Therapeutic agent for cancers comprising material specifically binding to VSIG2

Abstract

To develop a therapeutic agent and a therapeutic method for a specific cancer by identifying a membrane protein to be a therapeutic target in the specific cancer.SOLUTION: To provide a therapeutic agent comprising a substance that specifically binds to VSIG2, which is a cell-surface protein expressed in cancer cells of a specific cancer, or a nucleic acid that specifically suppresses the expression of VSIG2 expressed on the cell surface of the cancer cells.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to cancer treatment that targets the VSIG2 protein expressed in the cell membrane of specific cancers, as well as a method for determining the success of the treatment. [Background technology]

[0002] Pancreatic cancer is a solid tumor that develops in the pancreas. Despite ongoing advances in diagnostic and treatment methods, the prognosis for pancreatic cancer is poor, with a 5-year survival rate of less than 10% and a median survival time of only 3-6 months. One reason for this is that early detection is difficult because the disease is often asymptomatic, and it is often discovered only after the disease has progressed. While genomic information about pancreatic cancer has been accumulated, the development of effective molecular-targeted therapeutic drugs has yet to be achieved, and there is an urgent need to develop innovative approaches to treat pancreatic cancer.

[0003] Cancer cells can arise from any cell type, but their properties vary depending on the tissue of origin and the type of cell from which they originate, and the types and amounts of cell surface proteins they harbor also vary greatly. Given their localization and functional importance, cell surface proteins on cancer cells are promising targets for molecular targeted therapy and cancer immunotherapy, including antibodies and cancer vaccines. However, cell surface proteins are extremely rare compared to intracellular proteins, and the cell surface proteins of pancreatic cancer cells have not been fully characterized.

[0004] The VSIG2 protein belongs to the B7 family of proteins and is a member of the immunoglobulin superfamily (Non-Patent Document 1: Genome Biol. 2005;6(6):223. https: / / doi.org / 10.1186 / gb-2005-6-6-223) and has been reported to be involved in the infiltration of B cells and M1 macrophages (Non-Patent Document 2: Cancer Manag Res. 2021;13:5739-50). It has also been reported that VSIG2 functions as a scaffold that recruits LAMTOR2 and mTOR, increasing their interaction and enhancing LAMTOR2-mediated mTOR activation, thereby promoting the malignant progression of pancreatic ductal adenocarcinoma (Non-Patent Document 3: Cell Communication and Signaling, 2023 21:223). However, the above-mentioned reports on VSIG2 only report its function as a scaffold within the cytoplasm, and do not describe or suggest that it is a cell surface protein. In addition, the production of chimeric antigen receptor (CAR) NK cells using VSIG2 as an antigen has been reported (Patent Document 1: International Publication No. 2022 / 115565), but in this report, VSIG2 was merely used as a safety antigen that is selectively present in normal cells to evaluate the on-target and off-target toxicity of CAR-NK cells, and it was reported that VSIG2 is not expressed in tumor tissue. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2022 / 115565 [Non-patent literature]

[0006] [Non-Patent Document 1] Genome Biol. 2005;6(6):223 [Non-patent document 2] Cancer Manag Res. 2021;13:5739-50 [Non-patent document 3] Cell Communication and Signaling, 2023 21:223 Summary of the Invention [Problem to be solved by the invention]

[0007] The objective is to identify cell surface proteins that serve as therapeutic targets in specific cancers and to develop therapeutic agents, treatment methods, and / or diagnostic methods for specific cancers. [Means for solving the problem]

[0008] The present inventors generated patient-derived xenograft (PDX) models from clinical specimens collected from pancreatic cancer patients, followed by PDX-derived cell lines. Using PDX-derived cell lines, we identified VSIG2 as a cell surface protein expressed in pancreatic cancer cells and localized to the cell membrane. Furthermore, we found that VSIG2 was highly expressed in cancer cells compared with normal cells. Furthermore, VSIG2 expression was also observed in cancer cells other than pancreatic cancer, and was characteristically highly expressed in cancers of endoderm-derived organs, such as the pancreas, esophageal gland, stomach, colon, bladder, bile duct, lung, thyroid, and prostate. Based on the above findings, we have identified VSIG2 as a cell surface protein expressed in certain cancers, including pancreatic cancer, and discovered that it can be used as a new therapeutic target. We therefore provide a cancer treatment targeting VSIG2. Such treatment can use a substance (also called a binder) that specifically binds to VSIG2, or a nucleic acid that specifically inhibits the expression of VSIG2 expressed on the cell surface of cancer cells. We also provide a method for determining the efficacy of cancer treatment targeting VSIG2 using VSIG2 expression as an indicator. More specifically, the present invention relates to: [1] A cancer therapeutic agent comprising a substance that specifically binds to VSIG2. [2] The therapeutic agent described in Item 1, wherein the cancer is a cancer that expresses VSIG2 on the cell surface of cancer cells. [3] The therapeutic agent according to item 1 or 2, wherein the cancer is a cancer of an organ derived from the internal lung lobe. [4] The therapeutic agent according to any one of items 1 to 3, wherein the substance that specifically binds to VSIG2 is an antibody, an antibody fragment, an aptamer, a cell engager, or an immune cell comprising a chimeric antigen receptor (CAR). [5] The therapeutic agent according to any one of items 1 to 4, wherein the substance that specifically binds to VSIG2 is cytotoxic. [6] The therapeutic agent according to any one of items 1 to 5, wherein the substance that specifically binds to VSIG2 is a conjugate in which a drug is conjugated to either an antibody, an antibody fragment, or an aptamer that specifically binds to VSIG2. [7] The therapeutic agent according to Item 6, wherein the drug is at least one selected from the group consisting of MMAF, PNU159682, MMAE, Duocarmycin, DM1, PBD, and DX8951. [8] The therapeutic agent according to item 6 or 7, wherein the substance has internalization properties. [9] The therapeutic agent according to any one of items 1 to 5, wherein the substance that specifically binds to VSIG2 is an immune cell comprising a chimeric antigen receptor, and the immune cell comprises an ectodomain comprising a protein that specifically binds to the cancer cell membrane antigen VSIG2.

[10] The therapeutic agent of Item 9, wherein the ectodomain comprises a single-chain antibody that specifically binds to VSIG2.

[11] The therapeutic agent according to Aspect 9 or 10, wherein the immune cells are T cells, NK cells, or macrophages.

[12] The therapeutic agent according to any one of items 1 to 5, wherein the substance that specifically binds to VSIG2 is an antibody, and the antibody has antibody-dependent cellular cytotoxicity (ADCC) activity or complement-dependent cytotoxicity (CDC) activity.

[13] A cancer therapeutic agent comprising a nucleic acid that specifically inhibits the expression of VSIG2 expressed on the cell surface of cancer cells.

[14] The therapeutic agent according to Item 13, wherein the cancer is a cancer of an organ derived from endoderm.

[15] The therapeutic agent according to item 13 or 14, wherein the nucleic acid is an siRNA, miRNA, antisense oligonucleotide, or decoy that specifically suppresses the expression of VSIG2.

[16] The therapeutic agent according to any one of Aspects 13 to 15, wherein the cancer of an endoderm-derived organ is at least one cancer selected from the group consisting of pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, bladder cancer, lung cancer, and bile duct cancer.

