Death), Clostridium botulinum toxin( Clostridium botulinum toxin)(botulism), Francisela tularensis( Francisella tularensis Category A substances such as )(tularemia), filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagic fever), arenaviruses (Lassa fever, Junin hemorrhagic fever and related viruses); Coxiella brunettii ( Coxiella burnetti )(Q column), Brucella( Brucella ) species (brucellosis), Burkholderia malaya ( Burkholderia mallei )(paralytics), alphavirus (Venezuelan encephalomyelitis, eastern and western equine encephalomyelitis), ricin toxin of castor (Ricinus communis), Clostridium perfringens( Clostridium perfringens Category B substances such as epsilon toxin of ); Staphylococcus enterotoxin B ( Staphylococcus enterotoxin B), Salmonella species, Shigella discenteriae ( Shigella dysenteriae ), Iskerichia coli strain O157:H7( Escherichia coli strain O157:H7), Vibrio cholerae ( Vibrio cholerae), Cryptosporidium parvum; Category C substances such as Nipah virus, Hantavirus, Tick-borne hemorrhagic fever virus, Tick-borne encephalitis virus, yellow fever, and multidrug-resistant tuberculosis; Shestosoma ( Schistosoma ) and Tinia( Taenia Helminths such as ); Leishmania (e.g., L. mexicana ( L. mexicana )) and Plasmodium( Plasmodium It includes protozoa such as ).

[0387] In another embodiment, the antibody or antigen-binding fragment of the present invention is useful for therapeutic applications in addition to diagnostic, prognostic, and preventive applications regarding immunological resistance, organ transplant rejection, graft-versus-host disease (GVHD), allergic diseases, and diseases resulting from attenuation of the immune response mediated by HHLA2.

[0388] In the context of the present invention, as used herein, the terms “treating” or “treatment” mean reversing, alleviating, or inhibiting the progression of a disorder or condition to which such terms apply, or one or more symptoms of such disorder or condition, or preventing such disorder or condition or symptoms. As used herein, the term “cancer treatment” means inhibiting the growth and / or proliferation of cancer cells. Preferably, such treatment also induces regression of tumor growth (i.e., a measurable reduction in tumor size). Most preferably, such treatment induces complete regression of the tumor.

[0389] In some embodiments, the term “patient” or “patient requiring this” is intended for human or non-human mammals affected or likely to be affected by cancer associated with abnormal activation of HHLA2.

[0390] The "therapeutic effective dose" of the polypeptide of the present invention refers to a sufficient amount of antibody to treat a disorder of interest, such as cancer, with a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily use of the antibody and composition of the present invention will be determined by a physician within the scope of sound medical judgment. The specific therapeutic effective dose level for any particular patient will depend on various factors including the disorder to be treated and the severity of the disorder; the activity of the specific antibody used; the specific composition used, age, weight, general health, gender, and patient diet; the time of administration, the route of administration, and the efflux rate of the specific antibody used; the duration of treatment; drugs used in combination or simultaneously with the specific polypeptide used; and similar factors well known in the medical field. For example, it is well known to those skilled in the art to start administration of a compound at a level lower than necessary to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0391] Therapeutic formulations comprising one or more antibodies of the present invention are prepared in the form of lyophilized formulations or aqueous solutions by mixing antibodies having a desired degree of purity for storage with additional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th ed., Osol, A. Ed. (1980)). The antibody compositions will be formulated, quantified, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the substance, the method of administration, the administration schedule, and other factors known to the medical practitioner.

[0392] The therapeutic dose may be at least about 0.01 µg / kg body weight, at least about 0.05 µg / kg body weight; at least about 0.1 µg / kg body weight, at least about 0.5 µg / kg body weight, at least about 1 µg / kg body weight, at least about 2.5 µg / kg body weight, at least about 5 µg / kg body weight, and up to about 100 µg / kg body weight. Those skilled in the art will understand that these guidelines will be adjusted according to the molecular weight of the active substance, for example, in the use of antibody fragments or antibody conjugates. The dose may also vary depending on local administration, e.g., intranasal, inhalation, etc., or systemic administration, e.g., im, ip, iv, etc.

