HLA / cancer antigen pvt1-derived peptide complex-specific antibody

Abstract

The present invention addresses the problem of providing: an antibody that recognizes an HLA-A24 / HF10 peptide complex on a cancer cell surface; a pharmaceutical composition comprising the antibody, in particular, a pharmaceutical composition for treating cancer and a use thereof; and a method for treating cancer. Provided is a multispecific antibody having an antigen-binding site that recognizes a complex of a polypeptide represented by SEQ ID NO: 3 and an MHC molecule.

Description

HLA / cancer antigen PVT1-derived peptide complex-specific antibody

[0001] The present invention relates to an antibody that specifically recognizes a complex of a peptide derived from the cancer antigen PVT1 and an HLA molecule, and a pharmaceutical composition containing the antibody, particularly a pharmaceutical composition for treating cancer, and uses thereof.

[0002] Tumor antigen peptides are fragments of proteins expressed in tumor cells. Therefore, in the development of cancer immunotherapy, it is important to search for proteins expressed in tumor cells, especially tumor antigen proteins that are not expressed in normal cells but are expressed in tumor cells.

[0003] PVT1 (Plasmacytoma variant translocation 1) is a non-coding RNA that coexists with the proto-oncogene MYC on the same chromosome and was thought to function as a MYC activator. It has also been reported that copy number increases of MYC are also observed in many cancers, suggesting that increased transcription of the PVT1 gene is involved in cell proliferation and carcinogenesis.

[0004] Under these circumstances, it has been found that a partial peptide of a polypeptide predicted to be encoded by the open reading frame (ORF) of PVT1 is presented as an antigen in cancer cells, and HF10 derived from the ORF of PVT1 has been found to be useful in immunotherapy such as cancer vaccines (e.g., Patent Document 1, Non-Patent Document 1, etc.).

[0005] On the other hand, existing antibody drugs can only target cancer antigen proteins that are overexpressed on the cell surface, which has the problem of limiting the target antigens and diseases they can treat. In addition, the cancer antigen PVT1 has high cancer specificity, but its expression is localized intracellularly, meaning that it cannot be targeted by conventional antibodies.

[0006] International Publication No. 2017 / 115798

[0007] Cancer Immunology Research 2021 Nov;9(11):1342-1353. doi:10.1158 / 2326-6066.CIR-20-0964.

[0008] An objective of the present invention is to provide an antibody that recognizes the HLA-A24 / HF10 peptide complex on the surface of cancer cells, a pharmaceutical composition containing the antibody, particularly a pharmaceutical composition for treating cancer, uses thereof, a method for treating cancer, etc.

[0009] The present inventors focused on the fact that the PVT1 ORF-derived peptide HF10 is presented on MHC molecules on the cell surface, and developed an antibody (sometimes abbreviated as the antibody of the present disclosure) that recognizes the MHC molecule / HF10 peptide complex on the surface of cancer cells. In the course of continuing the development of such an antibody, the inventors succeeded in isolating an antibody fragment (scFv) that recognizes the complex of the PVT1 ORF-derived peptide HF10 and HLA-A24 using a phage display antibody library from peripheral blood derived from healthy individuals. Then, by flow cytometry (FACS) analysis, scFv clones with high specific reactivity to the HF10 peptide presented on HLA-A24 were selected, and furthermore, these were converted into scFv-hIgG1 (bivalent) antibodies to increase their binding strength and select those with high specificity. A highly specific HF10 scFv clone was used to prepare a bispecific antibody with CD3 scFv. The bispecific antibody exhibited T cell-mediated cytotoxic activity, was able to induce cytokine release, and was able to suppress tumor volume in vivo. Furthermore, the researchers discovered the novel finding that HF10 CAR-T cells recognized the HLA-A24 / HF10 peptide complex on the cell surface and produced the cytokine IFNg and CD107a-positive cytotoxic granules.

[0010] That is, the present invention relates to the following: [1] A multispecific antibody having an antigen-binding site that recognizes a complex between the polypeptide represented by SEQ ID NO: 3 and an MHC molecule. [2] The multispecific antibody according to [1], wherein the antigen-binding site that recognizes a complex between the polypeptide represented by SEQ ID NO: 3 and an MHC molecule comprises the amino acid sequence of any of SEQ ID NOs: 4 to 8 or the amino acid sequence of GRD, or an amino acid sequence in which one or two amino acids have been substituted in the amino acid sequence of any of SEQ ID NOs: 4 to 8. [3] The multispecific antibody according to [1], having the amino acid sequence of a variable region represented by SEQ ID NO: 19, optionally with one or two amino acid substitutions. [4] The multispecific antibody according to [1], further having an antigen-binding site that recognizes CD3. [5] A pharmaceutical composition comprising an antibody that recognizes a complex between the polypeptide represented by SEQ ID NO: 3 and an MHC molecule. [6] The pharmaceutical composition according to [5], which is a pharmaceutical composition for preventing and / or treating cancer. [7] The pharmaceutical composition according to [5], wherein the MHC molecule is an HLA molecule. [8] The pharmaceutical composition according to [5], wherein the antigen-binding site of the antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 4 to 18, or the amino acid sequence of GRD, GKN, or NDN. [9] The pharmaceutical composition according to [5], wherein the antibody is a multispecific antibody set forth in any one of [1] to [4].

[10] A bivalent artificial antibody comprising: (1) two antibody fragments (scFv) that recognize a complex between the polypeptide set forth in SEQ ID NO: 3 and an HLA molecule; and (2) an Fc region of human IgG.

[11] A chimeric antigen receptor comprising an antigen-binding site that recognizes a complex between the polypeptide set forth in SEQ ID NO: 3 and an MHC molecule, and a CD3ζ chain.

[12] The chimeric antigen receptor according to

[11] , further comprising a costimulatory molecule.

[13] A genetically modified T cell, genetically modified NK cell, or genetically modified macrophage (MΦ) (preferably a genetically modified T cell), characterized in that the chimeric antigen receptor set forth in

[11] or

[12] has been introduced.

[14] An artificial cytotoxic T cell comprising a T cell receptor that recognizes a complex of the polypeptide represented by SEQ ID NO: 3 and an MHC molecule.