[17] The therapeutic agent according to any one of items 1 to 16, which is administered to a patient having cancer cells that express VSIG2.

[18] The therapeutic agent according to Item 17, comprising a step of measuring the expression of VSIG2.

[19] A method for assisting in determining the efficacy of a substance that specifically binds to VSIG2, comprising a step of measuring VSIG2 expression.

[20] The method according to Item 19, wherein the step of measuring the expression of VSIG2 is a step of measuring the mRNA expression level or protein of VSIG2.

[21] The method according to item 19 or 20, wherein the step of measuring the expression of VSIG2 is a step of measuring the expression of VSIG2 in the cell membrane.

[22] A reagent kit comprising a reagent for measuring VSIG2 expression, used in a method for assisting in determining the efficacy of a substance that specifically binds to VSIG2 or a method for selecting a cancer patient for treatment with a substance that specifically binds to VSIG2. [Effects of the Invention]

[0009] It is possible to provide a cancer therapeutic agent and method that targets VSIG2 as a therapeutic target, and to determine the therapeutic efficacy of the treatment. [Brief explanation of the drawings]

[0010] [Figure 1A-B] Figure 1(A)-(B) shows the cell surface localization of VSIG2 measured by surfaceome analysis (A) and gene expression of VSIG2 measured by RNA sequencing (B) in cell lines derived from patient tumor tissue xenograft models. [Figure 1C]FIG. 1(C) shows the results of measuring VSIG2 expression by flow cytometry analysis (C). [Figure 2] Figure 2 shows the gene expression of VSIG2 in various cancer tissues using TCGA information. [Figure 3A] FIG. 3(A) shows the results of flow cytometry analysis of VSIG2 expression in each cancer cell line. [Figure 3B] FIG. 3(B) shows the gene expression of VSIG2 in each cancer cell line using DepMap information. [Figure 4] Figure 4 shows the protein expression levels of VSIG2 measured by Western blotting in each cancer cell line (A), each cancer cell line and a patient tumor tissue transplant model-derived cell line (B), and a patient tumor tissue transplant model-derived cell line (C). [Figure 5A] Figure 5(A) shows fluorescent and DIC images showing the internalization of VSIG2 immediately (0 h) after the addition of an anti-VSIG2 monoclonal antibody and an antibody labeling reagent (pH-red). [Figure 5B] Figure 5(B) shows fluorescent and DIC images showing the internalization of VSIG2 5 hours (5 h) after the application of anti-VSIG2 monoclonal antibody and the addition of an antibody labeling reagent (pH-red). [Figure 5C] Figure 5(C) shows fluorescent and DIC images showing the internalization of VSIG2 23 hours (23 h) after the application of anti-VSIG2 monoclonal antibody and the addition of an antibody labeling reagent (pH-red). [Figure 5D] FIG. 5(D) shows a graph of Red Area / Total Area in time series. [Figure 6] FIG. 6 shows the results of cell growth inhibition in various cancer cells when antibody-drug conjugates (ADCs) were administered ((A): PNU159682, (B): MMAF). [Figure 7]FIG. 7 shows the results of cell growth inhibition when antibody-drug conjugates (ADCs) were administered to cell lines derived from patient tumor tissue xenograft models ((A): PNU159682, (B): MMAF). [Figure 8] Figure 8 shows the effect of VSIG2 knockdown on cell proliferation ((A): ACCPAC026 cell line derived from patient tumor tissue xenograft model; (B): H6c7 normal pancreatic duct epithelial cell line). [Figure 9] Figure 9(A) shows the scores obtained by immunostaining VSIG2 in pancreatic cancer tissues, and Figure 9(B) shows the IHC scores of pancreatic cancer tissues. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to a therapeutic agent comprising a substance that specifically binds to VSIG2 or a nucleic acid that specifically inhibits the expression of VSIG2 expressed on the cell surface of cancer cells. It also relates to a method of using a substance that specifically binds to VSIG2 or a nucleic acid that specifically inhibits the expression of VSIG2 expressed on the cell surface of cancer cells. More specifically, it relates to a therapeutic and / or preventive method comprising administering a substance that specifically binds to VSIG2 or a nucleic acid that specifically inhibits the expression of VSIG2 expressed on the cell surface of cancer cells to a subject suffering from a cancer in which VSIG2 is expressed on the cell surface of cancer cells. It also relates to a method for predicting or diagnosing the therapeutic effect of a given treatment in a subject using a substance that specifically binds to VSIG2.

[0012] VSIG2 (V-set and immunoglobulin domain containing 2) is a gene located in the long region 2 of chromosome 11. The expressed VSIG2 protein belongs to the B7 family of proteins and is also a member of the immunoglobulin superfamily (IgSF). VSIG2 is localized to the cell membrane of pancreatic cancer cells, while its expression in normal pancreatic ductal cells is very low, making it a potential therapeutic target. Furthermore, it is localized on the surface of cancer cells derived from several types of cancer, not just pancreatic cancer, and can be used as a therapeutic target for VSIG2-positive cancer cells.

[0013] VSIG2 has intracellular internalization properties. Therefore, when a drug is conjugated to a substance (binder) that specifically binds to VSIG2, the drug can be delivered specifically to cells that express VSIG2 on the cell membrane. This allows the provision of antibody-drug conjugates (ADCs) based on anti-VSIG2 antibodies.

[0014] The cancer to be treated in the present invention may be any cancer, but is particularly preferably a cancer in which VSIG2 is expressed on the cell surface of cancer cells. Cancers in which VSIG2 is expressed on the cell surface include cancers in organs of endodermal origin. Specific examples of such cancers include pancreatic cancer, gastric cancer, esophageal cancer, colon cancer, bladder cancer, lung cancer, bile duct cancer, thyroid cancer, and prostate cancer. Given that other treatment options are limited, pancreatic cancer is preferably the target of treatment. In the present invention, the terms "expressed on the cell surface" and "localized on the cell surface" are used synonymously.

[0015] Substances that specifically bind to the therapeutic target VSIG2 can also be referred to as VSIG2-binding agents, and include any substance, particularly antibodies, antibody fragments, aptamers, cell engagers, or immune cells containing chimeric antigen receptors (CARs). The present invention provides methods of using substances that specifically bind to VSIG2 and pharmaceutical compositions containing substances that specifically bind to VSIG2 for use in treating, preventing, or diagnosing cancer. The present invention also provides cells (e.g., host cells) engineered to express substances that specifically bind to VSIG2.

[0016] [antibody] As used herein, the term "antibody" is used in the broadest sense and refers to any molecule derived from the variable region of an immunoglobulin molecule that exhibits the desired specific binding property, particularly an immunoglobulin molecule. The immunoglobulin may be IgA, IgD, IgE, IgG, or IgM. The antibody may be a monomer or a polymerized form. The antibody is an immunoglobulin molecule derived from any animal and includes not only monoclonal antibodies but also polyclonal antibodies, which are a collection of immunoglobulin molecules. Antibodies can also be classified into, for example, mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, and camel antibodies, depending on the animal from which they are derived. In the present invention, the antibody may be any antibody as long as it has specific binding property to VSIG2.