[0393] Although not necessary, the composition is formulated with one or more substances that selectively enhance activity or otherwise increase the therapeutic effect. These are generally used at the same dosage and route of administration as previously used, or at about 1 to 99% of the previously used dosage.

[0394] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohols; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; and amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Sacosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counter-ion-forming salts such as sodium; metal complexes (e.g., Zn-protein complexes); and / or TWEEN TM , PLURONICS TM Or it contains non-ionic surfactants such as polyethylene glycol (PEG). Formulations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter membrane.

[0395] The active ingredient may also be captured in microcapsules prepared in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, for example, by coacervation technology or interfacial polymerization. These technologies are described in the literature [Remington's Pharmaceutical Sciences 16th ed., Osol, A. Ed. (1980)].

[0396] The compositions described herein may be administered by any suitable method including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and nasal. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Additionally, the compositions may be appropriately administered, particularly by pulse infusion with a reduced dose of antibody.

[0397] For the prevention or treatment of disease, the appropriate dosage of antibodies will depend on the type of disease to be treated as defined above, the severity and course of the disease, whether antibodies are administered for prophylactic purposes, prior therapy, the patient's clinical history and response to antibodies, and the physician's discretion. Antibodies are administered to the patient appropriately, either at once or over a series of treatments.

[0398] The therapeutic substance of the present invention may be used alone or administered in combination with, for example, chemotherapy substances, hormones, anti-angiogenic agents, radiolabels, compounds, or in combination with surgery, cryotherapy, and / or radiotherapy. The aforementioned treatment methods may be administered in combination with other forms of conventional therapy (e.g., standard treatments for cancer well known to those skilled in the art), or sequentially with pre- or post-conventional therapy. For example, the substance of the present invention may be administered with a therapeutically effective amount of chemotherapy substance. In another embodiment, the substance of the present invention is administered with chemotherapy to enhance the activity and efficacy of the chemotherapy substance. The Physicians' Desk Reference (PDR) discloses dosages of chemotherapy substances that have been used to treat various cancers. The administration regimen and dosage of the aforementioned therapeutically effective chemotherapy drugs will vary depending on the specific cancer to be treated, the severity of the disease, and other factors familiar to those skilled in the art, and may be determined by a physician.

[0399] Cancer vaccines may also be administered in combination with targeted therapies, such as immunotherapy. The term "targeted therapy" refers to the administration of a substance that treats cancer by selectively interacting with selected biomolecules. For example, targeted therapy involving the inhibition of immune checkpoint inhibitors is useful in combination with the methods of the present invention. The term "immune checkpoint inhibitor" refers to a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by down-regulating or inhibiting the anti-tumor immune response. Immune checkpoint proteins are well known in this field and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR (see, e.g., WO 2012 / 177624). Inhibition by one or more immune checkpoint inhibitors can upregulate the immune response to treat cancer more effectively by blocking or neutralizing inhibitory signaling. In some embodiments, the cancer vaccine is administered in combination with one or more immune checkpoint inhibitors, such as PD1, PD-L1, and / or CD47 inhibitors.

[0400] Immunotherapy is a form of targeted therapy that may include, for example, the use of additional cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are potentially useful for cancer therapy because they can infect and lyse cancer cells but are harmless to normal cells. Replication of oncolytic viruses promotes tumor cell destruction and generates dose amplification at the tumor site. They can also act as vectors for anticancer genes, allowing for the specific delivery of anticancer genes to the tumor site. Immunotherapy may include passive immunity for short-term host protection achieved by administering pro-forming antibodies against cancer antigens or disease antigens (e.g., administration of monoclonal antibodies against tumor antigens selectively linked to chemotherapy substances or toxins). For example, anti-VEGF and mTOR inhibitors are known to be effective in the treatment of renal cell carcinoma. Immunotherapy may also focus on using cytotoxic lymphocyte-recognizing epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc., can be used to selectively control biomolecules associated with the initiation, progression, and / or pathology of tumors or cancer.

