WO2022210863A1 - Antigen peptide expressed in human bladder cancer stem cells - Google Patents

Abstract

The purpose of the present invention is to provide, inter alia: a detection agent for specifically detecting bladder cancer stem cells; a tumor antigen peptide that is specifically presented by bladder cancer stem cells; a drug composition that contains the aforementioned tumor antigen peptide as an active ingredient, and that is useful for preventing and/or treating treatment-resistant bladder cancer; and a method for screening such a tumor antigen peptide. Provided is a tumor antigen peptide, or a motif substitution product thereof, that has HLA connectivity and that is formed from 8-14 consecutive amino acids in the amino acid sequence of a protein coded by a gene selected from the group consisting of GRIK2 and Claspin.

Description

Antigenic peptides expressed in human bladder cancer stem cells

The present invention is useful as a detection agent for detecting treatment-resistant bladder cancer stem cells using a gene specifically expressed in bladder cancer stem cells, and as an agent for the prevention and/or treatment of treatment-resistant bladder cancer, The present invention relates to tumor antigen peptides derived from said gene and uses thereof.

Adjuvant therapies such as chemotherapy and radiotherapy play an important role in bladder cancer treatment. However, the therapeutic effects of chemotherapy and radiotherapy are limited, and recurrence occurs after treatment in many cases. This is because treatment-resistant cancer stem cells exist, and an effective therapeutic method for bladder cancer stem cells has been sought.

An immune checkpoint inhibitor (ICI) has also been approved for bladder cancer, and is expected to be the fourth treatment for bladder cancer. Effector cells for immunotherapy are cytotoxic T cells (CTL), and the presence of antigenic peptides recognized by CTL is essential for successful immunotherapy. However, an antigenic peptide that can be expressed in bladder cancer stem cells and recognized by CTL has not yet been reported.

Patent Document 1 describes a tumor antigen peptide having cytotoxic T cell inducing activity derived from proteins encoded by Or7c1 and Dnajb8 genes.
Patent Document 2 describes a tumor antigen peptide that is derived from the ASB4 protein and has cytotoxic T cell-inducing activity and that is presented specifically to cancer stem cells.
Patent Document 3 discloses tumor antigen peptides derived from PVT1, SUV39H2, ZNF724P, SNRNP40 and DYRK4 proteins and exhibiting high binding to HLA-A24.
However, References 1 to 3 do not teach tumor antigen peptides that are expressed in bladder cancer stem cells and can be recognized by CTL.

WO2010/050190 WO2016/093243 WO2017/115798

An object of the present invention is to provide a detection agent for specifically detecting bladder cancer stem cells, a tumor antigen peptide specifically presented to bladder cancer stem cells, and treatment-resistant bladder cancer containing this as an active ingredient. It is an object of the present invention to provide pharmaceutical compositions useful for prevention and/or treatment, methods for screening such tumor antigen peptides, and the like.

In order to solve the above problems, the present inventors continued intensive research, isolated cells with high aldehyde dehydrogenase activity (ALDH ^high ) from human bladder cancer cell line UM-UC-3, and isolated ALDH ^high cells. Surprisingly, it was found that the ALDH ^high clone cells exhibited a high content of cancer stem cells even after being cultured in vitro for one month or longer.

In further research, the present inventors used ALDH ^low clone cells as a comparison group as a non-cancer stem cell model, performed HLA ligandome analysis, and analyzed ALDH ^high clone cells and ALDH ^low clone cells. When gene expression analysis was performed by the CAGE method, it was found that ALDH ^high clone cells highly expressed the GRIK2 and Claspin genes and at the same time expressed antigen peptides encoded by the GRIK2 and Claspin proteins. was completed.

That is, the present invention relates to:
[1] A tumor antigen peptide or a motif substitute thereof, which consists of 8 to 14 consecutive amino acids in the amino acid sequence of a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin and has HLA binding properties.
[2] The tumor antigen peptide of [1], wherein the HLA is HLA-A02, or a motif-substituted product thereof.
[3] 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, or in the peptide, the second amino acid from the N-terminus is The tumor antigen peptide of [1] or [2], wherein the peptide is substituted with leucine, isoleucine or methionine and/or the C-terminal amino acid is substituted with valine, leucine or isoleucine.

[4] The tumor antigen peptide of [1] to [3] represented by SEQ ID NO:1 or SEQ ID NO:2.
[5] A polyepitope peptide in which a plurality of epitope peptides are linked, the polyepitope peptide comprising at least one tumor antigen peptide of [1] to [4] as the epitope peptide.
[6] A polynucleotide encoding at least one of the tumor antigen peptides of [1] to [4] or the polyepitope peptide of [5].

[7] An expression vector comprising the polynucleotide of [6].
[8] A composition for gene introduction, comprising the expression vector of [7].
[9] (A) the tumor antigen peptide of [1] to [4] or the polyepitope peptide of [5], or
(B) A method for producing an antigen-presenting cell, comprising contacting, in vitro, a polynucleotide encoding at least one of the peptide and/or polyepitope peptide of (A) with a cell capable of presenting an antigen. .

[10] The following (a) to (e):
(a) the antigenic peptide of [1] to [4] or the polyepitope peptide of [5],
(b) the polynucleotide of [6];
(c) the expression vector of [7],
(d) a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin, a polynucleotide encoding the protein, or a partial peptide of the protein including the antigen peptide represented by SEQ ID NO: 1 or 2, or the polynucleotide an expression vector comprising
(e) A cytotoxic T cell inducer containing, as an active ingredient, any one of antigen-presenting cells that present the antigenic peptides of [1] to [4] as antigens.

[11] (A) the tumor antigen peptide of [1] to [4] or the polyepitope peptide of [5],
(B) a polynucleotide encoding at least one of the peptides and/or polyepitopic peptides of (A); or
(C) A method for inducing cytotoxic T cells, which comprises contacting peripheral blood lymphocytes in vitro with antigen-presenting cells that present the antigenic peptides of [1] to [4] as antigens.

[12] The following (a) to (e):
(a) the antigenic peptide of [1] to [4] or the polyepitope peptide of [5],
(b) the polynucleotide of [6];
(c) the expression vector of [7],
(d) a protein selected from the group consisting of GRIK2 and Claspin, a polynucleotide encoding the protein or a partial peptide of the protein including the antigen peptide represented by SEQ ID NO: 1 or 2, or an expression vector comprising the polynucleotide;
(e) A pharmaceutical composition comprising, as an active ingredient, any one of cytotoxic T cells that specifically kill antigen-presenting cells that present the antigenic peptides of [1] to [4] as antigens.

[13] The pharmaceutical composition of [12], comprising the antigen peptide of [1] to [4] and/or the polyepitope peptide of [5] as an active ingredient.
[14] The pharmaceutical composition of [12] or [13], further comprising an adjuvant.
[15] The pharmaceutical composition of [12]-[14], which is a prophylactic and/or therapeutic agent for treatment-resistant bladder cancer.
[16] The pharmaceutical composition of [12] to [15], which is a preventive and/or therapeutic vaccine for treatment-resistant bladder cancer.

[17] The pharmaceutical composition of [12]-[16], which is used together with an immune checkpoint inhibitor.
[18] An HLA multimer comprising the antigenic peptides of [1] to [4] and HLA.
[19] A diagnostic agent comprising the HLA multimer of [18].
[20] A T-cell receptor-like antibody that recognizes a complex of the antigen peptide of [1] to [4] and HLA.
[21] A tumor-detecting agent containing the T-cell receptor-like antibody of [20].

[22] A chimeric antigen receptor that recognizes a complex of the antigen peptides of [1] to [4] and HLA.
[23] An artificial CTL comprising a T-cell receptor that recognizes the complex of the antigenic peptide of [1]-[4] and HLA.
[24] A bispecific antibody that specifically recognizes a complex of the antigen peptide of [1] to [4] and HLA and a lymphocyte surface antigen.
[25] A tumor cell detection agent comprising a detection agent for detecting an expression product of a gene selected from the group consisting of GRIK2 and Claspin.

[26] The tumor cell-detecting agent of [25], for detecting tumor cells in a cell population containing cells derived from a bladder biological sample.
[27] The agent for detecting tumor cells of [25] or [26], wherein the gene expression product is mRNA and/or an endogenous polypeptide.
[28] The agent for detecting tumor cells of [25] to [27], wherein the expression product of the gene is mRNA and contains a probe and/or primer having a nucleotide sequence complementary to the gene.
[29] The agent for detecting tumor cells of [25] to [28], wherein the expression product of the gene is an endogenous polypeptide, and a detection substance that specifically reacts with the endogenous polypeptide is included.

[30] The tumor cell detection agent of [29], wherein the detection substance is an antibody.
[31] A diagnostic agent for selecting a patient to be treated for whom a cancer treatment method using the pharmaceutical composition of [12] to [17] is effective, comprising the HLA multimer of [18], Said diagnostic agent comprising a T-cell receptor-like antibody and/or a tumor cell detection agent of [25]-[30].
[32] An antisense oligonucleotide against a gene selected from the group consisting of GRIK2 and Claspin.
[33] An siRNA comprising an antisense region complementary to a gene selected from the group consisting of GRIK2 and Claspin and a sense region at least partially complementary to the antisense region.

[34] A pharmaceutical composition comprising the antisense oligonucleotide of [32] and/or the siRNA of [33], and a pharmaceutically acceptable carrier.
[35] The pharmaceutical composition of [34], which is a prophylactic and/or therapeutic agent for treatment-resistant bladder cancer.
[36] A clone cell established from a cell population of human bladder cancer cell line UM-UC-3 containing cells with high aldehyde dehydrogenase enzyme activity (ALDH ^high ).
[37] A clone cell established from a cell population of human bladder cancer cell line UM-UC-3 containing cells with low aldehyde dehydrogenase enzyme activity (ALDH ^low ).
[38] The clone cell of [36] or [37], wherein the cell population of human bladder cancer cell line UM-UC-3 is separated by the ALDEFLUOR method.
[39] The chimeric antigen receptor of [22], further comprising a CD3ζ chain.
[40] The chimeric antigen receptor of [39], further comprising a co-stimulatory molecule.
[41] A genetically modified T cell into which the chimeric antigen receptor of any of [22], [39], and [40] has been introduced.
[42] A dendritic cell that presents a complex of the peptides of [1] to [4] and HLA on the cell surface.
[43] The agent for detecting tumor cells of [25]-[30], which is used for predicting the prognosis of bladder cancer.

According to the present invention, a tumor antigen peptide useful as an inducer of CTLs that specifically attack bladder cancer stem cells, and a medicament containing the same as an active ingredient useful for the prevention and/or treatment of treatment-resistant bladder cancer Compositions and the like are provided.

Figure 1 shows cells with high aldehyde dehydrogenase enzymatic activity (ALDH ^high ) and cells with low aldehyde dehydrogenase enzymatic activity (ALDH ^low ) for human bladder cancer cell line UM-UC-3 by the ALDEFLUOR method based on aldehyde dehydrogenase enzymatic activity. ) are separated. FIG. 2 shows that clonal cells established from ALDH ^high cells indeed exhibited high ALDH enzymatic activity. The DEAB(+) group is a negative control. In the DEAB(-) group, ALDH ^high cells exhibit high brightness in the x-axis direction. FIG. 3 shows that clonal cells established from ALDH ^low cells indeed exhibited low ALDH enzymatic activity.

Figure 4 shows the sphere formation rate (left side) of H clone cells (H-10), L clone cells (L-3) and wild type UM-UC-3 (WT), and the predicted presence of cancer stem cells rate (right). FIG. 5 shows in vivo tumor growth curves of H clone cells (H-10) and L clone cells (L-3). The vertical axis indicates tumor volume (mm ³ ), and the horizontal axis indicates days (weeks) after tumor cell implantation. FIG. 6 shows the sensitivity of H clone cells (H-10) and L clone cells (L-3) to cisplatin (CDDP). The vertical axis indicates cell viability, and the horizontal axis indicates the concentration of cisplatin. FIG. 7 shows the resistance of H clone cells (H-10) and L clone cells (L-3) to radiotherapy. The vertical axis indicates cell viability, and the horizontal axis indicates absorbed dose (Gy).

The left diagram of FIG. 8 is a schematic diagram of HLA of human bladder cancer cell line UM-UC-3. The central figure in FIG. 8 shows that each cell lysate was reacted with an anti-HLA-A*02:01-specific antibody, immunoprecipitated using protein-A Sepharose beads, and HLA-A*02:01 molecules were collected. It is a schematic diagram of a procedure. The right figure in FIG. 8 shows the results of amino acid sequence analysis by mass spectrometry analysis, and the amino acid length (horizontal axis) and the number of peptides (horizontal axis) of peptides analyzed from H-10 cells, L-3 cells, and wild type strains. vertical axis). FIG. 9 shows the procedure for analyzing the amino acid sequences of 9-mer peptides among the peptides recovered from H-10 cells, L-3 cells, and wild type. Among the 123 peptides specific to H-10 cells, the antigen peptide LMYDAVHVV (SEQ ID NO: 1) encoded by the GRIK2 gene product and the antigen peptide SLLNQPKAV encoded by the Claspin gene product were identified from the peptide-encoding gene expression information. (SEQ ID NO: 2) was selected.

FIG. 10 shows the results of GRIK2 peptide-induced cytotoxic T cell (CTL) induction experiments using peripheral blood mononuclear cells. T2A24 cells were used as target cells. The graph on the left is the GRIK2 peptide (-) negative control, and the graph on the right shows the group in which the GRIK2 peptide was developed in T2A24 cells. The Y-axis of the graph indicates the number of IFNγ spots. FIG. 11 shows the results of GRIK2 peptide tetramer-specific staining. Figure 12 shows the results of the IFNγ ELISPOT method of the obtained GRIK2 peptide-specific 9G23 clone. The graph on the left is the GRIK2 peptide (-) negative control, and the graph on the right shows the group in which the GRIK2 peptide was developed in T2A24 cells. The Y-axis of the graph indicates the number of IFNγ spots.