[0017] Naturally occurring antibodies consist of a Y-shaped heterotetramer formed by disulfide-bonded heterodimers of light and heavy chains. Each of the light and heavy chains is divided into a variable region (V region), which exhibits a high degree of amino acid variation, and a constant region (C region), which exhibits relatively little amino acid variation. The light and heavy chain variable regions typically contain three complementarity-determining regions (CDRs 1 to 3), also known as hypervariable regions, that are involved in antigen binding, and four framework regions (FRs 1 to 4) that surround the three CDRs. The amino acid positions of the complementarity-determining regions and framework regions in immunoglobulin molecules are determined according to the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0018] A monoclonal antibody is an antibody from an antibody population consisting of only a single clone (single molecular species) in a designed amino acid sequence, and can be identified by the full-length sequences of the heavy and light chains. Furthermore, a monoclonal antibody can be identified by the sequences of the heavy chain variable region and the light chain variable region, and further by the sequences of the three complementarity-determining regions of the heavy chain and the three complementarity-determining regions of the light chain. Monoclonal antibodies can be produced as chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies, and artificial antibodies by appropriately designing the sequences of the complementarity-determining regions and other sequences. Modifications may be made to the full-length sequences of the heavy and / or light chains, the sequences of the complementarity-determining regions, the framework regions, and / or other sequences, as long as they do not affect the binding properties of the antibody. Monoclonal antibodies may be produced, for example, by the hybridoma method, or they can be generated using any known method, such as phage display or genetic engineering, based on the above-specified sequences.

[0019] In the hybridoma method, B cells collected from the spleen or lymph nodes of an animal, particularly a rat or mouse, immunized with an immunogen are fused with immortalized cells, such as myeloma cells, to produce hybridomas. Hybridomas producing antibodies with the desired binding activity are then screened, and antibodies can be produced using the screened hybridomas. Human antibodies can also be obtained by using mice transfected with human antibody genes. Monoclonal antibodies can be obtained from hybridomas by culturing the hybridomas according to conventional methods and obtaining the culture supernatant, or by administering the hybridomas to a compatible mammal, allowing them to grow, and obtaining the ascites fluid. The former method is suitable for obtaining highly purified antibodies, while the latter method is suitable for mass production of antibodies. Monoclonal antibodies can be produced using known techniques, such as those described in Chapter 2 of *Current Protocols in Immunology*, Wiley and Sons Inc.

[0020] In the phage display method, phages selected from a phage antibody library are screened with the target immunogen to select phages with the desired binding affinity to the immunogen. Next, the antibody-specific sequences contained in the phages are isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding a monoclonal antibody is constructed based on the isolated or sequence information. Then, a cell line transfected with such an expression vector is cultured to produce a monoclonal antibody. By using a human antibody library as the phage antibody library, human antibodies with the desired binding affinity can be generated.

[0021] In genetic engineering techniques, a mutation is introduced into a sequence corresponding to a complementarity-determining region (CDR) or other sequence in a gene sequence encoding an antibody, and the sequence is incorporated into an expression vector, which is then transformed into a host cell to produce a recombinant antibody (see, for example, Borrebaeck CAK and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0022] Since the antibody of the present invention binds to VSIG2, a cell surface protein, antibodies that bind to the extracellular domain of VSIG2 are preferred. Furthermore, antibodies that induce cytotoxicity or internalization upon binding to the extracellular domain of VSIG2 are preferred. Examples of antibodies that induce cytotoxicity include antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity and complement-dependent cytotoxicity (CDC) activity. More specifically, the antibody or antibody fragment thereof used in the present invention comprises the amino acid sequences of a specific heavy chain complementarity-determining region (CDRH1-3) and a specific light chain complementarity-determining region (CDRL1-3). The antibody may be a newly generated antibody or an existing antibody. Examples of existing antibodies include clone #OTI11B3 (MA5-25355, Invitrogen; CF502162, ORIGENE) and clone #541522 (MAB51771, R&D SYSTEM).

[0023] The antibody of the present invention may be an antibody that competes with a monoclonal antibody specified by the above-mentioned complementarity-determining regions. Such a competitive antibody refers to an antibody that exhibits significantly reduced binding to VSIG2 compared to a specific monoclonal antibody, as measured by surface plasmon resonance (SPR).

[0024] A chimeric antibody is an antibody in which the variable and constant regions of the light chain, heavy chain, or both are of different origins. For example, a chimeric antibody refers to an antibody composed of a variable region of a non-human immunoglobulin and a constant region of a human immunoglobulin.

[0025] A humanized antibody is an antibody that consists of a variable region composed of a complementarity-determining region of a non-human immunoglobulin and a framework region derived from a human immunoglobulin, and a constant region derived from a human immunoglobulin. Humanized antibodies can have reduced immunogenicity compared to chimeric antibodies.

[0026] A human antibody refers to an antibody in which both the light and heavy chains are derived from human immunoglobulins. Depending on the differences in the heavy chain constant region, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) having γ heavy chains, IgM having μ heavy chains, IgA (including IgA1 and IgA2) having α heavy chains, IgD having δ heavy chains, and IgE having ε heavy chains. Furthermore, the light chain typically contains either a κ chain or a λ chain. Human antibodies can be engineered using germline sequences. Compared to chimeric and humanized antibodies, human antibodies can have reduced immunogenicity, and more preferably, do not cause immunogenicity.

[0027] Multispecific antibodies are potentially asymmetric antibodies possessing two or more independent antigen-recognition sites with two or more different antigen specificities. Examples include bispecific antibodies with two antigen specificities and trispecific antibodies with three antigen specificities. One or more antigens recognized by multispecific antibodies is VSIG2. Multispecific antibodies can also be used as cell engagers. Bispecific and other multispecific antibodies can be produced by genetic engineering techniques using the antigen-binding regions of two or more monoclonal antibodies. Such genetic engineering techniques have already been established in this field. For example, desired bispecific antibodies can be obtained using DVD-Ig technology (Wu et al., Nature Biotechnology 25(11), 1290(2007)), which links the antigen-binding regions of two monoclonal antibodies in series, or ART-Ig technology (Kitazawa et al., Nature Medicine 18(10), 1570(2012)), which combines the heavy chains of two antibodies that bind to different antigens by modifying the Fc region of the antibody.

[0028] Artificial antibodies are, for example, protein scaffolds that do not have the structure of immunoglobulins but have similar functions to immunoglobulins. Protein scaffolds include the Kunitz domain of human serine protease inhibitors, the extracellular domain of human fibronectin, ankyrin, and lipocalin. By modifying the sequence of the target binding site on the scaffold, it is possible to generate protein scaffolds that bind to epitopes (Clifford Mintz et al., BioProcess International, 2013, Vol. 11(2), pp. 40-48).

[0029] Antibody production systems can be either in vitro or in vivo. In vitro production systems include those using eukaryotic cells, such as animal cells, plant cells, or fungal cells, and those using prokaryotic cells, such as bacterial cells such as Escherichia coli and Bacillus subtilis. Animal cells may include commonly used mammalian cells, such as CHO, COS, myeloma, BHK, HeLa, and Vero cells, as well as insect and plant cells. In vivo production systems include those using animals and plants. Examples of animal production systems include mammalian and insect production systems. Examples of mammalian animals include goats, pigs, sheep, mice, and cows (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Examples of insect production systems include silkworms. Examples of plants include tobacco.

[0030] When producing antibodies in an in vitro or in vivo production system as described above, DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) may be separately incorporated into expression vectors and co-transformed into a host, or DNAs encoding the H chain and L chain may be incorporated into a single expression vector and used to transform a host.