[0011] The present invention may also relate to the following: [1A] An antibody having an antigen-binding site that recognizes a complex of a polypeptide represented by SEQ ID NO: 3 and an HLA-A24 molecule, wherein the antigen-binding site comprises a VH having (a) a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 6, and (d) a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, (e) a VL-CDR2 consisting of the amino acid sequence of GRD, and (f) a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, wherein any one or two amino acid residues in one or more of the CDRs of (a) to (f) may be substituted with other amino acids. [2A] The antibody according to [1A], wherein the antigen-binding site recognizing the complex of the polypeptide represented by SEQ ID NO: 3 and an HLA-A24 molecule comprises a VH consisting of an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 22 (VH of clone 1), and a VL consisting of an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 23 (VL of clone 1). [3A] The antibody according to [1A] or [2A], wherein (1) the antigen-binding site recognizing the complex of the polypeptide represented by SEQ ID NO: 3 and an HLA-A24 molecule is an scFv, and (2) the antibody is an artificial antibody comprising the Fc region of human IgG. [4A] The antibody according to [3A], wherein the scFv comprises the amino acid sequence represented by SEQ ID NO: 19. [5A] The antibody according to any of [1A] to [4A], further comprising an antigen-binding site recognizing CD3. [6A] The antibody according to [5A], wherein the antigen-binding site that recognizes CD3 is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 32. [7A] (1) The antibody according to [5A] or [6A], which is a bispecific T cell-inducing antibody.[8A] A chimeric antigen receptor comprising an antigen-binding site that recognizes a complex of the polypeptide represented by SEQ ID NO: 3 and an HLA-A24 molecule, and a CD3ζ chain, wherein the antigen-binding site is a VH having (a) a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 6, and (d) a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, (e) a VL-CDR2 consisting of the amino acid sequence of GRD, and (f) a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, wherein any one or two amino acid residues in one or more of the CDRs of (a) to (f) may be substituted with other amino acids. [9A] The chimeric antigen receptor according to [8A], wherein the antigen-binding site recognizing a complex of the polypeptide shown in SEQ ID NO: 3 and an HLA-A24 molecule has a VH consisting of an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 22 (VH of clone 1), and a VL consisting of an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 23 (VL of clone 1). [10A] The chimeric antigen receptor according to [8A] or [9A], further comprising a costimulatory molecule (preferably CD28). [11A] A genetically modified immune cell (preferably a genetically modified T cell (CAR-T), a genetically modified NK cell (CAR-NK), or a genetically modified macrophage (CAR-MΦ), more preferably a genetically modified T cell (CAR-T)), characterized in that the chimeric antigen receptor according to any one of [8A] to [10A] has been introduced. [12A] A pharmaceutical composition comprising the antibody according to any one of [1A] to [7A] or the genetically modified immune cell according to [11A]. [13A] The pharmaceutical composition according to [12A], further comprising at least one pharmaceutically acceptable carrier. [14A] The pharmaceutical composition according to [12A], which is a pharmaceutical composition for preventing and / or treating cancer.[15A] Use of the antibody described in any one of [1A] to [7A], the genetically modified immune cell described in [11A], or the pharmaceutical composition described in any one of [12A] to [14A] in the manufacture of a medicament for preventing or treating cancer. [16A] A method for preventing or treating cancer, comprising the step of administering an effective amount of the antibody described in any one of [1A] to [7A], the genetically modified immune cell described in [11A], or the pharmaceutical composition described in any one of [12A] to [14A] to a subject in need thereof. [17A] The antibody described in any one of [1A] to [7A], the genetically modified immune cell described in [11A], or the pharmaceutical composition described in any one of [12A] to [14A], for use in the prevention or treatment of cancer. [18A] An agent for preventing or treating cancer, comprising the antibody described in any one of [1A] to [7A], the genetically modified immune cell described in [11A], or the pharmaceutical composition described in any one of [12A] to [14A].

[0012] The antibodies of the present disclosure are capable of specifically recognizing the HLA-A24 / HF10 peptide complex. Furthermore, for example, the bispecific antibodies of the present disclosure are capable of inducing T cell-mediated cytokine production, suppressing tumor volume in vivo, and specifically targeting the intracellular cancer antigen PVT1. Furthermore, for example, because the antibodies of the present disclosure target PVT1, which has high cancer specificity, treatment with fewer side effects on normal organs is possible. Furthermore, because the antibodies of the present disclosure are highly specific, they may be able to improve the prognosis of terminal cancer patients who are unresponsive to existing treatments.

[0013] Figure 1A is a schematic diagram showing the chromosomal location of the PVT1 gene, and also shows an enlarged view of the nucleotide sequence of exons 1 and 2 of the PVT1 gene (SEQ ID NO: 1) and the amino acid sequence of the ORF (SEQ ID NO: 2). The HF10 peptide (SEQ ID NO: 3) is a 10-amino acid peptide encoded by a 447-base (149 amino acid) ORF. Figure 1B shows the relative expression of PVT1 mRNA in various cancer cell lines, including colon cancer, lung cancer, oral squamous cell carcinoma, and osteosarcoma. Figure 2A is a schematic diagram showing an overview of scFv antibody screening using the phage display method. An scFv phage display library is first screened for reactivity with HLA-HIV complexes, and reactive phages are removed as nonspecific antibody phages. Phages that react with the HLA-A24 / natural antigen peptide complex are then selected as specific antibody phages, while those that do not are selected as nonspecific antibody phages. This process is repeated three times. Soluble scFv is obtained from specific antibody phages. Figure 2B shows the results of screening 94 scFv clones for antigen reactivity. A1 / H12 shows a blank. The top 12 clones with high reactivity were selected (Figure 2B, left).

[0014] FIG. 3 shows the results of examining the reactivity of PVT1 HF10 scFv by flow cytometry (FACS) analysis. Antigen-presenting T2-A24 cells were pulsed with HF10 or virus-derived peptides (EBV, CMV) at 50 μM, then reacted with scFv clones and stained with a secondary antibody. FIG. 4 shows that the artificial antibody of the present disclosure recognizes the PVT1 peptide presented on the surface of cancer cells and binds to the cancer cells. The left figure is a schematic diagram of binding in the case of a T cell receptor of a cytotoxic T cell, and the right figure is a schematic diagram of binding in the case of an antibody of the present disclosure. FIG. 5 shows the results of affinity analysis of PVT1 HF10 scFv by surface plasmon resonance (SPR).

[0015] 6 shows the results of FACS analysis of the reaction specificity of the scFv-IgG1 artificial antibody of the present disclosure, which has been converted from scFv (monovalent) to hIgG1 (bivalent), to the HLA-A24 / PVT1 HF10 peptide complex. FIG. 7 shows a photograph of peptide-pulsed 293T-A24 cells (HLA-A24-positive 293T cells) reacted with the scFv-IgG1 artificial antibody of the present disclosure and observed under a fluorescence microscope. This photograph shows the case where scFv-IgG1 was used as the antibody and HF10 was used as the natural antigen peptide. It can be seen that the HF10 antigen peptide presented by HLA-A24 in the cell membrane of cells pulsed with the HF10 antigen peptide binds to the scFv-IgG1 artificial antibody (HF10scFv-IgG1 artificial antibody) and is stained with fluorescently labeled PE. Nuclei are stained with DAPI. The left diagram of Figure 8 corresponds to the right diagram of Figure 8, and shows the results of examining the reactivity of scFv-IgG1 with colon cancer cell lines and oral squamous cell carcinoma cell lines by flow cytometry (FACS) analysis and fluorescence microscopy.

[0016] FIG. 9 shows peripheral blood mononuclear cells (PBMCs) reacting to cancer cells via a bispecific antibody of the present disclosure. FIG. 9A shows that PBMCs produced cytokines (IFNg) in response to cancer cells. FIG. 9B shows that PBMCs produced cytokines (IFNg, IL-2, and TNFα) in response to cancer cells. FIG. 9C shows that CD3 scFv alone and HF10 scFv alone did not induce T cell-mediated cytokine production. FIG. 10 shows cytotoxic activity measured by impedance assay using Maestro Z, Axion Biosystems. FIG. 10A shows that T cells exhibited cytotoxic activity against OS-2000 via a bispecific antibody of the present disclosure. FIG. 10B shows the cytotoxic activity at 20 hours, and FIG. 10C shows the cytotoxic activity at 40 hours.

[0017] Figure 11 shows the generation of CAR-T cells. Figure 11A shows a schematic diagram of the HF10 CAR construct using HF10 scFv. Figure 11B shows the detection of CAR using HLA-A24 / HF10 peptide tetramers when a retroviral vector (pMX) encoding the HF10 CAR structure was constructed and used to infect peripheral blood mononuclear cells (PBMCs) from a healthy donor. Figure 12 shows that HF10 CAR-T cells recognized the HLA-A24 / HF10 peptide complex on the cell surface and produced the cytokine IFNg and CD107a-positive cytotoxic granules. Figure 13 shows that HF10 CAR-T cells exhibited cytotoxic activity against HF10 peptide-pulsed T2-A24 cells, and LDH (lactate dehydrogenase) release from the target cells was observed. Figure 14 shows the antitumor effect in mice using a bispecific antibody of the present disclosure. Figure 14A shows a schematic diagram of the administration schedule for this study. Figure 14B shows the change in tumor volume over 20 days after PBMC administration in the treatment group. Figure 14C shows the change in tumor volume over 20 days after PBMC administration in the control group. Figure 14D shows the change in tumor volume over 20 days in the no-treatment group. Figure 14E shows the average change in tumor volume in each group. Figure 14F shows a comparison of the groups (from left: treatment group, control group, no-treatment group) on Day 10.

[0018] The present invention will be described in detail below. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. All patents, applications and other publications and information referenced herein are incorporated herein by reference in their entirety. In addition, if there is any discrepancy between the publications referenced herein and the description in this specification, the description in this specification shall prevail.