FIG. 13 shows the results of specific staining of the 9G23 clone reanalyzed with GRIK2 tetramer. FIG. 14 shows reactivity between GRIK2 peptide-specific CTL clones and GRIK2 overexpressing strains. The left and right graphs are negative controls using cells that do not express the GRIK2 peptide, respectively, and the middle graph shows the group using cells that overexpress the GRIK2 peptide. The Y-axis of the graph indicates the number of IFNγ spots. FIG. 15 shows the reactivity of GRIK2 peptide-specific CTL clones with H-10 cells. The three graphs from the far right are negative controls using cells that do not express the GRIK2 peptide, and the leftmost graph shows the group using H-10 cells that express the GRIK2 peptide. The Y-axis of the graph indicates the number of IFNγ spots. Figure 16 shows a schematic and results of an in vitro therapeutic model using GRIK2 peptide-specific CTL clones. FIG. 17 shows the effect of combining GRIK2 peptide-specific CTL clones with the anticancer drug cisplatin (CDDP) or irradiation. The vertical axis of the graph on the left indicates cell viability, and the horizontal axis indicates the concentration of cisplatin. The vertical axis of the graph on the right indicates cell viability, and the horizontal axis indicates absorbed dose (Gy). FIG. 18 shows the results of the IFNγ ELISPOT method for the obtained Claspin peptide-specific cy3 clones. The left side is the negative control of Claspin peptide (-), and the right side shows the group in which T2A24 cells were expanded with Claspin peptide. FIG. 19 shows the results of specific staining of cy3 clones reanalyzed using Claspin tetramers.

The present invention will be described in detail below.
In the present invention, "epitope peptide" means a peptide that binds to MHC (HLA in humans), is presented as an antigen on the cell surface, and has antigenicity (that can be recognized by T cells). The epitope peptide includes the CTL epitope peptide, which is an epitope peptide that binds to MHC class I and is antigen-presented and is recognized by CD8-positive T cells, and the CTL epitope peptide, which is an epitope peptide that binds to MHC class II and is antigen-presented and is recognized by CD4-positive T cells. Included are helper epitope peptides, which are epitope peptides that are

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 a phenomenon in which intracellular peptides bind to MHC and this MHC/antigen peptide complex is localized on the cell surface. As described above, antigens presented on the cell surface are known to activate cell-mediated immunity and humoral immunity after being recognized by T cells, etc. Tumor antigen peptides of the MHC class are used for immunotherapy because they activate sexual immunity, are recognized by T-cell receptors of naive T-cells, and induce naive T-cells into CTLs having cytotoxic activity. Peptides that bind I and are antigen-presented are preferred.

Many of the peptides that bind to MHC are known to have certain characteristics. In the context of the present invention, this feature is referred to as a "binding motif". It is known in the art what MHC binds to peptides with what binding motifs. For example, the binding motif of HLA-A02, one of human MHC, has leucine, isoleucine or methionine as the second amino acid from the N-terminus, and valine, leucine or isoleucine as the C-terminal amino acid.

As used herein, the term "motif substitution" refers to a peptide having a binding motif in which the binding motif is substituted with another binding motif. A person skilled in the art will naturally understand that in the present invention, the motif-substituted product also exhibits the same effect as the peptide before substitution.

In the present invention, "tumor" includes benign and malignant tumors (cancer, malignant neoplasm). Cancer includes hematopoietic tumors, epithelial malignancies (carcinoma) and non-epithelial malignancies (sarcoma). In the present invention, cancer particularly refers to bladder cancer that is resistant to chemotherapy, radiation therapy, and the like.
In the present invention, the term “cancer stem cells” refers to cells present in cancer tissue that exhibit stem cell-like properties, and are considered to be causative cells involved in the development, recurrence and metastasis of cancer. cells that are Since "cancer stem cells" generally exist in only a small amount in cancer tissue, it is difficult to distinguish them from other cells. Examples include the SP fractionation method.

Cancer stem cells are known to have high aldehyde dehydrogenase (ALDH) enzymatic activity. In the present invention, cells with high aldehyde dehydrogenase enzyme activity (ALDH ^high ) and cells with low aldehyde dehydrogenase enzyme activity (ALDH ^low ) were separated by the ALDEFLUOR method based on aldehyde dehydrogenase enzyme activity. ALDH ^high cells are known to differentiate into ALDH ^low cells in in vitro culture, and are unsuitable for stable analysis. Therefore, in the present invention, in order to establish stable ALDH ^high cells, human bladder cancer cell line UM-UC-3 was used to prepare single cell clones from ALDH ^high cells.

In the present invention, clone cells established from ALDH ^high cells actually exhibited high ALDH enzyme activity, and clone cells established from ALDH ^low cells actually exhibited low ALDH enzyme activity. ALDH ^high clones (H clones) and ALDH ^low clones (L clones) of the present invention exhibit stable traits in in vitro culture for one month or more.

In the present invention, the natural peptides of the present invention were isolated/identified by using the following method, which can isolate/identify the natural peptides that are actually antigen-presented on the cell surface of such clones. In the present invention, "natural peptide" refers to a peptide that is actually presented as an antigen on the cell surface. "Natural antigenic peptide" refers to a natural peptide whose antigenicity has been confirmed. By isolating this natural antigen peptide from cancer cells and determining its sequence and origin, it is possible to obtain useful knowledge for targeted cancer therapy using CTL.

The method for isolating/identifying natural peptides used in the present invention includes the steps of lysing cancer stem cells presenting natural peptides and isolating complexes of MHC and natural peptides from the lysate. , separating the isolated complex into MHC molecules and natural peptides to isolate the natural peptides, and identifying the isolated natural peptides.

In the following examples, the isolation of the MHC-natural peptide complex was carried out by extracting the peptide/MHC complex by immunoprecipitation using a specific antibody against MHC. Any method may be used as long as it can isolate the complex with the peptide.
In the examples below, an antibody against HLA class I, such as an anti-HLA-A02 antibody, was used as a suitable anti-MHC antibody, but any antibody that can specifically recognize a complex of MHC and a natural peptide can be used. Antibodies may also be used.

In the following examples, peptide isolation was performed using a weak acid as the step of separating the complex into MHC molecules and natural peptides, but any method that can separate MHC and natural peptides may be used. good.
Furthermore, in the following examples, the sequences of the isolated natural peptides were analyzed using HLA ligandome analysis by mass spectrometry, and natural peptides actually presented as antigens on the cell surface were identified. Any method that allows identification may be used for identification.
HLA ligandome analysis using mass spectrometry requires a large number of cells, ie, 10 ⁹ cells. Since cancer stem cells account for only about 1% of all cancer cells, this method is seemingly inefficient and unsuitable for analysis of cancer stem cells. However, in the present invention, this disadvantage is overcome by using the bladder cancer cell line UM-UC-3 to generate single cell clones from ALDH ^high cells.

The present inventors analyzed natural antigen peptides presented as antigens in the human bladder cancer cell line UM-UC-3. As a result, 848 types of peptides for ALDH ^high clones (H clones), 1832 types of peptides for ALDH ^low clones (L clones), and 1073 types for wild-type peptides were found to be present as antigen-presented 9-amino acid natural peptides in cancer cells. A class of peptides was identified.
Of the 848 peptides for H clones, 123 peptides were H clone-specific peptides that were not common to L and wild-type clones. For 123 types of peptides specific to the H clone, genes that are expressed in bladder cancer and have low expression in major normal organs were selected from the gene expression information. As a result, a peptide derived from the GRIK2 protein (SEQ ID NO: 1) and a peptide derived from the Claspin protein (SEQ ID NO: 2) were identified as natural antigen peptides presented as antigens in the human bladder cancer cell line UM-UC-3. was done.

GRIK2 belongs to the ion channel type kainate type glutamate receptor and is expressed in the central nerve. In the central nervous system, GRIK2 protein bound to glutamic acid released as a neurotransmitter is activated and excites nerve cells.

Claspin was identified as a protein that binds to the CHK1 molecule, localizes to DNA replication forks, and promotes DNA synthesis.

<1> Gene expression product of the present invention In the present invention, when simply referred to as a gene name such as "GRIK2" and "Claspin", unless otherwise specified, known nucleic acid sequences represented by the gene name Although it typically represents a cDNA or mRNA sequence, it is not limited to this as long as a person skilled in the art can recognize it as the sequence of the gene. Examples include the following genes represented by the following sequences.
GRIK2: GenBank Accession No. NM_021956.5
Claspin: GenBank Accession No. NM_022111.4
Therefore, the mRNA as the gene expression product of the present invention may be expressed simply by the description of the gene name.

In the present invention, when "protein" is added to a gene name, such as "GRIK2 protein", it means the protein encoded by the gene, its isoforms, and its homologues. Examples of such isoforms include splicing variants, variants such as SNPs based on individual differences, and the like. Specifically, (1) a protein consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more homology with the protein encoded by the gene, (2) the gene Substitution or deletion of one or more, preferably 1 to several, more preferably 1 to 10, 1 to 5, 1 to 3, 1 or 2 amino acids in the amino acid sequence of the encoded protein , proteins consisting of added or inserted amino acid sequences.

A preferred protein as the gene expression product of the present invention is a protein comprising an amino acid sequence encoded by the gene (nucleic acid sequence) described above, or a protein in which 1 to 3, preferably 1 or 2 amino acids are substituted. Proteins consisting of amino acid sequences can be mentioned. More preferably, a protein consisting of an amino acid sequence encoded by the gene (nucleic acid sequence) described above can be mentioned.

<2> Peptide of the present invention In one aspect, the peptide of the present invention is a partial peptide of a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin, and preferably binds to MHC, particularly HLA. includes peptides that are presented by MHC, especially HLA, more preferably peptides that are presented by MHC, especially HLA, and are capable of inducing CTLs. Although there are several types of HLA, the peptide of the present invention can preferably bind to HLA class I, more preferably HLA-A02. The peptides of the present invention may undergo treatments such as processing before binding to MHC, and peptides that generate epitope peptides as a result of these treatments are also included in the peptides of the present invention. Therefore, the amino acid length of the peptide of the present invention is not particularly limited as long as the sequence contains the amino acid sequence of the epitope peptide. However, it is preferable that the peptide of the present invention itself is an epitope peptide, and therefore the amino acid length is preferably about 8 to 14 amino acids, more preferably about 8 to 11 amino acids, and particularly preferably about 9 to about 11 amino acids. 9 amino acids are most preferred.

The epitope peptide that binds to human MHC class I, HLA class I, is about 8-14 amino acids long, preferably about 9-11 amino acids long, and may have an HLA-specific binding motif in its sequence. Are known. For example, a peptide that binds to HLA-A02 has 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. Peptides that bind A24 have binding motifs in which the penultimate N-terminal amino acid is tyrosine, phenylalanine, methionine or tryptophan and/or the C-terminal amino acid is leucine, isoleucine or phenylalanine.

Therefore, in a preferred embodiment, the peptide of the present invention is a partial peptide of a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin, and consists of 8 to 14 consecutive amino acids in the amino acid sequence of the protein. , 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, more preferably the epitope peptide itself. Among them, an epitope peptide consisting of the amino acid sequence represented by either SEQ ID NO: 1 or SEQ ID NO: 2 is particularly preferred.

In another preferred embodiment, in the partial peptide, the second amino acid from the N-terminus is leucine, isoleucine or methionine, and/or the C-terminal amino acid is substituted with valine, leucine or isoleucine. It includes an epitope peptide, more preferably the epitope peptide itself. Among them, particularly preferred is a peptide consisting of the amino acid sequence represented by either SEQ ID NO: 1 or SEQ ID NO: 2, wherein the second amino acid from the N-terminus is substituted with leucine, isoleucine or methionine, and/or C It is an epitope peptide in which the terminal amino acid is replaced with valine, leucine or isoleucine.

The peptides of the invention may be modified at their N-terminus and/or C-terminus. Specific examples of such modifications include N-alkanoylation (eg, acetylation), N-alkylation (eg, methylation), C-terminal alkyl esters (eg, ethyl esters), and C-terminal amides (eg, carboxamides). are mentioned.
Synthesis of the peptide of the present invention can be carried out according to known methods used in ordinary peptide chemistry. Such known methods include literature (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; , Maruzen Co., Ltd., 1985; Drug Development, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991, all of which are incorporated herein by reference).

The peptide of the present invention is subjected to the CTL induction method described below, an assay using a human model animal (International Publication No. 02/47474, Int J. Cancer: 100, 565-570 (2002)), etc. activity can be confirmed.

The peptides of the present invention further include peptides (polyepitope peptides) in which multiple epitope peptides containing at least one of the peptides of the present invention are linked. Therefore, polyepitope peptides having CTL-inducing activity can also be exemplified as specific examples of the peptides of the present invention.
Specifically, the polyepitope peptide of the present invention is
(i) a peptide in which the peptide of the present invention (epitope peptide) and any one or more CTL epitope peptides other than the peptide of the present invention are linked directly or via an appropriate spacer;
(ii) a peptide in which the peptide of the present invention and any one or two or more helper epitope peptides are linked directly or via an appropriate spacer, or
(iii) a peptide obtained by linking the polyepitope peptide according to (i) above with one or more helper epitope peptides directly or via an appropriate spacer, which is processed in antigen-presenting cells, The resulting epitope peptides can be defined as peptides that are presented to antigen-presenting cells and lead to CTL-inducing activity.