[0031] The obtained antibodies can be purified to homogeneity. Antibodies can be separated and purified using methods commonly used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but these methods are not limited to these. Columns used in affinity chromatography include protein A columns and protein G columns. Columns using protein A columns include Hyper D, POROS, and Sepharose FF (Amersham Biosciences).

[0032] [Antibody fragment] Antibody fragments with antigen-specific binding ability are designed and produced based on the structure of antibodies, particularly the structure of the variable region. An antibody fragment is a protein containing the antigen-binding domain of an antibody and has antigen-binding ability. The antigen-binding domain includes portions of the heavy and light chains, particularly the heavy chain variable region and the light chain variable region. Examples of antibody fragments include Fab fragments, Fv fragments, F(ab')2 fragments, Fab' fragments, and scFv. A combination of VH and VL can be used as the smallest unit of an antigen-binding domain. These antibody fragments or antigen-binding domains may be used instead of antibodies or may be applied to cell engagers or chimeric antigen-binding receptors (CARs). Similar to antibodies, they can be produced in an in vitro production system by expressing nucleic acids encoding the amino acid sequences of the light and heavy chains.

[0033] Functionally modified antibodies or conjugated antibodies (antibody complexes) can be produced by chemically or genetically binding antibodies or antibody fragments thereof with functional molecules other than antibodies, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, drugs, toxins, low-molecular-weight compounds, cytokines, albumin, enzymes, etc. Functionally modified antibodies are antibodies whose functions other than antigen-binding function, such as cell-killing function, complement activation function, and blood half-life, have been adjusted by modifying the amino acids and sugar chains, mainly in the constant region of immunoglobulin.

[0034] [Aptamer] An aptamer refers to a nucleic acid molecule or peptide that has specific binding affinity to a specific molecule. Aptamers can be produced by selecting based on their ability to interact with a target molecule. Aptamers can be broadly classified into nucleic acid aptamers such as RNA or DNA, and peptide aptamers made of peptides, and selection methods known in the art can be used for each. For nucleic acid aptamers, SELEX and in vitro selection methods can be used, and for peptide aptamers, screening can be performed using the two-hybrid method or the like.

[0035] [Cell Engager] A cell engager is a substance that brings immune cells into close proximity with cancer cells and allows them to act. Cell engagers can be produced by fusing a substance that binds to a cell surface protein on immune cells with a substance that binds to a cell surface protein on cancer cells. Substances that bind to cell surface proteins include ligands, receptors, antibodies, and antibody fragments. Specific examples of cell engagers include multispecific fusion antibodies linking two or more types of antibodies or antibody fragments, or antigen-binding domains, or multispecific antibodies. Examples of cell engagers include, but are not limited to, BiTE, BiKE, and TriKE. As an example, an anti-VSIG2 antibody, anti-VSIG2 antibody fragment, or anti-VSIG2 antigen-binding domain that binds to VSIG2 can be used as an antibody fragment that interacts with cancer cells, while an antibody, antibody fragment, or antigen-binding domain that can bind to a surface protein expressed on cells other than VSIG2-expressing cells, particularly immune cells, can be used. This allows binding to VSIG2-expressing cancer cells and cells other than VSIG2-expressing cells, respectively, bringing these cells into close proximity.

[0036] Surface proteins expressed on immune cells can be any protein known in the art, and examples include CD16, CD16a, NKG2A, NKG2D, NKp30, NKp44, and NKp46 expressed on the surface of NK cells, and CD3 expressed on the surface of T cells. Immune cells brought into proximity with VSIG2-expressing cancer cells by applying a cell engager exert cytotoxic activity against nearby cancer cells by producing proteins such as perforin, granzymes, and cytokines, or by stimulating antibody-dependent cellular cytotoxicity, regardless of the presence of MHC1 or costimulatory molecules.

[0037] The cell engagers herein may be designed to include a linker between each antigen-binding domain. Any suitable linker may be used to design the cell engagers provided herein. Examples of linkers that can be used to create the cell engagers described herein include, but are not limited to, linkers of approximately 3 to 100 amino acid residues.

[0038] In some cases, a cell engager (e.g., a BiTE) that targets VSIG2 can be designed to include a heavy chain variable domain including the heavy chain complementarity determining regions (CDRH1-3) of an antibody that binds to VSIG2, a linker, a light chain variable domain including the light chain complementarity determining regions (CDRL1-3), followed by a linker, followed by an scFv having an antigen-binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0039] As used herein, a modified sequence refers to a sequence that is capable of exhibiting a function equivalent to that of a protein encoded by the original amino acid sequence or a portion thereof, and that has one or several, more specifically, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid deletions, additions, and / or substitutions relative to the original amino acid sequence. In yet another example, a modified sequence refers to a sequence that is capable of exhibiting a function equivalent to that of a protein encoded by the original amino acid sequence or a portion thereof, and that has at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the original amino acid sequence.

[0040] [Cells containing chimeric antigen receptors (CARs)] A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor engineered to recognize and bind to an antigen expressed by a tumor cell. In the present invention, cells containing a CAR may be any cell, particularly immune cells or stem cells. Generally, CARs are designed for T cells and are chimeras of the signaling domain of the T cell receptor (TCR) complex and an antigen-binding domain (e.g., an antibody single-chain fragment (scFv) or other antibody fragment) (Enblad et al., Human Gene Therapy. 2015;26(8):498-505). However, CARs can also be designed for other immune cells, such as NK cells and macrophages. A CAR may typically comprise an ectodomain containing the antigen-binding domain (e.g., an antibody such as scFv), a transmembrane domain, and an intracellular domain. Among immune cells expressing CARs, T cells are specifically referred to as CAR T cells. CARs have the ability to exert T cell specificity and reactivity against selected targets in an MHC-independent manner. Cells expressing a CAR can kill cells recognized by the antigen recognition domain of the CAR, such as cancer cells.

[0041] The chimeric antigen receptor may be a first-generation CAR, a second-generation CAR, or a third-generation CAR, or may be an improved CAR. In a first-generation CAR, an antibody-derived scFv is linked to the CD3 zeta (ζ or z) signaling domain of a T cell receptor via a transmembrane domain and expressed in a T cell. In a second-generation CAR, an additional costimulatory domain (e.g., CD28, 4-1BB (41BB), or ICOS) is incorporated into the first-generation CAR, and expression of such a CAR in a T cell enables the delivery of a costimulatory signal. A third-generation CAR contains two costimulatory domains fused to the TCR CD3ζ chain. Third generation costimulatory domains can include, for example, combinations of CD3ζ, CD27, CD28, 4-1BB, ICOS, or OX40 (Maude et al., Blood. 2015; 125(26):4017-4023; Kakarla and Gottschalk, Cancer J. 2014; 20(2):151-155).

[0042] The ectodomain is the region of the CAR exposed to extracellular body fluids. The ectodomain contains an antigen-binding domain, including scFv, and may optionally further contain a signal peptide, a spacer domain, and / or a hinge domain. A single-chain variable fragment (scFv) is a fusion protein of the variable regions of an immunoglobulin heavy chain (VH) and light chain (VL), linked by a short linker peptide of about 10 to about 25 amino acids or by a direct amide bond. The linker is usually glycine-rich for flexibility and serine- or threonine-rich for solubility, and links the N-terminus of VH to the C-terminus of VL, or the C-terminus of VH to the N-terminus of VL, thereby maintaining the specificity of the original antibody. From the viewpoint of reducing immunogenicity, scFv preferably consists of a humanized sequence, and more preferably, scFv consists of a fully human sequence. In yet another embodiment, scFv may be a chimeric (e.g., mouse and human) sequence. The signal peptide is a peptide that contributes to anchoring the CAR to the cell membrane and can enhance the antigen specificity of CAR binding.

