Method for selecting subject expected to receive effect of pharmaceutical composition for treating or preventing cancer

Abstract

To provide a method for selecting a subject for whom the effect of a pharmaceutical composition for treating or preventing cancer can be expected.SOLUTION: A step of determining an expression level of WT1mRNA using a sample collected from a subject, and a step of providing an indicator that the subject is a subject for whom the pharmaceutical composition is expected to be effective when the expression level of WT1mRNA is less than or equal to or less than 10000 copies / μ gRNA.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer. [Background technology]

[0002] Cellular immunity, particularly cytotoxic T cells (CTLs), plays an important role in the body's elimination of tumor cells, virus-infected cells, etc. CTLs are generated by the differentiation and proliferation of precursor T cells that recognize complexes between antigenic peptides (tumor antigen peptides) on tumor cells and MHC (Major Histocompatibility Complex) class I antigens, and attack tumor cells.

[0003] In humans, MHC is called human leukocyte antigen (HLA), and known types include HLA-A, B, and Cw. Tumor antigen peptides are generated when proteins highly expressed in tumors, i.e., tumor antigen proteins, are synthesized intracellularly and then degraded by proteases. The generated tumor antigen peptides bind to MHC class I antigens in the endoplasmic reticulum to form complexes, which are then transported to the cell surface and presented as antigens. Tumor-reactive CTLs recognize these presented tumor antigen peptides (killer peptides) and exert antitumor effects through cytotoxicity and lymphokine production.

[0004] The development of cancer immunotherapy agents (cancer vaccines) that utilize tumor antigen proteins or tumor antigen-derived killer peptides as the main components is currently being investigated, with the aim of enhancing cancer-specific CTLs in cancer patients. For example, cancer immunotherapy targeting WT1 (Wilm's tumor 1) is currently under development. WT1 is a transcription factor with a zinc finger structure and was identified as the gene responsible for Wilms' tumor, a type of pediatric renal cancer (see Non-Patent Document 1). Initially, the WT1 gene was considered to be a tumor suppressor gene, but subsequent studies have shown that it functions as an oncogene in hematopoietic tumors and solid tumors. Furthermore, it has been reported that the WT1 gene is highly expressed in many malignant tumors (see Non-Patent Document 2). WT1 is believed to be a novel tumor antigen protein in leukemia and solid tumors (see Non-Patent Document 3). Therefore, cancer vaccine therapy and dendritic cell therapy using the WT1 protein or peptides derived from the WT1 protein, TCR-like antibodies that recognize peptides derived from the WT1 protein and HLA complexes, and chimeric antigen receptor (CAR) gene-modified T cell therapy using TCR-like antibodies are currently under development.

[0005] Regarding the WT1 protein, for example, WT1 126-134 Peptide, WT1 235-243 Peptide, WT1 10-18 Peptide, WT1 187-195 Peptide, WT1 302-310 Peptides and WT1 37-45 Killer peptides that bind to and are presented by MHC class I, such as peptides, have been reported (see Patent Document 1, Patent Document 2, Non-Patent Documents 4 and 5).

[0006] In addition to CTLs, helper T (Th1) cells also play an important role in cancer immunotherapy. Generally, antigenic proteins are degraded in intracellular lysosomes, and a portion of the fragmented peptides, consisting of approximately 13 to 17 amino acid residues, binds to MHC class II molecules as antigenic peptides (helper peptides). Subsequently, the complex of the antigenic peptide and MHC class II molecule is presented to the TCR-CD3 complex, activating Th1 cells, which then promote the induction and activation of CTLs. Known examples of human MHC class II molecules include HLA-DR, DQ, and DP, and several helper peptides derived from the WT1 protein have been identified (see Non-Patent Documents 6 and 7).

[0007] To fully utilize the effects of cancer immunotherapy, it is important to predict the response of a subject in advance and select subjects who are likely to benefit from the treatment. However, while cancer immunotherapies targeting WT1 using killer peptides that bind to and are presented on MHC class I molecules and / or helper peptides that bind to MHC class II molecules are being developed, there have been no reports to date on a method for preselecting subjects who are likely to benefit from treatment or prevention using a WT1 peptide vaccine that uses antigen peptides derived from the WT1 antigen protein. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 00 / 06602 [Patent Document 2] International Publication No. 00 / 18795 [Non-patent literature]

[0009] [Non-Patent Document 1] Am J Hum Genet. 1993; 52: 192-203 [Non-patent document 2] Blood.1997;89:1405-1412 [Non-patent document 3] Immunogenetics. 2000; 51: 99-107 [Non-patent document 4] Clin Cancer Res. 2005; 11: 8799-807 [Non-Patent Document 5] Blood. 2008 Oct 1; 112(7): 2956-64 [Non-patent document 6] J Immunother. 2007; 30: 282-93 [Non-Patent Document 7] Cancer Immunol Immunother. 2010; 59: 1467-79 Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, an object of the present invention is to provide a method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, wherein the pharmaceutical composition comprises a WT1 killer peptide and / or a WT1 helper peptide. [Means for solving the problem]

[0011] As a result of intensive research aimed at solving the above problems, the present inventors discovered a method for selecting subjects who are expected to benefit from a pharmaceutical composition for treating or preventing cancer, based on the presence or absence of mutations in the tumor protein p53 (TP53) gene and / or the BCL6 co-repressor (BCOR) gene, the mRNA expression level of the WT1 gene, etc., and thus completed the present invention.

[0012] That is, for example, the present invention includes the following inventions. [1] A method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, comprising: Determining the presence or absence of a mutation in the TP53 gene and / or the BCOR gene using a sample collected from the subject; and providing an indication that, in the case of TP53 wild-type and / or BCOR wild-type, the subject is a subject who can be expected to benefit from the pharmaceutical composition; The pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof. [2] The method of [1], wherein the pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7) and VLDFAPPGA (SEQ ID NO: 9), or a pharmaceutically acceptable salt thereof. [3] The method according to [1] or [2], wherein the pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof. [4] The pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9); and The method according to any one of [1] to [3], which comprises a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof. [5] The pharmaceutical composition comprises a compound of formula (I): [ka] [where, X a and Y a represents a single bond, and tumor antigen peptide A has the following amino acid sequence: It represents a peptide consisting of any one of the amino acid sequences selected from RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9), and the amino group of the N-terminal amino acid of tumor antigen peptide A is Y in formula (1). a and the carbonyl group of the C-terminal amino acid of tumor antigen peptide A binds to the hydroxyl group in formula (1), R 1 represents a hydrogen atom or tumor antigen peptide B, Tumor antigen peptide B has a different sequence from tumor antigen peptide A and has the following amino acid sequence: The peptide has an amino acid sequence selected from CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), and the thioether group of the cysteine ​​residue of tumor antigen peptide B is bonded to the thioether group in formula (1). The method according to any one of [1] to [4], comprising a compound represented by the formula: [6] The method according to any one of [1] to [5], wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of RMFPNAPYL (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof. [7] The method according to any one of [1] to [5], wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of YMFPNAPYL (SEQ ID NO: 8) or a pharmaceutically acceptable salt thereof. [8] The method according to any one of [1] to [5], wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence C-CMTWNQMNL (C-C represents a disulfide bond, SEQ ID NO: 21) or a pharmaceutically acceptable salt thereof. [9] The method according to any one of [1] to [5], wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence C-CYTWNQMNL (the C-C bonds represent disulfide bonds, SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof.

[10] The method according to any one of [1] to [9], wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) or a pharmaceutically acceptable salt thereof.

[11] The method according to any one of [1] to [9], wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16) or a pharmaceutically acceptable salt thereof.

[12] The method according to any one of [1] to [9], wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[13] The compound represented by formula (1) is a compound represented by formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) or a pharmaceutically acceptable salt thereof.

[14] The compound represented by formula (1) is a compound represented by formula (3): [ka] (wherein the bond between C and C represents a disulfide bond) or a pharmaceutically acceptable salt thereof.

[15] The method according to any one of [1] to

[14] , wherein the pharmaceutical composition further comprises a peptide comprising an amino acid sequence selected from the group consisting of CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[16] The compound represented by formula (1) is a compound represented by formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) or a pharmaceutically acceptable salt thereof, The method according to [5], wherein the pharmaceutical composition further comprises WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[17] The compound represented by formula (1) is a compound represented by formula (3): [ka] (wherein the bond between C and C represents a disulfide bond) or a pharmaceutically acceptable salt thereof, The method according to [5], wherein the pharmaceutical composition further comprises WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[18] The method according to any one of [1] to

[17] , wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[19] The method according to any one of [1] to

[18] , which provides an indication that, in the case of wild-type TP53, the subject is a subject for whom the pharmaceutical composition can be expected to be effective.

[20] The method according to any one of [1] to

[19] , which provides an indication that the subject is a subject for whom the pharmaceutical composition can be expected to be effective, in the case of TP53 wild-type and BCOR wild-type. 〔twenty one〕 The method according to any one of [1] to

[20] , further comprising the steps of: determining the mRNA expression level of the WT1 gene using a sample collected from the subject; and, when the mRNA expression level of the WT1 gene is less than or equal to a reference value, providing an indication that the subject is one for whom the effects of the pharmaceutical composition can be expected. 〔twenty two〕 detecting WT1 antigen peptide-specific CD8 T cells using a sample collected from the subject to which the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[18] is administered; and The method of any of [1] to

[21] , further comprising the step of providing an indicator that the subject is one for whom the pharmaceutical composition can be expected to be effective, when the number of WT1 antigen peptide-specific CD8 T cells is increased compared to a sample collected from the subject before administration. 〔twenty three〕 The method of

[22] , wherein the step of detecting WT1 antigen peptide-specific CD8 T cells is carried out by reacting a complex of a WT1 peptide and an HLA molecule with the sample, and examining the presence or number of WT1 antigen peptide-specific CD8 T cells that recognize the complex contained in the sample. 〔twenty four〕 The method according to

[23] , wherein the complex of the WT1 peptide and the HLA molecule is in the form of a tetramer. 〔twenty five〕 The method according to

[23] or

[24] , wherein the HLA molecule is compatible with the HLA of the subject.

[26] The method according to any one of

[22] to

[25] , wherein the step of detecting WT1 antigen peptide-specific CD8 T cells comprises analysis by flow cytometry.

[27] The method according to any one of [1] to

[26] , further comprising the step of providing an indication that, when a delayed-type hypersensitivity reaction is detected in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of [1] to

[18] has been administered multiple times, the subject can be expected to benefit from the pharmaceutical composition.

[28] The method of

[27] further comprises the step of comparing a reaction at a site in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of [1] to

[18] has been administered with a reaction at a site in the subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof has not been administered, and providing an indication that the subject can be expected to benefit from the pharmaceutical composition if the difference between the reaction at the administered site and the reaction at the non-administered site is equal to or greater than a reference value.

[29] The method according to any one of [1] to

[28] , further comprising the step of providing an indication that the subject is a subject for whom the pharmaceutical composition can be expected to be effective, when the subject's karyotype based on the revised IPSS (IPSS-R) is other than Very Poor.

[30] The method according to any one of [1] to

[29] , further comprising the step of providing an indicator that the subject is one for whom the effects of the pharmaceutical composition can be expected, when the value obtained by dividing the percentage of myeloblasts in a sample collected from a subject to which the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[18] has been administered by the percentage of myeloblasts in a sample collected from the subject before the administration of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof is less than or equal to a reference value.

[31] The method according to any one of [1] to

[30] , wherein the sample is selected from the group consisting of body fluids, mucous membranes, cells, tissues, and cell or tissue cultures, and combinations thereof.

[32] The method according to any one of [1] to

[31] , wherein the cancer is selected from the group consisting of leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, and brain tumor.

[33] A step of selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer by the method according to any one of [1] to

[32] ; and A method for treating cancer, comprising the step of administering the pharmaceutical composition according to any one of [1] to

[18] to a selected subject.

[34] 1. A pharmaceutical composition for use in a method for treating cancer, comprising: a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15), and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof; The above-mentioned treatment method includes the steps of selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer by the method according to any one of [1] to

[33] ; A pharmaceutical composition comprising administering the pharmaceutical composition to a selected subject.

[35] A method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, comprising: determining the mRNA expression level of the WT1 gene using a sample collected from the subject; and providing an indicator that the subject is one for whom the pharmaceutical composition can be expected to be effective when the mRNA expression level of the WT1 gene is less than or equal to the reference value; The pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[36] The method of

[35] , wherein the pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7) and VLDFAPPGA (SEQ ID NO: 9), or a pharmaceutically acceptable salt thereof.

[37] The method according to

[35] or

[36] , wherein the pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.

[38] The pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9); and CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4) The method according to any one of

[35] to

[38] , wherein the method comprises a peptide comprising an amino acid sequence selected from the group consisting of:

[39] The pharmaceutical composition comprises a compound of formula (I): [ka] [where, X a and Y a represents a single bond, and tumor antigen peptide A has the following amino acid sequence: It represents a peptide consisting of any one of the amino acid sequences selected from RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9), and the amino group of the N-terminal amino acid of tumor antigen peptide A is Y in formula (1). a and the carbonyl group of the C-terminal amino acid of tumor antigen peptide A binds to the hydroxyl group in formula (1), R 1 represents a hydrogen atom or tumor antigen peptide B, Tumor antigen peptide B has a different sequence from tumor antigen peptide A and has the following amino acid sequence: The peptide has an amino acid sequence selected from CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), and the thioether group of the cysteine ​​residue of tumor antigen peptide B is bonded to the thioether group in formula (1). The method according to any one of

[35] to

[38] , comprising a compound represented by the following formula (I): or a pharmaceutically acceptable salt thereof.

[40] The method according to any one of

[35] to

[39] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of RMFPNAPYL (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof.

[41] The method according to any one of

[35] to

[39] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of YMFPNAPYL (SEQ ID NO: 8) or a pharmaceutically acceptable salt thereof.