[0401] The term "non-targeted therapy" refers to the administration of a substance that treats cancer without selectively interacting with selected biomolecules. Representative examples of non-targeted therapy include, but are not limited to, chemotherapy, gene therapy, and radiation therapy.

[0402] In one embodiment, chemotherapy is used. Chemotherapy involves the administration of a chemotherapy substance. Such chemotherapy substances may be selected from, but are not limited to, the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, anti-mitotic substances, alkylating agents, algenin compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogs, plant alkaloids, and toxins; and synthetic derivatives thereof. Examples of compounds include alkylating agents: cisplatin, treosulfan, and trophosphamide; plant alkaloids: vinblastine, paclitaxel, and docetaxol; DNA topoisomerase inhibitors: tenifoside, crisnatol, and mitomycin; anti-folic acid: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxyfluridine, and cytosine arabinoside; Purine analogs: mercaptopurines and thioguanine; DNA anmetabolites: 2'-deoxy-5-fluoro-uridine, aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halicondrin, colchicine, and rhizoxin are included, but not limited thereto. Compositions comprising one or more chemotherapy agents (e.g., FLAG, CHOP) may also be used. FLAG includes fludarabine, cytosine arabinoside (Ara-C), and G-CSF. CHOP includes cyclophosphamide, vincristine, doxorubicin, and prednisone. The foregoing examples of chemotherapy agents are illustrative and are not intended to be limiting.

[0403] In another embodiment, radiation therapy is used. The radiation used in radiation therapy may be ionizing radiation. Radiation therapy may also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external beam radiation therapy, interstitial implantation of radioisotopes (I-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or whole abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see the literature [Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al. You may refer to [ , eds., JB Lippencott Company, Philadelphia]. Radiation therapy may be applied as external beam radiation or remote therapy in which radiation is derived from a distant source. Radiation therapy may also be applied as internal therapy or brachytherapy in which the source of radiation is located within the body close to the cancer cells or tumor mass. Also included is the use of photodynamic therapy, which includes the administration of photosensitizers such as hematoporphyrin and its derivatives, vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrelin; and 2BA-2-DMHA.

[0404] In another embodiment, hormone therapy is used. Hormone therapy treatment may include, for example, hormone agonists, hormone antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing and steroids (e.g., dexamethasone, retinoids, deltoid, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestin), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestogens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0405] 5. Analysis and screening methods

[0406] Another aspect of the present invention relates to a screening analysis comprising a non-cell-based analysis and an analysis of a xenograft animal model. In one embodiment, the analysis provides a method for identifying substances that regulate HHLA2 signaling to identify substances that increase an immune response by decreasing HHLA2 signaling and / or substances that decrease an immune response by increasing HHLA2 signaling, as in human or animal model analysis.

[0407] In one embodiment, the present invention relates to an analysis for screening a substance that binds to or modulates the biological activity of at least one biomarker described herein (e.g., in tables, figures, examples, or the specification), such as HHLA2, TMIGD2, and KIR3DL3. In one embodiment, a method for identifying such a substance requires determining the ability of the substance to modulate, for example, inhibit, at least one biomarker described herein.

[0408] In one embodiment, the analysis is a cell-free or cell-based analysis comprising contacting a test substance with at least one biomarker described herein and determining the ability of the test substance to regulate (e.g., inhibit) the enzymatic activity of the biomarker, such as by measuring the direct binding of a substrate or measuring an indirect parameter described below.

[0409] For example, in direct binding analysis, a biomarker protein (or its individual target polypeptide or molecule) can be linked to a radioisotope or enzymatic label, and binding can be determined by detecting the labeled protein or molecule within the complex. For example, the target is 125 I, 35 S, 14 C or 3 It can be labeled with H, directly or indirectly, and the radioisotope can be detected by direct counting of radioactivity emission or scintillation counting. Alternatively, the target can be enzymatically labeled, for example, with mustard bean peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label can be detected through a method that determines the conversion of the appropriate substrate to the product. Determining the interaction between the biomarker and the substrate can be achieved using standard binding or enzymatic assays. In one or more embodiments of the assays described above, it may be desirable to immobilize the polypeptide or molecule to facilitate the separation of the complex from one or two non-complex forms of the protein or molecule and to promote the automation of the assay.