[0019] In the present disclosure, "epitope peptide" or "antigenic peptide" refers to a peptide that binds to a major histocompatibility complex (MHC (human leukocyte antigen (HLA) in humans)) molecule, is presented as an antigen on the cell surface, and has antigenicity (can be recognized by T cells). Epitope peptides include CTL (cytotoxic T cell) epitope peptides, which are epitope peptides that are presented as antigens by binding to MHC class I and recognized by CD8-positive T cells, and helper epitope peptides, which are epitope peptides that are presented as antigens by binding to MHC class II and recognized by CD4-positive T cells.

[0020] Among epitope peptides, peptides derived from proteins that are specifically or excessively expressed in tumor cells are particularly referred to as tumor antigen peptides. Antigen presentation refers to the phenomenon in which a peptide present in a cell binds to MHC, and this MHC / antigen peptide complex is localized on the cell surface. It is known that antigens presented on the cell surface activate cellular and humoral immunity after being recognized by T cells, etc., and antigens presented on MHC class I activate cellular immunity and are recognized by the T cell receptors of naive T cells, inducing naive T cells into CTLs with cytotoxic activity. Therefore, peptides that bind to MHC class I and are presented as antigens are generally used as tumor antigen peptides in immunotherapy.

[0021] Many peptides that bind to MHC are known to have certain characteristics. In the present disclosure, these characteristics are referred to as "binding motifs." It is known in the art which MHCs bind to peptides having certain binding motifs. For example, the binding motif of HLA-A24, a type of human MHC, has a second amino acid from the N-terminus that is tyrosine, phenylalanine, methionine, or tryptophan, and a C-terminal amino acid that is leucine, isoleucine, or phenylalanine. Furthermore, the binding motif of HLA-A02 has a second amino acid from the N-terminus that is leucine, isoleucine, or methionine, and / or a C-terminal amino acid that is valine, leucine, or isoleucine.

[0022] In the present disclosure, "natural peptide" refers to a peptide that is actually presented as an antigen on a cell surface. Furthermore, "natural antigen peptide" refers to a natural peptide whose antigenicity has been confirmed. By isolating a complex of a natural antigen peptide and an MHC molecule from a cancer cell and determining the sequence and origin of the natural antigen peptide, it is possible to select candidate cell surface antigens recognized by the antibody of the present disclosure. In the present disclosure, "tumor" includes benign tumors and malignant tumors (cancer, malignant neoplasms). Cancer includes hematopoietic tumors, epithelial malignant tumors (carcinoma), and non-epithelial malignant tumors (sarcoma). Cancer includes colorectal cancer (e.g., colon cancer), lung cancer (e.g., non-small cell lung cancer), breast cancer, myeloma, head and neck cancer (e.g., oral cancer, oral squamous cell carcinoma, head and neck squamous cell carcinoma, pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer, acoustic neuroma, etc.), osteosarcoma, pancreatic cancer, skin cancer (e.g., malignant melanoma), prostate cancer, ovarian cancer, lymphoma (e.g., B-cell lymphoma, T-cell lymphoma, etc.), ), uveal malignant melanoma, thymoma, mesothelioma, esophageal cancer, gastric cancer, duodenal cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, renal cell carcinoma, renal pelvis / ureter cancer, bladder cancer, penile cancer, testicular cancer, uterine cancer, vaginal cancer, vulvar cancer, malignant bone tumor, soft tissue sarcoma, chondrosarcoma, leukemia (e.g., acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, etc.), myelodysplastic syndrome, or multiple myeloma, etc.

[0023] PVT1 (Plasmacytoma variant translocation 1) is a non-coding RNA present on the same chromosome as MYC, a known proto-oncogene, and is thought to function as a MYC activator. It has also been reported that copy number increases of MYC are also observed in many cancers where increased copy number is observed, suggesting that increased transcription of the PVT1 gene is involved in cell proliferation and carcinogenesis. While PVT1 was previously thought to be a non-coding RNA, the HF10 peptide predicted to be encoded by PVT1 was found to be presented as an antigen in cancer cells, suggesting that the peptide may be translated and expressed from the PVT1 ORF in cancer cells.

[0024] PVT1 mRNA expression has been detected in many colon cancers, and although mild expression is observed in the ovaries in normal tissues, the expression level is low (Non-Patent Document 1). The HF10 peptide (SEQ ID NO: 3) derived from PVT1 exhibited high binding affinity to HLA-A24 and is therefore considered to be a useful target for immunotherapy (Patent Document 1). Furthermore, since this gene is specifically expressed in cancer cells, it may be involved in cancer cell transformation and cancer cell survival, and therefore is expected to be effective as a target for nucleic acid therapy by suppressing expression, etc.

[0025] In the present disclosure, when a gene is referred to simply by its name, for example, "PVT1," unless otherwise specified, it refers to a gene having a known nucleic acid sequence represented by that gene name, and typically represents a cDNA or mRNA sequence, but is not limited to these, as long as one skilled in the art can recognize it as the sequence of the gene. For example, an example of a preferred gene and its nucleic acid sequence in the present disclosure is the base sequence represented by SEQ ID NO: 1, but genetic polymorphisms thereof (e.g., SNPs), and sequences that can be recognized as the sequence of the gene, like these genes, are also included in the genes of the present disclosure. Therefore, mRNA as a gene expression product of the present disclosure may be referred to simply by the name of the gene.

[0026] In the present disclosure, an "antigenic peptide derived from the ORF of PVT1" refers to a partial peptide consisting of a continuous portion of the amino acid sequence constituting the ORF of PVT1, which has the properties of the above-mentioned antigenic peptide. In a preferred embodiment of the present disclosure, the "antigenic peptide derived from the ORF of PVT1" specifically refers to HF10, a polypeptide represented by SEQ ID NO:3.

[0027] <1> Antibodies of the Present Disclosure One aspect of the present disclosure relates to antibodies that specifically recognize a complex between an antigenic peptide HF10 derived from the ORF of a gene specifically expressed in cancer cells, such as PVT1, and an MHC molecule. In principle, such antibodies have an antigen-binding site that recognizes the complex between the antigenic peptide and an MHC molecule.

[0028] In the present disclosure, the term "antibody" refers to a protein that has an antigen-binding site and has the property of binding to a molecule recognized by the antigen-binding site, typically, but not limited to, an immunoglobulin. Therefore, not only immunoglobulin molecules but also functional fragments (antigen-binding fragments) of antibodies that can be generated from the antigen-binding site of immunoglobulins are included in the antibodies of the present disclosure. Such antigen-binding fragments typically include F(ab') ２ fragments, Fab' fragments, Fab fragments, Fv fragments, rIgG fragments, as well as scFv, dsFv, diabodies and sc(Fv) ２and the like are also included. For example, these fragments may be linked by disulfide bonds in the constant region or hinge region, or may be single-chain fragments (scFv) formed by linking each region with a linker. In a preferred embodiment, the antibody of the present disclosure is a single-chain antibody (scFv). In a preferred embodiment, the antibody of the present disclosure is an scFv-IgG artificial antibody. Examples of scFv-IgG artificial antibodies include (1) an antibody fragment (scFv) having an antigen-binding site that recognizes a complex between the polypeptide represented by SEQ ID NO: 3 and an HLA molecule, and (2) an artificial antibody comprising the Fc region of human IgG. In another preferred embodiment, the antibody of the present disclosure is an IgG type, preferably an IgG1 type. Furthermore, the antibodies of the present disclosure also include multimers (e.g., dimers, trimers, tetramers, and polymers) of immunoglobulins or functional fragments thereof.

[0029] The antibody of the present disclosure is not particularly limited as long as it can recognize a complex between a cancer cell antigen peptide and an MHC molecule present on the cell surface, and may be a polyclonal or monoclonal antibody. Furthermore, it is not particularly limited as long as it has an antigen-binding site that recognizes the complex between a cancer cell antigen peptide and an MHC molecule, and may have, for example, another antigen-binding site or a binding region for binding to another protein. Thus, in one embodiment, the antibody of the present disclosure may be a multispecific antibody. In a preferred embodiment, the antibody of the present disclosure is a bispecific antibody.