[42] The method according to any one of

[35] to

[39] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence C-CMTWNQMNL (the C-C bonds represent disulfide bonds, SEQ ID NO: 21) or a pharmaceutically acceptable salt thereof.

[43] The method according to any one of

[35] to

[39] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence C-CYTWNQMNL (the C-C bonds represent disulfide bonds, SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof.

[44] The method according to any one of

[35] to

[43] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) or a pharmaceutically acceptable salt thereof.

[45] The method according to any one of

[35] to

[43] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16) or a pharmaceutically acceptable salt thereof.

[46] The method according to any one of

[35] to

[43] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[47] The compound represented by formula (1) is a compound represented by formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) or a pharmaceutically acceptable salt thereof.

[48] The compound represented by formula (1) is a compound represented by formula (3): [ka] (wherein the bond between C and C represents a disulfide bond), or a pharmaceutically acceptable salt thereof.

[49] The method according to any one of

[35] to

[48] , wherein the pharmaceutical composition further comprises a peptide consisting of an amino acid sequence selected from the group consisting of CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[50] The compound represented by formula (1) is a compound represented by formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) or a pharmaceutically acceptable salt thereof, The method according to

[39] , wherein the pharmaceutical composition further comprises WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[51] The compound represented by formula (1) is a compound represented by formula (3): [ka] (wherein the bond between C and C represents a disulfide bond) or a pharmaceutically acceptable salt thereof, The method according to

[39] , wherein the pharmaceutical composition further comprises WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[52] The method according to any one of

[35] to

[50] , wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[53] Determining the presence or absence of a mutation in the TP53 gene and / or the BCOR gene using a sample collected from the subject; and The method according to any one of

[35] to

[52] , further comprising the step of providing an indication that, in the case of TP53 wild-type and / or BCOR wild-type, the subject is a subject for whom the pharmaceutical composition can be expected to be effective.

[54] The method according to

[53] , which provides an indication that, in the case of wild-type TP53, the subject is one for whom the pharmaceutical composition can be expected to be effective.

[55] The method according to

[53] or

[54] , which provides an indication that the subject is a subject who can be expected to benefit from the pharmaceutical composition in the case of TP53 wild-type and BCOR wild-type.

[56] detecting WT1 antigen peptide-specific CD8 T cells using a sample collected from the subject to which the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof according to any one of

[35] to

[52] is administered; and The method of any of

[35] to

[55] , further comprising the step of providing an indicator that the subject is one in whom the pharmaceutical composition can be expected to be effective, when the number of WT1 antigen peptide-specific CD8 T cells is increased compared to a sample collected from the subject before administration.

[57] The method of

[56] , wherein the step of detecting WT1 antigen peptide-specific CD8 T cells is carried out by reacting a complex of a WT1 peptide and an HLA molecule with the sample, and examining the presence or number of WT1 antigen peptide-specific CD8 T cells that recognize the complex contained in the sample.

[58] The method according to

[57] , wherein the complex of the WT1 peptide and the HLA molecule is in the form of a tetramer.

[59] The method according to

[57] or

[58] , wherein the HLA molecule is compatible with the HLA of the subject.

[60] The method according to any one of

[56] to

[59] , wherein the step of detecting WT1 antigen peptide-specific CD8 T cells comprises analysis by flow cytometry.

[61] The method according to any one of

[35] to

[60] , further comprising the step of providing an indication that, when a delayed hypersensitivity reaction is detected in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of

[35] to

[52] has been administered multiple times, the subject is one for whom the effects of the pharmaceutical composition can be expected.

[62] The method of

[61] further comprises the step of comparing a reaction at a site in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of

[35] to

[52] has been administered with a reaction at a site in the subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof has not been administered, and providing an indication that the subject can be expected to benefit from the pharmaceutical composition if the difference between the reaction at the administered site and the reaction at the non-administered site is equal to or greater than a reference value.

[63] The method according to any one of

[35] to

[62] , further comprising the step of providing an indication that the subject is a subject for whom the pharmaceutical composition can be expected to be effective, when the subject's karyotype based on the revised IPSS (IPSS-R) is other than Very Poor.

[64] The method according to any one of

[35] to

[63] , further comprising the step of providing an indicator that the subject is one for whom the effects of the pharmaceutical composition can be expected, when the value obtained by dividing the percentage of myeloblasts in a sample collected from a subject to which the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof according to any one of

[35] to

[52] has been administered by the percentage of myeloblasts in a sample collected from the subject before the administration of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof is less than or equal to a reference value.

[65] The method according to any one of

[35] to

[64] , wherein the sample is selected from the group consisting of body fluids, mucous membranes, cells, tissues, and cell or tissue cultures, and combinations thereof.

[66] The method according to any one of

[35] to

[65] , wherein the cancer is selected from the group consisting of leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, and brain tumor.

[67] A step of selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer by the method according to any one of

[35] to

[66] ; and A method for treating cancer, comprising the step of administering to a selected subject the pharmaceutical composition according to any one of

[35] to

[52] .

[68] 1. A pharmaceutical composition for use in a method for treating cancer, comprising: a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15), and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof; The above-mentioned treatment method includes the steps of selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer by the method according to any one of

[35] to

[66] ; A pharmaceutical composition comprising administering the pharmaceutical composition to a selected subject.

[69] Use of the TP53 gene and / or the BCOR gene as a marker for providing an indication of whether or not a subject is expected to be effective in a pharmaceutical composition for treating or preventing cancer, The pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[70] The use according to

[69] , which provides an indicator of whether or not a subject is likely to benefit from a pharmaceutical composition for treating or preventing cancer, based on the presence or absence of a mutation in the gene.

[71] Use of the WT1 gene as a marker for providing an indicator of whether or not a subject is a candidate for whom a pharmaceutical composition for treating or preventing cancer can be expected to be effective, based on the level of expression of WT1 gene mRNA, The pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[72] The use according to

[71] , wherein when the mRNA expression level of the WT1 gene is less than or equal to the reference value, an indicator is provided that the subject is one for whom the effects of a pharmaceutical composition for treating or preventing cancer can be expected.

[73] A method for evaluating the effect of a candidate substance of a pharmaceutical composition for treating or preventing cancer, comprising: detecting WT1 antigen peptide-specific CD8 T cells using a sample collected from a subject administered with the pharmaceutical composition, or a peptide or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition; and providing an indicator that the candidate substance is expected to be effective in treating and preventing cancer when the number of WT1 antigen peptide-specific CD8 T cells is increased compared to a sample collected from the subject before administration; The pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[74] A method for evaluating the effect of a candidate substance of a pharmaceutical composition for treating or preventing cancer, comprising: and providing an indication that the candidate substance is expected to be effective in treating and preventing cancer when a delayed-type hypersensitivity reaction is detected in a subject to which the pharmaceutical composition or the peptide or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition has been administered multiple times; The pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[75] A method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, comprising: and providing an indication that the subject is a subject who can be expected to benefit from the pharmaceutical composition when the subject's karyotype based on the revised IPSS (IPSS-R) is other than Very Poor; The pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[76] The method of

[75] , wherein the pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7) and VLDFAPPGA (SEQ ID NO: 9), or a pharmaceutically acceptable salt thereof.

[77] The method according to

[75] or

[76] , wherein the pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.

[78] The pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9); and The method according to any one of

[75] to

[77] , comprising a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.

[79] The pharmaceutical composition comprises a compound of formula (I): [ka] [where, X a and Y a represents a single bond, and tumor antigen peptide A has the following amino acid sequence: It represents a peptide consisting of any one of the amino acid sequences selected from RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9), and the amino group of the N-terminal amino acid of tumor antigen peptide A is Y in formula (1). aand the carbonyl group of the C-terminal amino acid of tumor antigen peptide A binds to the hydroxyl group in formula (1), R 1 represents a hydrogen atom or tumor antigen peptide B, Tumor antigen peptide B has a different sequence from tumor antigen peptide A and has the following amino acid sequence: The peptide has an amino acid sequence selected from CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), and the thioether group of the cysteine ​​residue of tumor antigen peptide B is bonded to the thioether group in formula (1). The method according to any one of

[75] to

[78] , comprising a compound represented by the following formula (I): or a pharmaceutically acceptable salt thereof.

[80] The method according to any one of

[75] to

[79] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of RMFPNAPYL (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof.

[81] The method according to any one of

[75] to

[79] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of YMFPNAPYL (SEQ ID NO: 8) or a pharmaceutically acceptable salt thereof.

[82] The method according to any one of

[75] to

[79] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence C-CMTWNQMNL (the C-C bonds represent disulfide bonds, SEQ ID NO: 21) or a pharmaceutically acceptable salt thereof.

[83] The method according to any one of

[75] to

[79] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence C-CYTWNQMNL (the C-C bonds represent disulfide bonds, SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof.

[84] The method according to any one of

[75] to

[83] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) or a pharmaceutically acceptable salt thereof.

[85] The method according to any one of

[75] to

[83] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16) or a pharmaceutically acceptable salt thereof.

[86] The method according to any one of

[75] to

[83] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[87] The compound represented by formula (1) is a compound represented by formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) The method according to

[79] , wherein the compound according to [1] is a compound represented by the formula:

[88] The compound represented by formula (1) is a compound represented by formula (3): [ka] (wherein the bond between C and C represents a disulfide bond), or a pharmaceutically acceptable salt thereof.

[89] The method according to any one of

[75] to

[88] , wherein the pharmaceutical composition further comprises a peptide comprising an amino acid sequence selected from the group consisting of CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[90] The compound represented by formula (1) is a compound represented by formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) or a pharmaceutically acceptable salt thereof, The method of

[79] , wherein the pharmaceutical composition further comprises WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[91] The compound represented by formula (1) is a compound represented by formula (3): [ka] (wherein the bond between C and C represents a disulfide bond) or a pharmaceutically acceptable salt thereof, The method of

[79] , wherein the pharmaceutical composition further comprises WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[92] The method according to any one of

[75] to

[91] , wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[93] Determining the presence or absence of a mutation in the TP53 gene and / or the BCOR gene using a sample collected from the subject; and The method according to any one of

[75] to

[92] , further comprising the step of providing an indication that, in the case of TP53 wild-type and / or BCOR wild-type, the subject is a subject for whom the pharmaceutical composition can be expected to be effective.

[94] The method according to

[93] , which provides an indication that, in the case of wild-type TP53, the subject is one for whom the pharmaceutical composition can be expected to be effective.

[95] The method according to

[92] or

[93] , which provides an indication that the subject is a subject who can be expected to benefit from the pharmaceutical composition in the case of TP53 wild-type and BCOR wild-type.

[96] The method of any of

[75] to

[95] , further comprising the steps of: determining the mRNA expression level of the WT1 gene using a sample collected from the subject; and, when the mRNA expression level of the WT1 gene is less than or equal to a reference value, providing an indication that the subject is one for whom the effects of the pharmaceutical composition can be expected.

[97] detecting WT1 antigen peptide-specific CD8 T cells using a sample collected from the subject to which the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof according to any one of

[75] to

[92] is administered; and The method of any of

[75] to

[96] , further comprising the step of providing an indicator that the subject is one for whom the pharmaceutical composition can be expected to be effective, when the number of WT1 antigen peptide-specific CD8 T cells is increased compared to a sample collected from the subject before administration.

[98] The method of

[97] , wherein the step of detecting WT1 antigen peptide-specific CD8 T cells is carried out by reacting a complex of a WT1 peptide and an HLA molecule with the sample, and examining the presence or number of WT1 antigen peptide-specific CD8 T cells that recognize the complex contained in the sample.

[99] The method according to

[98] , wherein the complex of the WT1 peptide and the HLA molecule is in the form of a tetramer.

[100] The method according to

[98] or

[99] , wherein the HLA molecule is compatible with the HLA of the subject.

[101] The method of any one of

[97] to

[100] , wherein the step of detecting WT1 antigen peptide-specific CD8 T cells comprises analysis by flow cytometry.

[102] The method according to any one of

[75] to

[101] , further comprising the step of providing an indication that, when a delayed hypersensitivity reaction is detected in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of

[75] to

[92] has been administered multiple times, the subject is one for whom the effects of the pharmaceutical composition can be expected.

[103] The method of

[102] further comprises the step of comparing a reaction at a site in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of

[75] to

[92] has been administered with a reaction at a site in the subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof has not been administered, and providing an indication that the subject can be expected to benefit from the pharmaceutical composition if the difference between the reaction at the administered site and the reaction at the non-administered site is equal to or greater than a reference value.

[104] The method of any of

[75] to

[103] , further comprising the step of providing an indicator that the subject is one for whom the effects of the pharmaceutical composition can be expected, when the value obtained by dividing the percentage of myeloblasts in a sample collected from a subject to which the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof according to any of

[75] to

[92] has been administered by the percentage of myeloblasts in a sample collected from the subject before the administration of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof is less than or equal to a reference value.

[105] The method according to any one of

[75] to

[104] , wherein the sample is selected from the group consisting of body fluids, mucous membranes, cells, tissues, and cell or tissue cultures, and combinations thereof.

[106] The method according to any one of

[75] to

[105] , wherein the cancer is selected from the group consisting of leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, and brain tumor.

[107] A step of selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, by the method according to any one of

[75] to

[106] ; and A method for treating cancer, comprising the step of administering to a selected subject the pharmaceutical composition of any one of

[75] to

[92] .

[108] 1. A pharmaceutical composition for use in a method for treating cancer, comprising: a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15), and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof; The above-mentioned treatment method includes the steps of selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer by the method according to any one of

[75] to

[107] ; A pharmaceutical composition comprising administering the pharmaceutical composition to a selected subject.