[0410] Target binding of the test substance may be performed in any container suitable for containing the reactant. Non-limiting examples of such containers include microtitration plates, test tubes, and microcentrifuge tubes. The immobilized forms of the antibody described herein may also comprise antibodies bound to a solid phase such as a porous, microporous (having an average pore diameter of less than about 1 micron) or macroporous (having an average pore diameter of more than about 10 microns) material, such as a membrane, cellulose, nitrocellulose, or glass fiber; beads made of agarose, polyacrylamide, or latex; or the surface of a dish, plate, or well made of polystyrene.

[0411] In an alternative embodiment, determining the ability of a substance to regulate the interaction between a biomarker and a substrate or between a biomarker and its natural binding partner can be performed by determining the ability of a test substance to regulate the activity of a polypeptide or other product that acts downstream or upstream of its location within a signaling pathway (e.g., a feedback loop). Such feedback loops are well known in the field (e.g., Chen and Guillemin (2009) Int. J. Tryptophan Res. (See 2:1-19).

[0412] HHLA2 status can be measured using the anti-HHLA2 antibody described herein. A decrease in HHLA2 expression indicates that the substance inhibits HHLA2 activity / signaling and indicates that the substance is useful for inhibiting HHLA2 activity / signaling and increasing the immune response. A decrease in HHLA2 binding to TMIGD2, KIR3DL3, and / or inhibitory receptors indicates that the substance inhibits HHLA2 activity / signaling and indicates that the substance is useful for inhibiting HHLA2 activity / signaling and increasing the immune response. Conversely, an increase in HHLA2 expression indicates that the substance promotes HHLA2 activity / signaling and indicates that the substance is useful for promoting HHLA2 activity / signaling and decreasing the immune response. Increased HHLA2 binding to TMIGD2, KIR3DL3 and / or inhibitory receptors indicates that the substance promotes HHLA2 activation / signaling and is a useful substance for promoting HHLA2 activation / signaling and reducing immune responses.

[0413] The present invention also relates to novel substances identified through the screening analysis described above. Accordingly, using the identified substances as described herein, as in appropriate animal models, is also within the scope of the present invention. For example, the identified substances as described herein may be used in animal models to determine the therapeutic efficacy, toxicity, or side effects of such substances. Alternatively, the identified antibodies as described herein may be used in animal models to determine the mechanism of action of such substances.

[0414] One aspect of the present invention relates to a screening analysis comprising a non-cell-based analysis and a xenograft animal model analysis. In one embodiment, the analysis provides a method for determining, for example using a xenograft animal model analysis, whether cancer in humans is likely to respond to anti-HHLA2 antibody therapy, and / or whether a substance can inhibit the growth of cancer cells that are unlikely to respond to anti-HHLA2 antibody therapy or kill these cancer cells.

[0415] 6. Preventive methods

[0416] In one aspect, the present invention provides a method for preventing a disease or condition associated with an unwanted immune response or a less-than-desirable immune response in a subject. A subject at risk of the disease who will receive therapeutic benefits using the claimed substance or method may be identified, for example, by any diagnostic or prognostic assay known in the art or a combination thereof. The administration of the prophylactic substance may occur prior to the onset of symptoms associated with an unwanted immune response or a less-than-desirable immune response. The appropriate substance to be used for treatment (e.g., antibody, peptide, fusion protein, or small molecule) may be determined based on clinical indications and may be identified, for example, using the screening assays described herein.