Here, the CTL epitope peptide other than the peptide of the present invention in (i) is not particularly limited. (Peptides described in Publication No. 2010/050190), epitope peptides derived from human FAM83B (International Application No. PCT/JP2014/076625), and the like.
The spacer is not particularly limited as long as it does not adversely affect processing in antigen-presenting cells, and is preferably a linker linked to each epitope peptide via a peptide bond, such as a peptide linked with several amino acids. Examples include linkers and linkers having amino groups and carboxyl groups at both ends. Specific examples include glycine linkers and PEG (polyethylene glycol) linkers, and examples of glycine linkers include polyglycine (for example, a peptide consisting of 6 glycines; Cancer Sci, vol.103, p150-153), and PEG linkers. These include linkers derived from compounds having amino and carboxy groups at both ends of PEG (eg, H ₂ N—(CH ₂ ) 2—(OCH ₂ CH ₂ ) ₃ —COOH; Angew. Chem. Int. Ed. 2008, 47, 7551-7556).

One or more of the epitope peptides of the present invention may be selected from the polyepitope peptides of the present invention. That is, a plurality of identical epitope peptides may be linked, or a plurality of different epitope peptides may be linked. Of course, even when two or more epitope peptides are selected, one or two or more of the selected epitope peptides may be linked. For epitope peptides other than the peptide of the present invention, multiple types and/or multiple epitope peptides may be similarly linked. The polyepitope peptide of the present invention may be one in which 2 to 12 epitope peptides are linked, preferably 2, 3, 4, 5, 6, 7, 8 or 9. , 10, 11, 12 epitope peptides are linked, most preferably 2 epitope peptides are linked.
Here, when the epitope peptide linked to the peptide of the present invention is a helper epitope peptide, the helper epitope peptide to be used includes, for example, hepatitis B virus-derived HBV c128-140 and tetanus toxin-derived TT947-967. The length of the helper epitope peptide is about 13 to 30 amino acids, preferably about 13 to 17 amino acids.

Such a peptide (polyepitope peptide) in which multiple epitope peptides are linked can also be produced by a general peptide synthesis method as described above. It can also be produced using conventional DNA synthesis and genetic engineering techniques based on the sequence information of a polynucleotide encoding a polyepitope peptide in which multiple epitope peptides are linked.
That is, the polynucleotide is inserted into a well-known expression vector, the resulting recombinant expression vector is used to transform a host cell, the resulting transformant is cultured, and multiple epitopes of interest are ligated from the culture. It can be produced by recovering the polyepitope peptide. These techniques should be carried out according to the methods described in the literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM.Glover, IRL PRESS (1985)) as described above. can be done.

A polyepitope peptide in which a plurality of epitope peptides produced as described above are linked is subjected to the above-mentioned in vitro assay, International Publication No. WO 02/47474 and Int J. Cancer: 100, 565-570 (2002) (these The CTL-inducing activity can be confirmed by subjecting it to the in vivo assay using a human model animal described in the literature, which is incorporated herein by reference.
As described herein, the peptides (including polyepitope peptides) of the present invention are useful for the prevention and/or treatment of treatment-resistant bladder cancer, and can be used as active ingredients of pharmaceutical compositions. . Also, the peptides of the present invention may be for the prevention and/or treatment of treatment-resistant bladder cancer. Furthermore, the invention relates to the use of the peptides of the invention for the manufacture of a medicament for the prevention and/or treatment of treatment-resistant bladder cancer.

<3> Polynucleotide of the Present Invention The polynucleotide of the present invention includes a polynucleotide encoding at least one of the peptides of the present invention. Polynucleotides of the present invention may be cDNA, mRNA, cRNA, or synthetic DNA. In addition, it may be in either single-stranded or double-stranded form. Specifically, but not limited to, for example, a partial peptide of a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin, which is a MHC-peptide binding prediction program BIMAS (http http://www-bimas.cit.nih.gov/molbio/hla_bind/), SYFPEITHI (http://www.syfpeithi.de/) and IEDB (MHC-I processing predictions; http://www.iedb.org /), etc., and polynucleotides comprising a nucleotide sequence encoding an amino acid sequence predicted to have binding properties. In another specific embodiment, a polynucleotide consisting of a nucleotide sequence encoding the amino acid sequences set forth in SEQ ID NOs: 1 and 2, and any two or more peptides selected from SEQ ID NOs: 1 and 2, or SEQ ID NO: 1 and 2, and a polynucleotide comprising a nucleotide sequence encoding a polyepitope peptide linked with a helper epitope so that it can be expressed, respectively.

The polynucleotides of the present invention can be in either single-stranded or double-stranded form. When the polynucleotide of the present invention is double-stranded, a recombinant expression vector for expressing the peptide of the present invention can be constructed by inserting the polynucleotide of the present invention into an expression vector. That is, the polynucleotide of the present invention also includes a recombinant expression vector produced by inserting the double-stranded polynucleotide of the present invention into an expression vector.
As described herein, the polynucleotide of the present invention is useful for prevention and/or treatment of treatment-resistant bladder cancer, and can be used as an active ingredient of a pharmaceutical composition. Also, the polynucleotide of the present invention may be for prevention and/or treatment of treatment-resistant bladder cancer. Furthermore, the invention relates to the use of the polynucleotide of the invention for the manufacture of a medicament for the prevention and/or treatment of treatment-resistant bladder cancer.

Various expression vectors can be used in the present invention depending on the host, purpose, etc., and can be appropriately selected by those skilled in the art. Examples of expression vectors that can be used in the present invention include plasmids, phage vectors, virus vectors and the like. For example, when the host is Escherichia coli, vectors include plasmid vectors such as pUC118, pUC119, pBR322 and pCR3, and phage vectors such as λZAPII and λgt11. When the host is yeast, vectors include pYES2, pYEUra3 and the like. Examples include pAcSGHisNT-A when the host is an insect cell. When the host is an animal cell, plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV and pRc/CMV, and viral vectors such as retroviral vectors, adenoviral vectors and adeno-associated viral vectors is mentioned.

The vector may appropriately have factors such as an expression-inducible promoter, a signal sequence-encoding gene, a selection marker gene, a terminator, and the like. Also, to facilitate isolation and purification, a sequence expressed as a fusion protein with thioredoxin, a His tag, or GST (glutathione S-transferase) may be added. In this case, a GST fusion protein vector (such as pGEX4T) having an appropriate promoter (lac, tac, trc, trp, CMV, SV40 early promoter, etc.) that functions in the host cell, or a vector having a tag sequence such as Myc or His (pcDNA3.1/Myc-His, etc.), a vector (pET32a) that expresses a fusion protein with thioredoxin and a His tag, and the like can be used.

By transforming a host with the expression vector prepared above, a transformed cell containing the expression vector can be prepared. Therefore, the present invention includes a composition for gene transfer containing the expression vector.
As a host used for transformation, any cell may be used as long as it does not impair the functions of the polypeptide of the present invention, and examples thereof include E. coli, yeast, insect cells and animal cells. Examples of Escherichia coli include HB101 strain, C600 strain, JM109 strain, DH5α strain, and AD494 (DE3) strain of E. coli K-12 strain. Examples of yeast include Saccharomyces cerevisiae. Animal cells include L929 cells, BALB/c3T3 cells, C127 cells, CHO cells, COS cells, Vero cells, HeLa cells, 293-EBNA cells and the like. Insect cells include sf9 and the like.
As a method for introducing the expression vector into the host cell, a conventional introduction method suitable for the host cell may be used. Specific examples include a calcium phosphate method, a DEAE-dextran method, an electroporation method, and a method using lipids for gene introduction (Lipofectamine, Lipofectin; Gibco-BRL). After the introduction, by culturing in a normal medium containing a selection marker, transformed cells into which the expression vector has been introduced can be selected.

The peptide of the present invention can be produced by continuing to culture the transformed cells obtained as described above under suitable conditions. The resulting peptide can be further isolated and purified by common biochemical purification means. Examples of purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography and the like. When the peptide of the present invention is expressed as a fusion protein with the aforementioned thioredoxin, His tag, GST or the like, it can be isolated and purified by a purification method utilizing the properties of these fusion proteins or tags.
A polynucleotide encoding the peptide of the present invention may be in the form of DNA or RNA. These polynucleotides of the present invention can be easily produced using conventional methods known in the art based on the amino acid sequence information of the peptide of the present invention and the sequence information of the DNA encoded thereby. Specifically, it can be produced by ordinary DNA synthesis, amplification by PCR, or the like.
A polynucleotide encoding the peptide of the present invention includes a polynucleotide encoding the epitope peptide.

<4> CTL Inducer/Pharmaceutical Composition Containing the Peptide of the Present Invention as an Active Ingredient The peptide of the present invention has CTL inducing activity and can serve as a CTL inducer as a tumor antigen peptide. Further, as described above, the present inventors determined that a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin is a tumor antigen, and that a peptide derived from the protein binds to an HLA class I antigen on the surface of tumor cells. It was found for the first time that a complex is formed with the cell surface and is transported to the cell surface for antigen presentation. Therefore, the protein itself encoded by a gene selected from the group consisting of GRIK2 and Claspin can also be a CTL inducer.

That is, peripheral blood lymphocytes are isolated from HLA-A02 antigen-positive humans and stimulated in vitro by adding the peptide of the present invention and/or a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin. CTLs that specifically recognize HLA-A02 antigen-positive cells pulsed with the peptide can be induced (J. Immunol., 154, p2257, 1995). Here, the presence or absence of CTL induction can be confirmed, for example, by measuring the amount of various cytokines (eg, IFN-γ) produced by CTLs in response to antigen peptide-presenting cells, for example, by ELISA or the like. can. It can also be confirmed by a method of measuring CTL toxicity against ⁵¹ Cr-labeled antigen peptide-presenting cells ( ⁵¹ Cr release assay, Int. J. Cancer, 58: p317, 1994).
CTL clones can also be established by the methods described in Int. J. Cancer, 39, 390-396, 1987, N. Eng. J. Med, 333, 1038-1044, 1995 and the like.

CTLs induced by a peptide of the invention and/or a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin are induced by a peptide of the invention and/or another gene selected from the group consisting of GRIK2 and Claspin It has a toxic effect on cells that present epitope peptides derived from the protein encoded by as an antigen and the ability to produce lymphokines. The peptide of the present invention is a tumor antigen peptide as described above, and the protein encoded by the gene selected from the group consisting of GRIK2 and Claspin is intracellularly degraded to produce the tumor antigen peptide. It can exert an antitumor effect, preferably an anticancer effect. Therefore, the peptide of the present invention and/or the protein encoded by the gene selected from the group consisting of GRIK2 and Claspin, and the CTL induced thereby can be used as pharmaceuticals and pharmaceutical compositions for the prevention and/or treatment of cancer. can be used as an active ingredient of
When a CTL inducer containing, as an active ingredient, the peptide of the present invention and/or a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin is administered to a cancer patient, HLA antigens, preferably HLA, of antigen-presenting cells - A02 antigen is presented with the peptide of the present invention and / or an epitope peptide derived from a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin, and specifically the complex of the HLA antigen and the presented peptide CTLs that recognize can proliferate and destroy cancer cells, thereby preventing and/or treating cancer. Therefore, the CTL inducer containing, as an active ingredient, the peptide of the present invention and/or a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin is preferably used in HLA-A02 antigen-positive subjects, It can be used for subjects suffering from GRIK2 or Claspin positive cancer. Examples of GRIK2- or Claspin-positive cancers include cancers (tumors) such as resistant bladder cancer, and the CTL inducer of the present invention is used for the prevention and/or treatment of these cancers. can do.

Here, “prevention” of cancer includes not only the prevention of patients from contracting cancer, but also the prevention of recurrence in patients who have had their primary tumor removed by surgery, cancer treatment such as surgery, radiation therapy, or drug therapy. including prevention of metastasis of tumors that could not be completely removed by In addition, "treatment" of cancer includes not only cure and improvement of symptoms to shrink cancer, but also progression to suppress cancer cell growth, tumor expansion, or metastasis of cancer cells from the primary focus. prevention, etc.

A CTL inducer containing, as an active ingredient, the peptide of the present invention and/or a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin is HLA-A02, which is affected by GRIK2- or Claspin-positive cancer, for example. It is especially effective for positive cancer patients. Specifically, for example, it can be used for prevention or treatment of cancer (tumor) such as resistant bladder cancer. Therefore, the present invention also includes a pharmaceutical composition containing, as an active ingredient, the peptide of the present invention and/or a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin. Such a pharmaceutical composition is preferably a composition for prevention and/or treatment of treatment-resistant bladder cancer, ie, an agent for prevention and/or treatment of treatment-resistant bladder cancer. In addition, the pharmaceutical composition of the present invention can prevent and/or prevent cancer by inducing CTLs specific to cancer cells (preferably cancer stem cells), that is, by activating cancer cell-specific cellular immunity. Alternatively, it is a vaccine for the prevention and/or treatment of cancer, since it is for treatment. When the cancer preventive and/or therapeutic vaccine is an mRNA vaccine, the pharmaceutical composition of the present invention can use pseudouridine instead of uridine to suppress inflammatory reactions.