[109] A method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, comprising: The pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15), and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof; providing an indicator that the subject is one for whom the effects of the pharmaceutical composition can be expected, when the value obtained by dividing the percentage of myeloblasts in a sample collected from the subject to which the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof has been administered by the percentage of myeloblasts in a sample collected from the subject before the administration of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof is less than or equal to a reference value.

[110] The method of

[109] , wherein the pharmaceutical composition comprises a peptide or a pharmaceutically acceptable salt thereof comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7) and VLDFAPPGA (SEQ ID NO: 9).

[111] The method according to

[109] or

[110] , wherein the pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.

[112] The pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9); and The method according to any one of

[109] to

[111] , comprising a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.

[113] The pharmaceutical composition comprises a compound of formula (I): [ka] [where, X a and Y a represents a single bond, and tumor antigen peptide A has the following amino acid sequence: It represents a peptide consisting of any one of the amino acid sequences selected from RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9), and the amino group of the N-terminal amino acid of tumor antigen peptide A is Y in formula (1). a and the carbonyl group of the C-terminal amino acid of tumor antigen peptide A binds to the hydroxyl group in formula (1), R 1 represents a hydrogen atom or tumor antigen peptide B, Tumor antigen peptide B has a different sequence from tumor antigen peptide A and has the following amino acid sequence: The peptide has an amino acid sequence selected from CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), and the thioether group of the cysteine ​​residue of tumor antigen peptide B is bonded to the thioether group in formula (1). The method according to any one of

[109] to

[112] , comprising a compound represented by the formula:

[114] The method according to any one of

[109] to

[113] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of RMFPNAPYL (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof.

[115] The method according to any one of

[109] to

[113] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of YMFPNAPYL (SEQ ID NO: 8) or a pharmaceutically acceptable salt thereof.

[116] The method according to any one of

[109] to

[113] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence C-CMTWNQMNL (the C-C bonds represent disulfide bonds, SEQ ID NO: 21) or a pharmaceutically acceptable salt thereof.

[117] The method according to any one of

[109] to

[113] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence C-CYTWNQMNL (the C-C bonds represent disulfide bonds, SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof.

[118] The method according to any one of

[109] to

[117] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) or a pharmaceutically acceptable salt thereof.

[119] The method according to any one of

[109] to

[117] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16) or a pharmaceutically acceptable salt thereof.

[120] The method according to any one of

[109] to

[117] , wherein the pharmaceutical composition comprises a peptide consisting of the amino acid sequence WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[121] The compound represented by formula (1) is a compound represented by formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) The method according to

[113] , wherein the compound according to [1] is a compound represented by the formula:

[122] The compound represented by formula (1) is a compound represented by formula (3): [ka] (wherein the bond between C and C represents a disulfide bond), or a pharmaceutically acceptable salt thereof. 〔one two three〕 The method according to any one of

[109] to

[122] , wherein the pharmaceutical composition further comprises a peptide comprising an amino acid sequence selected from the group consisting of CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[124] The compound represented by formula (1) is a compound represented by formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) or a pharmaceutically acceptable salt thereof, The method according to

[113] , wherein the pharmaceutical composition further comprises WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[125] The compound represented by formula (1) is a compound represented by formula (3): [ka] (wherein the bond between C and C represents a disulfide bond) or a pharmaceutically acceptable salt thereof, The method according to

[113] , wherein the pharmaceutical composition further comprises WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[126] The method according to any one of

[109] to

[125] , wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[127] Determining the presence or absence of a mutation in the TP53 gene and / or the BCOR gene using a sample collected from the subject; and The method according to any one of

[109] to

[126] , further comprising the step of providing an indication that, in the case of TP53 wild-type and / or BCOR wild-type, the subject is a subject for whom the pharmaceutical composition can be expected to be effective.

[128] The method according to

[127] , which provides an indication that, in the case of wild-type TP53, the subject is one for whom the pharmaceutical composition can be expected to be effective.

[129] The method according to

[127] or

[128] , which provides an indication that the subject is a subject who can be expected to benefit from the pharmaceutical composition in the case of TP53 wild-type and BCOR wild-type.

[130] The method of any of

[109] to

[129] , further comprising the steps of determining the mRNA expression level of the WT1 gene using a sample collected from the subject, and providing an indication that the subject is one for whom the pharmaceutical composition can be expected to be effective, if the mRNA expression level of the WT1 gene is less than or equal to a reference value.

[131] detecting WT1 antigen peptide-specific CD8 T cells using a sample collected from the subject to which the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof according to any one of

[109] to

[126] is administered; and The method of any of

[109] to

[130] , further comprising the step of providing an indicator that the subject is one in whom the pharmaceutical composition can be expected to be effective, when the number of WT1 antigen peptide-specific CD8 T cells is increased compared to a sample collected from the subject before administration.

[132] The method of

[131] , wherein the step of detecting WT1 antigen peptide-specific CD8 T cells is carried out by reacting a complex of a WT1 peptide and an HLA molecule with the sample, and examining the presence or number of WT1 antigen peptide-specific CD8 T cells that recognize the complex contained in the sample.

[133] The method according to

[132] , wherein the complex of the WT1 peptide and the HLA molecule is in the form of a tetramer.

[134] The method according to

[132] or

[133] , wherein the HLA molecule is compatible with the HLA of the subject.

[135] The method of any one of

[131] to

[134] , wherein the step of detecting WT1 antigen peptide-specific CD8 T cells comprises analysis by flow cytometry.

[136] The method according to any one of

[109] to

[135] , further comprising the step of providing an indication that, when a delayed hypersensitivity reaction is detected in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of

[109] to

[126] has been administered multiple times, the subject is one for whom the effects of the pharmaceutical composition can be expected.

[137] The method of

[136] further comprises the step of comparing a reaction at a site in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of

[109] to

[126] has been administered with a reaction at a site in the subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof has not been administered, and providing an indication that the subject can be expected to benefit from the pharmaceutical composition if the difference between the reaction at the administered site and the reaction at the unadministered site is equal to or greater than a reference value.

[138] The method according to any one of

[109] to

[137] , further comprising the step of providing an indication that the subject is a subject for whom the pharmaceutical composition can be expected to be effective, when the subject's karyotype based on the revised IPSS (IPSS-R) is other than Very Poor.

[139] The method according to any one of

[109] to

[138] , wherein the sample is selected from the group consisting of body fluids, mucous membranes, cells, tissues, and cell or tissue cultures, and combinations thereof.

[140] The method according to any one of

[109] to

[139] , wherein the cancer is selected from the group consisting of leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, and brain tumor.

[141] A step of selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer by the method according to any one of

[109] to

[140] ; and A method for treating cancer, comprising the step of administering to a selected subject the pharmaceutical composition of any one of

[109] to

[126] .

[142] 1. A pharmaceutical composition for use in a method for treating cancer, comprising: a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15), and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof; The above-mentioned treatment method includes the steps of selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer by the method according to any one of

[109] to

[140] ; A pharmaceutical composition comprising administering the pharmaceutical composition to a selected subject.

[143] Determining the presence or absence of a mutation in the TP53 gene and / or the BCOR gene using a sample collected from the subject; and administering to a subject who is TP53 wild-type and / or BCOR wild-type an effective amount of a pharmaceutical composition for treating or preventing cancer; A method for treating cancer, wherein the pharmaceutical composition comprises a peptide or a pharmaceutically acceptable salt thereof having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16).

[144] determining the mRNA expression level of the WT1 gene using a sample collected from the subject; and administering an effective amount of a pharmaceutical composition for treating or preventing cancer to a subject whose mRNA expression level of the WT1 gene is lower than or equal to a reference value, A method for treating cancer, wherein the pharmaceutical composition comprises a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

[145] administering to a subject an effective amount of a pharmaceutical composition for treating or preventing cancer when the subject's karyotype based on the revised IPSS (IPSS-R) is other than Very Poor; A method for treating cancer, wherein the pharmaceutical composition comprises a peptide or a pharmaceutically acceptable salt thereof having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16).

[146] RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAY A method for treating cancer, comprising the step of administering to a subject an effective amount of a pharmaceutical composition for treating or preventing cancer when the percentage of myeloblasts in a sample collected from a subject to which a pharmaceutical composition containing a peptide or a pharmaceutically acceptable salt thereof comprising an amino acid sequence selected from the group consisting of GSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or the peptide or a pharmaceutically acceptable salt thereof, is divided by the percentage of myeloblasts in a sample collected from the subject before administration of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof, and the value obtained is less than or equal to a standard value. [Effects of the Invention]

[0013] The present invention provides a method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer. The pharmaceutical composition contains a WT1 killer peptide and / or a WT1 helper peptide, or a pharmaceutically acceptable salt thereof. By selecting a subject who is expected to benefit from each pharmaceutical composition, the pharmaceutical composition can be used to effectively treat or prevent cancer. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows the results of a comparison between the test results of the WT1 peptide cocktail vaccine and the control (BSC) in the ONTIME study of Rigosertib. [Figure 2]Survival curves for TP53 wild-type and BCOR wild-type, and TP53 mutant or BCOR mutant, are shown. [Figure 3] 1 shows the results of comparing survival curves based on whether WT1 antigen peptide-specific immune responses are positive or negative for TP53 wild-type and BCOR wild-type, and TP53 mutant or BCOR mutant. [Figure 4] 1 shows the results of comparing survival curves based on the expression level of WT1 mRNA in Example 5 (Results (1)). [Figure 5] 1 shows the results (Results (1)) of comparing survival curves based on whether the WT1 antigen peptide-specific immune response was positive or negative with respect to the expression level of WT1 mRNA. [Figure 6] 1 shows the results of comparing survival curves based on the expression level of WT1 mRNA in Example 5 (Results (2)). [Figure 7] 1 shows the results (Results (2)) of comparing survival curves based on whether the WT1 antigen peptide-specific immune response was positive or negative with respect to the expression level of WT1 mRNA. [Figure 8] The results of a two-way analysis of the results of the HLA tetramer assay and the DTH test using a WT1 killer peptide conjugate are shown. [Figure 9] The graph shows the results of comparing survival curves for positive or negative WT1 antigen peptide-specific immune responses and stabilization of myeloblasts. [Figure 10] 1 shows the results of comparing the transition period to acute myeloid leukemia (AML) between positive and negative WT1 antigen peptide-specific immune responses. [Figure 11] 1 shows the results of comparing survival curves based on whether the WT1 antigen peptide-specific immune response was positive or negative. [Figure 12] 1 shows the results of comparing survival curves based on whether WT1 antigen peptide-specific immune responses were positive or negative for IPSS-R karyotypes. [Figure 13] The median survival time for each gender difference is compared with the median survival time for BSC in historical data and the Rigosertib study. [Figure 14]1 shows the results of comparing survival curves based on the expression level of WT1 mRNA in Example 11. [Figure 15] 1 shows the results of comparing survival curves based on whether the WT1 antigen peptide-specific immune response was positive or negative, with respect to the expression level of WT1 mRNA. [Figure 16] 1 shows the relationship between the level of WT1 mRNA expression in peripheral blood and that in bone marrow fluid. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described in detail.

[0016] As used herein, the term "amino acid residue" refers to a portion of a peptide or protein molecule that corresponds to one unit of an amino acid constituting the peptide or protein. Examples of "amino acid residues" include natural or unnatural α-amino acid residues, β-amino acid residues, γ-amino acid residues, and δ-amino acid residues. Specific examples include natural α-amino acid residues, ornithine residues, homoserine residues, homocysteine ​​residues, β-alanine, γ-aminobutanoic acid, and δ-aminopentanoic acid. When the "amino acid residue" may be optically active, it may be either the L-form or the D-form, with the L-form being preferred.

[0017] When "amino acid residues" are abbreviated in this specification, they are written using the following abbreviations. Ala or A: alanine residue Arg or R: arginine residue Asn or N: asparagine residue Asp or D: aspartic acid residue Cys or C: cysteine ​​residue Gln or Q: glutamine residue Glu or E: glutamic acid residue Gly or G: glycine residue His or H: histidine residue Ile or I: isoleucine residue Leu or L: leucine residue Lys or K: lysine residue Met or M: methionine residue Phe or F: phenylalanine residue Pro or P: proline residue Ser or S: serine residue Thr or T: threonine residue Trp or W: tryptophan residue Tyr or Y: tyrosine residue Val or V: valine residue Abu: 2-aminobutyric acid residue (also called α-aminobutyric acid residue) Orn: ornithine residue Cit: citrulline residue

[0018] In this specification, the amino acid sequence of a "peptide" is written according to the conventional method, with the N-terminal amino acid residue located on the left and the C-terminal amino acid residue located on the right. Furthermore, in a "peptide," unless otherwise specified, the amino group of the N-terminal amino acid residue is bonded to a hydrogen atom, and the carbonyl group of the C-terminal amino acid residue is bonded to a hydroxyl group. A divalent group of a peptide refers to a group bonded via the amino group of the N-terminal amino acid residue and the carbonyl group of the C-terminal amino acid residue.

[0019] In the compounds herein, for example, in the compounds represented by formulas (2) to (3), and also in peptides corresponding to their partial structures, unless otherwise specified, the amino group of the N-terminal amino acid residue is bonded to a hydrogen atom, and the carbonyl group of the C-terminal amino acid residue is bonded to a hydroxyl group.

[0020] In this specification, "R 1 " represents a hydrogen atom or tumor antigen peptide B, and preferably tumor antigen peptide B. R 1 For compounds of formula (1) in which R is a hydrogen atom, the sequence is not completely identical to the partial sequence of the WT1 protein. 1The compound of formula (1) in which is a hydrogen atom has a cysteine ​​residue added to the N-terminus of tumor antigen peptide A, and is therefore not a partial peptide consisting of 8 to 35 consecutive amino acid residues in the amino acid sequence of human WT1 set forth in SEQ ID NO: 1.