[0030] The antibodies of the present disclosure can recognize and bind to a complex between a cancer cell antigen peptide and an MHC molecule present on the surface of cancer cells, and thus can be used in a variety of applications. In one embodiment, the antibodies of the present disclosure can be used for detecting cancer cells. In another embodiment, the antibodies of the present disclosure can be used for treating cancer cells (i.e., treating cancer). When the antibodies of the present disclosure are used for treating cancer cells, the method may utilize cellular immunity or humoral immunity. An embodiment utilizing cellular immunity includes, for example, adoptive cell therapy using immune cells (e.g., T cells (CAR-T cells), NK cells (CAR-NK cells), macrophages (MΦ) (CAR-MΦ), etc.) introduced with a chimeric antigen receptor (CAR) incorporating the antigen-binding site of an antibody of the present disclosure. An embodiment utilizing humoral immunity includes, for example, a method utilizing the antibody-dependent cellular cytotoxicity (ADCC) activity or complement-dependent cytotoxicity (CDCC) activity of an antibody. In another aspect, the antibody of the present disclosure encompasses a chimeric antigen receptor (CAR) incorporating an antigen-binding site that recognizes a complex of a cancer cell antigen peptide and an MHC molecule.

[0031] As described above, the antibody of the present disclosure has an antigen-binding site that specifically recognizes a complex between an MHC molecule and the antigen peptide HF10 derived from the ORF of PVT1, which is an expression product of a gene specifically expressed in cancer cells. Any method known in the art may be used to select an antigen-binding site that recognizes a specific antigen or an antibody having such an antigen-binding site. Such methods include, for example, phage display.

[0032] The antigen peptide is not particularly limited as long as it binds to MHC. Peptides that bind to MHC have different characteristics depending on the type of MHC. For example, in the case of HLA, which is a human MHC, peptides that bind to HLA class I are about 8 to 14 amino acids long, preferably about 8 to 10 amino acids long, while HLA class II binds to peptides that are 10 amino acids or longer, for example, about 10 to 30 amino acids long. Furthermore, among HLA class I, for example, peptides that bind to HLA-A02 have a binding motif in which the second amino acid from the N-terminus is leucine, isoleucine, or methionine and / or the C-terminal amino acid is valine, leucine, or isoleucine, while peptides that bind to HLA-A24 have a binding motif in which the second amino acid from the N-terminus is tyrosine, phenylalanine, methionine, or tryptophan and / or the C-terminal amino acid is leucine, isoleucine, or phenylalanine.

[0033] The antigenic peptide may be determined by predicting an MHC-restricted peptide based on a binding motif from the full-length protein sequence, or by identifying a natural peptide that is actually presented as an antigen. Methods for identifying natural peptides may be methods known in the art or a combination thereof, such as those described in International Publication Nos. 2015 / 050259 and 2017 / 115798. Since the antibody of the present disclosure recognizes antigenic peptides presented on cell surfaces, the antigenic peptide is preferably a natural peptide. The present inventors have identified HF10 (HWNDTRPAHF (SEQ ID NO: 3)) as a natural peptide of PVT1 in human cancer cells. Therefore, in a more preferred embodiment, the antigenic peptide is HF10 represented by SEQ ID NO: 3.

[0034] In a preferred embodiment of the present disclosure, the MHC is HLA. In a more preferred embodiment, the MHC is HLA class I. In a further preferred embodiment, the MHC is HLA-A02. In another further preferred embodiment, the MHC is HLA-A24.

[0035] As described above, the antibody of the present disclosure is not particularly limited as long as it can specifically recognize the complex of the antigen peptide HF10 and an MHC molecule, and may also be an antibody that recognizes and binds to another molecule. Therefore, in a preferred embodiment, the antibody of the present disclosure may have a binding region for another molecule in addition to the antigen-binding site that recognizes the complex of the cancer cell antigen peptide HF10 and an MHC molecule. Such a binding region is typically an antigen-binding site, but is not limited thereto and may be, for example, a ligand for a cell surface receptor. In a preferred embodiment, the antibody of the present disclosure is an antibody that specifically binds to two or more molecules, i.e., a multispecific antibody. Here, the term "multispecific antibody" refers to an antibody that has at least one antigen-binding site and specifically binds to two or more molecules. Therefore, for example, a bispecific antibody of the present disclosure refers to an antibody that has at least one antigen-binding site and specifically binds to two molecules, where at least one antigen-binding site is an antigen-binding site that recognizes the complex of the cancer cell antigen peptide HF10 and an MHC molecule and can also specifically bind to another molecule. Specific binding to another molecule may be achieved by another antigen binding site or by other means, such as by a ligand domain for another molecule.

[0036] When the antibody of the present disclosure is a multispecific antibody, the additional antigen recognized by the antibody of the present disclosure is not particularly limited, but is preferably one that contributes to the treatment of cancer cells. Examples of such antigens include proteins expressed on the cell surface of immune cells, such as CD3, CD28, CD40, PD1, CTLA4, TIGIT, OX40, and CD137. In a preferred embodiment of the present disclosure, the additional antigen includes a T cell surface protein such as CD3 or CD28. Therefore, in a preferred embodiment, the multispecific antibody of the present disclosure has a binding region for CD3 and / or CD28 in addition to an antigen-binding site that recognizes a complex between a cancer cell antigen peptide and an MHC molecule. Such a binding region may be an antigen-binding site, or may be, for example, CD80, a ligand for CD28.

[0037] The present inventors screened for antibodies having an antigen-binding site that recognizes a complex between the above-mentioned natural antigen peptide HF10 and HLA-A24. Accordingly, in a preferred embodiment of the present disclosure, the antigen-binding site that recognizes a complex between the cancer cell antigen peptide HF10 and an MHC molecule comprises the amino acid sequence set forth in any one of SEQ ID NOS: 4 to 18, or the amino acid sequence GRD, GKN, or NDN. In another preferred embodiment of the present disclosure, the antigen-binding site that recognizes a complex between the cancer cell antigen peptide HF10 and an MHC molecule may comprise an amino acid sequence in which one or two amino acids have been substituted in the amino acid sequence set forth in any one of SEQ ID NOS: 4 to 18, or the amino acid sequence GRD, GKN, or NDN. The amino acid substitution is preferably a conservative substitution, such as a substitution between acidic amino acids, a substitution between basic amino acids, or a substitution between neutral amino acids.

[0038] Typical examples of antibodies of the present disclosure include antibodies having an antigen-binding site that recognizes a complex of a cancer cell antigen peptide (HF10) consisting of the amino acid sequence set forth in SEQ ID NO: 3 and HLA-A24; bispecific antibodies having an antigen-binding site that recognizes a complex of a cancer cell antigen peptide (HF10) consisting of the amino acid sequence set forth in SEQ ID NO: 3 and HLA-A24 and an antigen-binding site that recognizes CD3 (a preferred embodiment is a BiTE (bispecific T cell-inducing antibody)); trispecific antibodies having an antigen-binding site that recognizes a complex of a cancer cell antigen peptide (HF10) consisting of the amino acid sequence set forth in SEQ ID NO: 3 and HLA-A24, an antigen-binding site that recognizes CD3, and an antigen-binding site that recognizes CD28; and trispecific antibodies having an antigen-binding site that recognizes a complex of a cancer cell antigen peptide (HF10) consisting of the amino acid sequence set forth in SEQ ID NO: 3 and HLA-A24, an antigen-binding site that recognizes CD3, and a CD80 region.