[0043] In some embodiments, the spacer domain or hinge domain is located between the extracellular domain (including the antigen-binding domain) and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. A spacer domain is any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain and / or an intracellular domain in a polypeptide chain. A hinge domain is any oligopeptide or polypeptide that functions to provide flexibility to the CAR or a domain thereof or to prevent steric hindrance of the CAR or a domain thereof. In some embodiments, the spacer domain or hinge domain can comprise up to 300 amino acids (e.g., 10-100 amino acids or 5-20 amino acids). Hinge domains derived from immunoglobulins can be used as hinge domains. In some embodiments, one or more spacer domains can be included in other regions of the CAR. In some embodiments, the hinge domain is a CD8 hinge domain. Other hinge domains can be used.

[0044] The transmembrane domain mainly comprises a hydrophobic alpha helix spanning the membrane. The transmembrane domain provides stability to the CAR. Examples of the transmembrane domain include, but are not limited to, the CD3ζ transmembrane domain, the CD4 transmembrane domain, the CD8α transmembrane domain, the CD28 transmembrane domain, and the 4-1BB transmembrane domain, as well as chimeras thereof. Furthermore, transmembrane domains with one, two, three, four, five, six, seven, eight, or nine amino acid deletions, additions, and / or substitutions can be used.

[0045] The intracellular domain is the functional end of the receptor and functions to transduce receptor clusters and signals into the cell after antigen recognition. The intracellular domain contains one or more signaling domains. Signaling domains include the CD3ζ intracellular signaling domain, the CD27 intracellular signaling domain, the CD28 intracellular signaling domain, the OX40 (CD134) intracellular signaling domain, the 4-1BB (CD137) intracellular signaling domain, the CD278 intracellular signaling domain, the DAP10 intracellular signaling domain, and the DAP12 intracellular signaling domain, as well as chimeras thereof. Depending on the design of the signaling domain, CARs can be classified as first-generation, second-generation, or third-generation CARs. First-generation CARs are designed to contain the CD3ζ intracellular signaling domain. Second-generation CARs can use the CD3ζ intracellular signaling domain and the CD28 signaling domain as a costimulatory domain. Third-generation CARs can utilize intracellular domains that incorporate the CD28 costimulatory domain as well as signaling domains such as 4-1BB, ICOS, or OX40. Introduction of these costimulatory domains can confer memory function to CTLs. Furthermore, these intracellular domains can be used with deletion, addition, and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, as long as the function is not impaired.

[0046] As one specific example, a CAR that targets VSIG2 can be designed to include a heavy chain variable domain of an antibody that binds to VSIG2, followed by a linker, followed by a light chain variable domain (e.g., an scFv), followed by a hinge, followed by a transmembrane domain, followed by one or more intracellular domains.

[0047] Cells containing chimeric antigen receptors (CARs) are prepared by introducing the CAR gene into the host genome using viral and non-viral gene transfer methods. The introduced CAR gene can be randomly integrated into the host genome or site-specifically integrated using genome modification techniques. Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery into cells. Non-viral nucleic acid delivery methods include electroporation, lipofection, microinjection, gene guns, liposomes, and any other method available in the art.

[0048] The CAR cells of the present invention may be autologous or non-autologous immune cells. In the case of non-autologous immune cells, CAR-immune cells (also called universal CAR-immune cells) can be used, which suppress the host attack (GVHD) reaction caused by the administered CAR-immune cells and modify the immunocompatibility of CAR-T cells, thereby suppressing the reaction of the patient's immune cells attacking the transplanted CAR-immune cells.

[0049] CAR cells include, in particular, immune cells, but may also be stem cells capable of differentiating into immune cells. Stem cells may include ES cells, iPS cells, somatic stem cells, etc. Immune cells may be obtained from any tissue or differentiated from stem or progenitor cells. Sources of immune cells include, but are not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, immune cells can be obtained from blood collected from a subject using any number of techniques known to those skilled in the art, such as centrifugal sedimentation (e.g., FICOLL™ separation).

[0050] Examples of immune cells that can be used include NK cells, T cells, macrophages, and the like. Subpopulations of T cells can also be used, such as cytotoxic T cells, helper T cells, naive T cells, memory T cells, and effector T cells. Immune cells can be separated using one or more cell surface markers, such as TCRab, CD3, CD4, CD8, CD27, CD28, CD38, CD45RA, CD45RO, CD62L, CD127, CD122, CD95, CD197, CCR7, KLRG1, MCH-I protein, and / or MCH-II protein. As an example, a population of T cells to be used as CAR-T cells can be CD3+, CD4+, CD8+, or a combination thereof. The T cells thus separated can be first activated and stimulated to proliferate in vitro before undergoing gene editing.

[0051] [Complex] A drug may be further conjugated to a substance that specifically binds to VSIG2, particularly an antibody, antibody fragment, or aptamer, to form a conjugate. A conjugate between an antibody and a drug is particularly called an antibody-drug conjugate (ADC). A radioisotope may also be further conjugated to a substance that specifically binds to VSIG2, particularly an antibody, antibody fragment, or aptamer, to form a conjugate (radioimmunotherapy (RIT)). Conjugation may be direct or via an appropriate linker. For example, conjugation of a drug may result in the toxicity of the drug when the substance that specifically binds to VSIG2 interacts with cancer cells. More preferably, when a substance that specifically binds to VSIG2 interacts with VSIG2 in cancer cells, VSIG2 is internalized. Internalization of VSIG2 may result in the drug-conjugated conjugate being taken up into cells, which may cause toxicity. When the drug-conjugated conjugate is taken up into cells, the drug may be cleaved from the conjugate and released in response to a change in pH. Such drugs may be compounds or radioisotopes, and are preferably substances that induce cytotoxicity in cells. Photosensitizers such as IRDye700DX can be used as such compounds, and can exert their toxicity by externally irradiating light after administration. Examples of radioisotopes include: 131 iodine, 177 ruthenium, 90Compounds containing yttrium can kill cancer cells through the action of radiation. Toxic compounds include maytansines, such as mertansine (DM1) and ravtansine (DM4), diphtheria toxins, such as dmDT390 and DT388, topoisomerase I inhibitors, such as DXd / DX8951, melphalan, auristatins, such as MMAE and MMAF, Pseudomonas aeruginosa exotoxins, such as PE38, SG3199, SN-38, PNU159682, Duocarmycin, such as Duocarmycin DM and DX8951, and DNA-damaging agents, such as PBD, with MMAF, PNU159682, MMAE, Duocarmycin, DM1, PBD, and DX8951 being preferred.