[0417] VII. Pharmaceutical composition

[0418] Substances that modulate (e.g., inhibit or promote) the interaction between HHLA2 and one or more natural binding partners (e.g., TMIGD2 and / or KIR3DL3), such as blocking antibodies, peptides, fusion proteins, or small molecules, may be included in a pharmaceutical composition suitable for administration to a target. Such compositions typically comprise antibodies, peptides, fusion proteins, or small molecules and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption retardants, etc., suitable for pharmaceutical administration. The use of such media and materials for pharmaceutically active substrates is well known in the art. Their use in the composition is considered, except where any existing media or material is incompatible with the active compound. A supplementary active compound may also be included in the composition.

[0419] The pharmaceutical compositions of the present invention are formulated to be suitable for the intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, salt solutions, fixatives, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and tonics such as sodium chloride or dextrose. The pH may be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be contained in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0420] Pharmaceutical compositions suitable for injectable use comprise a sterile aqueous solution (if water-soluble) or dispersion and a sterile powder for the immediate preparation or dispersion of a sterile injectable solution. For intravenous administration, suitable carriers are physiological saline, bacteriostatic water, Cremophor EL. TM It includes (BASF, Parsipanye, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and preserved against the action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained through the use of coatings, for example, lecithin, maintaining the necessary particle size in the case of dispersion, and the use of surfactants. Prevention of microbial action can be achieved with various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is desirable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Long-term absorption of the injectable composition can be achieved by including an absorption retardant, for example, aluminum monostearate and gelatin, in the composition.

[0421] Sterile injectable solutions can be prepared by incorporating the required amount of an active compound into a suitable solvent with one or a combination of the components listed above, if necessary, and filtering and sterilizing. Generally, dispersions are prepared by incorporating the active compound into a base dispersion medium and a sterile medium containing the required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, a preferred method of preparation is to obtain powders of the active component and any desired additional component from a solution that has been pre-sterilized and filtered by vacuum drying and freeze-drying.

[0422] Oral compositions generally comprise an inactive diluent or an edible carrier. These may be contained in gelatin capsules or compressed into tablets. For the purpose of oral therapy administration, the active compound may be incorporated with an excipient and available in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier for use in oral rinsing, wherein the compound within the fluid carrier is applied orally and can be rinsed, spat out, or swallowed. Pharmaceutically suitable binders and / or auxiliary substances may be included as part of the composition. Tablets, pills, capsules, troches, etc., may contain any of the following components or compounds of similar properties: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginate, primogel, or corn starch; lubricants such as magnesium stearate or steroids; fluidizing agents such as colloidal silicon dioxide; Sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavoring.

[0423] For inhalation administration, the compound is delivered in the form of an aerosol spray or nebulizer from a pressure vessel or dispenser containing a suitable compressed gas, for example, carbon dioxide.

[0424] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrating agent suitable for the barrier to be penetrated is used in the formulation. Such penetrating agents are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration may be performed using a nasal spray or suppository. For transdermal administration, the active compound is formulated into an ointment, plaster, gel, or cream as is generally known in the art.

[0425] The compound can be formulated as a suppository (e.g., with a conventional suppository base such as cocoa butter and other glycerides) or a delayed enema for rectal delivery.

[0426] In one embodiment, the modifier is prepared with a carrier that protects the compound from rapid clearance from the body, such as in a controlled-release formulation comprising an implantable and microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyothoesters, and polylactic acid may be used. Methods for preparing such formulations should be obvious to those skilled in the art. Additionally, the materials may be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (containing liposomes targeted to infected cells together with a monoclonal antibody against a viral antigen) may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, the methods described in U.S. Patent No. 4,522,811.

[0427] It is particularly advantageous to formulate oral or parenteral compositions in the form of dosage units for ease of administration and uniformity of dosage. As used herein, a dosage unit refers to a physically separated unit suitable as a single dose for a subject to be administered; each unit contains a predetermined amount of an active compound calculated to produce a desired therapeutic effect in relation to the required pharmaceutical carrier. The dosage unit form of the present invention is dictated by and directly depends on the unique characteristics of the active compound, the specific therapeutic effect to be achieved, and the inherent limitations of the field in which such active compound is formulated for the treatment of the subject.