[0039] The antibody of the present disclosure preferably comprises at least one of complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 in the antigen-binding site, and both the heavy chain and the light chain preferably comprise at least one of complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3. Furthermore, in a preferred embodiment, the antibody of the present disclosure may comprise an antigen-binding site comprising an amino acid sequence in which one or two amino acids have been substituted in the amino acid sequence set forth in any of SEQ ID NOS: 4 to 18. In addition, in a preferred embodiment, the antibody of the present disclosure comprises, in particular, the amino acid sequence set forth in SEQ ID NOS: 4 and / or 7 as complementarity-determining region (CDR) 1, the amino acid sequence of SEQ ID NOS: 5 and / or GRD as complementarity-determining region (CDR) 2, and the amino acid sequence set forth in SEQ ID NOS: 6 and / or 8 as complementarity-determining region (CDR) 3. In another preferred embodiment, the antibody of the present disclosure comprises, in the antigen-binding site, the amino acid sequence of SEQ ID NO: 9 and / or SEQ ID NO: 12, particularly as complementarity-determining region (CDR) 1, the amino acid sequence of SEQ ID NO: 10 and / or GKN, as complementarity-determining region (CDR) 2, and the amino acid sequence of SEQ ID NO: 11 and / or SEQ ID NO: 13, particularly as complementarity-determining region (CDR) 3. In a further preferred embodiment, the antibody of the present disclosure comprises, in the antigen-binding site, the amino acid sequence of SEQ ID NO: 14 and / or SEQ ID NO: 17, particularly as complementarity-determining region (CDR) 1, the amino acid sequence of SEQ ID NO: 15 and / or NDN, as complementarity-determining region (CDR) 2, and the amino acid sequence of SEQ ID NO: 16 and / or SEQ ID NO: 18, particularly as complementarity-determining region (CDR) 3. In a further preferred embodiment, the antibody of the present disclosure may comprise, in the antigen-binding site, an amino acid sequence in which one or two amino acids have been substituted in the amino acid sequence of any of SEQ ID NOs: 4 to 18.

[0040] Furthermore, in a preferred embodiment, the antibody of the present disclosure may have a VH consisting of an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 22 (VH of clone 1), and a VL consisting of an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 23 (VL of clone 1). Furthermore, in a preferred embodiment, the antibody of the present disclosure may have a VH consisting of an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 24 (VH of clone 2), and a VL consisting of an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 25 (VL of clone 2). Furthermore, in a preferred embodiment, the antibody of the present disclosure may have a VH consisting of an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 26 (VH of clone 5), and a VL consisting of an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 27 (VL of clone 5). Another preferred embodiment of the antibody of the present disclosure is an scFv comprising a VH and a VL, and preferred embodiments of the VH and VL are as described above. The scFv preferably has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 19 to 21. Furthermore, the antibody of the present disclosure may be derived from any of chimeric, humanized, and human antibodies. Of the three CDRs in the antibody of the present disclosure, CDR3 is particularly important for antigen binding.

[0041] <2> Pharmaceutical Composition of the Present Disclosure One aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody of the present disclosure. As described above, the antibody of the present disclosure can specifically recognize cancer cell antigen peptides antigenically presented on the surface of cancer cells, and therefore can be used as an active ingredient in pharmaceutical compositions for various applications. Therefore, any of the antibodies detailed in <1> above can be used as the antibody of the present disclosure contained as an active ingredient in the pharmaceutical composition of the present disclosure. The pharmaceutical composition of the present disclosure contains the antibody of the present disclosure as an active ingredient. The pharmaceutical composition of the present disclosure may further contain at least one pharmaceutically acceptable carrier. Here, examples of pharmaceutically acceptable carriers include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, antiseptics, pH adjusters, and antioxidants.

[0042] The pharmaceutical composition of the present disclosure can be used not only as a treatment agent for treating cancer cells (i.e., a cancer therapeutic agent), but also as, for example, a cancer cell detection agent, a companion diagnostic agent for diagnosing the effectiveness of cancer vaccine immunotherapy in a patient being treated, etc. That is, the antibody of the present disclosure recognizes a cancer cell antigen peptide displayed on the surface of a cancer cell and binds to the cancer cell surface, thereby exerting cytotoxic activity against the cancer cell. Therefore, in a preferred embodiment, the pharmaceutical composition of the present disclosure is a pharmaceutical composition for preventing and / or treating cancer.

[0043] Here, "prevention" of cancer includes not only preventing patients from contracting cancer, but also preventing recurrence in patients whose primary tumor has been surgically removed, and preventing metastasis of tumors that could not be completely removed by cancer treatments such as surgery, radiation therapy, or chemotherapy. Furthermore, "treatment" of cancer includes not only curing cancer or improving symptoms by shrinking the cancer, but also preventing progression by suppressing the proliferation of cancer cells, tumor expansion, or metastasis of cancer cells from the primary tumor.

[0044] The pharmaceutical composition of the present disclosure can be administered to any individual organism that may have a tumor, but is preferably administered to humans and non-human mammals (e.g., rodents such as mice, rats, guinea pigs, and hamsters; primates such as chimpanzees; even-toed ungulates such as cows, goats, and sheep; perissodactyla such as horses; rabbits, dogs, and cats), and more preferably to humans.

[0045] Furthermore, when the pharmaceutical composition of the present disclosure is used as a tumor detection agent, the cell population to be detected can be a cell population derived from any biological sample obtained from the above-mentioned individual organism, but is preferably a cell population derived from a biological sample obtained from a human, and more preferably a cell population containing cells derived from one or more biological samples selected from the group consisting of tissues other than the ovary in which it has been confirmed that PVT1 is hardly expressed in the cells of the tissue, such as the heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, small intestine, large intestine, and blood.

[0046] As described above, a preferred embodiment of the antibody of the present disclosure has an antigen-binding site that recognizes a complex between the cancer cell antigen peptide HF10 derived from the ORF of PVT1 and HLA-A24. Therefore, the pharmaceutical composition of the present disclosure can be particularly suitably used for subjects suffering from cancers that express PVT1. It can also be particularly suitably used for subjects whose HLA is HLA-A24. Specifically, the composition can be used for the prevention or treatment of cancers (tumors), such as colon cancer, lung cancer, breast cancer, myeloma, oral cancer, oral squamous cell carcinoma, osteosarcoma, pancreatic cancer, skin cancer, and prostate cancer.

[0047] In recent years, it has also been discovered that cancer cells avoid elimination by the immune system by shielding themselves from attacks by immune cells, and that this shielding utilizes a mechanism known as an "immune checkpoint," which is originally designed to suppress excessive immune responses against the self and damage to normal tissues. Therefore, by suppressing the function of immune checkpoints in cancer cells, it is possible to make attacks by immune cells more effective. In one aspect, the pharmaceutical composition of the present disclosure exerts an antitumor effect by utilizing immune cells that can damage cancer cells bound to antibodies, which are active ingredients. Therefore, it is believed that a greater therapeutic effect can be achieved by simultaneously suppressing the function of immune checkpoints. Therefore, in a preferred aspect, the pharmaceutical composition of the present disclosure is used together with an immune checkpoint inhibitor.

[0048] In the present disclosure, when a certain agent A and another agent B are "used together" or "used in combination," it means that the agent B is in a state in which it exerts its effect while the agent A is exerting its effect. Therefore, agent B may be administered simultaneously with the administration of agent A, or agent B may be administered at a certain interval after the administration of agent A. Furthermore, agent A and agent B may be administered in the same dosage form or in different dosage forms. Furthermore, agent A and agent B may be mixed to form a single composition, as long as the effect of agent A or agent B is not lost.

[0049] In this embodiment, any known immune checkpoint inhibitor can be used as the immune checkpoint inhibitor, as long as it does not inhibit the antigen recognition ability of the pharmaceutical composition of the present disclosure. Known immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-B7-H5 antibodies, and anti-TIGIT antibodies.

[0050] Furthermore, the dosage form of the pharmaceutical composition of the present disclosure is not particularly limited, and examples thereof include oil emulsions (emulsion formulations), polymeric nanoparticles, liposome formulations, particulate formulations bound to beads with a diameter of several microns, lipid-bound formulations, microsphere formulations, and microcapsule formulations. Examples of the administration method include any known administration method such as intradermal administration, subcutaneous administration, intramuscular administration, and intravenous administration. The dosage of the pharmaceutical composition of the present disclosure in the formulation can be adjusted appropriately depending on the disease to be treated, the age and weight of the patient, etc., but 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.