[0021] R 1 Examples of compounds of formula (1) in which is a hydrogen atom include the following amino acid sequences: CRMFPNAPYL (SEQ ID NO: 40), CCMTWNQMNL (SEQ ID NO: 41), CCYTWNQMNL (SEQ ID NO: 42), CALLPAVPSL (SEQ ID NO: 43), CSLGEQQYSV (SEQ ID NO: 44) and CRVPGVAPTL (sequence number: 45).

[0022] In this specification, "X a " and "Y a " independently represents a single bond or a divalent group of a peptide consisting of 1 to 4 amino acid residues. a and the number of amino acid residues in Y a The sum of the numbers of amino acid residues in X is an integer of 0 to 4. For example, the sum is an integer of 0 when X a and Y a is a single bond. In addition, when the sum is an integer of 4, for example, X a and Y a are independently divalent radicals of peptides consisting of two amino acid residues, X a is a divalent radical of a peptide consisting of three amino acid residues, and Y a is a divalent group of a peptide consisting of one amino acid residue, X a is a divalent radical of a peptide consisting of four amino acid residues, and Y a is a single bond.

[0023] The integer of the sum is preferably 0 to 2, more preferably 0 to 1, and most preferably 0. That is, X a and Ya It is most preferable that both of them are single bonds.

[0024] If the sum is an integer of 2, then X a is a divalent radical of a peptide consisting of two amino acid residues, and Y a If is a single bond, X a and Y a are independently divalent radicals of a peptide consisting of one amino acid residue, or X a is a single bond and Y a is a divalent group of a peptide consisting of two amino acid residues.

[0025] If the sum is 1, then X a is a divalent radical of a peptide consisting of one amino acid residue, and Y a is a single bond, or X a is a single bond and Y a is a divalent group of a peptide consisting of one amino acid residue. a is a single bond and Y a is an alanine residue, a leucine residue, or a methionine residue.

[0026] In this embodiment, the pharmaceutical composition contains specific WT1 killer peptides and / or WT1 helper peptides or pharmaceutically acceptable salts thereof, i.e., RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), C The pharmaceutical composition of this embodiment includes a peptide comprising an amino acid sequence selected from the group consisting of NKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15), and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof. This does not preclude the pharmaceutical composition of this embodiment from containing a peptide or a pharmaceutically acceptable salt thereof other than those described above, and the pharmaceutical composition may further contain a peptide other than those described above, for example, another WT1 killer peptide and / or a WT1 helper peptide.

[0027] As used herein, the term "WT1 peptide" refers to a peptide that includes a portion consisting of consecutive amino acids present in the amino acid sequence of human WT1 set forth in SEQ ID NO:1.

[0028] The WT1 killer peptide means an MHC class I-restricted WT1 peptide. As used herein, "MHC class I restriction" refers to the property of inducing CTLs by binding to MHC class I molecules, which are class I major histocompatibility complex (MHC) antigens. "MHC class I-restricted WT1 peptide" refers to a peptide that binds to MHC class I antigens in vitro and / or in vivo and is presented as a complex, and that induces CTLs as a result of the complex being recognized by precursor T cells.

[0029] In humans, MHC is called human leukocyte antigen (HLA). HLA, which corresponds to MHC class I molecules, is classified into subtypes such as HLA-A, B, Cw, F, and G. "MHC class I restriction" preferably includes HLA-A restriction, HLA-B restriction, and HLA-Cw restriction.

[0030] Polymorphisms (alleles) are known for each HLA subtype. Examples of HLA-A polymorphisms include 27 or more types, such as HLA-A1, HLA-A2 (A0201, A0206, etc.), and HLA-A24. Examples of HLA-B polymorphisms include 59 or more types, such as HLA-B7, HLA-B40, and HLA-B4403. Examples of HLA-Cw polymorphisms include 10 or more types, such as HLA-Cw0301, HLA-Cw0401, and HLA-Cw0602. Among these polymorphisms, HLA-A2 and HLA-A24 are preferred.

[0031] An MHC class I-restricted WT1 peptide (WT1 killer peptide) is also referred to as an "MHC class I-restricted WT1 epitope." As used herein, the term "MHC class I-restricted WT1 epitope" refers to the peptide itself that binds to an MHC class I antigen and is presented as a complex. Specifically, the MHC class I-restricted WT1 peptide generates an MHC class I-restricted WT1 epitope in vitro and / or in vivo by intracellular degradation of the conjugate (proteolysis, reductive cleavage of disulfide bonds) by proteosomes and / or proteases such as gamma-interferon-inducible lysosomal thiol reductase (GILT, GLT) and / or cleavage to an optimal number of residues (also referred to as trimming) by endoplasmic reticulum aminopeptidase 1 (ERAP1, ER-aminopeptidase 1). In this generation, the main thought is that the C-terminal amino acid of the MHC class I-restricted WT1 epitope is first generated as a result of degradation by proteosomes and / or proteases, and then the N-terminal amino acid of the MHC class I-restricted WT1 epitope is generated as a result of trimming (cleavage) by ERAP1. However, this generation may also involve processes other than this generation process. ERAP1 is currently also called ERAAP (ER aminopeptidase associated with antigen presentation), and was formerly called A-LAP, PILS-AP, or ARTS-1.

[0032] Therefore, the MHC class I-restricted WT1 peptide is preferably a peptide consisting of amino acids generated by adding an amino acid to the carbonyl group of the C-terminal amino acid of the MHC class I-restricted WT1 epitope.

[0033] The length of the WT1 killer peptide is not particularly limited as long as it functions as a WT1 killer peptide, and may be, for example, 7 to 30, 7 to 15, 8 to 12, 8 to 11, 8, or 9 amino acid residues, or a conjugate thereof. The WT1 killer peptide may be 7 or more or 8 or more amino acid residues, or a conjugate thereof, or 30 or less, 25 or less, 22 or less, 20 or less, 18 or less, 15 or less, 12 or less, 11 or less, 10 or less, or 9 or less amino acid residues, or a conjugate thereof.

[0034] Examples of WT1 killer peptides include peptides containing the amino acid sequences set forth in RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), and CYTWNQMNL (SEQ ID NO: 4), or peptides containing modified amino acid sequences containing modified amino acid residues in any of the amino acid sequences selected from SEQ ID NOs: 1 to 9 and having CTL-inducing activity. The pharmaceutical composition in this embodiment may contain, for example, a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof. Furthermore, the pharmaceutical composition in this embodiment may comprise, for example, a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9), which correspond to HLA subtype A2 type (A-0201, A0206, etc.), or a pharmaceutically acceptable salt thereof; or may comprise, for example, a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), which correspond to HLA subtype A24 type (A-2402, etc.), or a pharmaceutically acceptable salt thereof.Furthermore, the pharmaceutical composition in this embodiment may, for example, comprise a peptide consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof, a peptide consisting of the amino acid sequence YMFPNAPYL (SEQ ID NO: 8) or a pharmaceutically acceptable salt thereof, a peptide consisting of the amino acid sequence C-CYTWNQMNL (CC represents a disulfide bond, SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof, or a peptide consisting of the amino acid sequence C-CMTWNQMNL (CC represents a disulfide bond, SEQ ID NO: 21) or a pharmaceutically acceptable salt thereof.

[0035] As used herein, a "peptide comprising an amino acid sequence" encompasses a peptide consisting of the amino acid sequence, as well as a peptide in which additional amino acids are added to the N-terminal amino acid and / or C-terminal amino acid of the amino acid sequence. When an "MHC class I-restricted WT1 peptide" is added, the peptide is preferably added to the C-terminus. When an "MHC class I-restricted WT1 epitope" is added, the peptide is preferably added to the C-terminus.

[0036] In the present invention, a "peptide comprising a modified amino acid sequence containing modified amino acid residues in the amino acid sequence and having CTL-inducing activity" is also referred to as a "modified killer peptide." The modified killer peptide refers to a peptide consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted, and / or added, which binds to MHC class I, and induces CTLs. In the case of a peptide consisting of 9 amino acid residues, the substitution positions of the substituted amino acids include positions 1 (N-terminus), 2, 3, and 9. The number of added (including inserted) amino acids is preferably 1 or 2, more preferably 1. A preferred addition position is the C-terminus. The number of deleted amino acids is preferably 1. In the modification, the added or substituted amino acid may be a non-natural amino acid other than the 20 amino acids encoded by genes.

[0037] It is known that there is a regularity (binding motif) in the amino acid sequence of peptides that can bind to HLA antigens for each polymorphism of HLA subtypes. For example, it is known that, as a binding motif for HLA-A24, in a peptide consisting of 8 to 11 amino acid residues, the amino acid at position 2 is Tyr, Phe, Met, or Trp, and the amino acid at the C-terminus is Phe, Leu, Ile, Trp, or Met (J. Immunol., 152, p3913, 1994; J. Immunol., 155, p4307, 1994; Immunogenetics, 41, p178, 1995). Therefore, for example, in the case of a peptide consisting of 9 amino acid residues, position 2 can be substituted with Tyr, Phe, Met, or Trp, and / or position 9 can be substituted with Phe, Leu, Ile, Trp, or Met, and peptides with such substitutions are preferred as modified killer peptides. Similarly, HLA-A * It is known that the binding motif of 02:01 is a peptide consisting of 8 to 11 amino acid residues, in which the amino acid at position 2 is Leu or Met and the C-terminal amino acid is Val or Leu. Therefore, for example, in the case of a peptide consisting of 9 amino acid residues, position 2 can be substituted with Leu or Met and / or position 9 with Val or Leu, and peptides with such substitutions are preferred as modified killer peptides.

[0038] Examples of modified killer peptides include the following peptides: A modified killer peptide of RMFPNAPYL (SEQ ID NO: 2) RYFPNAPYL (SEQ ID NO: 22) (see WO 03 / 106682); FMFPNAPYL (SEQ ID NO: 23), RLFPNAPYL (SEQ ID NO: 24), RMMPNAPYL (SEQ ID NO: 25), RMFPNAPYV (SEQ ID NO: 26) or YMFPNAPYL (SEQ ID NO: 8) (see WO 2009 / 072610); A modified killer peptide of CMTWNQMNL (SEQ ID NO: 3) CYTWNQMNL (SEQ ID NO: 4) (see WO 02 / 79253); Xaa-Met-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 27) (Xaa in this sequence represents Ser or Ala) or Xaa-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 28) (In this sequence, Xaa represents Ser, Ala, Abu, Arg, Lys, Orn, Cit, Leu, Phe, or Asn) (see WO 2004 / 026897); A modified killer peptide of ALLPAVPSL (SEQ ID NO: 5) AYLPAVPSL (SEQ ID NO: 29) (see WO 2003 / 106682); SLGEQQYSV (SEQ ID NO: 6) is a modified killer peptide FLGEQQYSV (SEQ ID NO: 30), SMGEQQYSV (SEQ ID NO: 31) or SLMEQQYSV (SEQ ID NO: 32) (see WO 2009 / 072610); or A modified killer peptide of RVPGVAPTL (SEQ ID NO: 7) RYPGVAPTL (SEQ ID NO: 33) (see WO 2003 / 106682).

[0039] From the viewpoint of covering a wide range of HLA subtypes, the pharmaceutical composition of this embodiment preferably contains a plurality of peptides corresponding to different HLA subtypes. For example, it preferably contains both the above peptide corresponding to HLA subtype A2 or a pharmaceutically acceptable salt thereof and the above peptide corresponding to HLA subtype A24 or a pharmaceutically acceptable salt thereof. In such a case, for example, the pharmaceutical composition may contain a peptide represented by formula (I): [ka] [where, X a and Y arepresents a single bond, and tumor antigen peptide A has the following amino acid sequence: RMFPNAPYL (SEQ ID NO: 2), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), YMFPNAPYL (SEQ ID NO: 8), and VLDFAPPGA (SEQ ID NO: 9), wherein the amino group of the N-terminal amino acid of tumor antigen peptide A is Y in formula (1). a and the carbonyl group of the C-terminal amino acid of tumor antigen peptide A binds to the hydroxyl group in formula (1), R 1 represents a hydrogen atom or tumor antigen peptide B, Tumor antigen peptide B has a different sequence from tumor antigen peptide A and has the following amino acid sequence: The peptide has an amino acid sequence selected from CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), and the thioether group of the cysteine ​​residue of tumor antigen peptide B is bonded to the thioether group in formula (1). or a pharmaceutically acceptable salt thereof.

[0040] The compound represented by the above formula (I) has excellent stability against oxidizing agents in solution due to the formation of a disulfide bond between the cysteine ​​residues, and has a certain quality as a pharmaceutical raw material.

[0041] Furthermore, when the pharmaceutical composition of this embodiment contains the compound represented by formula (I) (WT1 killer peptide conjugate) (R 1is a hydrogen atom), the conjugate is decomposed by reductive cleavage of the disulfide bond between the N-terminal cysteine ​​residues by ERAP1 in vivo, and two types of epitopes corresponding to different HLA subtypes are generated. A conjugate in which multiple types of epitopes corresponding to different HLA subtypes are generated in vivo, such as the conjugate represented by formula (I), can widely correspond to different HLA subtypes depending on the subject, and can cover a large population with one conjugate, so that CTLs can be efficiently induced in the subject (see WO 2014 / 157692).

[0042] In this embodiment, the "tumor antigen peptide A" is an MHC class I-restricted WT1 peptide consisting of 7 to 30 amino acid residues. In formula (1), tumor antigen peptide A is a peptide in which the amino group of the N-terminal amino acid is Y in formula (1). a and the carbonyl group of the C-terminal amino acid binds to the hydroxyl group in formula (1).

[0043] The compound represented by the above formula (1) can also be represented by the formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) It may be a compound represented by Formula (3): [ka] (wherein the bond between C and C represents a disulfide bond).