[0052] [Nucleic acid that specifically suppresses the expression of VSIG2 expressed on the cell surface of cancer cells] Examples of nucleic acids that specifically suppress the expression of VSIG2 expressed on the cell surface of cancer cells include siRNA, miRNA, antisense oligonucleotides, and decoys. Inhibition of VSIG2 expression in specific cancer cells can suppress cell proliferation. Such siRNA, miRNA, and antisense oligonucleotides can be prepared by referring to the sequence of the gene encoding VSIG2 or its complementary sequence. By interacting with transcribed mRNA, they can suppress mRNA degradation or translation. It is preferable to suppress VSIG2 expression, particularly VSIG2 membrane localization. Furthermore, VSIG2 transcription can be suppressed by using a decoy nucleic acid against a transcription factor that contributes to VSIG2 transcription.

[0053] [Cancer treatment] The present invention provides a therapeutic agent containing a substance that specifically binds to VSIG2 or a nucleic acid that specifically inhibits the expression of VSIG2 on the cell surface of cancer cells (hereinafter sometimes abbreviated as the substance of the present invention, etc.), and a method for treating cancer comprising administering the substance of the present invention, etc. to a subject suffering from cancer. As used herein, cancer treatment includes, for example, treatments performed to (a) reduce cancer cell proliferation, (b) reduce symptoms caused by cancer and improve the quality of life of cancer patients, (c) reduce the dose of other anticancer drugs or adjuvant cancer treatment drugs already administered, and / or (d) extend the survival time of cancer patients. Furthermore, cancer treatment as used herein also includes suppression of cancer progression and / or suppression of recurrence. Suppression of cancer progression means delaying cancer progression, stabilizing cancer-related symptoms, and reversing the progression of symptoms. Suppression of recurrence means preventively preventing cancer recurrence in patients whose cancerous lesions have completely or substantially disappeared or been removed by cancer treatment or surgical resection. Furthermore, the present invention provides a cancer preventive agent comprising the substance of the present invention, or a method for preventing cancer, which comprises administering the substance of the present invention to a subject who may suffer from cancer. The substance of the present invention may be formulated in combination with one or more other drugs for the following purposes: (a) to enhance the therapeutic effect of cancer, (b) to reduce the dosage of other drugs prescribed in combination, (c) to reduce the side effects of other drugs prescribed in combination, and / or (d) to enhance the immune-enhancing effect of other drugs prescribed in combination, i.e., as an adjuvant. When formulated in combination with other drugs, the dosage form may be a combination formulation in which both components are combined in a single formulation, or may be a separate formulation. This combination can complement the therapeutic effect of the other drug or maintain or reduce the dosage or frequency of administration. When the substance of the present invention and other drugs are formulated separately, they may be co-administered for a certain period of time, followed by administration of the substance of the present invention or other drugs alone. Alternatively, the substance of the present invention may be administered first, followed by administration of the other drug, or the other drug may be administered first, followed by administration of the substance of the present invention. Furthermore, during the above administration, both drugs may be administered simultaneously for a certain period of time. The administration methods for each drug may be the same or different. Depending on the properties of the drug, a kit containing a formulation containing the present compound and another drug may also be provided. The dosage of the other drug can be appropriately selected based on the clinically used dose. Furthermore, any two or more other drugs may be administered in combination at an appropriate ratio. Furthermore, the other drugs include not only those discovered to date but also those that will be discovered in the future. Examples of anticancer drugs that can be used in combination with the substance of the present invention in cancer treatment include alkylating agents, platinum compounds, antimetabolites, topoisomerase inhibitors, microtubule polymerization inhibitors, microtubule depolymerization inhibitors, antitumor antibiotics, cytokine preparations, antihormones, molecularly targeted drugs, cancer immunotherapeutic drugs, and other antibody drugs.

[0054] [Pharmaceutical composition] Pharmaceutical compositions of the present invention containing a substance that specifically binds to VSIG2 or a nucleic acid that specifically inhibits the expression of VSIG2 expressed on the cell surface of cancer cells may contain, in addition to the active ingredient, the substance that specifically binds to VSIG2 or the nucleic acid that specifically inhibits the expression of VSIG2 expressed on the cell surface of cancer cells, or a salt thereof, a pharmacologically acceptable carrier, diluent, or excipient. Such compositions are provided in dosage forms suitable for parenteral or oral administration, although parenteral administration is preferred from the perspective of their use as antibody drugs, cellular drugs, or nucleic acid drugs. Examples of parenteral administration include, but are not limited to, intravenous, intraarterial, subcutaneous, topical, intraperitoneal, intramuscular, nasal, ophthalmic, transdermal, transmucosal, intrameningeal, rectal, intramuscular, and intracerebral administration.

[0055] The pharmaceutical composition can be formulated in an appropriate dosage form depending on the route of administration, and may be in any form, such as an injection, powder, infusion preparation, granules, tablets, suppositories, etc. From the viewpoint of parenteral administration, however, injection, infusion preparation, powder that dissolves when used, etc. are preferred. Furthermore, these preparations may contain various adjuvants used in medicine, i.e., carriers and other auxiliary agents, such as stabilizers, preservatives, soothing agents, emulsifiers, and other additives.

[0056] The substance of the present invention that specifically inhibits VSIG2 expression or the substance that specifically inhibits VSIG2 expression can be administered by continuous infusion or by administration, for example, once daily, once weekly, once monthly, or 1 to 7 times per year. Administration can be intravenous, subcutaneous, topical, oral, nasal, rectal, intramuscular, intracerebroventricular, or by inhalation. A preferred dosing protocol involves the maximum dose or dosing frequency that avoids serious undesirable side effects. The total weekly dose is typically 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, and more preferably 0.1 mg to 10 mg, and is preferably administered once every few days to several months.

[0057] [Method for determining efficacy] In one embodiment, the efficacy of a substance that specifically binds to VSIG2 can be evaluated by measuring the expression level of VSIG2 in cancer cells. Therefore, measuring VSIG2 expression in a subject with cancer can determine the efficacy of a substance that specifically binds to VSIG2, and a method for assisting this determination is provided. This allows for determining whether a subject with cancer should be treated with a substance that specifically binds to VSIG2, thereby enabling the selection of cancer patients to be treated. VSIG2 expression can be measured in any biological sample containing cancer cells, including blood samples and tissue samples. VSIG2 expression may be measured by mRNA expression or protein expression. Since substances that specifically bind to VSIG2 act via VSIG2 present on the cell membrane, it is preferable to measure proteins expressed on the cell membrane. For example, membrane protein expression can be detected using flow cytometry. Cells labeled with a fluorescently labeled anti-VSIG2 antibody can be counted by flow cytometry to determine the number of positive cells. Cells may also be labeled with known cancer cell markers to identify cancer cells. Known membrane protein measurement methods can be used, such as on-surface reconstitution (OSR). Alternatively, a biological sample can be obtained, mRNA reverse transcribed, and quantified using real-time PCR. Alternatively, protein expression levels can be measured using conventional immunostaining or Western blotting.

[0058] A reagent kit containing a reagent for measuring VSIG2 expression, which is used in a method for assisting in determining the efficacy of a substance that specifically binds to VSIG2 or a method for selecting a cancer patient for treatment with a substance that specifically binds to VSIG2, may contain a labeled anti-VSIG2 antibody. Depending on the analytical method, the kit may further contain detergents, other cancer markers, solubilizing agents, gels, transfer membranes, and the like.

[0059] All documents mentioned herein are incorporated by reference in their entirety.