A pharmaceutical composition containing the peptide of the present invention as an active ingredient may contain a single CTL epitope (the peptide of the present invention) as an active ingredient, or may be linked to other peptides (CTL epitope or helper epitope). It may contain a polyepitope peptide as an active ingredient. In recent years, it has been shown that a polyepitope peptide in which multiple CTL epitopes (antigen peptides) are linked has an efficient CTL-inducing activity in vivo. For example, Journal of Immunology 1998, 161: 3186-3194 (this document is incorporated herein by reference) describes HLA-A2, -A3, -A11, -B53-restricted CTLs derived from the cancer antigen protein PSA. It has been described that about 30-mer polyepitope peptides linked with epitopes (antigen peptides) induced CTLs specific to each CTL epitope in vivo. It has also been shown that CTLs are efficiently induced by a polyepitope peptide in which a CTL epitope and a helper epitope are linked. When administered in the form of such polyepitope peptides, the polyepitope peptides are taken up into antigen-presenting cells, and then individual antigen peptides produced by intracellular degradation bind to HLA antigens to form complexes. Then, the complex is displayed at high density on the surface of antigen-presenting cells, and CTL specific to this complex efficiently proliferate in the body to destroy cancer cells. In this way cancer treatment or prevention is facilitated.

A pharmaceutical composition containing, as an active ingredient, the peptide of the present invention and/or a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin is pharmaceutically acceptable so as to effectively establish cell-mediated immunity. It can be administered in admixture or in combination with a carrier such as a suitable adjuvant.

As the adjuvant, adjuvants known in the art such as those described in literature (e.g., Clin Infect Dis.: S266-70, 2000) can be applied. Aluminum, aluminum phosphate and calcium phosphate, CpG, monophosphoryl lipid A (MPL), cholera toxin, E. coli heat-labile toxin, pertussis toxin and muramyl dipeptide (MDP) as cell types, oil Freund's incomplete adjuvant, MF59 and SAF as emulsion type (emulsion formulation), Immunostimulatory complexes (ISCOMs), liposomes, biodegradable microspheres and saponin as polymer nanoparticle type Synthetic types include nonionic block copolymers, Muramyl peptide analogues, polyphosphazenes and synthetic polynucleotides, Cytokine types include IFN-γ, IL-2 and IL-12. be able to.
In addition, the dosage form of the CTL inducer/pharmaceutical composition containing, as active ingredients, the peptide of the present invention and/or a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin is not particularly limited. Emulsions (emulsion formulations), polymeric nanoparticles, liposome formulations, particulate formulations bound to beads with a diameter of several μm, formulations bound to lipids, microsphere formulations, microcapsule formulations, and the like can be mentioned.

Administration methods include any known administration methods such as intradermal administration, subcutaneous administration, intramuscular administration, and intravenous administration. The dose of the peptide of the present invention in the formulation can be appropriately adjusted depending on the disease to be treated, the patient's age, body weight, etc., and is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably 0.001 mg to 1000 mg. The dose is 0.1 mg to 10 mg, and is preferably administered once every several days to several months.
Techniques for making the peptide of the present invention actually act as a drug include an in vivo method in which the peptide is directly introduced into the body, as well as certain cells collected from humans and the peptide of the present invention acting outside the body. There is an ex vivo method for returning cells to the body (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Special Edition, 12 (15), ( 1994), and references therein and others, which are incorporated herein by reference), the skilled artisan will be able to select appropriate cells, modes of administration, dosage forms and dosages for such procedures. can be done.

<5> CTL inducer/pharmaceutical composition containing the polynucleotide of the present invention as an active ingredient Cells expressing the polynucleotide of the present invention and/or a polynucleotide encoding a protein selected from the group consisting of GRIK2 and Claspin , the peptide of the present invention and/or other epitope peptides derived from a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin as antigens. It has the feature of being recognized by Accordingly, polynucleotides of the present invention and/or polynucleotides encoding proteins selected from the group consisting of GRIK2 and Claspin can also be inducers of CTLs. The induced CTL are anti-tumor through cytotoxicity and lymphokine production, similar to CTL induced by the peptide of the present invention and/or a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin. It can exert an action, preferably an anticancer action. Therefore, the polynucleotide of the present invention and/or a polynucleotide encoding a protein selected from the group consisting of GRIK2 and Claspin is used as an active ingredient of a drug or pharmaceutical composition for treating or preventing treatment-resistant bladder cancer. be able to. A CTL inducer containing, as an active ingredient, a polynucleotide encoding a protein selected from the group consisting of the polynucleotide of the present invention and/or GRIK2 and Claspin is, for example, the polynucleotide of the present invention and/or GRIK2 and Claspin Treatment-resistant bladder cancer can be treated and/or prevented by administering to a cancer patient and expressing a polynucleotide encoding a protein selected from the group consisting of.

For example, when the polynucleotide of the present invention incorporated into an expression vector and/or a polynucleotide encoding a protein selected from the group consisting of GRIK2 and Claspin is administered to a cancer patient by the following method, tumor antigens are expressed in antigen-presenting cells. Peptides are highly expressed. Thereafter, the resulting tumor antigen peptide binds to an HLA antigen such as HLA-A02 antigen to form a complex, and the complex is presented at high density on the surface of antigen-presenting cells, thereby generating cancer-specific CTL. It multiplies efficiently in the body and destroys cancer cells. Treatment or prevention of cancer is achieved as described above. Pharmaceutical compositions comprising a polynucleotide of the invention and/or a polynucleotide encoding a protein selected from the group consisting of GRIK2 and Claspin are therefore also encompassed by the invention. Such a pharmaceutical composition is preferably a composition for cancer prevention and/or treatment, ie, a cancer prevention and/or treatment agent. In addition, the pharmaceutical composition of the present invention can prevent and/or prevent cancer by inducing CTLs specific to cancer cells (preferably cancer stem cells), that is, by activating cancer cell-specific cellular immunity. Alternatively, it is a vaccine for the prevention and/or treatment of cancer, since it is for treatment.

The CTL inducer/pharmaceutical composition containing the polynucleotide of the present invention as an active ingredient can be preferably used for subjects who are HLA-A02 antigen-positive and have GRIK2 and Claspin-positive cancer. can. Examples of GRIK2- and Claspin-positive cancers include cancers (tumors) such as resistant bladder cancer, and the CTL inducer of the present invention can be used for the prevention or treatment of these cancers. can be done.
Methods for administering and introducing into cells the polynucleotide of the present invention and/or a polynucleotide encoding a protein selected from the group consisting of GRIK2 protein and Claspin protein include methods using viral vectors and other methods (Nikkei Science, April 1994, pp. 20-45, Gekkan Yakuji, 36(1), 23-48 (1994), Jikken Igaku Supplement, 12(15), (1994), and references cited therein. incorporated herein by reference) can be applied. Therefore, in one aspect of the pharmaceutical composition of the present invention, a vector comprising the polynucleotide of the present invention and/or a polynucleotide encoding a protein selected from the group consisting of GRIK2 protein and Claspin protein is contained as an active ingredient.

As a method using a viral vector, for example, the DNA of the present invention is introduced by incorporating it into a DNA virus or RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus, and Simbis virus. method. Among these, methods using retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and the like are particularly preferred.
Other methods include direct intramuscular administration of an expression plasmid (DNA vaccine method), liposome method, lipofectin method, microinjection method, calcium phosphate method, electroporation method, and the like, particularly DNA vaccine method and liposome method. method is preferred.

In order to make the polynucleotide of the present invention and/or a polynucleotide encoding a protein selected from the group consisting of GRIK2 protein and Claspin protein actually act as a medicine, an in vivo method of directly introducing the polynucleotide into the body, and There is an ex vivo method in which certain cells are collected from humans, the polynucleotide of the present invention is introduced into the cells outside the body, and the cells are returned to the body (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs). , 36(1), 23-48 (1994), Experimental Medicine Extra, 12(15), (1994), and references cited therein, which are incorporated herein by reference). In vivo methods are more preferred.
When administering the polynucleotide of the present invention and/or a polynucleotide encoding a protein selected from the group consisting of GRIK2 protein and Claspin protein by an in vivo method, an appropriate administration route according to the disease to be treated, symptoms, etc. and the administration form can be appropriately selected and administered. For example, it can be administered in an injectable form intravenously, arterially, subcutaneously, intradermally, intramuscularly, and the like. When administered by an in vivo method, for example, it can be in the form of a liquid formulation or the like, but it is generally an injection containing the polynucleotide of the present invention, which is an active ingredient. An acceptable carrier may be added. In addition, liposomes or membrane-fusion liposomes (Sendai virus (HVJ)-liposomes, etc.) containing the polynucleotide of the present invention may be in the form of liposome preparations such as suspensions, freezing agents, and centrifugation-concentrating freezing agents. can.

The content of the polynucleotide of the present invention in the preparation can be appropriately adjusted according to the disease to be treated, the patient's age, body weight, etc. Generally, the content of the polynucleotide is 0.0001 mg to 100 mg, preferably 0.001 mg to 100 mg. 001 mg to 10 mg of the polynucleotide of the invention is preferably administered once every few days to several months.
A person skilled in the art can appropriately select suitable cells, vectors, administration methods, dosage forms and dosages.

Moreover, in recent years, a polynucleotide encoding a polyepitope peptide in which a plurality of CTL epitopes (tumor antigen peptides) are linked, or a polynucleotide encoding a polyepitope peptide in which a CTL epitope and a helper epitope are linked has been found to be effective in vivo. has been shown to have CTL-inducing activity. For example, Journal of Immunology 1999, 162: 3915-3925 (this document is incorporated herein by reference) describes six HBV-derived HLA-A2-restricted antigenic peptides, three HLA-A11-restricted antigenic peptides, and a DNA (minigene) encoding an epitope peptide linked with a helper epitope effectively induced CTL against each epitope in vivo.
Thus, polynucleotides produced by ligating one or more polynucleotides encoding the peptides of the present invention, and optionally also polynucleotides encoding other peptides, can be appropriately expressed. By incorporating it into a vector, it can be used as an active ingredient of a CTL inducer. Such a CTL inducer can also be administered in the same administration method and administration form as described above.

In recent years, it has also been found that cancer cells avoid elimination by the immune system by shielding against attacks by immune cells. It has become clear that it uses a mechanism called "immune checkpoint" that Therefore, by suppressing the functions of immune checkpoints in cancer cells, the attack by immune cells can be made effective. Since the pharmaceutical composition of the present invention exhibits an antitumor effect by inducing tumor-specific immune cells, it exhibits a higher therapeutic effect by suppressing immune checkpoint functions as well. be able to. Therefore, in one preferred aspect, the pharmaceutical composition of the present invention is used together with an immune checkpoint inhibitor.

In the present invention, when a certain agent A and another agent B are “used together” or “used in combination”, it means that while agent A is exerting its effect, agent B is in a state of exerting its effect. . Therefore, the agent B may be administered simultaneously with the administration of the agent A, or the agent B may be administered at a certain interval after the administration of the agent A. In addition, agent A and agent B may have the same dosage form, or may have different dosage forms. Furthermore, agent A and agent B may be mixed into one composition as long as agent A or agent B does not lose its effect.

Any agent known as an immune checkpoint inhibitor can be used as the immune checkpoint inhibitor in this embodiment, as long as it does not inhibit the ability of the composition of the present invention to induce CTL. Known immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-TIM-3 antibody, anti-LAG- 3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-B7-H5 antibody, anti-TIGIT antibody and the like.

<6> Antigen-presenting cells of the present invention The peptides and polynucleotides of the present invention described above can be used in vitro, for example, as follows. That is, antigen-presenting cells that present the antigenic peptide of the present invention as an antigen can be prepared by contacting in vitro either the peptide or polynucleotide of the present invention with a cell capable of presenting an antigen. Accordingly, one aspect of the present invention provides an antigen-presenting cell that presents a complex of the HLA-A02 antigen and the peptide of the present invention on the cell surface, and a method for producing the same. As mentioned above, the peptides and polynucleotides of the invention can be used to prevent and/or treat cancer. Therefore, the antigen-presenting cells of this embodiment or the method for producing the same preferably utilize isolated cells derived from cancer patients. Specifically, by contacting in vitro either the peptide or polynucleotide of the present invention with an isolated cell having antigen-presenting ability derived from a cancer patient, HLA-A02 is present on the cell surface of the cell. Antigen-presenting cells that present the complex of the antigen and the peptide of the present invention are produced.

Here, "cells having antigen-presenting ability" are not particularly limited as long as they express MHC capable of presenting the peptide of the present invention, preferably HLA, more preferably HLA-A02 antigen on the cell surface. However, among these, professional antigen-presenting cells are preferred, and dendritic cells, which are said to have particularly high antigen-presenting ability, are more preferred.
Moreover, the substance added to prepare the antigen-presenting cells of the present invention from the cells capable of presenting antigens may be either the peptide or the polynucleotide of the present invention.
The antigen-presenting cells of the present invention can be obtained, for example, by isolating cells having antigen-presenting ability from cancer patients, pulsing the cells with the peptide of the present invention in vitro, and obtaining the HLA-A02 antigen and the peptide of the present invention. It is obtained by presenting a complex (Cancer Immunol. Immunother., 46:82, 1998, J. Immunol., 158, p1796, 1997, Cancer Res., 59, p1184, 1999). When using dendritic cells, for example, lymphocytes are separated from the peripheral blood of cancer patients by the Ficoll method, then non-adherent cells are removed, and the adherent cells are cultured in the presence of GM-CSF and IL-4 to produce dendritic cells. The antigen-presenting cells of the present invention can be prepared by inducing dendritic cells, culturing and pulsing the dendritic cells with the peptide of the present invention, and the like.

When the antigen-presenting cell of the present invention is prepared by introducing the polynucleotide of the present invention into the cell capable of presenting an antigen, the polynucleotide may be in the form of DNA or RNA. may Specifically, in the case of DNA, refer to Cancer Res., 56: p5672, 1996 and J. Immunol., 161: p5607, 1998 (these documents constitute a part of the present application by reference). In the case of RNA, J. Exp. Med., 184: p465, 1996 (this document constitutes a part of the present application by reference) can be referred to.