[0044] The compound represented by the above formula (1) can also be represented by the formula (2): [ka] (In the formula, the bond between C and C represents a disulfide bond.) or a pharmaceutically acceptable salt thereof, The pharmaceutical composition may further comprise WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof; The compound represented by formula (1) is a compound represented by formula (3): [ka] (wherein the bond between C and C represents a disulfide bond) or a pharmaceutically acceptable salt thereof, The pharmaceutical composition may further comprise WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[0045] The pharmaceutical composition of this embodiment may further comprise a WT1 helper peptide. When the pharmaceutical composition of this embodiment comprises a peptide comprising an amino acid sequence selected from the group consisting of CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15), and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), the pharmaceutical composition may further comprise a peptide comprising another amino acid sequence selected from the above group and / or another WT1 helper peptide.

[0046] The WT1 helper peptide means an MHC class II-restricted WT1 peptide. As used herein, the term "MHC class II restriction" refers to the property of inducing helper T cells by binding to MHC class II molecules.

[0047] HLA, which corresponds to MHC class II molecules, is classified into subtypes such as HLA-DR, DQ, and DP. "MHC class II restriction" preferably includes HLA-DR restriction, HLA-DQ restriction, or HLA-DP restriction.

[0048] As used herein, the term "MHC class II-restricted WT1 peptide" refers to a peptide that binds to an MHC class II antigen in vitro and / or in vivo and induces helper T cells.

[0049] The length of the WT1 helper peptide is not particularly limited as long as it functions as a WT1 helper peptide, and may be, for example, 7 to 30 or 14 to 30 amino acid residues. The WT1 helper peptide may be composed of 7 or more, 8 or more, 10 or more, 12 or more, or 14 or more amino acid residues, or 30 or less, 25 or less, 22 or less, or 20 or less amino acid residues.

[0050] Examples of WT1 helper peptides include CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15), WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 17) (see WO 2007 / 120673), RSDELVRH Examples include peptides comprising the amino acid sequences of HNMHQRNMTKL (SEQ ID NO: 18) (see WO 2007 / 120673), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 19) (see WO 2007 / 120673), and KRYFKLSHLQMHSRKH (SEQ ID NO: 20) (see WO 2005 / 045027), or peptides comprising a modified amino acid sequence containing an amino acid residue modification in any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 11 to 20, and having helper T cell-inducing activity. The pharmaceutical composition in this embodiment may contain, for example, a peptide comprising an amino acid sequence selected from the group consisting of CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof. Furthermore, the pharmaceutical composition in this embodiment may, for example, comprise a peptide consisting of the amino acid sequence of CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) or a pharmaceutically acceptable salt thereof, or may comprise a peptide consisting of the amino acid sequence of WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16) or a pharmaceutically acceptable salt thereof, or may comprise a peptide consisting of the amino acid sequence of WAPVLDFAPPGASAYGSL (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof.

[0051] As described above, the term "peptide containing an amino acid sequence" refers to a peptide consisting of the amino acid sequence and a peptide in which additional amino acids are added to the N-terminal amino acid and / or C-terminal amino acid of the amino acid sequence. The WT1 helper peptide may contain one or more cysteine ​​residues in the amino acid sequence. For example, when cysteine ​​residues are added to the amino acid sequence, they may be added to the N-terminal and / or C-terminal sides of the amino acid sequence.

[0052] As used herein, a "peptide comprising a modified amino acid sequence containing modified amino acid residues in the amino acid sequence and having helper T cell-inducing activity" is also referred to as a "modified helper peptide." The modified helper peptide refers to a peptide consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted, and / or added, and which binds to MHC class II and induces helper T cells. The number of added (including inserted) amino acids is preferably 1 to 3. The number of deleted amino acids is preferably 1 to 5. In the modification, the added or substituted amino acids may be unnatural amino acids other than the 20 amino acids encoded by genes.

[0053] Examples of modified helper peptides include the following peptides: A modified helper peptide of SGQARMFPNAPYLPSCLES (SEQ ID NO: 34) SGQAYMFPNAPYLPSCLES (SEQ ID NO: 35) (see Patent Document 6), SGQARMFPNAPYLPSC (SEQ ID NO: 36) or SGQAYMFPNAPYLPSC (SEQ ID NO: 37); or A modified helper peptide of PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 19) PGCNKRYFKLSHLQMHSRK (SEQ ID NO: 38), PGCNKRYFKLSHLQMHSRKH (SEQ ID NO: 39), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12) or CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13).

[0054] The peptide or compound of this embodiment can be produced by the method described in the Examples herein or by a method similar to that used in conventional peptide synthesis. Examples of production methods include those described in literature (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1985; Pharmaceutical Development, Continued, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991). For a method for producing the compound represented by formula (1), see, for example, International Publication No. WO 2014 / 157692.

[0055] For example, there are mentioned a production method using a solid-phase synthesizer by the Fmoc method or the Boc method, and a production method in which Boc-amino acids or Z-amino acids are successively condensed by a liquid-phase synthesis method (Fmoc represents a 9-fluorenylmethoxycarbonyl group, Boc represents a t-butoxycarbonyl group, and Z represents a benzyloxycarbonyl group, respectively).

[0056] In the intermediates for producing the peptide or compound of this embodiment, functional groups such as amino, carboxy, and mercapto groups can be protected with and deprotected from suitable protecting groups using protection and deprotection techniques as needed. Suitable protecting groups, protection methods, and deprotection methods are described in detail in "Protective Groups in Organic Synthesis 2nd Edition (John Wiley & Sons, Inc.; 1990)" and the like. For example, protecting groups for mercapto groups include acetamidomethyl and trityl groups.

[0057] When the peptide or compound of this embodiment has a disulfide bond, the disulfide bond can be formed between two different peptides containing cysteine ​​residues, or between a peptide containing a cysteine ​​residue and cysteine, according to a method commonly used in peptide chemistry. Examples of methods for forming disulfide bonds include those described in literature (e.g., Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1985; Pharmaceutical Development, Continued, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991).

[0058] Specifically, when a peptide contains one cysteine ​​residue, a compound having a disulfide bond (disulfide compound) can be produced by removing all protecting groups, including the protecting group for the mercapto group on the cysteine ​​side chain, followed by oxidation in an inert solvent. Alternatively, a disulfide compound can be produced by mixing two intermediates having a mercapto group in an appropriate solvent and oxidizing the mixture. The oxidation method can be selected from known methods for forming disulfide bonds in conventional peptide synthesis. Examples include iodine oxidation, air oxidation under alkaline conditions, and the addition of an oxidizing agent under alkaline or acidic conditions to form disulfide bonds. Examples of oxidizing agents include iodine, dimethyl sulfoxide (DMSO), and potassium ferricyanide. Examples of solvents that can be used include water, acetic acid, methanol, chloroform, DMF, DMSO, and mixtures thereof. Oxidation often yields a mixture of symmetric and asymmetric disulfide compounds. The desired asymmetric disulfide compound can be obtained by purification using various chromatography techniques or recrystallization. Alternatively, selective disulfide bonds can be formed by mixing an intermediate having an activated mercapto group with another intermediate having a mercapto group. Examples of intermediates having an activated mercapto group include a mercapto group bonded to an Npys group (3-nitro-2-pyridinesulfenyl group). Alternatively, selective disulfide bonds can be formed by first activating the mercapto group by mixing one intermediate with, for example, 2,2'-dithiobis(5-nitropyridine), and then adding the other intermediate (Tetrahedron Letters, Vol. 37, No. 9, pp. 1347-1350).

[0059] The same method can be used when a peptide contains two or more cysteine ​​residues. In this case, isomers with different disulfide bond patterns can be obtained. By using a specific combination of protecting groups for the cysteine ​​side chains, a dimer in which a disulfide bond is formed between the desired cysteine ​​residues can be obtained. Examples of such protecting group combinations include MeBzl (methylbenzyl) and Acm (acetamidomethyl), Trt (trityl) and Acm, Npys (3-nitro-2-pyridylthio) and Acm, and S-Bu-t (S-tert-butyl) and Acm. For example, in the case of a combination of MeBzl and Acm, one method involves first removing all protecting groups other than the MeBzl group and the cysteine ​​side chain, then subjecting a solution containing the peptide monomer to air oxidation to form a disulfide bond between the deprotected cysteine ​​residues, followed by deprotection and oxidation with iodine to form a disulfide bond between the cysteine ​​residues previously protected with Acm.

[0060] The resulting peptide or compound of this embodiment can be purified according to methods known to those skilled in the art or methods commonly used in peptide chemistry. For example, purification can be achieved by various types of chromatography (e.g., silica gel column chromatography, ion exchange column chromatography, gel filtration, or reverse-phase chromatography) or recrystallization. Examples of recrystallization solvents that can be used include alcoholic solvents such as methanol, ethanol, or 2-propanol; ether solvents such as diethyl ether; ester solvents such as ethyl acetate; aromatic hydrocarbon solvents such as benzene or toluene; ketone solvents such as acetone; hydrocarbon solvents such as hexane; aprotic solvents such as dimethylformamide or acetonitrile; water; or a mixture of these solvents. Other purification methods that can be used include those described in Volume 1 of "Experimental Chemistry Lectures" (edited by the Chemical Society of Japan, published by Maruzen).

[0061] Methods for purifying disulfide compounds are described in the literature (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Fundamentals and Experiments in Peptide Synthesis, Maruzen Co., Ltd., 1985; Pharmaceutical Development, Continued, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991), etc. Among these, HPLC is preferred.

[0062] When the compound of this embodiment has one or more asymmetric centers, it can be produced by a conventional method using a starting material (amino acid) having the asymmetric center. Furthermore, to increase the optical purity of the compound of this embodiment, optical resolution or the like may be performed at an appropriate stage in the production process. For example, optical resolution can be performed by a diastereomeric method in which the compound of this embodiment or an intermediate thereof is salted with an optically active acid (e.g., a monocarboxylic acid such as mandelic acid, N-benzyloxyalanine, or lactic acid; a dicarboxylic acid such as tartaric acid, o-diisopropylidenetartaric acid, or malic acid; or a sulfonic acid such as camphorsulfonic acid or bromocamphorsulfonic acid) in an inert solvent (e.g., an alcoholic solvent such as methanol, ethanol, or 2-propanol; an ether solvent such as diethyl ether; an ester solvent such as ethyl acetate; a hydrocarbon solvent such as toluene; or an aprotic solvent such as acetonitrile; or a mixture thereof). When the compound or intermediate of this embodiment has an acidic functional group such as a carboxy group, optical resolution can also be performed by forming a salt with an optically active amine (e.g., an organic amine such as α-phenethylamine, quinine, quinidine, cinchonidine, cinchonine, or strychnine).

[0063] The temperature for salt formation is selected from the range of room temperature to the boiling point of the solvent. To improve optical purity, it is desirable to first raise the temperature to near the boiling point of the solvent. When filtering out the precipitated salt, cooling can be performed as needed to improve yield. The amount of optically active acid or amine used is approximately 0.5 to approximately 2.0 equivalents relative to the substrate, preferably approximately 1 equivalent. If necessary, the crystals can be recrystallized in an inert solvent (e.g., alcoholic solvents such as methanol, ethanol, and 2-propanol; etheric solvents such as diethyl ether; esteric solvents such as ethyl acetate; hydrocarbon solvents such as toluene; aprotic solvents such as acetonitrile; and mixtures thereof) to obtain a highly pure optically active salt. Furthermore, if necessary, the optically resolved salt can be treated with an acid or base by a conventional method to obtain a free form.

[0064] In the present specification, "pharmaceutically acceptable salts" include acid addition salts and base addition salts. For example, acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, etc., and organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc.; base addition salts include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, etc., and organic base salts such as triethylammonium salt, triethanolammonium salt, pyridinium salt, diisopropylammonium salt, etc.; and amino acid salts such as basic or acidic amino acids such as arginine, aspartic acid, glutamic acid, etc.

[0065] This embodiment also includes solvates such as hydrates and ethanol solvates of the peptide or compound of this embodiment or a pharmaceutically acceptable salt thereof. Furthermore, the pharmaceutical composition of this embodiment also encompasses all possible stereoisomers such as all diastereomers and enantiomers, and all crystalline forms of the compound represented by formula (I).

[0066] The pharmaceutical composition of this embodiment can be used to treat or prevent cancers in which the WT1 gene is expressed or cancers accompanied by elevated WT1 gene expression levels (WT1-related cancers). Examples of such cancers include leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, extragonadal germ cell tumor, brain tumor, extracranial germ cell tumor, bone cancer, pancreatic cancer, head and neck cancer, jaw cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, lip and oral cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma (such as pleural mesothelioma, pericardial mesothelioma, and peritoneal mesothelioma), osteosarcoma, and leukemia. Sarcomas (such as leiomyosarcoma, Kaposi's sarcoma, malignant fibrous histiocytoma, liposarcoma, Ewing's sarcoma, and dermatofibrosarcoma protuberans), neuroblastoma, retinoblastoma, hepatoblastoma, nephroblastoma, glial tumors, malignant melanoma of the skin or orbit, squamous cell carcinoma, squamous cell carcinoma of the cervix, intraocular melanoma, biliary tract cancer, colon cancer, duodenal cancer, small intestine cancer, rectal cancer, anal cancer, appendix cancer, bile duct cancer, extrahepatic bile duct cancer, pancreatic islet cell carcinoma, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma Lucinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, lymphoplasmacytic lymphoma, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor, cerebellar astrocytoma, chronic nasal and paranasal cancer, nasopharyngeal cancer, salivary gland cancer, sublingual gland cancer, parotid gland cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, astrocytoma, basal cell carcinoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia Cancers include, for example, leukemia, chronic or acute leukemia including chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, kidney or ureter cancer, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell carcinoma, squamous cell carcinoma, T-cell lymphoma, glioblastoma multiforme, malignant melanoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, AIDS-related cancer, and asbestos-induced cancer. The cancer may be selected from the group consisting of the above-mentioned examples, for example, from the group consisting of leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, and brain tumor.The cancer may be selected from the group consisting of, for example, leukemia, myelodysplastic syndrome, multiple myeloma, bladder cancer, brain tumor, breast cancer, lung cancer, colon cancer, malignant lymphoma, esophageal cancer, head and neck cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer and gastric cancer, or may be selected from the group consisting of leukemia, myelodysplastic syndrome and multiple myeloma, or may be selected from the group consisting of myelodysplastic syndrome, breast cancer, lung cancer, colon cancer and bladder cancer.