[0060] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention. [Example]

[0061] Example 1: Preparation of patient tumor tissue transplant model-derived cell lines Pancreatic cancer PDX mouse models were generated by subcutaneously implanting biopsy specimens obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) or surgical specimens from pre-treatment pancreatic cancer patients into Rag-2 / Jak3 double-deficient mice. For cases where PDX was successfully generated, after one passage, the engrafted PDX tumor was minced into approximately 1 cm cubes, added to 1 mL of medium (RPMI-1680), and minced with a razor. Another 1 mL of medium and collagenase were added, and the cells were incubated at 37°C for 40 minutes. After pipetting, cell clumps were removed using a 100 μm filter. After three cycles of centrifugation at 1800 rpm for 5 minutes and washing with PBS, the cells were cultured in 10 cm dishes, and 20 PDX model-derived cell lines were generated.

[0062] Example 2: Surfaceome analysis of cell lines derived from patient tumor tissue transplant models (1) Surface Ohmic Analysis In PDX model-derived cell lines and the normal pancreatic duct epithelial cell line H6c7 (Kerafast), biotin was added to label the cell surface proteins of live cells, and the cell surface proteins were then isolated using a streptavidin column. A protein profile (surfaceome) of the cell surface proteins was obtained using a mass spectrometer (Orbitrap Fusion).

[0063] (2) RNA sequencing analysis For RNA sequencing (SEQ), total RNA was extracted from the PDX model-derived cell lines and the normal pancreatic ductal epithelial cell line H6c7, and analysis was outsourced to NOVOGENE. The RNA SEQ data obtained from the PDX model-derived cell lines was purified using Xenofilter® to remove potentially contaminating data from mouse cells, then mapped with hisat2 and normalized and counted using DESeq2.

[0064] Based on surface expression and RNA sequencing data of proteins expressed on the cell membrane of PDX model-derived cell lines and normal pancreatic ductal epithelial cell lines, we selected approximately 30 genes as potential new therapeutic targets based on their high expression levels on the cell membrane and gene expression levels, as well as the size of the extracellular domain of the protein, compared to the normal pancreatic ductal epithelial cell line H6c7. From these, we focused on VSIG2. Figure 1 shows the surface protein levels (A) and gene expression levels (B) of VSIG2 in the normal pancreatic ductal epithelial cell line H6c7 and each pancreatic cancer PDX model-derived cell line. In the surface domain, VSIG2 expression was overexpressed in most PDX model-derived cell lines compared to normal pancreatic ductal epithelial cells H6c7, and correlated well with gene expression.

[0065] (3) Flow cytometry analysis PDX model-derived cell lines PAC034, PAC126, PAC103, and PAC026, normal fibroblast cell line WI-38, and normal pancreatic duct epithelial cell line H6c7 were plated in a 6-well plate at 1 × 10 6Cells were seeded individually and cultured for 72 hours, after which they were harvested and cell suspensions were prepared. Anti-VSIG2 monoclonal antibody (clone #OTI11B3) was added 1 hour, followed by secondary antibody (A-11029, Invitrogen). Alexa-488 fluorescence signals were measured 30 minutes later using a flow cytometer (BD FACSCalibur, hereafter abbreviated as FACS). The flow cytometry results are shown in Figure 1(C). In the PDX model-derived cell lines PAC034, PAC126, PAC103, and PAC026, which express high levels of VSIG2, the fluorescence intensity of the anti-VSIG2 antibody was higher than that of the control IgG, confirming VSIG2 expression on the cell surface. In contrast, the fluorescence intensity of the anti-VSIG2 antibody was unchanged from the control in the normal cell lines WI-38 and H6c7, suggesting that VSIG2 expression was either absent or below the sensitivity of the FACS.

[0066] Example 3: Gene expression analysis of VSIG2 using information such as DepMap VSIG2 gene expression information was obtained for 1,450 cancer cell lines from the DepMap portal (https: / / depmap.org / portal / ). VSIG2 gene expression was ranked in order of highest expression, and the top 41 cell lines with expression levels (Log2(TPM+1)) of 5 or higher are shown in Tables 1 and 2. Examining these cell lines by cancer type revealed that VSIG2 was clearly overexpressed in certain cancer types: 16 pancreatic cancers, 11 esophageal adenocarcinomas, 6 bladder cancers, and 3 biliary tract cancers. Therefore, we analyzed gene expression in each cancer type using TCGA (Figure 2). Figure 2 shows that VSIG2 expression was particularly high in bladder cancer, pancreatic intraductal papillary mucinous neoplasm, pancreatic cancer, thyroid cancer, prostate cancer, lung cancer, esophagogastric cancer, and colorectal cancer. These cancers all originate from the endoderm, suggesting that gene expression control mechanisms involved in endoderm development and differentiation may also be involved in the regulation of VSIG2 expression. [Table 1] [Table 2]

[0067] Example 4: Flow cytometry analysis of each cancer cell line Esophageal adenocarcinoma cell lines SKGT4 and OE19, liver cancer cell line Huh6, colon cancer cell line HT29, pancreatic cancer cell lines AsPC1 and BxPC3, and lung cancer cell line PC9 were cultured in a 6-well plate at 1 × 10 6 Cells were seeded individually and cultured for 72 hours, after which they were harvested and a cell suspension was prepared. Anti-VSIG2 monoclonal antibody (clone #OTI11B3) was added 1 hour later, followed by secondary antibody (A-11029, Invitrogen). Alexa-488 fluorescence signals were measured 30 minutes later using a flow cytometer (FACSCalibur). The results of flow cytometry analysis are shown in Figure 3(A), and gene expression in the DepMap for the corresponding cell lines is shown in Figure 3(B). Fluorescence intensity in each cell line correlated well with gene expression. VSIG2 was not detectable on the cell surface in cell lines with VSIG2 gene expression levels lower than that of BXPC3 (Huh6, PC9, SKGT4, and AsPC1).

[0068] Example 5: Western blotting analysis of VSIG2 expression Pancreatic cancer PDX model-derived cell lines PAC026, PAC041, PAC003, PAC005, PAC081, PAC038, PAC034, PAC126, and PAC103; esophageal adenocarcinoma cell lines SKGT4, OE19, and OE33; liver cancer cell line Huh6; gastric cancer cell line AGS; colon cancer cell line HT29; pancreatic cancer cell lines AsPC1 and BxPC3; lung cancer cell line PC9; normal fibroblast cell line WI38; and normal pancreatic ductal epithelial cell line H6c7 were cultured in a 6-well plate at 1 × 10 6Cells were seeded individually and cultured for 72 hours, after which lysates were collected. Western blotting using an anti-VSIG2 monoclonal antibody (clone #OTI11B3) was performed, and the results are shown in Figure 4 (A), (B), and (C). The findings from Western blotting were consistent with those from flow cytometry and gene expression analysis. VSIG2 expression was also detectable by Western blotting in BxPC3, HT29, and OE19, where it was detected by flow cytometry. While the level of VSIG2 expression in BxPC3 was considered to be at the lower limit of detection for flow cytometry or Western blotting, the PDX-derived cell line PAC041 showed VSIG2 expression at a similar level to that of BxPC3. Furthermore, VSIG2 expression equivalent to or greater than that of PAC041 was confirmed in PAC034, PAC126, PAC103, and PAC026. Combined with the VSIG2 expression data in the surface domain shown in Figure 1(A), this suggests that VSIG2 expression on the cell surface is at a level detectable by flow cytometry analysis or Western blotting in approximately 40% of cell lines derived from pancreatic cancer PDX models.