[0428] The toxicity and therapeutic efficacy of these compounds can be determined in cell cultures or experimental animals, for example, according to standard pharmaceutical procedures for determining the LD50 (lethal dose for 50% of the population) and ED50 (therapeutic dose for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds with a large therapeutic index are preferred. Although compounds exhibiting toxic side effects may be used, care must be taken in designing a delivery system that reduces side effects by targeting the compound to the infected tissue site to minimize potential damage to uninfected cells.

[0429] Data obtained from cell culture analysis and animal studies can be used to formulate dosage ranges for use in humans. Dosages of these compounds are preferably within a range of circulating concentrations containing an ED50 that is low or non-toxic. Dosages may vary within a range depending on the applied dosage form and the route of administration utilized. For any compound used in the method of the present invention, the therapeutically effective dose can be initially estimated from cell culture analysis. A single dose may be prescribed to an animal model to achieve a range of circulating plasma concentrations containing an IC50 (i.e., the concentration of the test compound that achieves half-maximum inhibition of symptoms) as determined in cell culture. This information can be used to more accurately determine a dosage useful for humans. Plasma levels can b...

Claims

Claim 1 A monoclonal antibody capable of binding to HHLA2 or an antigen-binding fragment thereof, wherein the monoclonal antibody comprises: a) a heavy chain CDR 1 amino acid sequence of SEQ ID NO. 206; a heavy chain CDR 2 amino acid sequence of SEQ ID NO. 210; a heavy chain CDR 3 amino acid sequence of SEQ ID NO. 214; and b) a light chain CDR 1 amino acid sequence of SEQ ID NO. 224; a light chain CDR 2 amino acid sequence of SEQ ID NO. 228; and a light chain CDR 3 amino acid sequence of SEQ ID NO.