[0067] In this embodiment, the subject may be a human or a non-human animal. The non-human animal is preferably a mammal. The subject may be a human with cancer, a human suspected of having cancer, or a human at risk of developing cancer, and is preferably a human with cancer. When the subject has cancer, the subject may be referred to as a patient.

[0068] The peptide or compound or pharmaceutically acceptable salt thereof of this embodiment can be used as an active ingredient of a CTL inducer in cancer cellular immunotherapy, an active ingredient of a cancer vaccine, and / or an active ingredient of a pharmaceutical composition by being prepared in an appropriate form depending on each peptide or compound or each salt.

[0069] The pharmaceutical composition, peptide, or compound, or a pharmaceutically acceptable salt thereof, of this embodiment can be administered with a pharmaceutically acceptable carrier, such as a suitable adjuvant, to effectively activate cellular immunity of the peptide or compound. For the same reason, the pharmaceutical composition of this embodiment can also contain a pharmaceutically acceptable carrier, such as a suitable adjuvant. Examples of adjuvants include precipitating adjuvants and oily adjuvants. Precipitating adjuvants are inorganic suspensions to which peptides adsorb. Specific examples of precipitating adjuvants include sodium hydroxide, aluminum hydroxide (Alum), calcium phosphate, aluminum phosphate, alum, Pepes, and carboxyvinyl polymers. Oily adjuvants are oil emulsions that emulsify an aqueous solution containing a peptide by enveloping it in mineral oil to form micelles. Specific examples of oil-based adjuvants include liquid paraffin, lanolin, Freund's adjuvant (complete Freund's adjuvant, incomplete Freund's adjuvant), Montanide, W / O emulsion, etc. Furthermore, for example, adjuvants such as those described in the literature (Clin. Microbiol. Rev., 7:277-289, 1994) can be used, and specific examples include bacterial cell-derived components, cytokines such as GM-CSF, interleukin-2, interleukin-7, and interleukin-12, plant-derived components, marine organism-derived components, mineral gels such as aluminum hydroxide, lysolecithin, surfactants such as pluronic polyol, polyanions, peptides, and oil emulsions (emulsion preparations). Examples of bacterial cell-derived components include lipid A, its derivative monophosphoryl lipid A, killed bacteria (including Mycobacterium bacteria such as BCG), bacterial-derived proteins, polynucleotides, Freund's incomplete adjuvant, Freund's complete adjuvant, cell wall skeletal components (such as BCG-CWS), and trehalose dimycolate (TDM).

[0070] The peptide or compound of this embodiment can also be administered in the form of a liposome preparation, a particulate preparation bound to beads with a diameter of several μm, a lipid-bound preparation, a W / O emulsion preparation, or the like.

[0071] Furthermore, the peptide or compound (conjugate) of this embodiment can be administered together with an MHC class II-restricted WT1 peptide (i.e., a helper peptide). Although the conjugate and the helper peptide can be administered separately, a cocktail preparation (cocktail agent, cocktail) containing the conjugate and the helper peptide in a single pharmaceutical composition is more preferred. This cocktail preparation contains a conjugate capable of generating an MHC class I-restricted WT1 peptide (i.e., a killer peptide) and an MHC class II-restricted WT1 peptide (i.e., a helper peptide). Therefore, administration of this cocktail preparation containing a helper peptide as a cancer vaccine in cancer immunotherapy can activate helper T cells, which are important for enhancing the function of other T cells, including CTLs, thereby improving the function and efficacy (e.g., cellular immunity) of the conjugate.

[0072] In the method for selecting a subject for whom a pharmaceutical composition for treating or preventing cancer is expected to be effective according to this embodiment (hereinafter also referred to as the "selection method of this embodiment"), an index indicating that the subject is expected to be effective can be provided based on one or a combination of two or more selected from the group consisting of the following (1) to (6). By providing an index indicating that the subject is expected to be effective according to the pharmaceutical composition, it is also possible to predict the response of the subject to the pharmaceutical composition. (1) Mutations in the TP53 gene and / or BCOR gene (2) WT1 gene mRNA expression level (3) Karyotype based on the revised IPSS (IPSS-R) (4) Whether or not there is an increase in WT1 antigen peptide-specific CD8 T cells (5) Presence or absence of delayed hypersensitivity reaction (6) Changes in the proportion of bone marrow blasts

[0073] When providing an indicator that a subject is expected to be effective based on a combination of two or more selected from the group consisting of (1) to (6), the order of steps is not limited to the above. Those skilled in the art can appropriately determine the steps and order of steps to be performed, taking into consideration the efficiency of selection, the condition and burden of the subject, etc. Furthermore, it is also possible to predict and select effective combinations using artificial intelligence, machine learning, and / or statistical methods from big data related to patient treatment, such as disease, medical condition, genomic information, risk factors for treatment, and test data. For example, it is possible to first perform selection based on one or more selected from (1) to (3) that does not require administration of the pharmaceutical composition or peptide, and then narrow down the subjects for whom the pharmaceutical composition is expected to be effective based on the results, and then perform selection based on one or more selected from (4) to (6) that require administration of the pharmaceutical composition, thereby selecting subjects for whom the pharmaceutical composition is expected to be effective. Alternatively, it is also possible to first perform selection based on one or more selected from (4) to (6), and then narrow down the subjects for whom the pharmaceutical composition is expected to be effective based on the results, and then perform selection based on one or more selected from (1) to (3). Furthermore, for example, when the karyotype of a subject has already been determined based on IPSS-R, it is possible to narrow down subjects for whom the pharmaceutical composition is expected to be effective by selection based on (3), and then perform selection based on one or more selected from (1) to (2) and (4) to (6).If selection requiring administration of a pharmaceutical composition or peptide is performed, followed by selection that is preferably performed on the same subject without administration of the pharmaceutical composition or peptide, it is also possible to perform the selection after a predetermined period of time has passed and the effects of administration of the pharmaceutical composition or peptide have sufficiently decreased.

[0074] In this embodiment, the sample is not particularly limited as long as it can be collected from a subject, and examples include body fluids such as blood, lymph, ascites, pleural effusion, sputum, cerebrospinal fluid (CSF), tears, nasal discharge, saliva, urine, vaginal fluid, semen, and synovial fluid, as well as mucous membranes, cells, tissues, and cell or tissue cultures. Blood includes plasma, serum, and interstitial fluid. Cells include blood cells such as red blood cells, white blood cells, platelets, hematopoietic stem cells, bone marrow blood, and myeloblasts, circulating tumor cells, leukemia cells, blasts associated with dysplasia, and malignant tumor (cancer) cells such as brain tumors, colon cancer cells, lung cancer cells, breast cancer cells, uterine cancer cells, gastric cancer cells, liver cancer cells, prostate cancer cells, renal cancer cells, pancreatic cancer cells, sarcoma cells, malignant mesothelioma cells, and lymphoma cells. Tissue containing cancer is called cancer tissue.

[0075] Samples can be collected from subjects using methods known in the art. For example, blood and lymph can be collected using known blood collection methods. For example, cells and tissues can be collected using known methods such as puncture, fine needle aspiration, scraping, peritoneal lavage, needle biopsy, and surgical biopsy. The sample in this embodiment may be selected from the group consisting of body fluids, mucous membranes, cells, tissues, and cell or tissue cultures, and combinations thereof; blood, cerebrospinal fluid, blood cells, cancer cells, cancer tissues, and cell or cancer tissue cultures, and combinations thereof; blood, cerebrospinal fluid, cancer cells, cancer tissues, and cancer cell or cancer tissue cultures, and combinations thereof; or blood or cerebrospinal fluid.

[0076] The effects of a pharmaceutical composition for treating or preventing cancer may vary depending on the type of cancer and the condition of the subject, but examples include extension of survival time, stabilization of myeloblasts, reduction of cancer cells, prevention of metastasis and delay of progression (in the case of myelodysplastic syndrome (MDS) patients, extension of the transition period to acute myeloid leukemia (AML)), etc. Subjects for whom the pharmaceutical composition can be expected to be effective include, for example, subjects for whom the pharmaceutical composition is likely to be effective, subjects for whom administration of the pharmaceutical composition is likely to extend survival time, etc.

[0077] The term "WT1 antigenic peptide-specific immune response" refers to an immune response that is specifically induced by administration of a WT1 antigenic peptide, etc. The induction of a "WT1 antigenic peptide-specific immune response" can be confirmed, for example, by a positive result in an HLA tetramer assay described below and / or a positive delayed hypersensitivity reaction.

[0078] (1) Mutations in the TP53 gene and / or BCOR gene The TP53 gene is a tumor suppressor gene that encodes the nuclear protein p53, which consists of 393 amino acids. The BCOR gene is a corepressor of BCL6, and specifically inhibits gene expression by binding to transcription factors. Both genes have been reported to be poor prognostic factors.

[0079] As used herein, a mutation in the TP53 gene and / or the BCOR gene refers to a mutation in both the TP53 gene and the BCOR gene, a mutation in the TP53 gene, or a mutation in the BCOR gene. The mutation in the TP53 gene and / or the BCOR gene may be a substitution, deletion, insertion, or a combination thereof of nucleotide bases in the respective base sequences. The mutation in the TP53 gene and / or the BCOR gene may be a substitution, deletion, insertion, or a combination thereof of 1 to 20, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide base.

[0080] As used herein, the term "TP53 wild-type" refers to a case in which there is no mutation in the TP53 gene, or a case in which there is a mutation in the TP53 gene but the original function is not lost or abnormal (including silent mutations, synonymous mutations, etc.). The term "TP53 mutant-type" refers to a case in which there is a mutation in the TP53 gene but the original function is lost or abnormal (including silent mutations, synonymous mutations, etc.). The term "BCOR wild-type" refers to a case in which there is no mutation in the BCOR gene, or a case in which there is a mutation in the BCOR gene but the original function is lost or abnormal (including silent mutations, synonymous mutations, etc.). The term "BCOR mutant-type" refers to a case in which there is a mutation in the BCOR gene but the original function is lost or abnormal. Therefore, the terms "TP53 wild-type" and / or "BCOR wild-type" include cases in which either TP53 or BCOR is wild-type, and cases in which both TP53 and BCOR are wild-type.

[0081] The selection method of this embodiment includes the steps of determining the presence or absence of a mutation in the TP53 gene and / or the BCOR gene using a sample collected from a subject; and providing an indication that, when the subject is TP53 wild-type and / or BCOR wild-type, the subject is one who can be expected to benefit from the pharmaceutical composition.

[0082] The subject, sample, pharmaceutical composition, etc. are as described above.

[0083] The process of determining the presence or absence of mutations in the TP53 gene and / or BCOR gene involves determining the presence or absence of mutations in the TP53 gene and / or BCOR gene in a subject by comparing the nucleotide sequence of the corresponding gene with that of the wild-type gene, if the mutation is found to be different from the wild-type nucleotide sequence and is a mutation that abolishes or is abnormal in a manner that abolishes the original function of the wild-type TP53 gene, as "mutant TP53 and / or BCOR." Alternatively, determining the presence or absence of mutations from the wild-type nucleotide sequence or the mutations that do not affect the transcription level of each gene or the function of the protein encoded by each gene (including silent mutations and synonymous mutations) as "wild-type TP53 and BCOR." Differences from the wild-type nucleotide sequence may be detected by comparing the nucleotide sequence of the corresponding gene with that of the wild-type TP53 and / or BCOR gene. The determination of the nucleotide sequence of the corresponding gene with that of the wild-type TP53 and / or BCOR gene can be performed by methods known to those skilled in the art. For example, DNA can be extracted from a sample by conventional methods, the sequence of each gene determined by next-generation sequencing (NGS), or the like, and the presence or absence of a mutation can be determined by comparing it with the corresponding wild-type gene sequence. Depending on the mutation, for example, the presence or absence of a mutation can also be determined by PCR-RFLP (Restriction Fragment Length Polymorphism) without determining the base sequence. This can also be performed using, for example, a commercially available DNA mutation / polymorphism detection kit.

[0084] The selection method of this embodiment may include, for example, a step of determining the presence or absence of mutations in the TP53 gene and the BCOR gene, and as a result, in the case of TP53 wild-type, may include a step of providing an indicator that the subject is a subject for whom the effects of the pharmaceutical composition can be expected, or in the case of BCOR wild-type, may include a step of providing an indicator that the subject is a subject for whom the effects of the pharmaceutical composition can be expected, or in the case of TP53 wild-type and BCOR wild-type, may include a step of providing an indicator that the subject is a subject for whom the effects of the pharmaceutical composition can be expected.

[0085] Furthermore, the selection method of this embodiment may include, for example, a step of determining the presence or absence of a mutation in the TP53 gene, and as a result, if the TP53 is wild-type, a step of providing an indicator that the subject is a subject for whom the pharmaceutical composition can be expected to be effective.Similarly, the selection method of this embodiment may include, for example, a step of determining the presence or absence of a mutation in the BCOR gene, and as a result, if the BCOR is wild-type, a step of providing an indicator that the subject is a subject for whom the pharmaceutical composition can be expected to be effective.

[0086] The selection method of this embodiment may, for example, conversely include providing an indication that, in the case of a TP53 mutant and / or a BCOR mutant, the subject is not one for whom the pharmaceutical composition is expected to be effective.