[0051] As described above, cancer can also be treated by adoptive cell therapy using T cells (CAR-T cells) transfected with a chimeric antigen receptor (CAR) incorporating an antigen-binding site of an antibody of the present disclosure. In the present disclosure, a "chimeric antigen receptor" is a chimeric protein molecule designed to have, at its N-terminus, a single-chain fragment (scFv) formed by tandemly linking the light and heavy chains of an antibody variable region of an antibody that recognizes a molecule present on the surface of a cancer cell, and a CD3ζ chain from among the molecules that constitute the T cell receptor (TCR) / CD3 complex, at its C-terminus. When this chimeric antigen receptor recognizes a specific antigen via the scFv region, T cells are activated via the CD3ζ chain. To enhance T cell activation, one or more costimulatory molecules (e.g., CD28, 4-1BB, ICOS, etc.) may be incorporated between the scFv and the ζ chain. Therefore, a CAR can be produced using an antibody of the present disclosure as the scFv. A CAR that recognizes a complex of a cancer cell antigen peptide and MHC can recognize cancer cells presenting cancer cell antigen peptides that can be targeted by CTLs, dendritic cells that have phagocytosed cancer cells and present tumor antigen peptides on MHC class I, and the like, and therefore pharmaceutical compositions comprising genetically modified T cells into which the CAR has been introduced (CAR-T cells) are also encompassed by the pharmaceutical compositions of the present disclosure. Furthermore, pharmaceutical compositions of the present disclosure also include pharmaceutical compositions comprising genetically modified NK cells into which the CAR has been introduced (CAR-NK cells), pharmaceutical compositions comprising genetically modified macrophages (MΦ) into which the CAR has been introduced (CAR-MΦ), and the like.

[0052] <3> Tumor Detection Method (Testing Method, Diagnostic Method) One aspect of the present disclosure relates to a tumor detection method (testing method, diagnostic method) using an antibody of the present disclosure. The detection method (diagnostic method) of the present disclosure using an antibody of the present disclosure typically involves collecting blood from a subject or collecting a portion of a test tissue suspected of having a tumor by biopsy or the like, and detecting and measuring the amount of cells contained therein that have a complex between a cancer cell antigen peptide and an MHC molecule using the antibody of the present disclosure, thereby detecting, testing, or diagnosing the presence or absence, or the severity, of cancer (tumor) such as colon cancer, lung cancer, breast cancer, myeloma, oral cancer, pancreatic cancer, skin cancer, or prostate cancer.

[0053] A specific embodiment of the detection (test) method of the present disclosure using the antibody of the present disclosure comprises the following steps (a) and (b), and optionally (c): (a) contacting a biological sample obtained from a subject with the tumor detection agent of the present disclosure; (b) measuring the amount of cells in the biological sample that present a complex of a cancer cell antigen peptide and an HLA antigen, using the amount of cells bound to the tumor detection agent as an indicator; and (c) determining the presence of cancer based on the results of step (b). A specific embodiment of the diagnostic method of the present disclosure using the antibody of the present disclosure comprises the above steps (a), (b), and (c). The biological sample used here can be a sample prepared from the subject's biological tissue (such as a tissue suspected of containing cancer cells and its surrounding tissue, or blood). Specifically, a sample containing tissue cells collected from the tissue can be included.

[0054] Prediction, determination, judgment, or diagnosis of the presence or absence of a tumor can be performed, for example, by measuring the amount of cells bound by the antibody of the present disclosure in a subject's blood or a test tissue suspected of having a tumor. In some cases, the level of cells bound by the antibody of the present disclosure in a normal counterpart tissue can be used as a reference value, and the reference value can be compared with the level in a sample obtained from the subject to determine the difference between the two. The comparison of the levels in the subject's test tissue and the normal counterpart tissue can be performed by performing parallel measurements on the subject's biological sample and a biological sample from a normal individual. If parallel measurements are not performed, the average or statistical median of the amount of cells bound by the antibody of the present disclosure obtained by measuring multiple (at least two, preferably three or more, more preferably five or more) normal tissues under uniform measurement conditions can be used as the normal individual value, i.e., the reference value, for comparison.

[0055] Whether or not a subject is suffering from cancer can be determined, for example, by using as an indicator whether the number of cells to which the antibody of the present disclosure binds in the tissue of the subject is, for example, at least two-fold, preferably at least three-fold, higher than the level of cells in a normal subject.

[0056] <4> Method for Preventing and / or Treating Cancer One aspect of the present disclosure also relates to a method for preventing and / or treating cancer in a subject, the method comprising administering an effective amount of an antibody or CAR-T cell of the present disclosure to a subject in need thereof. The "subject" in the present disclosure may be any individual organism that can be affected by cancer, but is preferably a human or non-human mammal (e.g., rodents such as mice, rats, guinea pigs, and hamsters; primates such as chimpanzees; even-toed ungulates such as cows, goats, and sheep; perissodactyls such as horses; rabbits, dogs, cats, etc.), and more preferably a human. In the present disclosure, the subject may be healthy or suffering from a disease, but when cancer prevention and / or treatment is intended, the subject typically refers to a subject that is affected by or at risk of developing cancer. In one embodiment of the present disclosure, the subject is HLA-A24- or HLA-A02-positive. In a preferred embodiment of the present disclosure, the subject has or is at risk of having a PVT1-positive cancer. In one embodiment of the present disclosure, the subject is HLA-A24-positive and has or is at risk of having a PVT1-positive cancer.

[0057] The antibodies and CAR-T cells of the present disclosure used in the prevention / treatment methods of the present disclosure include any of those described herein. The effective amount in the present disclosure refers, for example, to an amount that reduces the symptoms of cancer or delays or stops its progression, preferably an amount that suppresses or cures cancer. An amount that does not cause adverse effects that exceed the benefits of administration is also preferred. Such amounts can be appropriately determined by in vitro tests using cultured cells or tests in model animals such as mice and rats, and such test methods are well known to those skilled in the art. The specific dose of the active ingredient can be determined taking into consideration various conditions related to the subject in need thereof, such as the severity of symptoms, the subject's general health condition, age, body weight, sex, diet, timing and frequency of administration, concomitant medications, responsiveness to treatment, dosage form, and compliance with treatment.

[0058] Specific doses, for example, for antibodies of the present disclosure, are typically 0.0001 mg to 2000 mg, preferably 0.001 mg to 2000 mg, and are preferably administered once every 1 to 4 weeks. For CAR-T cells of the present disclosure, typically 1 x 10 ４ ~1 x 10 ８ pieces, preferably 1 x 10 ５ ~1 x 10 ７ The dosage is preferably administered once a day to once every four weeks. Any known appropriate administration method can be used, such as intradermal administration, subcutaneous administration, intramuscular administration, or intravenous administration.

[0059] One embodiment of the prevention / treatment method of the present disclosure further comprises the step of selecting an HLA-A24- or HLA-A02-positive subject as a subject for prevention / treatment prior to the administering step. This embodiment of the present disclosure may further comprise the step of determining the subject's HLA type prior to the selecting step. Determining the subject's HLA type can be performed by any known technique. Furthermore, one embodiment of the prevention / treatment method of the present disclosure further comprises the step of selecting a subject with a PVT1-positive cancer as a subject for prevention / treatment prior to the administering step. This embodiment of the present disclosure may further comprise the step of detecting PVT1-positive cancer in the subject prior to the selecting step. PVT1-positive cancer in the subject can be detected using the tumor detection method described in <3> above. One embodiment of the prevention / treatment method of the present disclosure further comprises the step of selecting a subject who is HLA-A24-positive and has a PVT1-positive cancer as a subject for prevention / treatment prior to the administering step. This aspect of the disclosure may further comprise, prior to said selecting step, determining the subject's HLA type and detecting PVT1-positive cancer in the subject.

[0060] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Various changes and modifications can be made by those skilled in the art based on the description of the present invention, and these changes and modifications are also included in the present invention.