The antigen-presenting cells can be used as a CTL inducer and/or an active ingredient of a pharmaceutical composition. The CTL inducer and/or pharmaceutical composition containing the antigen-presenting cells as an active ingredient contains physiological saline, phosphate-buffered saline (PBS), medium, etc. in order to stably maintain the antigen-presenting cells. preferably included. Administration methods include intravenous administration, subcutaneous administration, and intradermal administration. By returning a CTL inducer and/or pharmaceutical composition containing such antigen-presenting cells as an active ingredient to the patient's body, the body of a patient suffering from GRIK2- or Claspin-positive cancer, CTLs specific to cancer cells that present the peptide of the present invention as antigens are efficiently induced, and as a result, GRIK2- or Claspin-positive cancers that present the peptide of the present invention as antigens can be prevented and/or treated.

<7> Cytotoxic T cells (CTL) of the present invention
Peptides and polynucleotides of the invention can be utilized in vitro, for example, as follows. That is, CTLs can be induced by contacting peripheral blood lymphocytes in vitro with either the peptide or polynucleotide of the present invention. Accordingly, one aspect of the present invention provides CTLs that specifically damage cells that present the peptide of the present invention as antigens, and methods for inducing the same. As mentioned above, the peptides and polynucleotides of the invention can be used to prevent and/or treat cancer. Therefore, peripheral blood lymphocytes derived from cancer patients are preferably used in the CTLs of this embodiment and the method for inducing them. Specifically, by contacting in vitro either the peptide or polynucleotide of the present invention with peripheral blood lymphocytes derived from a cancer patient, the peptide of the present invention is specifically damaged by antigen-presenting cells. induce CTLs;

For melanoma, for example, adoptive immunotherapy, in which a large amount of the patient's own tumor-infiltrating T cells are cultured outside the body and returned to the patient, has been shown to have therapeutic effects (J.Natl.Cancer.Inst., 86: 1159, 1994). In mouse melanoma, metastasis was suppressed by stimulating splenocytes with the tumor antigen peptide TRP-2 in vitro to proliferate CTL specific to the tumor antigen peptide, and administering the CTL to melanoma-transplanted mice. (J. Exp. Med., 185:453, 1997). This is based on the results of in vitro proliferation of CTLs that specifically recognize complexes of MHC of antigen-presenting cells and tumor antigen peptides. Therefore, it is believed that therapeutic methods in which the peptides or polynucleotides of the present invention are used to stimulate patient peripheral blood lymphocytes in vitro to increase tumor-specific CTLs, and then these CTLs are returned to the patient.

The CTL can be used as an active ingredient of a cancer therapeutic or preventive agent. The therapeutic or prophylactic agent preferably contains physiological saline, phosphate-buffered saline (PBS), medium, etc., in order to stably maintain CTLs. Administration methods include intravenous administration, subcutaneous administration, and intradermal administration. By returning such a cancer therapeutic or preventive agent containing CTL as an active ingredient to the patient's body, cancer cells by CTL in the body of a patient suffering from GRIK2- or Claspin-positive cancer of the present invention. can treat cancer by promoting the cytotoxic effects of and destroying cancer cells.

The CTL of the present invention can exert cytotoxic activity by targeting the complex of the peptide of the present invention and HLA, which is presented as an antigen to tumor cells. That is, the T cell receptor (TCR) of the CTL of the present invention recognizes the complex of the peptide of the present invention and HLA. In recent years, a TCR gene that recognizes a specific peptide-HLA complex expressed in CTL has been cloned, and the TCR gene has been introduced into CD8 ⁺ T cells collected from cancer patients to artificially produce CTL, and a large amount of Adoptive immunotherapy has been devised in which cells are cultured for 10 days and then transferred back into the patient (eg, Ochi et al., Blood. 2011 Aug 11;118(6):1495-503). In the present invention, the term "artificial CTL" means a CTL produced by introducing a gene encoding a TCR that recognizes a complex of a peptide and HLA into T cells as described above. Similar to the natural CTL described above, it can be used for the treatment of cancer. Therefore, such artificial CTLs are also included in the CTLs of the present invention. In this embodiment, the TCR that recognizes the complex of the peptide of the present invention and HLA, which is introduced into the artificial CTL, may be appropriately modified in order to increase the binding affinity and cytotoxic activity for the complex. . Therefore, "artificial CTL" also includes CTLs produced by appropriately genetically modifying a gene encoding a TCR that recognizes the complex of the peptide of the present invention and HLA, and then introducing the gene into patient-derived T cells. be done. Methods known in the art can be used to generate artificial CTLs.

<8> Agent for Detecting Tumor-Specific CTL Using the Peptide of the Present Invention The peptide of the present invention is useful as a component of an agent for detecting tumor-specific CTL because it can be recognized by tumor-specific CTL. Accordingly, the present invention also relates to tumor-specific CTL detection agents comprising the peptides of the present invention. In one aspect, the tumor-specific CTL detection agent of the present invention comprises HLA multimers (monomers, dimers, tetramers, pentamers and dextramers) containing the peptide of the present invention and HLA-A02.

For example, HLA tetramer refers to a tetramer obtained by biotinylating a complex (HLA monomer) in which HLA α chain and β2 microglobulin are associated with a peptide (epitope peptide) and binding to avidin ( Science 279: 2103-2106 (1998), Science 274: 94-96 (1996)). At present, HLA tetramers containing various antigenic peptides are commercially available (for example, from Medical and Biological Laboratories, Inc.), and HLA tetramers containing the peptide of the present invention and HLA-A02 can be easily produced. can. HLA dimers and HLA pentamers are also based on similar principles, in which the HLA monomers are dimerized and pentamerized, respectively. Therefore, HLA multimers containing a peptide of the invention and HLA-A02 are also an aspect of the invention.

Specifically, for example, an HLA tetramer containing a peptide consisting of the amino acid sequence of either SEQ ID NO: 1 or 2 and HLA-A02 can be mentioned. The HLA tetramer is preferably fluorescently labeled so that bound CTLs can be easily selected or detected by known detection means such as flow cytometry and fluorescence microscopy. Specific examples include HLA tetramers labeled with phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), and the like.

Examples of methods for producing HLA tetramers include those described in literatures such as Science 279: 2103-2106 (1998) and Science 274: 94-96 (1996), which are briefly described below.
First, an HLA-A02 α-chain expression vector and a β2-microglobulin expression vector are introduced into Escherichia coli or mammalian cells capable of expressing proteins and expressed. E. coli (eg, BL21) is preferably used here. The obtained monomeric HLA-A02 and the peptide of the present invention are mixed to form a soluble HLA-peptide complex. Next, the sequence of the C-terminal region of the α chain of HLA-A02 in the HLA-peptide complex is biotinylated with BirA enzyme. HLA tetramers can be prepared by mixing this biotinylated HLA-peptide complex and fluorescently labeled avidin at a molar ratio of 4:1. In each of the steps described above, it is preferable to perform protein purification by gel filtration or the like.

<9> Agent for Detecting Cancer Stem Cells As described above, the present inventors discovered for the first time that a gene selected from the group consisting of GRIK2 and Claspin is a tumor antigen highly expressed in bladder cancer stem cells. That is, the present inventors have determined that GRIK2 and Claspin are genes that are highly expressed in bladder cancer stem cells, although their expression is not observed in bladder cancer-derived non-cancer stem cells or normal somatic cells. became clear for the first time. Based on these findings, it was found that genes selected from the group consisting of GRIK2 and Claspin can be used as markers for identifying bladder cancer stem cells. Accordingly, in one aspect, the present invention relates to a bladder cancer stem cell detection agent comprising a detection agent for detecting an expression product of a gene selected from the group consisting of GRIK2 and Claspin.

In the present invention, simply referring to GRIK2 and the like means the GRIK2 gene and the like unless otherwise specified. It is preferably a human gene, but may be a homolog thereof.
In the present invention, "expression of a gene" refers to a series of biological reactions originating from transcription of the gene, and "expression product" refers to, for example, mRNA and endogenous polypeptides produced by this series of biological reactions. A molecule that An endogenous polypeptide that is the expression product of a gene is preferably the protein ultimately produced by expression of the gene.
In the present invention, the "agent for detecting gene expression products" means an agent for qualitatively and/or quantitatively detecting a gene expression product selected from the group consisting of GRIK2 and Claspin.

Cancer stem cell detection agents of the present invention include detection agents for detecting expression products of genes selected from the group consisting of GRIK2 and Claspin. When an expression product of a gene selected from the group consisting of GRIK2 and Claspin is detected in the detection target, it can be determined that the detection target has cancer stem cells, that is, cancer stem cells have been detected. The agent for detecting cancer stem cells of the present invention can be used both in vivo and in vitro. Used in vitro. In this case, the detection of cancer stem cells in the cell population derived from the biological sample to be detected means that the cancer stem cells are also detected in the individual organism from which the biological sample to be examined was collected, i.e., the organism It means that the individual has cancer stem cells. Therefore, as will be described later, the present invention also includes a method for detecting cancer stem cells in a test subject using the agent for detecting cancer stem cells of the present invention.
The organism to be tested may be any organism that can have a tumor in the bladder, for example, but preferably humans and non-human mammals (e.g., mice, rats, guinea pigs, hamsters, etc.) rodents, primates such as chimpanzees, artiodactyls such as cattle, goats and sheep, perissodactyla such as horses, rabbits, dogs, cats, etc.), and more preferably human individuals.
The cell population to be detected can be a cell population derived from any biological sample obtained from the test subject, but is preferably a cell population derived from a biological sample obtained from a human, More preferably, it has been confirmed that genes selected from the group consisting of GRIK2 and Claspin are hardly expressed in tissue cells, bladder and tissues other than bladder, heart, brain, placenta, lung, liver, skeletal muscle, A cell population containing cells derived from one or more biological samples selected from the group consisting of kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, large intestine and blood.

The agent for detecting the expression product of a gene selected from the group consisting of GRIK2 and Claspin, which is included in the agent for detecting cancer stem cells of the present invention, can vary depending on the expression product to be detected, and can be appropriately optimized by those skilled in the art. can choose something. Specifically, for example, when the expression product is mRNA, any mRNA detection method known in the art can be used, including, but not limited to, RT-PCR, in situ high Bridization method, Northern blotting method, real-time RT-PCR, and the like can be mentioned. Among them, RT-PCR method is preferable because of its high detection sensitivity, simplicity of experimental procedures, and the like. For example, when the expression product is an endogenous polypeptide (preferably GRIK2 protein and/or Claspin protein), examples include, but are not limited to, Western blotting, immunohistochemical staining, and the like. The detection agent for the gene expression product selected from the group consisting of GRIK2 and Claspin to be used can vary depending on the expression product to be detected and the detection method to be employed, and those skilled in the art can appropriately select the optimum one.

Specifically, for example, a specific antibody (preferably a monoclonal antibody) against GRIK2 protein and/or Claspin protein when detecting an endogenous polypeptide, or a gene selected from the group consisting of GRIK2 and Claspin when detecting mRNA Examples include, but are not limited to, probes and/or primers having a nucleotide sequence complementary to a portion of the nucleotide sequence of . The expression product to be detected may be a single expression product or a combination of multiple expression products.

<10> Antibody that recognizes the peptide of the present invention As described above, the peptide of the present invention is presented as a CTL epitope peptide by cancer cells, particularly cancer stem cells. At this time, since a complex with MHC is formed and presented on the cell surface, the peptide of the present invention can be used as a tumor marker by using an antibody that recognizes the peptide of the present invention or a complex of the peptide and MHC. can. Such antibodies include, for example, an antibody (preferably a monoclonal antibody) that specifically recognizes the peptide of the present invention, a complex of the peptide of the present invention and HLA, preferably a complex that recognizes HLA-A02. and TCR (T-cell antigen receptor)-like antibodies. Accordingly, the present invention also relates to antibodies, in particular monoclonal antibodies and T-cell antigen receptor-like antibodies, which recognize the peptides of the present invention or complexes of said peptides and MHC.
In the present invention, the "TCR-like antibody" is a TCR-like binding force (antigen recognition ability) to a complex (pMHC) of a fragmented antigen-derived peptide and a major histocompatibility complex (MHC) molecule. is a molecule having For example, as reported by Eur J Immunol. It can recognize presenting cancer cells, dendritic cells presenting tumor antigen peptides on MHC class I by phagocytosing cancer cells, and the like.

The TCR-like antibody can be prepared by the method described in Eur J Immunol. 2004;34:2919-29. For example, complex-specific antibodies can be obtained by immunizing animals such as mice with MHC and peptide complexes. It is also possible to obtain a complex-specific antibody using a phage display method.

As described above, by recognizing the peptide of the present invention and/or the MHC complex presenting the peptide, it is possible to detect tumor cells presenting the MHC complex containing the peptide of the present invention on the cell surface. be. The present invention therefore also relates to a tumor detection agent comprising the above TCR-like antibody. In addition to tumor cells, the peptide of the present invention is similarly presented to antigen-presenting cells, particularly professional antigen-presenting cells such as dendritic cells. It is also useful for detection of cells and the like.
In the present invention, the term "antibody" refers not only to immunoglobulin molecules, but also to functional antibodies such as Fab, Fab', F(ab')2, Fv, scFv, dsFv, Diabody and sc(Fv)2. Fragments are also included. Multimers (eg, dimers, trimers, tetramers, polymers) of these functional fragments are also included in the antibodies of the present invention.

As described above, the peptide of the present invention is presented as a CTL epitope peptide by cancer cells, particularly cancer stem cells. A TCR-like antibody that recognizes the complex of can bind to said complex present on the cell surface in a subject. When the TCR-like antibody binds to the tumor cell surface, the Fc region of the antibody binds to the Fc receptor of effector cells such as macrophages and NK cells, and the effector cells attack tumor cells Antibody-dependent cellular cytotoxicity (ADCC) The resulting activity can treat tumors. Therefore, the above TCR-like antibody is also useful for cancer prevention and/or treatment. Therefore, the present invention also relates to preventive and/or therapeutic agents for cancer comprising the TCR-like antibody of the present invention.