[0087] When the original function is not lost or abnormality is not involved, it means that each gene is identical or substantially identical to the wild type (including silent mutations and synonymous mutations, etc.).

[0088] In this way, the TP53 gene and / or the BCOR gene can be used as a marker to provide an indication of whether or not a subject is likely to benefit from a pharmaceutical composition for treating or preventing cancer.

[0089] (2) WT1 gene mRNA expression level In another aspect, the present invention provides a method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, based on the expression level of WT1 gene mRNA.

[0090] The selection method of this embodiment includes the steps of determining the mRNA expression level of the WT1 gene using a sample collected from a subject; and providing an indicator that the subject is one for whom the pharmaceutical composition can be expected to be effective when the mRNA expression level of the WT1 gene is less than or equal to the reference value.

[0091] The step of determining the mRNA expression level of the WT1 gene can be carried out by methods known to those skilled in the art, such as quantitative PCR. Alternatively, it can be carried out using a commercially available kit, such as the WT1 mRNA Measurement Kit II "Otsuka" (Otsuka Pharmaceutical Co., Ltd.). For example, RNA is extracted from a sample, and quantitative PCR, such as RT-PCR, is performed using WT1-specific primers in a real-time PCR device or the like. Based on a standard curve, the measured values ​​of WT1 mRNA and the mRNA of housekeeping genes (GAPDH, β-actin, etc.) serving as endogenous controls can be calculated. Then, for example, as shown in the following formula (1), the WT1 mRNA expression level (copies / μg RNA) can be calculated by multiplying the value obtained by dividing the measured WT1 mRNA value by the measured housekeeping gene mRNA value (WT1 mRNA copy number per housekeeping gene mRNA copy) by the average mRNA copy number of the housekeeping gene per μg RNA in healthy adults (housekeeping gene mRNA expression level). Therefore, the WT1 mRNA expression level (copies / μg RNA) can also be calculated using the following formula (1):

number

[0092] When GAPDH is used as the housekeeping gene, for example, RNA is extracted from a sample, and a quantitative PCR reaction such as RT-PCR is performed using WT1-specific primers in a real-time PCR device or the like, and the measured values ​​of WT1 mRNA and GAPDH mRNA can be calculated based on a standard curve. Then, for example, as shown in the following formula, the measured WT1 mRNA value is divided by the measured GAPDH mRNA value (WT1 mRNA copy number per GAPDH mRNA copy) and multiplied by the average GAPDH mRNA copy number per μg RNA in healthy adults (GAPDH mRNA expression level), to calculate the WT1 mRNA expression level (copies / μg RNA). Therefore, the WT1 mRNA expression level (copies / μg RNA) can also be calculated using the following formula (Equation 2). Note that, when 2.7×107 (copies / μg RNA) is the average GAPDH mRNA measurement per μg RNA in healthy adults.

number

[0093] The WT1 mRNA expression level (copies / μg RNA) can also be calculated from the above formula (Equation 2) using the WT1 mRNA Measurement Kit II "Otsuka" (Otsuka Pharmaceutical Co., Ltd.) and the accompanying protocol.

[0094] In the selection method of this embodiment, a WT1 gene mRNA expression level below or equal to a reference value can provide an indicator that the subject is one for whom the pharmaceutical composition is likely to be effective. The reference value for the WT1 gene mRNA expression level may be, for example, 50 to 100,000 copies / μg RNA, 100 to 50,000 copies / μg RNA, 1,000 to 20,000 copies / μg RNA, 2,000 to 10,000 copies / μg RNA, 3,000 to 10,000 copies / μg RNA, or 4,000 to 10,000 copies / μg RNA.Furthermore, the reference value for the mRNA expression level of the WT1 gene is, for example, a value of 50 (copies / μg RNA) or more, a value of 100 (copies / μg RNA) or more, a value of 250 (copies / μg RNA) or more, a value of 500 (copies / μg RNA) or more, a value of 750 (copies / μg RNA) or more, a value of 1000 (copies / μg RNA) or more, a value of 1000 (copies / μg RNA) or more, a value of 1250 (copies / μg RNA) or more, a value of 1500 (copies / μg RNA) or more, a value of 1750 (copies / μg RNA) or more, a value of 2000 (copies / μg RNA) or more, a value of 2250 (copies / μg RNA) or more, a value of 2500 (copies / μg RNA) or more, a value of 2750 (copies / μg RNA) or more, a value of 3000 (copies / μg RNA) or more, a value of 3250 (copies / μg RNA) or more, a value of 350 ... The RNA may be a value of 15,000 (copies / μg RNA) or less, a value of 14,000 (copies / μg RNA) or less, a value of 13,000 (copies / μg RNA) or less, a value of 12,000 (copies / μg RNA) or less, a value of 11,000 (copies / μg RNA) or less, a value of 10,000 (copies / μg RNA) or less, and may be 50 (copies / μg RNA), 100 (copies / μg RNA), 250 (copies / μg RNA), 500 (copies / μg RNA), 750 (copies / μg RNA), 1000 (copies / μg RNA), 1000 (copies / μg RNA), 1250 (copies / μg RNA), 1500 (copies / μg RNA), 1750 (copies / μg RNA), The number of copies per μg of RNA may be 2000 (copies / μg RNA), 2000 (copies / μg RNA), 2250 (copies / μg RNA), 2500 (copies / μg RNA), 2750 (copies / μg RNA), 3000 (copies / μg RNA), 3250 (copies / μg RNA), 3500 (copies / μg RNA), 3750 (copies / μg RNA), 4000 (copies / μg RNA), 15000 (copies / μg RNA), 14000 (copies / μg RNA), 13000 (copies / μg RNA), 12000 (copies / μg RNA), 11000 (copies / μg RNA), or 10000 (copies / μg RNA).The selection method of this embodiment may provide, for example, an indicator that the subject is one for whom the effects of a pharmaceutical composition can be expected when the mRNA expression level of the WT1 gene is less than or equal to 4000 (copies / μg RNA), or an indicator that the subject is one for whom the effects of a pharmaceutical composition can be expected when the mRNA expression level of the WT1 gene is less than or equal to 10000 (copies / μg RNA).The selection method of this embodiment preferably provides an indicator that the subject is one for whom the effects of a pharmaceutical composition can be expected when the mRNA expression level of the WT1 gene is less than or equal to a value between 4000 and 10000 (copies / μg RNA).

[0095] When the subject is a myelodysplastic syndrome (MDS) patient, the reference value for the mRNA expression level of the WT1 gene is preferably 100,000 (copies / μg RNA) or less, more preferably 4,000 to 10,000 (copies / μg RNA), and even more preferably 10,000 (copies / μg RNA) or less. When the mRNA expression level of the WT1 gene in a sample collected from the subject is less than or equal to the reference value, OS tends to be longer.

[0096] Thus, the WT1 gene can be used as a marker to provide an indication of whether or not a subject is one who can be expected to benefit from a pharmaceutical composition for treating or preventing cancer.

[0097] (3) Karyotype based on the revised IPSS (IPSS-R) In another aspect, the present invention provides a method for selecting a subject who is likely to benefit from a pharmaceutical composition for treating or preventing cancer, based on a karyotype based on the revised IPSS (IPSS-R).

[0098] The selection method of this embodiment includes a step of providing an indication that a subject is one for whom the pharmaceutical composition is likely to be effective, when the subject's karyotype based on the revised IPSS-R ("Greenberg et al., Blood 120, no. 12, pp. 2454-2465 (2012)") is other than Very Poor. The selection method of this embodiment may include, prior to this step, a step of determining the subject's karyotype based on the IPSS-R using a sample collected from the subject.

[0099] The IPSS-R-based karyotype can be determined by analysis using methods known to those skilled in the art, such as G-banding and Q-banding. If a subject's IPSS-R-based karyotype is anything other than Very Poor, an indicator is provided that the subject is a candidate for whom the pharmaceutical composition may be effective. In the selection method of this embodiment, for example, if a subject's IPSS-R-based karyotype is Good / Very Good, Intermediate, or Poor, an indicator may be provided that the subject is a candidate for whom the pharmaceutical composition may be effective. If the karyotype is Intermediate or Poor, an indicator may be provided that the subject is a candidate for whom the pharmaceutical composition may be effective. If the karyotype is Poor, an indicator may be provided that the subject is a candidate for whom the pharmaceutical composition may be effective. If the karyotype is Good / Very Good, an indicator may be provided that the subject is a candidate for whom the pharmaceutical composition may be effective. If the karyotype is Good / Very Good, or Good / Very Good, or Intermediate, an indicator may be provided that the subject is a candidate for whom the pharmaceutical composition may be effective. Furthermore, if a subject's karyotype based on IPSS-R is Very Poor, an indication may be provided that the subject is not one for whom the pharmaceutical composition is expected to be effective.

[0100] (4) Whether or not there is an increase in WT1 antigen peptide-specific CD8 T cells In another aspect, the present invention provides a method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, based on the presence or absence of an increase in WT1 antigen peptide-specific CD8 T cells.

[0101] The selection method of this embodiment includes the steps of detecting WT1 antigen peptide-specific CD8 T cells using a sample collected from a subject administered with a pharmaceutical composition, or a peptide or a pharmaceutically acceptable salt thereof; and providing an indicator that the subject is one for whom the pharmaceutical composition is expected to be effective, when the number of WT1 antigen peptide-specific CD8 T cells is increased compared to a sample collected from the subject before administration. The subject, sample, pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, etc. are as described above.

[0102] WT1 antigenic peptide-specific CD8 T cells can be detected by measuring the presence or number of WT1 antigenic peptide-specific CD8 T cells using, for example, the HLA monomer method, HLA dimer method, HLA tetramer method (Int. J. Cancer: 100, 565-570 (2002)), HLA pentamer method, HLA dextramer method, ELISPOT method, real-time RT-PCR, and limiting dilution method (Nat. Med.: 4, 321-327 (1998)). Therefore, the step of detecting WT1 antigenic peptide-specific CD8 T cells may be a step of measuring the presence or number of WT1 antigenic peptide-specific CD8 T cells. WT1 antigenic peptide-specific CD8 T cells are preferably detected by the HLA tetramer method. HLA tetramers are prepared by biotinylating a complex (HLA monomer) comprising an HLA α chain and β2-microglobulin associated with a peptide, and tetramerizing the complex by binding to fluorescently labeled avidin. The presence or number of WT1 antigen peptide-specific CD8 T cells can be measured by staining them with HLA tetramers and analyzing them with a flow cytometer. The presence or number of WT1 antigen peptide-specific CD8 T cells can also be measured based on the same principle using the HLA monomer method, HLA dimer method, HLA pentamer method, and HLA dextramer method.

[0103] The step of detecting WT1 antigen peptide-specific CD8 T cells may be carried out by reacting a complex of a WT1 peptide and an HLA molecule with the sample and examining the presence or number of WT1 antigen peptide-specific CD8 T cells that recognize the complex contained in the sample. The complex of a WT1 peptide and an HLA molecule may be selected from the group consisting of an HLA monomer, an HLA dimer, an HLA tetramer, an HLA pentamer, and an HLA dextramers. The HLA molecule preferably matches the HLA of the subject. The HLA molecule may be, for example, an HLA-A24 antigen or an HLA-A2 antigen. The step of detecting WT1 antigen peptide-specific CD8 T cells may include analysis by flow cytometry.

[0104] Furthermore, the presence or number of WT1 antigen peptide-specific CD8 T cells that recognize the complex contained in the sample may be determined, for example, by measuring the ratio of HLA tetramer-binding cells to CD8-positive or CD8 / CD3-positive WT1 antigen peptide-specific CD8 T cells.

[0105] The presence or number of WT1 antigen peptide-specific CD8 T cells bound to HLA tetramers can be determined, for example, by measuring the ratio of HLA tetramer-binding cells to CD8-positive cells or CD8 / CD3-positive cells.

[0106] Here, CD8-positive cells can be labeled and detected using, for example, a fluorescently labeled mouse anti-human CD8 monoclonal antibody, and CD3-positive cells can be labeled and detected using a fluorescently labeled mouse anti-human CD3 monoclonal antibody.

[0107] The fluorescent dye used here must be different from that used in the HLA tetramer. For example, when using a PE-labeled HLA tetramer, the fluorescent dye must be different from that used in the HLA tetramer. For example, when using a PE-labeled HLA tetramer, an FITC-labeled mouse anti-human CD8 monoclonal antibody and a PerCP-labeled mouse anti-human CD3 monoclonal antibody must be used.

[0108] Specifically, when measuring the ratio of HLA tetramer-binding cells to CD8-positive cells, for example, a biological sample is contacted with PE-labeled HLA tetramer, and then an FITC-labeled mouse anti-human CD8 monoclonal antibody is added to react with the sample, and the stained cells are analyzed using a flow cytometer or a fluorescent microscope. + ) and select tetramer-positive cells (CD8 + tetramer + ) can be used as the percentage of WT1 antigen peptide-specific CD8 T cells (see below): WT1 antigen peptide-specific CD8 T cells (%) = (CD8 + tetramer + Cell count / CD8 + number of cells) x 100.

[0109] In addition, when measuring the ratio of HLA tetramer-binding cells to CD3-positive and CD8-positive cells, for example, after contacting a biological sample with a PE-labeled HLA tetramer, an FITC-labeled mouse anti-human CD8 monoclonal antibody and a PerCP-labeled mouse anti-human CD3 antibody are further added and reacted, and the stained cells are analyzed using a flow cytometer or a fluorescent microscope. + CD8 + ) and select tetramer-positive cells (CD3 + CD8 + tetramer + ) can be used as the percentage of WT1 antigen peptide-specific CD8 T cells (see below): WT1 antigen peptide-specific CD8 T cells (%) = (CD3 + CD8 + tetramer + Cell count / CD3 + CD8 + number of cells) x 100.