[0061] Example 1. Expression of PVT1 gene CD8 ＋T cells recognize peptides presented by HLA class I molecules. Proteogenomic analysis of HLA ligandomes has shown that there is a subset of peptides derived from non-coding regions of the genome. One such peptide is PVT1, a long noncoding RNA (lncRNA), which is partially translated in many colorectal cancer tissues and binds to HLA class I molecules on the cell surface to form a complex as PVT1 peptide (HF10), which then activates CD8. ＋ It has been suggested that PVT1 is a target of T cells (Patent Document 1, Non-Patent Document 1). Therefore, we investigated the expression of PVT1 mRNA in various cancer cells. Overexpression of PVT1 was observed in various carcinomas, including colorectal cancer (Figure 1B). High expression was observed in the colorectal cancer cell line Colo320, the lung cancer cell line LHK2, the oral squamous cell carcinoma cell line MO-1000, and the osteosarcoma cell line OS-2000. In normal tissues, expression levels were low, with mild expression observed in the ovary (Non-Patent Document 1).

[0062] Example 2. Screening for antibodies specific to the HLA-A24 / natural antigen peptide complex (1) Preparation of antibody phage Using the natural antigen peptide HF10 (SEQ ID NO: 3) derived from the PVT1 ORF as the cancer cell antigen peptide, scFvs recognizing the HLA-A24 / HF10 complex were identified using a method similar to the phage display method described in Tsukahara et al., J Biol Chem., 2014, Aug 8;289(32):22035-47. In the following Examples 3 to 6, "cancer cell antigen peptide" refers to HF10. Specifically, a phagemid vector incorporating DNA encoding a peptide formed by linking the heavy chain variable region (VH region) and light chain variable region (VL region) of an antibody with a linker was first prepared from the scFv library, and then used to infect Escherichia coli. This was further infected with a helper phage to produce M13 phage (antibody phage) displaying scFv in which the VH and VL regions were linked by a linker.

[0063] (2) Biopanning Prior to biopanning, non-specifically binding phages were removed from the phage library using a biotin-modified complex of HLA-A24 and an HIV peptide (RYLRDQQLLGI (SEQ ID NO: 28)) and streptavidin-coupled magnetic beads. From the remaining antibody phages, antibody phages that bind to each complex were selected (positive panning) using biotin-modified HLA-A24 / HF10 complexes and streptavidin-coupled magnetic beads. This process was repeated three times to obtain candidate antibody phages that specifically bind to the HLA-A24 / HF10 complex.

[0064] (3) Conversion of scFv Antibody to IgG1 Type and FACS Analysis The scFv portion was cleaved from the obtained candidate antibody phage, and a FLAG tag was attached to the end of the VL region. The reactivity to the HLA-A24 / HF10 complex was analyzed using ELISA. As a result of the analysis, 12 clones with high reactivity were selected. The affinity to the HF10 peptide was in the order of scFv clone 1 < clone 2 < clone 5, with clone 5 having the highest affinity. In addition, analysis was performed using FACS and SPR, and as a result of the analysis, scFv clones (clones 1, 2, and 5) with high specific reactivity to the HF10 peptide were selected ( Figures 3 and 5 ). The heavy chain Fc region of human IgG1 (CH2 and CH3 regions, SEQ ID NO: 30 (the nucleotide sequence encoding this is shown in SEQ ID NO: 29)) was linked to the scFv antibody in place of the FLAG tag to convert it to the IgG1 type. Further FACS analysis was performed using the antibody converted to the IgG1 type (scFv-hIgG1), and clone 1, which had low affinity by SPR but particularly high reactivity, was selected (Figure 6), and used in the following examples.

[0065] The sequences of the CDR1 to CDR3 regions of these clones (clones 1, 2, and 5) were as follows: Clone 1 VH region CDR1 region sequence: GYSFTTYG (SEQ ID NO: 4) CDR2 region sequence: ISAYTGDT (SEQ ID NO: 5) CDR3 region sequence: CARGSSNFYGMDVW (SEQ ID NO: 6) Clone 1 VL region CDR1 region sequence: SLRNYY (SEQ ID NO: 7) CDR2 region sequence: GRD CDR3 region sequence: CQSRDISGNPQNVVF (SEQ ID NO: 8) Clone 2 VH region CDR1 region sequence: GYSFTSYW (SEQ ID NO: 9) CDR2 region sequence: IYPGDSDT (SEQ ID NO: 10) CDR3 region sequence: CARQSGSYGGTFDIW (SEQ ID NO: 11) Clone 2 VL region CDR1 region sequence: SLRNYY (SEQ ID NO: 12) CDR2 region sequence: GKN Sequence of CDR3 region: CSSRDSAGKHLVF (SEQ ID NO: 13) Clone 5 VH region Sequence of CDR1 region: GFTFSSYA (SEQ ID NO: 14) Sequence of CDR2 region: SSYGGGTT (SEQ ID NO: 15) Sequence of CDR3 region: CASSITVRGSLRNW (SEQ ID NO: 16) Clone 5 VL region Sequence of CDR1 region: SLRSYY (SEQ ID NO: 17) Sequence of CDR2 region: NDN Sequence of CDR3 region: CSSRDSSGDPVIF (SEQ ID NO: 18)

[0066] The amino acid sequences of the ScFVs containing the variable regions (VH, VL) of clones 1, 2, and 5 were as follows: Clone 1 QVQLVQSGAEVKKPGASVKVSCKASGYSFTTYGISWVRQAPGQGLEWMGWISAYTGDTNYAQKFQGRVTVTTDTSTGTVYMELRTLRSDDSAVYYCARGSSNFYGMDVWGQGTTVTVSSGGGGSGGGGGSGGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQRAGQAPVLVFYGRDNRPSGIPDRFSGSTSGSTASLTITGAQAEDEADYYCQSRDISGNPQNVVFGGGTKVTVL (SEQ ID NO: 19) Clone 2 QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQSGSYGGTFDIWGQGTMVTVSSGGGGSGGGGGSGGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQTPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADFYCSSRDSAGKHLVFGTGTKVPVL (SEQ ID NO: 20) clone 5 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGAGVGLNSSYGGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSITVRGSLRNWGQGTLVTVSSGGGGGSGGGGSGGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPTLVLYNDNNNRPSGIPDRFSGSTSGTTASLTITGAQAEDDADYYCSSRDSSGDPVIFGGGTKLTVL (SEQ ID NO: 21)

[0067] The amino acid sequences of the variable regions (VH, VL) of clones 1, 2, and 5 were as follows: VH of clone 1 QVQLVQSGAEVKKPGASVKVSCKASGYSFTTYGISWVRQAPGQGLEWMGWISAYTGDTNYAQKFQGRVTVTTDTSSTGTVYMELRTLRSDDSAVYYCARGSSNFYGMDVWGQGTTVTVSS (SEQ ID NO: 22) VL of clone 1 SSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQRAGQAPVLVFYGRDNRPSGIPDRFSGSTSGSTASLTITGAQAEDEADYYCQSRDISGNPQNVVFGGGTKVTVL (SEQ ID NO: 23) VH of clone 2 QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQSGSYGGTFDIWGQGTMVTVSS (SEQ ID NO: 24) VL of clone 2 SSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQTPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADFYCSSRDSAGKHLVFGTGTKVPVL (SEQ ID NO: 25) VH of clone 5 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGAGVGLNSSYGGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSITVRGSLRNWGQGTLVTVSS (SEQ ID NO: 26) Clone 5 VL SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPTLVLYNDNNNRPSGIPDRFSGSTSGTTASLTITGAQAEDDADYYCSSRDSSGDPVIFGGGTKLTVL (SEQ ID NO: 27)

[0068] Example 3. (1) Detection of HLA-A24 / HF10 peptide complex on the cell surface of peptide-pulsed 293T-A24 cells. We tested whether the scFv-hIgG1 antibody obtained in Example 2 actually reacts with cells that present antigens on their surface. 293T-A24 cells were pulsed with HF10 adjusted to a final concentration of 50 μg / mL, and then reacted with the scFv-hIgG1 antibody obtained in Example 2 at a final concentration of 10 μg / mL. Observation was performed using a PE-conjugated anti-human IgG antibody as a fluorescent label and DAPI for nuclear staining. When the HLA-A24 / HF10 peptide complex is present on the cell surface and the scFv-hIgG1 antibody binds to it, the periphery of the cell is stained red. The nuclei of cancer cells are stained blue by DAPI. The results are shown in Figure 7. An HLA-A24 / HF10 peptide complex was detected on the cell surface of 293T-A24 cells pulsed with the cancer cell antigen peptide HF10.