In recent years, bispecific antibodies have also been developed in which the two antigen-binding sites are modified to bind to different antigens. A bispecific antibody that recognizes a cancer cell surface antigen such as an MHC-antigen peptide complex at one antigen-binding site and a lymphocyte surface antigen such as CD3 at the other antigen-binding site is a cancer cell It becomes possible to bind and accumulate cells having lymphocyte surface antigens such as CTLs and effector cells in the vicinity of the cells. Lymphocytes confined to the vicinity of cancer cells not only exhibit anti-tumor activity such as ADCC activity themselves, but also act as anti-tumor cells by secreting cytokines to activate naive immune cells around cancer cells. It can attack cancer cells by exerting its standard effect.

Therefore, the present invention also includes bispecific antibodies that specifically recognize the peptide of the present invention and/or a complex of the peptide and HLA, and a lymphocyte surface antigen. The specifically recognized lymphocyte surface antigen is not particularly limited as long as it is an antigen specifically expressed on the surface of lymphocytes, but preferably includes CD3, CD16, CD64 and the like. In particular, CD3 is a cell surface antigen that is involved in inducing the cytotoxic activity of CTL, and when an antibody binds to CD3, CTL can be activated in an HLA-unrestricted manner without recognizing HLA-cancer antigen complexes. It is preferable because it can be expected to exhibit strong cytotoxic activity.

Furthermore, in recent years, a chimeric antigen receptor (CAR), which is a part of a tumor antigen-specific monoclonal antibody that has been genetically modified, has been introduced into patient-derived T cells, and the genetically modified T cells have been amplified in vitro. A new immune cell therapy method has been devised that is cultured and then infused into patients (Nat Rev Immunol. 2012;12:269-81). Specifically, after activating T cells by culturing peripheral blood mononuclear cells collected from patients in the presence of anti-CD3 antibody and IL-2, etc. Genetically modified T cells are generated by introducing a gene encoding CAR into T cells using a transforming vector.
In the present invention, the term “chimeric antigen receptor” refers to a single-chain antibody (scFv) in which the light chain and heavy chain of the antibody variable region of an antibody that recognizes molecules present on the cell surface of cancer cells are linked in series. It is a chimeric protein molecule designed to have the CD3ζ chain on the C-terminal side among the molecules that constitute the T cell receptor (TCR)/CD3 complex on the terminal side. When this chimeric antigen receptor recognizes a specific antigen in its scFv region, it activates T cells via the CD3ζ chain. One or more co-stimulatory molecules (eg, CD28, 4-1BB, ICOS, etc.) may be incorporated between the scFv and the ζ chain to enhance T cell activation. In the present invention, a CAR can be produced using the TCR-like antibody of this embodiment (including antibody molecules or fragments thereof that can be designed from the TCR-like antibody) as the scFv. CAR, which recognizes a complex of a peptide derived from a tumor antigen and MHC, phagocytizes cancer cells presenting a tumor antigen peptide that can be targeted by CTL, and the tumor antigen peptide on MHC class I. Since the CAR-introduced genetically modified T cells can recognize presenting dendritic cells and the like, the CAR-introduced genetically modified T cells can be used as a preventive and/or therapeutic agent for cancer specific to the tumor antigen, similar to artificial CTL. Useful. Therefore, the present invention also relates to preventive and/or therapeutic agents for cancer, comprising gene-modified T cells or artificial CTLs introduced with CAR that recognize the complex of the peptide derived from the tumor antigen of the present invention and MHC. .

<11> Tumor detection method (examination method, diagnosis method)
The present invention provides a tumor detection method (examination method, diagnosis method) using the above-described CTL detection agent, cancer stem cell detection agent, or tumor detection agent of the present invention.
In the detection method (diagnosis method) of the present invention using the CTL detection agent of the present invention, typically, blood of a subject is collected, or a part of a test tissue suspected of having a tumor is collected by biopsy or the like. GRIK2- or Claspin-positive GRIK2- or Claspin-positive diseases such as bladder cancer are detected and measured by the CTL detection agent of the present invention by detecting and measuring the amount of CTLs that recognize the complex of the GRIK2- or Claspin-derived tumor antigen peptide and HLA antigen contained in the It detects, examines, or diagnoses the presence or absence or degree of cancer (tumor).

The detection method (examination method, diagnosis method) of the present invention using the agent for detecting cancer stem cells of the present invention typically involves collecting the blood of a subject, or biopsying a portion of a tissue suspected of having a tumor. and the amount of GRIK2 or Claspin expression product contained therein is detected and measured by the agent for detecting cancer stem cells of the present invention, thereby detecting the incidence of GRIK2 or Claspin positive cancer (tumor) such as bladder cancer. It detects, examines or diagnoses the presence or absence or degree of
The detection method (examination method, diagnosis method) of the present invention using the tumor-detecting agent of the present invention typically involves collecting the blood of a subject, or collecting a part of a test tissue suspected of having a tumor by biopsy or the like. Then, the amount of cells presenting a complex of GRIK2 or Claspin-derived tumor antigen peptide and HLA antigen contained therein is detected and measured by the tumor-detecting agent of the present invention, thereby detecting GRIK2 or GRIK2 such as bladder cancer or It detects, examines, or diagnoses the presence or absence or degree of Claspin-positive cancer (tumor).

The detection (testing, diagnosing) method of the present invention detects (testing, diagnosing) the presence or absence or degree of improvement of a tumor when a therapeutic agent is administered to improve the tumor, for example, in a patient with a tumor. You can also Furthermore, the detection (examination, diagnosis) method of the present invention includes selection of patients to be treated to whom the drug containing the peptide or polynucleotide of the present invention as an active ingredient can be effectively applied, prediction and determination of the therapeutic effect and prognosis of the drug, and the like. also available. In addition, in the embodiment using the tumor-detecting agent of the present invention, a tumor antigen peptide that can be actually targeted by CTL induced in vivo by administering a cancer vaccine containing the peptide of the present invention as an active ingredient is used. It is possible to detect presenting cancer cells.

A specific embodiment of the detection (examination) method of the present invention using the CTL detection agent of the present invention comprises the following steps (a) and (b), and optionally (c):
(a) contacting a biological sample obtained from a subject with the CTL detection agent of the present invention;
(b) measuring the amount of CTLs in the biological sample that recognize a complex of a tumor antigen peptide derived from GRIK2 or Claspin and an HLA antigen, using the amount of cells to which the CTL detection agent binds as an index;
(c) A step of judging the presence of cancer based on the result of (b).
A specific embodiment of the diagnostic method of the present invention using the CTL detection agent of the present invention includes steps (a), (b) and (c) above.

A specific embodiment of the detection (examination) method of the present invention using the cancer stem cell detection agent of the present invention comprises the following steps (d) and (e), and optionally (f):
(d) contacting a biological sample obtained from a subject with the agent for detecting cancer stem cells of the present invention;
(e) measuring the amount of GRIK2 or Claspin expression product in said biological sample;
(f) A step of judging the presence of cancer based on the result of (e).
A specific embodiment of the diagnostic method of the present invention using the agent for detecting cancer stem cells of the present invention includes the steps (d), (e) and (f) above.
Embodiments of the method for detecting cancer stem cells using the agent for detecting cancer stem cells of the present invention include steps (d) and (e) above, and the following step (f′) instead of (f):
(f') A step of determining the presence or absence of cancer stem cells in the biological sample based on the result of (e).
The biological sample used here includes a sample prepared from a subject's biological tissue (tissue suspected to contain cancer cells, surrounding tissue, blood, etc.). Specifically, a sample containing tissue cells collected from the tissue can be used.

A specific embodiment of the detection (examination) method of the present invention using the cancer stem cell detection agent of the present invention comprises the following steps (g) and (h), and optionally (i):
(g) contacting a biological sample obtained from a subject with the tumor-detecting agent of the present invention;
(h) measuring the amount of cells presenting a complex of a GRIK2- or Claspin-derived tumor antigen peptide and an HLA antigen in the biological sample, using the amount of cells bound to the tumor-detecting agent as an index;
(i) A step of judging the presence of cancer based on the result of (h).
A specific embodiment of the diagnostic method of the present invention using the tumor detecting agent of the present invention comprises steps (g), (h) and (i) above.
The biological sample used here includes a sample prepared from a subject's biological tissue (a tissue suspected of containing cancer cells and its surrounding tissue, blood, etc.). Specifically, a sample containing tissue cells collected from the tissue can be used.

One embodiment of the detection method (test method, diagnostic method) of the present invention using the CTL detection agent of the present invention is performed by detecting the peptide-specific CTL of the present invention in a biological sample and measuring the amount thereof. . Specifically, according to the method described in the literature (Science, 274: p94, 1996, which is incorporated herein by reference), 4 amounts of complexes of fluorescently labeled HLA antigens and peptides of the present invention HLA tetramers are prepared and used to quantify antigen peptide-specific CTLs in peripheral blood lymphocytes of patients suspected of having cancer using a flow cytometer.

Prediction, judgment, judgment or diagnosis of the presence or absence of a tumor is, for example, by measuring the amount of the peptide-specific CTL of the present invention or the amount of cells presenting the peptide of the present invention in the blood of a subject or a test tissue suspected of having a tumor. It can be done by In this case, GRIK2 or Claspin gene expression level, peptide level or CTL level of the present invention, etc., in a normal corresponding tissue may be used as a reference value, and the reference value is compared with the level in the sample obtained from the subject, This can be done by determining the difference between the two.
Here, the comparison of the levels between the test tissue of the subject and the corresponding normal tissue can be carried out by performing measurements in parallel on the biological sample of the subject and the biological sample of the normal subject. If not performed in parallel, the peptide-specific CTL of the present invention obtained by measurement under uniform measurement conditions using multiple (at least 2, preferably 3 or more, more preferably 5 or more) normal tissues or the average or statistical mean value of the amount of cells presenting the peptide of the present invention can be used for comparison as the normal person's value, ie, the reference value.

Determination of whether a subject is suffering from cancer is, for example, the amount of the peptide-specific CTL of the present invention in the subject's tissue, or the amount of cells presenting the peptide of the present invention is higher than those of a normal person. For example, it can be measured by using a ratio of 2 times or more, preferably 3 times or more, as an index.
It is also possible to determine whether or not CTL are actually induced by measuring the amount of peptide-specific CTL of the present invention in subjects administered with the peptide or polynucleotide of the present invention. . For example, the amount of the peptide-specific CTL of the present invention in the tissue of the subject is, for example, 2-fold or more, preferably 3-fold or more, as compared to the level of those in normal subjects. Treatment with nucleotides can be determined to be effective.

<12> Method for preventing and/or treating cancer The present invention also provides a method for preventing and/or treating cancer in a subject, comprising the peptide, polynucleotide, CTL, antigen-presenting cell, and TCR-like antibody of the present invention. , artificial CTL, and genetically modified T cells, to a subject in need thereof.
A “subject” in the present invention may be any organism as long as it can be affected by cancer. primates such as teeth and chimpanzees; artiodactyls such as cows, goats and sheep; perissodactyla such as horses; rabbits, dogs and cats), and more preferably human individuals. In the present invention, the subject may be healthy or suffer from some disease. means a subject who has or is at risk of being affected. In one aspect of the invention, the subject is HLA-A02 positive. In one aspect of the invention, the subject has or is at risk of having a GRIK2 or Claspin positive cancer. In one aspect of the invention, the subject is HLA-A02 positive and has or is at risk of having a GRIK2 or Claspin positive cancer.

Peptides, polynucleotides, CTLs, antigen-presenting cells, TCR-like antibodies, artificial CTLs, and genetically modified T cells of the invention for use in the prophylactic/therapeutic methods of the invention include any described herein. . An effective amount in the present invention is, for example, an amount that reduces symptoms of cancer or delays or stops the progression thereof, preferably an amount that suppresses or cures cancer. Also, an amount that does not cause adverse effects that exceed the benefits of administration is preferred. Such an amount can be appropriately determined by in vitro tests using cultured cells or the like, or tests using 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 depends on various conditions related to the subject requiring it, such as severity of symptoms, general health condition of the subject, age, weight, sex of the subject, diet, timing and frequency of administration, It can be determined in consideration of concomitant drugs, responsiveness to treatment, dosage form, compliance to treatment, and the like.

As a specific dose, for example, in the case of the peptide of the present invention, it is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably 0.1 mg to 10 mg. A single dose is preferred. In the case of the polynucleotide of the present invention, it is usually 0.0001 mg to 100 mg, preferably 0.001 mg to 10 mg, and is preferably administered once every several days to several months. In the case of the TCR-like antibody of the present invention, the dose is usually 0.0001 mg to 2000 mg, preferably 0.001 mg to 2000 mg, and is preferably administered once every 1 to 4 weeks. In the case of the genetically modified T cells or artificial CTLs of the present invention, it is usually 1×10 ⁴ to 1×10 ⁸ , preferably 1×10 ⁵ to 1×10 ⁷ , and is administered once every 1 day to 4 weeks. preferably. Moreover, as an administration method, any known appropriate administration method such as intradermal administration, subcutaneous administration, intramuscular administration, and intravenous administration can be used. In addition to the in vivo method of directly administering the peptides and nucleotides of the present invention into the body, certain cells were collected from humans and the peptides and polynucleotides of the present invention were used to induce CTLs and antigen-presenting cells in vitro. An ex vivo method can also be used to later put these cells back into the body.