232. Claim 2 delete Claim 3 delete Claim 4 In claim 1, the monoclonal antibody comprises: a) a heavy chain variable region (V) comprising the sequence of SEQ ID NO. 199 H ); and b) a light chain variable region (V) comprising the sequence of sequence number 217 L A monoclonal antibody capable of binding to HHLA2, comprising ) or an antigen-binding fragment thereof. Claim 5 The monoclonal antibody or its antigen-binding fragment capable of binding to HHLA2 is (i) chimeric, humanized, synthetic, rat, or human; (ii) labeled for detection, containing an effector domain, containing an Fc domain, or selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments; (iii) inhibiting a) binding of HHLA2 to TMIGD2, b) binding of HHLA2 to KIR3DL3, or c) binding of HHLA2 to TMIGD2 and binding of HHLA2 to KIR3DL3; or (iv) a monoclonal antibody or its antigen-binding fragment capable of binding to HHLA2 that specifically binds to HHLA2. Claim 6 A complement of a nucleic acid encoding a monoclonal antibody capable of binding to HHLA2 of any one of claims 1, 4 and 5 or an antigen-binding fragment thereof, or an isolated nucleic acid molecule hybridized under strict conditions with a nucleic acid encoding a monoclonal antibody capable of binding to HHLA2 of any one of claims 1, 4 and 5 or an antigen-binding fragment thereof. Claim 7 A vector comprising the isolated nucleic acid of paragraph 6. Claim 8 As an isolated host cell, comprising: (a) an isolated nucleic acid molecule that is hybridized under strict conditions with a complement of a nucleic acid encoding a monoclonal antibody capable of binding to HHLA2 of any one of claims 1, 4, and 5 or an antigen-binding fragment thereof; (b) a nucleic acid encoding a monoclonal antibody capable of binding to HHLA2 of any one of claims 1, 4, and 5 or an antigen-binding fragment thereof; (c) a vector comprising the isolated nucleic acid of (a) or (b); and (d) an isolated host cell expressing an antibody capable of binding to HHLA2 of any one of claims 1, 4, and 5 or an antigen-binding fragment thereof. Claim 9 An apparatus comprising a monoclonal antibody capable of binding to at least one HHLA2 of any one of claims 1, 4, and 5, for diagnosing cancer by detecting the presence or level of an HHLA2 polypeptide, or an antigen-binding fragment thereof. Claim 10 A kit comprising a monoclonal antibody capable of binding to at least one HHLA2 of any one of claims 1, 4, and 5, or an antigen-binding fragment thereof, for diagnosing cancer by detecting the presence or level of an HHLA2 polypeptide. Claim 11 A method for producing a monoclonal antibody or an antigen-binding fragment thereof capable of binding to at least one HHLA2 according to any one of claims 1, 4 and 5, comprising: (i) culturing isolated host cells transformed with a nucleic acid comprising a sequence encoding at least one monoclonal antibody or an antigen-binding fragment thereof capable of binding to at least one HHLA2 according to any one of claims 1, 4 and 5 under conditions suitable for the expression of said monoclonal antibody or an antigen-binding fragment thereof capable of binding to HHLA2; and (ii) recovering the expressed monoclonal antibody or an antigen-binding fragment thereof capable of binding to HHLA2. Claim 12 A method for detecting the presence or level of an HHLA2 polypeptide, comprising the step of detecting said polypeptide in a sample using a monoclonal antibody capable of binding to at least one HHLA2 according to any one of claims 1, 4, and 5, or an antigen-binding fragment thereof. Claim 13 A method for detecting the presence or level of an HHLA2 polypeptide according to claim 12, wherein at least one monoclonal antibody capable of binding to HHLA2 or an antigen-binding fragment thereof forms a complex with the HHLA2 polypeptide and detects the complex using an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical assay, or Western blot, or by using an intracellular flow assay. Claim 14 A method for providing information for monitoring the progression of a disorder characterized by abnormal HHLA2 expression in a subject, comprising: a) detecting a level of HHLA2 in a subject sample at a first time point using a monoclonal antibody or an antigen-binding fragment thereof capable of binding to at least one HHLA2 according to any one of claims 1, 4 and 5; 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 disorder in a subject. Claim 15 A method for providing information for monitoring the progression of a disorder characterized by abnormal HHLA2 expression in a subject, wherein the subject receives treatment to improve the disorder between the first time point and the subsequent time point. Claim 16 A method for predicting the clinical outcome of a subject suffering from a disorder characterized by abnormal HHLA2 expression, comprising: a) determining the level of HHLA2 in a sample of the subject using a monoclonal antibody capable of binding to at least one HHLA2 according to any one of claims 1, 4, and 5, or an antigen-binding fragment thereof; b) determining the level of HHLA2 in a sample of a control subject having good clinical outcome using the monoclonal antibody capable of binding to at least one HHLA2, or an antigen-binding fragment thereof; and c) comparing the level of HHLA2 in a sample of the subject and a sample of the control subject, wherein if the level of HHLA2 in the sample of the subject is significantly higher than the level in the sample of the control subject, the subject has poor clinical outcome. Claim 17 A method for predicting clinical outcomes of a subject suffering from a disorder