[0069] (2) Detection of HLA-A24 / HF10 peptide complexes on the cell surface of HF10-expressing cancer cells. Next, we tested the reactivity against actual cancer cells. The human colon cancer cell line colo320 (HLA-A24(+) and HF10(+)) and the human oral squamous cell carcinoma cell line MO-1000 (HLA-A24(+) and HF10(+)) were tested in the same manner as in (1). The results are shown in Figure 8. Similar to 293T-A24 cells, the HLA-A24 / HF10 peptide complexes on the cell surface of actual cancer cells were detected by flow cytometry (Figure 8, left) and fluorescence microscopy (Figure 8, right). Therefore, the HLA-A24 / HF10 peptide complexes were also detected on the cell surface of cancer cells that endogenously present cancer cell antigens. Thus, the antibodies of the present disclosure were shown to be capable of detecting HLA-A24 / HF10 peptide complexes on the cell surface of cells that endogenously present natural antigen peptides.

[0070] Example 4. Analysis of Bispecific Antibody BiTEs (1) Construction of BiTEs (Bispecific T Cell-Inducing Antibodies) and Their Binding to Various Cells Using a method similar to that described in Stadler et al., Nature Medicine volume 23, pages 815-817 (2017), a bispecific antibody BiTE was constructed by linking an anti-CD3 scFv (SEQ ID NO: 32) (the encoding nucleotide sequence is shown in SEQ ID NO: 31) to the scFv of the clone 1 antibody. Using this BiTE, binding to various cancer cells and PBMCs pulsed with cancer cell antigen peptides was tested. The BiTE reacted with the colon cancer cell line colo320 and lung cancer cell line LHK2 pulsed with cancer cell antigen peptides, resulting in the production of IFN-γ (Figure 9A).

[0071] (2) Cytotoxic Activity The BiTEs prepared in (1) were mixed with PBMCs and co-cultured with 293T-A24 cells pulsed with cancer cell antigen peptides. The induction of CTLs can be confirmed, for example, by measuring the levels of various cytokines (e.g., IFN-γ, IL-2, and TNFα) produced by CTLs in response to antigen peptide-presenting cells using, for example, the V-Plex Cytokine Panel 1 Human Kit. As shown in Figure 9B, the bispecific antibody BiTEs increased the levels of various cytokines in 293T-A24 cells and the colon cancer cell line colo320. However, as shown in Figure 9C, CD3 scFv and HF10 scFv alone did not induce T cell-mediated cytokine production.

[0072] Example 5. Cytotoxicity Measurement The cytotoxic activity of T cells against cancer cells was tested using the bispecific antibody BiTE obtained in Example 4 using Maestro Z (Axion Biosystems). Specifically, a 96-well plate with electrodes attached to the bottom was used. When target cells were seeded, electrical resistance increased as they adhered to the bottom and proliferated. When cell death occurred, the target cells detached from the bottom, resulting in a decrease in electrical resistance. This was measured in real time. The results are shown in Figures 10A-C. The bispecific antibody BiTE exhibited cytotoxic activity against the osteosarcoma OS-2000 cell line. (A) shows the change in impedance over time. (B, C) show the cytotoxic activity calculated from the change in impedance. (B) The left side shows cytotoxic activity after 20 hours, and (C) the right side shows cytotoxic activity after 40 hours.

[0073] Example 6. Measurement of Cytotoxic Activity of HF10 CAR-T Cells As shown in Figure 11A, an HF10 CAR construct was constructed using HF10 scFv. A retroviral vector (pMX) encoding the HF10 CAR construct was constructed and used to infect peripheral blood mononuclear cells (PBMCs) from healthy donors. The CAR was then detected using an HLA-A24 / HF10 peptide tetramer (Figure 11B). HF10 CAR-T cells recognized the HLA-A24 / HF10 peptide complex on the cell surface and produced the cytokine IFNg and CD107a-positive cytotoxic granules (Figure 12). HF10 CAR-T cells also exhibited cytotoxic activity against HF10 peptide-pulsed T2-A24 cells, and release of LDH (lactate dehydrogenase) from the target cells was observed (Figure 13).

[0074] Example 7. In vivo antitumor effect of bispecific antibody BiTE. Human oral squamous cell carcinoma cell line MO1000 cells were subcutaneously implanted into the right dorsum of NSG mice at 2 x 10e6 cells per mouse (Day -12). Tumor engraftment was confirmed 11 days later (Day -1), and the following day, 1 x 10e7 PBMCs per mouse were intraperitoneally administered (Day 0). Starting the following day, the antibody was administered at 3 mg / kg / day per mouse for 10 days (Day 1-Day 10). The bispecific antibody BiTE obtained in Example 4 was used, and the mice were assigned to the following groups: No treatment (untreated group): 6 mice; Control (PBMC + PBS administration group): 7 mice; Treatment (PBMC + antibody administration group): 7 mice. From Day 1 to Day 10 (antibody administration period), tumor volume was suppressed in the treatment group compared to the no treatment group and the PBS control (PBMC + PBS) group (Figures 14B-E). After antibody administration was completed, a tendency for tumor growth was observed even in the treatment group (Fig. 14B).

[0075] The antibodies disclosed herein can react with endogenously presented natural antigen peptides and further exhibit cytotoxic activity through humoral immune responses. Therefore, it is possible to perform immunotherapy targeting cancer cells in cancer patients who express PVT1. Furthermore, because the cytotoxic mechanism is humoral, cancer vaccine immunotherapy can be performed independently of the immune strength of the recipient, and the antibodies are expected to be highly effective both as anticancer agents and as cancer immunotherapy.

Claims

1. A multispecific antibody having an antigen-binding site that recognizes a complex between the polypeptide represented by sequence number 3 and an MHC molecule.

2. The multispecific antibody according to claim 1, wherein the antigen-binding site recognizing a complex between the polypeptide represented by SEQ ID NO:3 and an MHC molecule comprises an amino acid sequence set forth in any one of SEQ ID NOs:4 to 8, or an amino acid sequence of GRD, or an amino acid sequence in which one or two amino acids have been substituted in an amino acid sequence set forth in any one of SEQ ID NOs:4 to 8.

3. The multispecific antibody described in claim 1, which has the amino acid sequence of the variable region shown in SEQ ID NO: 19, and in which one or two amino acids may be substituted.

4. The multispecific antibody of claim 1, further comprising an antigen-binding site that recognizes CD3.

5. A pharmaceutical composition comprising an antibody that recognizes a complex between the polypeptide represented by SEQ ID NO:3 and an MHC molecule.

6. The pharmaceutical composition according to claim 5, which is a pharmaceutical composition for preventing and / or treating cancer.

7. The pharmaceutical composition according to claim 5, wherein the MHC molecule is an HLA molecule.

8. The pharmaceutical composition according to claim 5, wherein the antigen-binding site of the antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 4 to 18, or an amino acid sequence of GRD, GKN, or NDN.

9. The pharmaceutical composition according to claim 5, wherein the antibody is a multispecific antibody according to any one of claims 1 to 4.

10. A bivalent artificial antibody comprising: (1) two antibody fragments (scFv) that recognize a complex between the polypeptide represented by sequence number 3 and an HLA molecule; and (2) an Fc region of human IgG.

11. A chimeric antigen receptor comprising an antigen-binding site that recognizes a complex of the polypeptide represented by sequence number 3 and an MHC molecule, and a CD3ζ chain.

12. The chimeric antigen receptor of claim 11, further comprising a costimulatory molecule.

13. A genetically modified T cell, characterized in that the chimeric antigen receptor according to claim 11 or 12 has been introduced therein.

14. An artificial cytotoxic T cell comprising a T cell receptor that recognizes a complex of a polypeptide represented by sequence number 3 and an MHC molecule.

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