One aspect of the prophylactic/therapeutic method of the present invention further comprises a step of selecting an HLA-A02-positive subject as a prophylactic/therapeutic target before the administering step. This aspect of the invention may further comprise determining the HLA type of the subject prior to the selecting step. Determining a subject's HLA type can be done by any known technique. In addition, one aspect of the preventive/therapeutic method of the present invention further comprises the step of selecting a subject having GRIK2- or Claspin-positive cancer as a preventive/therapeutic target before the administering step. This aspect of the invention may further comprise detecting GRIK2 or Claspin positive cancers in the subject prior to the selecting step. The tumor detection method described in <11> above can be used to detect GRIK2- or Claspin-positive cancer in a subject. One aspect of the preventive/therapeutic method of the present invention further comprises the step of selecting a subject who is HLA-A02 positive and has GRIK2 or Claspin-positive cancer as a preventive/therapeutic target before the administering step. include. This aspect of the invention may further comprise, prior to the selecting step, determining the HLA type of the subject and detecting GRIK2 or Claspin positive cancer in the subject.

<13> Screening method for cancer therapeutic drug targeting cancer cells In the embodiment using the agent for detecting cancer stem cells of the present invention, the expression level of the GRIK2 or Claspin expression product in the detection target is determined by the cancer stem cells in the detection target. is considered to be positively correlated with the amount of Therefore, by comparing the expression level of the GRIK2 or Claspin expression product before and after administration of the candidate compound of the cancer therapeutic drug to the detection target, it is possible to determine whether the administered candidate compound is a cancer therapeutic drug targeting cancer stem cells. useful or not.

The screening method of the present invention comprises the following steps (I), (II) and optionally (III):
(I) measuring the detected amount A of the expression product of the GRIK2 or Claspin gene in a subject before administering a candidate compound for a cancer therapeutic drug to the subject;
(II) after administering the candidate compound to the subject cell population, measuring the detected amount B of the expression product of the gene in the subject; and (III) comparing the detected amounts A and B, and detecting the If the amount A is significantly greater than B, determining that the candidate compound is a cancer therapeutic drug candidate characterized by targeting cancer stem cells.
A particular embodiment of the screening method of the present invention comprises steps (I) to (III) above. Here, the steps (I) and (II) of measuring the amount to be detected include steps (d) and (e) in the detection (examination, diagnosis) method described above, respectively.

<14> Polynucleotide for Suppressing Gene Expression As described above, it was first found that a gene selected from the group consisting of GRIK2 and Claspin is expressed in bladder cancer stem cells. This suggests that the expression of these genes is involved in the canceration of cells, and therefore it is thought that suppressing the expression of these genes can treat cancer.

That is, in one aspect, the present invention relates to a gene expression suppressor that suppresses the expression of a gene selected from the group consisting of GRIK2 and Claspin.
Methods for selectively suppressing the expression of specific genes in cells are not particularly limited, and examples thereof include antisense RNA method, RNA interference (RNAi) method, CRISPR-Cas method, ZFN method, TALEN method and the like. Among them, the antisense RNA method and the RNAi method are preferable, and the RNAi method is more preferable, from the viewpoint of bioavailability and low off-target effect.

Therefore, in a preferred embodiment of the present invention, the gene expression inhibitor is an antisense oligonucleotide against a gene selected from the group consisting of GRIK2 and Claspin. In the present invention, the term "antisense oligonucleotide" against a certain gene means an oligonucleotide capable of suppressing the expression of the gene by hybridizing to mRNA, which is the expression product of the gene. can be any nucleic acid such as DNA or RNA. Such oligonucleotides are typically oligonucleotides having a sequence complementary to a portion of the mRNA sequence of the gene. Here, the term "complementary" means that a nucleic acid can form a hydrogen bond with another nucleic acid sequence, and a specific "sequence complementary to (a portion of) a sequence" refers to a nucleotide having that sequence. It means a sequence having complementarity to the extent that it can hybridize with in the intracellular environment. Thus, not all of the sequences need be complementary (ie perfectly complementary).

The antisense oligonucleotides of the present invention typically have a length of about 15-30 nucleotides. In addition, modifications known in the art may be applied for purposes such as improving in vivo stability and expression-suppressing activity and reducing off-target effects.

Also, in a preferred embodiment of the present invention, the gene expression inhibitor is siRNA against a gene selected from the group consisting of GRIK2 and Claspin. In the present invention, "siRNA" against a certain gene means a double-stranded structural RNA that can inhibit the expression of the gene, the double-stranded structural RNA has a sense region and an antisense region, The sense region is complementary to the sequence of the mRNA for a particular gene, and the sense region is complementary to the sequence of the antisense region. Each sense region and antisense region of the siRNA of the present invention has a length of about 15-30 nucleotides, preferably about 19-27 nucleotides. Also, the sense region and the antisense region may form a double-stranded structure with two strands, the sense strand and the antisense strand, respectively. Alternatively, the sense region and the antisense region may be linked to form one nucleotide chain, in which case the single-stranded RNA is folded into a hairpin to form a double-stranded sense region and an antisense region. to form a structure.

In the art, methods are known for improving the expression-suppressing effect of siRNA, improving bioavailability, and reducing off-target effects. The siRNA of the present invention can appropriately apply these known modifications and alterations for improving the function as siRNA.
The polynucleotides can be easily synthesized by methods known in the art, such as commercially available DNA synthesizers.

In another aspect, the present invention provides a pharmaceutical composition comprising the antisense oligonucleotide and/or the siRNA. Other ingredients that can be contained in the pharmaceutical composition of this embodiment include, for example, pharmaceutically acceptable carriers, diluents, excipients, etc., and pharmaceutically acceptable carriers are particularly preferred. Pharmaceutically acceptable carriers include, but are not limited to, liposomes, hydrophilic polymers, and the like.

As described above, it is believed that cancer can be treated by suppressing the expression of a gene selected from the group consisting of GRIK2 and Claspin. Therefore, the pharmaceutical composition containing the polynucleotide of this embodiment can be used as a preventive and/or therapeutic agent for cancer.

In another aspect, the present invention also relates to a method for preventing and/or treating cancer, comprising suppressing the expression of a gene selected from the group consisting of GRIK2 and Claspin. In this method, the active ingredient to be administered is a gene expression inhibitor that suppresses the expression of a gene selected from the group consisting of GRIK2 and Claspin, preferably suppresses the expression of a gene selected from the group consisting of GRIK2 and Claspin. It can be carried out according to the method described in <12> above, except that it is an antisense oligonucleotide or siRNA.

That is, the above method is a method for preventing and/or treating cancer, comprising the step of administering an effective amount of a gene expression inhibitor selected from the group consisting of GRIK2 and Claspin to a subject in need thereof. can be done. The subject may be healthy or afflicted with any disease, but typically is afflicted with cancer or, where cancer prevention and/or treatment is contemplated, It means a subject who is at risk of being affected. Thus, the subject has or is at risk of having a GRIK2- or Claspin-positive cancer.

In addition, one aspect of the preventive/therapeutic method of the present invention may further include a step of selecting a subject with GRIK2- or Claspin-positive cancer as a preventive/therapeutic target before the administering step. When selecting a subject, a method for detecting GRIK2- or Claspin-positive cancer in the subject <11> above can be used.

An effective amount in this aspect is, for example, an amount that reduces symptoms of cancer or delays or stops the progression thereof, preferably an amount that suppresses or cures cancer. Also, an amount that does not cause adverse effects that exceed the benefits of administration is preferred. Such an amount can be appropriately determined by in vitro tests using cultured cells or the like, or tests using 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 depends on various conditions related to the subject requiring it, such as severity of symptoms, general health condition of the subject, age, weight, sex of the subject, diet, timing and frequency of administration, It can be determined in consideration of concomitant drugs, responsiveness to treatment, dosage form, compliance to treatment, and the like.

All patents, applications and other publications mentioned herein are hereby incorporated by reference in their entirety.
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1. Isolation of Cells with High Aldehyde Dehydrogenase Activity from Bladder Cancer Cell Line UM-UC-3 Cancer stem cells are known to have high aldehyde dehydrogenase (ALDH) enzymatic activity. Therefore, cells with high aldehyde dehydrogenase enzymatic activity (ALDH ^high ) and cells with low aldehyde dehydrogenase enzymatic activity (ALDH ^low ) were separated by the ALDEFLUOR method based on aldehyde dehydrogenase enzymatic activity ( FIG. 1 ). The cell line used was the bladder cancer cell line UM-UC-3.
2007 Nov;1(5):555-67. doi: 10.1016/j.stem.2007.08.014.(https://pubmed.ncbi.nlm.nih.gov/18371393/ ) and Oncotarget. 2017 Apr 25;8(17):28826-28839. doi: 10.18632/oncotarget.16259.
ALDH ^high cells are known to differentiate into ^{ALDH low} cells in in vitro culture, and are unsuitable for stable analysis. Single cell clones were generated from ^high cells.
As a result, clone cells (H-1, H-6, H-10) established from ALDH ^high cells exhibited high ^ALDH enzymatic activity (Fig. 2), and clone cells (L- 1, L-3, L-8) showed low ALDH enzymatic activity (Fig. 3).
Surprisingly, ALDH ^high clones (H clones) and ALDH ^low clones (L clones) exhibit stable traits in in vitro culture for more than one month, respectively, and were established from bladder cancer cell line UM-UC-3. These cloned cells were suggested to be suitable for bladder cancer stem cell analysis. Although cancer stem cells generally differentiate when cultured in vitro, these clones do not differentiate much even when cultured in vitro, so they have the unique property of maintaining the state of cancer stem cells. was considered to have
Therefore, in Examples 2 to 5 below, it was decided to confirm whether or not ALDH ^high clones (H clones) are cancer stem cells. In the following experiments, H-10 clones with high ALDH enzyme activity and high tumorigenicity in vivo were used as representative H clone cells, and L-10 cells with low ALDH enzyme activity and low tumorigenicity in vivo were used. 3 clones were used as representative L clone cells.

Example 2. Confirmation of carcinogenicity (sphere formation) of ALDH ^high clone (H clone) In order to evaluate the in vitro tumorigenicity, sphere formation was confirmed as follows.
1.0 to 10 ³ H clone cells (H-10), L clone cells (L-3) and wild type (WT) were seeded in a 96-well plate to confirm sphere formation. As a result, H-10 cells showed sphere formation in 7 out of 24 wells in the group in which 1 cell/well was seeded, whereas L-3 cells did not form spheres in 24 wells ( Figure 4). From the sphere formation rate, the abundance rate of stem cells was calculated by calculation (ELDA method, https://pubmed.ncbi.nlm.nih.gov/19567251/).
As a result, in H-10 cells, it was found that stem cells were present in 1 out of 7.56 cells. The ratio of the wild type was 1 out of 49.07 cells and the ratio of L-3 cells was 1 out of 67.32 cells (Fig. 4).
Therefore, it was confirmed that the ALDH ^high clone (H clone) has high carcinogenicity.

Example 3. Confirmation of carcinogenicity (tumorigenicity in immunodeficient mice) of ALDH ^high clones (H clones) Sphere formation is an in vitro tumorigenicity evaluation method. H-10 cells and L-3 cells were transplanted into deficient mice, and their tumorigenic potential was examined. Immunodeficient mice (nude mice) were implanted with 1000 H-10 clone cells and L-3 cells and tumor growth curves were drawn.
As a result, the H-10 clone cells showed higher tumorigenicity than the L-3 cells, and no tumorigenesis was observed in the L-3 cells (Fig. 5).

Example 4. Confirmation of carcinogenicity (treatment resistance to anticancer drugs) of ALDH ^high clones (H clones) Cancer stem cells exhibit resistance to anticancer drugs. Therefore, sensitivity to cisplatin (CDDP), a key drug for bladder cancer, was examined. After H-10 cells and L-3 cells were cultured in the presence of CDDP adjusted to each concentration for 2 days, the cell viability was examined by the WST-8 method.
As a result, H-10 cells showed resistance to CDDP compared to L-3 cells (Fig. 6).

Example 5. Confirmation of carcinogenicity (treatment resistance to radiotherapy) of ALDH ^high clones (H clones) Whether or not ALDH ^high clones (H clones) are cancer stem cells was confirmed as follows.
Cancer stem cells are resistant to radiotherapy. Therefore, H-10 cells and L-3 cells were exposed to each dose of radiation and cultured for 2 days, and cell viability was examined by the WST-8 method.
As a result, H-10 cells showed resistance to radiation compared with L-3 cells (Fig. 7).

Therefore, as shown in the results of Examples 2 to 5 above, H-10 cells show higher sphere formation (Example 2), higher tumorigenicity (Example 3), and anti-tumor activity than L-3 cells. It was suggested that cancer stem cells were enriched in H-10 cells because they exhibited cancer drug resistance (Example 4) and radiotherapy resistance (Example 5).
For bladder cancer, no cancer stem cell model that can be stably cultured in vitro has been reported other than H-10 cells.

Example 6. Search for antigenic peptides expressed in bladder cancer stem cells We comprehensively searched for antigenic peptides expressed in bladder cancer stem cells that could serve as immunotherapeutic targets by mass spectrometry analysis.
The HLA of UM-UC-3 cells is A*02:01/A*33:03, B*07:02/B*14:02, C*07:02/C*08:02 (Fig. 8 ). Since HLA-A*02:01 is a frequent HLA allele, we decided to focus on HLA-A*02:01.
10 ⁹ H-10 cells, L-3 cells, and wild-type cells were each cultured to prepare a cell lysate. The cell lysate was reacted with an anti-HLA-A*02:01 specific antibody, immunoprecipitated using protein-A Sepharose beads, and HLA-A*02:01 molecules were recovered. The collected antigen peptides presented on the HLA-A*02:01 molecules were extracted with an acidic buffer, and the amino acid sequences were analyzed by mass spectrometry analysis.
A summary of the amino acid lengths of peptides analyzed from H-10 cells, L-3 cells, and wild type is shown in FIG. As shown in FIG. 8, peptides with a length of 9 amino acids were most abundant in both H-10 cells, L-3 cells and wild type.