characterized by abnormal HHLA2 expression, wherein the significantly higher level of HHLA2 comprises at least a 20% increase between the level of HHLA2 in the subject's sample and the level of normal HHLA2 in the sample of the control subject. Claim 18 A method for evaluating the efficacy of a treatment for a disorder characterized by abnormal HHLA2 expression in a subject, comprising: a) determining the level of HHLA2 in a first sample obtained from a subject before providing at least a portion of the treatment to the subject using a monoclonal antibody or an antigen-binding fragment thereof capable of binding to at least one HHLA2 according to any one of claims 1, 4, and 5; and b) determining the level of HHLA2 in a second sample obtained from the subject after providing the portion of the treatment, wherein if the level of HHLA2 in the second sample is significantly lower than that in the first sample, the treatment is indicated as effective in inhibiting the disorder in the subject. Claim 19 A method for evaluating the efficacy of a treatment for a disorder characterized by abnormal HHLA2 expression in a subject, wherein the significantly lower level of HHLA2 described in claim 18 comprises a reduction of at least 20% in the level of HHLA2. Claim 20 A method for evaluating the efficacy of a test compound for inhibiting a disorder characterized by abnormal HHLA2 expression in a subject, comprising: a) determining the level of HHLA2 in a first sample obtained from the subject and exposed to the test compound using a monoclonal antibody or an antigen-binding fragment thereof capable of binding to at least one HHLA2 according to any one of claims 1, 4 and 5; and b) determining the level of HHLA2 in a second sample obtained from the subject, wherein the second sample is not exposed to the test compound, and the test compound is indicated as effective in inhibiting the disorder in the subject when the level of HHLA2 is significantly lower compared to the second sample. Claim 21 A method for evaluating the efficacy of a test compound, wherein, in paragraph 20, the first sample and the second sample are part of a single sample obtained from the subject or part of a mixed sample obtained from the subject. Claim 22 In claim 14, (a) the disorder is cancer; (b) the disorder is cancer selected from the group consisting of lung cancer, renal cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, glioma, glioblastoma, neuroblastoma, breast cancer, pancreatic ductal carcinoma, thymoma, B-CLL, leukemia, B-cell lymphoma, and cancer infiltrated by immune cells expressing receptors for HHLA2; (c) the sample comprises cells, serum, peritumoral tissue, or intratumoral tissue; and (d) the subject is a human. Claim 23 A pharmaceutical composition comprising a monoclonal antibody capable of binding to HHLA2 of any one of claims 1, 4, and 5, or an antigen-binding fragment thereof, for use in the treatment of cancer characterized by abnormal HHLA2 expression in a subject. Claim 24 In claim 23, a pharmaceutical composition corresponding to one, two, three, four, five, six, seven, eight, nine or more of (a) to (n): (a) a monoclonal antibody capable of binding to at least one HHLA2 or an antigen-binding fragment thereof is conjugated to a cytotoxic substance; (b) a monoclonal antibody capable of binding to at least one HHLA2 or an antigen-binding fragment thereof reduces the number of proliferating cells within the cancer or the tumor volume or size of the cancer; (c) a monoclonal antibody capable of binding to at least one HHLA2 or an antigen-binding fragment thereof may be administered in a pharmaceutically acceptable formulation; (d) additionally, a therapeutic agent or therapy for cancer treatment may be prescribed to the subject; (e) additional therapy selected from the group consisting of immunotherapy, checkpoint blockers, cancer vaccines, chimeric antigen receptors, chemotherapy, radiation, targeted therapy, and surgery may be prescribed to the subject; (f) the cancer is the cancer of a subject expressing HHLA2 (g) 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 infiltrated by immune cells expressing receptors for HHLA2; (h) 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; (i) the subject is an animal model of cancer; (j) the subject is a mouse model of cancer; (k) the subject is a humanized mouse model of cancer; (l) the subject is a mammal; (m) the subject is a humanized mouse; or (n) the subject is a human. Claim 25 A pharmaceutical composition according to claim 24, wherein the cytotoxic substance is selected from the group consisting of chemotherapy agents, biological agents, toxins, and radioactive isotopes. Claim 26 A pharmaceutical composition according to claim 23, wherein (a) blocking the interaction between HHLA2 and KIRDL3 for use in gate block cancer immunotherapy; and (b) inhibiting or blocking the interaction between HHLA2 and KIRDL3 using a monoclonal antibody capable of binding to HHLA2 or an antigen-binding fragment thereof. Claim 27 A pharmaceutical composition according to claim 26, wherein a monoclonal antibody capable of binding to HHLA2 or an antigen-binding fragment thereof is a checkpoint inhibitor for T cell activation for cancer immunotherapy. Claim 28 A pharmaceutical composition according to claim 23 that modulates an immune response by selectively inhibiting the interaction between HHLA2 and KIR3DL3 without blocking or significantly inhibiting the interaction between HHLA2 and TMIGD2. Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete

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