[0069] Example 6: Internalization by anti-VSIG2 monoclonal antibodies Esophageal adenocarcinoma cell line OE19 was cultured in a 96-well plate at 2.5 x 10 5 After seeding and culturing for 24 hours, primary antibody (anti-VSIG2 monoclonal antibody, clone #OTI11B3) and antibody labeling reagent (pH-red) were added. DIC and fluorescent images were taken immediately (0 h), 5 h (5 h), and 23 h (23 h) using an Incucyte system and quantified. The results are shown in Figure 5 (A: 0 h, B: 5 h, C: 23 h) and Figure 5 (D). The anti-VSIG2 monoclonal antibody was rapidly internalized into the cells, reaching a plateau approximately 10 h after the start of the experiment. This suggests that antibody-drug conjugates (ADCs) targeting VSIG2 may be promising for cancer cells expressing VSIG2 on their cell surface.

[0070] Example 7: Cell proliferation inhibitory effect of antibody-drug conjugates (ADCs) Pancreatic cancer PDX model-derived cell lines PAC026, PAC103, and PAC034, bladder cancer cell line UMUC9, esophageal adenocarcinoma cell line OE19, cholangiocarcinoma cell line EGI-1, colon cancer cell line HT29, and pancreatic cancer cell line MAPACHS77 were plated in a 96-well plate at 1 × 10 3 After 24 hours of incubation, cells were seeded and treated with a primary antibody (anti-VSIG2 monoclonal antibody, clone #OTI11B3) and a secondary antibody (MMAF: AM202-AF, Moradec; PNU159682: AM-102PN, Moradec). Cell proliferation was assessed by WST-8 assay after 120 hours. The results are shown in Figures 6(A) and 7(A) for PNU159682 and Figures 6(B) and 7(B) for MMAF. The secondary ADC using PNU15968 exhibited cytostatic effects in all cell lines, while the secondary ADC using MMAF exhibited cytostatic effects in all cell lines except PAC026. These results suggest that VSIG2-targeting ADCs are effective against at least pancreatic cancer, bladder cancer, esophageal adenocarcinoma, bile duct cancer, and colorectal cancer, and that efficacy can be further enhanced by modifying the payload.

[0071] Example 8: Suppression of VSIG2 gene expression siRNA (s24139, Thermo) or control (4390843, Thermo) was added to the pancreatic cancer PDX model-derived cell line PAC026 or the normal pancreatic ductal epithelial cell line H6c7 at a final concentration of 5 nM. Cell proliferation was assessed by WST-8 assay after 120 hours. The results are shown in Figure 8(A) for the pancreatic cancer PDX model-derived cell line PAC026 and Figure 8(B) for the normal pancreatic ductal epithelial cell line H6c7. VSIG2 siRNA exhibited a cell growth inhibitory effect only on PAC026, suggesting the importance of VSIG2 for pancreatic cancer cell proliferation and the efficacy of nucleic acid drugs targeting VSIG2.

[0072] Example 9: Tissue staining of VSIG2 in pancreatic cancer FFPE specimens of pancreatic cancer tissue were deparaffinized and then retrieval was performed with TE buffer (pH 9). Blocking was performed for 1 hour, and rabbit anti-VSIG2 polyclonal antibody (HPA050147, Sigma-Aldrich) was incubated overnight at 4°C. After treatment with oxygen peroxide, secondary antibody was added and incubated for 1 hour at room temperature. Then, DAB was developed for 3 minutes, counterstained, and mounted. VSIG2 expression intensity was scored on a 5-point scale (0 to 4+). A representative staining example is shown in Figure 9(A), and the staining results for 59 pancreatic cancer cases are shown in Figure 9(B). VSIG2 overexpression (IHC score 3 or 4) was observed in 13 of the 59 cases (22%) for which evaluation was completed. Compared with flow cytometry analysis and Western blotting results using cell lines, cases with an IHC score of 3 or 4 were considered to have detectable levels of VSIG2 expression on the cell surface, indicating that ADCs targeting VSIG2 are effective.

Claims

1. A cancer therapeutic agent comprising a substance that specifically binds to VSIG2.

2. The method of claim 1, wherein the cancer expresses VSIG2 on the cell surface of cancer cells.

3. The therapeutic agent according to claim 1 or 2, wherein the cancer is a cancer of an organ derived from the internal lobe of the lung.

4. The therapeutic agent according to any one of claims 1 to 3, wherein the substance that specifically binds to VSIG2 is an antibody, an antibody fragment, an aptamer, a cell engager, or an immune cell comprising a chimeric antigen receptor (CAR).

5. The therapeutic agent of claim 1 , wherein the substance that specifically binds to VSIG2 is cytotoxic.

6. The therapeutic agent according to any one of claims 1 to 5, wherein the substance that specifically binds to VSIG2 is a conjugate in which a drug is conjugated to an antibody, antibody fragment, or aptamer that specifically binds to VSIG2.

7. The therapeutic agent according to claim 6, wherein the drug is at least one selected from the group consisting of MMAF, PNU159682, MMAE, Duocarmycin, DM1, PBD, and DX8951.

8. The therapeutic agent according to claim 6 or 7, wherein the VSIG2 has internalization properties.

9. The therapeutic agent according to any one of claims 1 to 5, wherein the substance that specifically binds to VSIG2 is an immune cell comprising a chimeric antigen receptor and comprises an ectodomain comprising a protein that specifically binds to the cancer cell membrane antigen VSIG2.

10. The therapeutic agent of claim 9 , wherein the ectodomain comprises a single-chain antibody that specifically binds to VSIG2.

11. The therapeutic agent according to claim 9 or 10, wherein the immune cells are T cells, NK cells, or macrophages.

12. A cancer therapeutic agent comprising a nucleic acid that specifically inhibits the expression of VSIG2 expressed on the cell surface of cancer cells.

13. The therapeutic agent according to claim 12, wherein the cancer is a cancer of an organ derived from endoderm.

14. The therapeutic agent according to claim 12 or 13, wherein the nucleic acid is an siRNA, miRNA, antisense oligonucleotide, or decoy that specifically suppresses the expression of VSIG2.

15. The therapeutic agent according to claim 3 or 13, wherein the cancer of an endoderm-derived organ is at least one cancer selected from the group consisting of pancreatic cancer, gastric cancer, esophageal cancer, colon cancer, bladder cancer, lung cancer, bile duct cancer, thyroid cancer, and prostate cancer.

16. The therapeutic agent according to any one of claims 1 to 15, which is administered to a patient having cancer cells that express VSIG2.

17. A method for assisting in determining the efficacy of a substance that specifically binds to VSIG2, comprising a step of measuring the expression of VSIG2.

18. The method according to claim 17, wherein the step of measuring the expression of VSIG2 is a step of measuring the expression level of VSIG2 mRNA or protein.

19. The method according to claim 17 or 18, wherein the step of measuring the expression of VSIG2 is a step of measuring the expression of VSIG2 in the cell membrane.

20. A reagent kit comprising a reagent for measuring VSIG2 expression, used in a method for assisting in determining the efficacy of a substance that specifically binds to VSIG2 or a method for selecting a cancer patient for treatment with a substance that specifically binds to VSIG2.

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