Example 7. Sequence analysis of 9-amino acid long peptides Among peptides recovered from H-10 cells, L-3 cells, and wild-type strains, the amino acid sequences of 9-mer peptides were analyzed.
As a result, it was found that the 2nd amino acid of the peptide was leucine (L), and the 9th C-terminal amino acid was valine (V) and leucine (L) (Fig. 9). This result matched the HLA-A2 subtype binding motif (https://pubmed.ncbi.nlm.nih.gov/8254189/), indicating that the HLA-A*02:01 binding peptide analysis was functional. shown.
A Venn diagram was constructed using antigenic peptides identified from H-10 cells, L-3 cells, and wild-type cells (Fig. 9). As a result, 123 peptides were found to be specifically extracted from H-10 cells. Of the 123 peptides, from the peptide-encoding gene expression information, 2 of the 123 antigen peptides (LMYDAVHVV (SEQ ID NO: 1) and SLLNQPKAV (SEQ ID NO: 2) specifically expressed in H-10 clone cells )) were determined to be suitable immunotherapeutic targets.

Example 8. Antigenicity Test of GRIK2 Peptide To investigate whether the GRIK2 peptide has antigenicity, a cytotoxic T cell (CTL) induction experiment was performed using peripheral blood mononuclear cells.
GRIK2 peptide was added to peripheral blood mononuclear cells (PBMC) collected from HLA-A*02:01 positive donors in the presence of interleukin-2 (IL-2) and cultured.
As a result, interferon γ (IFNγ) ELISPOT method confirmed a high response to the GRIK2 peptide (Fig. 10).

Example 9. Generation of GRIK2 Peptide-Specific CTL Clones To analyze GRIK2 peptide-specific CTLs, GRIK2 peptide-specific CTL clones were generated.
PBMC were stained with PE-labeled HLA-A*02:01+GRIK2 peptide complex tetramer.
As a result, GRIK2 peptide tetramer-specific staining was observed in about 0.099% of the cells (Fig. 11), and the same cells were isolated and single cell clones were generated.
The obtained GRIK2 peptide-specific 9G23 clone showed high reactivity in the GRIK2 peptide-added group (right panel) by the IFNγ ELISPOT method (FIG. 12).
The 9G23 clone was also reanalyzed using the GRIK2 tetramer. As a result, the 9G23 clone showed a high tetramer staining rate (Fig. 13), indicating that it was a GRIK2 peptide-specific CTL clone.

Example 10. Functional analysis of GRIK2 peptide-specific CTL clones To examine whether GRIK2 peptide-specific CTL clones can recognize the GRIK2 peptide endogenously expressed in cancer cells, the GRIK2 gene was overexpressed in wild-type UM-UC-3 cells. rice field. A retroviral vector (pMXs-puro) was used for overexpression.
As a result, the GRIK2 peptide-specific CTL clones showed high reactivity to the GRIK2 overexpressing strain (center graph in FIG. 14).
In addition, GRIK2 peptide-specific CTL clones showed high response to H-10 cells compared to L-3 cells and wild type.
As a result, the GRIK2 peptide-specific CTL clone was found to exhibit high reactivity with H-10 cells (leftmost graph in FIG. 15).
These results suggested that the GRIK2 antigen peptide expressed in treatment-resistant bladder cancer cells could be a therapeutic target for immunotherapy.

Example 11. In Vitro Treatment Model Using GRIK2 Peptide-Specific CTL Clones As a standard model of bladder cancer cell population, H-10 cells and L-3 cells were mixed at a ratio of 1:9. A group treated with CTL clone 9G23 (CTL+) and a group not treated with CTL clone 9G23 (CTL-) were each cultured for 2 days. After 2 days, the percentage of ALDH ^high cells was 10.9% for CTL− and 1.16% for CTL+. A schematic and results of the in vitro therapeutic model are shown in FIG.
Therefore, it was shown that ALDH ^high cells (cancer stem cells) decreased in the group (CTL+) treated with CTL clone 9G23.

Example 12. Combined Effects of GRIK2 Peptide- Specific CTL Clones and Other Therapies The effects of combined use of GRIK2 peptide-specific CTL clones with the anticancer drug cisplatin (CDDP) or irradiation were investigated.
A group treated with CTL clone 9G23 (CTL+) and a group not treated with CTL clone 9G23 (CTL-) were cultured for 2 days in the presence of CDDP adjusted to each concentration, and cell viability was examined by the WST-8 method. Furthermore, the cell viability was examined by the WST-8 method after culturing for 2 days after culturing for 2 days after exposure to each dose of CTL clone 9G23 (CTL+) and non-acting group (CTL-).
As a result, it was shown that the cell viability of the CTL+ group was further reduced by the anticancer drug cisplatin (CDDP) and irradiation (Fig. 17).
Immunotherapy using the CTL clone 9G23 of the present invention targets cancer stem cells, and anticancer agents and radiotherapy target non-cancer stem cells, and further effects were obtained by the combination of these.

Example 13. Generation of Claspin Peptide-Specific CTL Clones To analyze Claspin peptide-specific CTLs, Claspin peptide-specific CTL clones were generated.
PBMCs were stained with PE-labeled HLA-A*02:01+Claspin peptide complex tetramer.
As a result, a high percentage of Claspin peptide tetramer-specific staining was observed (not shown), and the same cells were isolated and single cell clones were generated.
The resulting Claspin peptide-specific yc3 clones showed high reactivity in the Claspin peptide-added group (right panel) in the IFNγ ELISPOT method (Fig. 12).
The yc3 clone was also reanalyzed using the Claspin tetramer. As a result, the yc3 clone showed a high tetramer staining rate (Fig. 13), indicating that it was a Claspin peptide-specific CTL clone.

In the present invention, by identifying GRIK2- and Claspin-derived natural peptides that are actually presented as antigens to bladder cancer stem cells, CTLs induced by peptide vaccines can reliably kill cancer cells, and highly effective cancer vaccines can be developed. It contributes to development. In addition, from the identified bladder cancer stem cell-specific natural peptides, it was possible to identify that GRIK2 and Claspin are specifically expressed in cancer stem cells, so GRIK2 and Claspin are used as markers to identify bladder cancer stem cells. becomes possible. Furthermore, the natural antigen peptide derived from the same gene is useful as a preventive and/or therapeutic agent for cancer, which is highly effective even in a small amount. The present invention also provides GRIK2-derived tumor antigen peptides and the like having CTL-inducing activity. The peptide of the present invention is useful as a prophylactic and/or therapeutic agent for treatment-resistant bladder cancer.

Claims (43)

A tumor antigen peptide or a motif substitute thereof, consisting of 8 to 14 consecutive amino acids in the amino acid sequence of a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin, and having HLA binding properties. The tumor antigen peptide or its motif substitute according to claim 1, wherein HLA is HLA-A02. 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, or in the peptide, the second amino acid from the N-terminus is leucine, isoleucine 3. The tumor antigen peptide according to claim 1 or 2, which is a peptide substituted with methionine, and/or substituted with valine, leucine or isoleucine at the C-terminal amino acid. The tumor antigen peptide according to any one of claims 1 to 3, represented by SEQ ID NO: 1 or SEQ ID NO: 2. A polyepitope peptide in which a plurality of epitope peptides are linked, said polyepitope peptide comprising at least one tumor antigen peptide according to any one of claims 1 to 4 as said epitope peptide. A polynucleotide encoding at least one of the tumor antigen peptide according to any one of claims 1 to 4 or the polyepitope peptide according to claim 5. An expression vector comprising the polynucleotide according to claim 6. A composition for gene transfer, comprising the expression vector according to claim 7. (A) the tumor antigen peptide according to any one of claims 1 to 4 or the polyepitope peptide according to claim 5, or
(B) A method for producing an antigen-presenting cell, comprising contacting in vitro a polynucleotide encoding at least one of the peptide and/or polyepitope peptide of (A) with a cell having antigen-presenting ability. . (a) to (e) below:
(a) the antigenic peptide according to any one of claims 1 to 4 or the polyepitope peptide according to claim 5;
(b) the polynucleotide of claim 6;
(c) the expression vector of claim 7;
(d) a protein encoded by a gene selected from the group consisting of GRIK2 and Claspin, a polynucleotide encoding the protein, or a partial peptide of the protein including the antigen peptide represented by SEQ ID NO: 1 or 2, or the polynucleotide an expression vector comprising
(e) An inducer of cytotoxic T cells, containing as an active ingredient any of antigen-presenting cells that present the antigenic peptide according to any one of claims 1 to 4 as an antigen. (A) the tumor antigen peptide according to any one of claims 1 to 4 or the polyepitope peptide according to claim 5;
(B) a polynucleotide encoding at least one of the peptides and/or polyepitopic peptides of (A); or
(C) the production of cytotoxic T cells, comprising contacting peripheral blood lymphocytes in vitro with antigen-presenting cells that present the antigenic peptide of any one of claims 1 to 4 as antigens; induction method. (a) to (e) below:
(a) the antigenic peptide according to any one of claims 1 to 4 or the polyepitope peptide according to claim 5;
(b) the polynucleotide of claim 6;
(c) the expression vector of claim 7;
(d) a protein selected from the group consisting of GRIK2 and Claspin, a polynucleotide encoding the protein or a partial peptide of the protein including the antigen peptide represented by SEQ ID NO: 1 or 2, or an expression vector containing the polynucleotide;
(e) A pharmaceutical composition comprising, as an active ingredient, any one of cytotoxic T cells that specifically injure antigen-presenting cells that present the antigen peptide according to any one of claims 1 to 4 as an antigen. The pharmaceutical composition according to claim 12, comprising the antigenic peptide according to any one of claims 1 to 4 and/or the polyepitope peptide according to claim 5 as an active ingredient. The pharmaceutical composition according to claim 12 or 13, further comprising an adjuvant. The pharmaceutical composition according to any one of claims 12 to 14, which is a preventive and/or therapeutic agent for treatment-resistant bladder cancer. The pharmaceutical composition according to any one of claims 12 to 15, which is a preventive and/or therapeutic vaccine for treatment-resistant bladder cancer. The pharmaceutical composition according to any one of claims 12 to 16, which is used with an immune checkpoint inhibitor. An HLA multimer comprising the antigenic peptide according to any one of claims 1 to 4 and HLA. A diagnostic agent comprising the HLA multimer according to claim 18. A T-cell receptor-like antibody that recognizes the complex of the antigen peptide according to any one of claims 1 to 4 and HLA. A tumor-detecting agent containing the T-cell receptor-like antibody according to claim 20. A chimeric antigen receptor that recognizes a complex of the antigen peptide according to any one of claims 1 to 4 and HLA. An artificial CTL comprising a T-cell receptor that recognizes the complex of the antigen peptide according to any one of claims 1 to 4 and HLA. A bispecific antibody that specifically recognizes the complex of the antigen peptide according to any one of claims 1 to 4 and HLA and a lymphocyte surface antigen. A tumor cell detection agent comprising a detection agent for detecting an expression product of a gene selected from the group consisting of GRIK2 and Claspin. The tumor cell detection agent according to claim 25, for detecting tumor cells in a cell population containing cells derived from a bladder biological sample. The tumor cell detection agent according to claim 25 or 26, wherein the gene expression product is mRNA and/or endogenous polypeptide. The tumor cell detection agent according to any one of claims 25 to 27, wherein the expression product of the gene is mRNA and contains a probe and/or primer having a nucleotide sequence complementary to the gene. The tumor cell detection agent according to any one of claims 25 to 28, wherein the expression product of the gene is an endogenous polypeptide and contains a detection substance that specifically reacts with the endogenous polypeptide. The tumor cell detection agent according to claim 29, wherein the detection substance is an antibody. The HLA multimer of claim 18, which is a diagnostic agent for selecting a patient to be treated for whom a cancer treatment method using the pharmaceutical composition of any one of claims 12 to 17 is effective, Said diagnostic agent comprising the T-cell receptor-like antibody according to claim 20 and/or the tumor cell detection agent according to any one of claims 25-30. An antisense oligonucleotide against a gene selected from the group consisting of GRIK2 and Claspin. An siRNA comprising an antisense region complementary to a gene selected from the group consisting of GRIK2 and Claspin and a sense region at least partially complementary to the antisense region. A pharmaceutical composition comprising the antisense oligonucleotide of claim 32 and/or the siRNA of claim 33, and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 34, which is a preventive and/or therapeutic agent for treatment-resistant bladder cancer. A clone cell established from a cell population of human bladder cancer cell line UM-UC-3 containing cells with high aldehyde dehydrogenase enzyme activity (ALDH ^high ). A clone cell established from a cell population of human bladder cancer cell line UM-UC-3 containing cells with low aldehyde dehydrogenase enzyme activity (ALDH ^low ). The clone cell according to claim 36 or 37, wherein the cell population of human bladder cancer cell line UM-UC-3 is separated by the ALDEFLUOR method. The chimeric antigen receptor according to claim 22, further comprising a CD3ζ chain. The chimeric antigen receptor of claim 39, further comprising a co-stimulatory molecule. A genetically modified T cell into which the chimeric antigen receptor according to any one of claims 22, 39 and 40 has been introduced. A dendritic cell that presents a complex of the peptide according to any one of claims 1 to 4 and HLA on the cell surface. The tumor cell detection agent according to any one of claims 25 to 30, which is used for predicting the prognosis of bladder cancer.

Images