[0110] The selection method of this embodiment includes a step of providing an indicator that a subject is one for whom the effects of the pharmaceutical composition can be expected, when the number of WT1 antigen peptide-specific CD8 T cells is increased compared to a sample collected from the subject before administration. The sample collected from the subject before administration of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof is the same type as the sample collected from the subject after administration. For example, if the sample collected from the subject before administration is blood, the sample collected from the subject after administration is also blood. The timing of sample collection can be determined appropriately, and examples that can be used include samples collected after the most recent administration, immediately after administration to 12 months, immediately after administration to 6 months, immediately after administration to 3 months, immediately after administration to 2 months, immediately after administration to 1 month, immediately after administration to 4 weeks, immediately after administration to 3 weeks, immediately after administration to 2 weeks, immediately after administration to 1 week, immediately after administration to 3 days, or immediately after administration to 1 day. Furthermore, samples may be collected immediately after administration and / or within 12 months. For example, samples collected less than or within 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or 6 months after administration can be used. For example, samples collected 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or 6 months or more after administration can be used. The number of administrations is not particularly limited, and may be 1 to 1000 times, 1 to 100 times, 1 to 50 times, 1 to 10 times, 1 to 5 times, 1 to 3 times, 1 to 2 times, or 1 time. The number of administrations may be, for example, 1 or more or less than 1000 times, or may be less than or equal to 100, 50, 10, 5, 4, 3, or 2 times, or may be 100, 50, 10, 5, 4, 3, or 2 times or more.

[0111] An increase in WT1 antigen peptide-specific CD8 T cells also includes a case where WT1 antigen peptide-specific CD8 T cells are not detected in a sample collected from the subject before administration, but are detected in a sample collected from the subject after administration.

[0112] For example, when the ratio (positive rate) of WT1 antigen peptide-specific CD8 T cells among CD8 T cells in a sample collected from a subject after administration is increased by a predetermined ratio or more compared to the ratio (positive rate) of WT1 antigen peptide-specific CD8 T cells among CD8 T cells in a sample collected from the subject before administration, it can be determined that the WT1 antigen peptide-specific CD8 T cells have increased. (a) WT1 antigen peptide-specific CD8 T cells are not detected in a sample collected from the subject before administration, but are detected in a sample collected from the subject after administration, and the rate (positive rate) of WT1 antigen peptide-specific CD8 T cells is equal to or higher than a reference value; and / or (b) the ratio (positive rate) of WT1 antigen peptide-specific CD8 T cells among CD8 T cells in a sample collected from the subject after administration is equal to or greater than a predetermined ratio compared with the ratio (positive rate) of WT1 antigen peptide-specific CD8 T cells among CD8 T cells in a sample collected from the subject before administration. Furthermore, it can be said that WT1 antigen peptide-specific CD8 T cells increased.

[0113] Regarding (a) If WT1 antigenic peptide-specific CD8 T cells are not detected (are not present) in a sample collected from a subject before administration, it is not possible to calculate the fold increase in the number of WT1 antigenic peptide-specific CD8 T cells compared to the number of WT1 antigenic peptide-specific CD8 T cells in a sample collected from the subject after administration. In this case, for example, if the number is equal to or greater than a predetermined reference value, it can be determined that the WT1 antigenic peptide-specific CD8 T cells have increased (are positive). For example, those skilled in the art can determine a range in which the number of WT1 antigenic peptide-specific CD8 T cells is determined to be positive, based on the results of previous tests, and appropriately set a reference value for determining that the number of WT1 antigenic peptide-specific CD8 T cells falls within that range as an increase (positive). For example, when the step of detecting WT1 antigenic peptide-specific CD8 T cells includes analysis by flow cytometry, a gate is set that includes a cell population that is positive for CD8 and WT1 peptide. If the number of WT1 antigenic peptide-specific CD8 T cells (events) in a sample collected from a subject after administration that falls within that range is equal to or greater than a reference value, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more, the WT1 antigenic peptide-specific CD8 T cells can be determined to have increased (positive).

[0114] Regarding (b) When WT1 antigenic peptide-specific CD8 T cells are detected in a sample collected from a subject before administration, if the ratio (positive rate) of WT1 antigenic peptide-specific CD8 T cells among CD8 T cells in a sample collected from the subject after administration is equal to or greater than a predetermined ratio compared to the ratio (positive rate) of WT1 antigenic peptide-specific CD8 T cells among CD8 T cells in a sample collected from the subject before administration, the WT1 antigenic peptide-specific CD8 T cells can be determined to have increased (positive). For example, a person skilled in the art can determine a range within which WT1 antigenic peptide-specific CD8 T cells are determined to be positive, based on the results of previous tests, and appropriately set a standard value for determining an increase (positive) in WT1 antigenic peptide-specific CD8 T cells based on the ratio of the number of WT1 antigenic peptide-specific CD8 T cells falling within that range before and after administration. For example, when the step of detecting WT1 antigenic peptide-specific CD8 T cells includes analysis by flow cytometry, a gate is set that includes a population of cells that are positive for CD8 and WT1 tetramer. If the ratio of WT1 antigenic peptide-specific CD8 T cells (positive rate) among CD8 T cells in a sample collected from a subject after administration that falls within that range is equal to or greater than the ratio considered to be maintained compared to the ratio before administration, it can be determined that the WT1 antigenic peptide-specific CD8 T cells have increased (positive). If the ratio is equal to or less than the ratio considered to be maintained, it can be determined that the WT1 antigenic peptide-specific CD8 T cells have decreased (negative). The ratio considered to be maintained can be set, for example, at more than 0.1 to less than 5.0, more than 0.5 to less than 2.0, more than 0.8 to less than 1.2, more than 0.9 to less than 1.1, or 1.0. The ratio that is considered to be maintained may be, for example, not less than or greater than 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0, and may be less than or equal to 5.0 times, 4.5 times, 4.0 times, 3.5 times, 3.0 times, 2.5 times, 2.0 times, 1.8 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1.1 times, or 1.0 times.

[0115] When the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof is administered multiple times, for example, if the number of WT1 antigen peptide-specific CD8 T cells is determined to have increased (positive) or remained unchanged (maintained) at any time point after each administration, the result may be determined to be positive or maintained, whereas if the number of WT1 antigen peptide-specific CD8 T cells is determined to have decreased (negative) at any time point, the result may be determined to be negative.

[0116] As described above, it is also possible to evaluate the effect of a candidate substance for a pharmaceutical composition for treating or preventing cancer based on the presence or absence of an increase in WT1 antigen peptide-specific CD8 T cells. That is, in this embodiment, a method for evaluating the effect of a candidate substance for a pharmaceutical composition for treating or preventing cancer comprises the steps of: detecting WT1 antigen peptide-specific CD8 T cells using a sample collected from a subject administered with the pharmaceutical composition or a peptide or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition; and providing an indicator that the candidate substance is expected to be effective in treating and preventing cancer, when the number of WT1 antigen peptide-specific CD8 T cells is increased compared to a sample collected from the subject before administration. The subject, sample, pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, etc. are as described above.

[0117] (5) Presence or absence of delayed hypersensitivity reaction In another aspect, the present invention provides a method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, based on the presence or absence of a delayed-type hypersensitivity reaction.

[0118] The selection method of this embodiment includes a step of providing an indicator that the subject is one for whom the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof can be expected to be effective when a delayed-type hypersensitivity reaction is detected in the subject to multiple administrations of the pharmaceutical composition, or peptide or pharmaceutically acceptable salt thereof. The subject, sample, pharmaceutical composition, peptide or pharmaceutically acceptable salt thereof, etc. are as described above.

[0119] When a subject to which a pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof has been administered is subsequently administered the same pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, a delayed-type hypersensitivity reaction may be detected. A delayed-type hypersensitivity reaction (DTH reaction) is an allergic reaction caused by a cellular immune mechanism, which belongs to Type IV of the Coombs-Gell classification. The presence or absence of this delayed-type hypersensitivity reaction can be used as an indicator of whether an immune response of WT1 antigen peptide-specific CD8 T cells has been induced.

[0120] A delayed-type hypersensitivity test (DTH test) can be performed by administering a pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, to a subject who has received the same pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, one or more times. In other words, the selection method of this embodiment provides an indicator that a subject who has received a pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof one or more times can be expected to benefit from the pharmaceutical composition if a delayed-type hypersensitivity reaction is detected when the same pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof is administered to the subject. The one or more administrations described above are not intended for the DTH test, whereas the latter administration of the same pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof corresponds to the administration for the DTH test. The same pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof may be substantially the same. For example, if the pharmaceutical composition administered one or more times is a vaccine containing two or more peptides, the two or more peptides may be administered separately in the DTH test. For example, if a pharmaceutical composition administered one or more times contains a peptide consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 2), a peptide consisting of the amino acid sequence C-CYTWNQMNL (SEQ ID NO: 10), and a peptide consisting of the amino acid sequence WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), a DTH test may be performed by separately administering the peptide consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 2) and the peptide consisting of the amino acid sequence C-CYTWNQMNL (SEQ ID NO: 10). Furthermore, the peptide consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 2) and the peptide consisting of the amino acid sequence C-CYTWNQMNL (SEQ ID NO: 10) may be in the form of a conjugate, as shown in Formula 3.

[0121] In a DTH test, the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof is usually administered intradermally. It is preferable that the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof is administered to a site different from the site where the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof was initially administered to the subject. The timing of post-administration assessment for the DTH test can be appropriately determined by those skilled in the art, and can be, for example, 1 hour to 1 week, 12 hours to 5 days, 1 day to 3 days, or 2 days after the most recent administration. Furthermore, the assessment can be performed, for example, immediately after administration and / or within 1 week, or within 1, 2, 3, 4, 5, or 6 days or less after administration, or 1, 2, 3, 4, 5, or 6 days or more after administration. It is also preferable to perform the DTH test multiple times at different times and determine the presence or absence of a delayed-type hypersensitivity reaction based on the average of the results. The number of DTH tests may be, for example, two or more and / or 20 or less, for example, 2, 3, 4, 5, 7, 10, 15 or less than 20 times, or for example, 2, 3, 4, 5, 7, 10 or 15 or more times. Those skilled in the art can appropriately determine the dosage of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof sufficient to detect a delayed-type hypersensitivity reaction.

[0122] To determine whether a delayed-type hypersensitivity reaction has been detected in a DTH test (the presence or absence of a delayed-type hypersensitivity reaction), for example, a reaction at a site where the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof has been administered for the DTH test to a subject who has been administered a pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof at least once is compared with a reaction at a site in the subject where the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof for the DTH test has not been administered, and if the difference between the reaction at the administered site and the reaction at the non-administered site is equal to or greater than a reference value, it can be determined that a delayed-type hypersensitivity reaction has been detected (the presence of a delayed-type hypersensitivity reaction). The site in the subject where the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof has not been administered may be a site to which nothing has been administered, such as the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, or may be a site to which only a carrier, solvent, etc. (vehicle) other than the peptide or pharmaceutically acceptable salt thereof in the pharmaceutical composition has been administered.

[0123] Furthermore, the difference in reaction between an administration site and a non-administered site can be derived, for example, from the difference in the major axis of redness between the administration site and a non-administered site (control). Those skilled in the art can appropriately set a reference value for determining a positive WT1 antigen peptide-specific immune response. For example, a delayed-type hypersensitivity reaction may be detected when the major axis of redness in the skin of an administration site relative to a non-administered site is +0.1 mm to 100 mm or more, +0.5 mm to 50 mm or more, +1 mm to 10 mm or more, or 2 mm or more. The lower limit of the range of the major axis of redness, above which a delayed-type hypersensitivity reaction is detected, may be 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1.0 mm, and the upper limit may be 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 40 mm, 30 mm, 20 mm, 15 mm, or 10 mm. A score corresponding to the major axis of redness on the skin at the administration site relative to a non-administered site may be preset, and the presence or absence of a delayed-type hypersensitivity reaction may be determined based on the score. For example, a major axis of redness less than 2 mm may be assigned a score of 0, 2 mm or more but less than 5 mm a score of + / -, 5 mm or more but less than 10 mm a score of 1, 10 mm or more but less than 15 mm a score of 2, and 15 mm or more a score of 3. The above determination may be made using the average score of the subjects after multiple administrations, or the maximum score of the subjects after administration.

[0124] As described above, it is also possible to evaluate the effect of a candidate substance of a pharmaceutical composition for treating or preventing cancer based on the presence or absence of a delayed-type hypersensitivity reaction. That is, in this embodiment, the method for evaluating the effect of a candidate substance of a pharmaceutical composition for treating or preventing cancer includes the step of providing an indication that the candidate substance of the pharmaceutical composition is expected to be effective in treating or preventing cancer when a delayed-type hypersensitivity reaction is detected in a subject to multiple administrations of the pharmaceutical composition or a peptide or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition.

[0125] The method further includes a step of providing an indication that the candidate substance is expected to be effective in treating and preventing cancer when a delayed-type hypersensitivity reaction is detected in a subject to multiple administrations of the pharmaceutical composition, or the peptide or pharmaceutically acceptable salt thereof contained in the pharmaceutical composition. The subject, sample, pharmaceutical composition, or peptide or pharmaceutically acceptable salt thereof is as described above.

[0126] (6) Changes in the proportion of bone marrow blasts The selection method of this embodiment includes a step of providing an indicator that the subject is one in whom the effects of the pharmaceutical composition can be expected, when the value obtained by dividing the percentage of myeloblasts in a sample collected from a subject to which a pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof has been administered by the percentage of myeloblasts in a sample collected from the subject before administration of the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof is less than or equal to a reference value. The subject, sample, pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, etc. are as described above.

[0127] The smaller the change in the proportion of myeloblasts in a sample such as bone marrow fluid before and after administration of a pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, the more stable the myeloblasts can be said to be. For example, a ratio (hereinafter also referred to as the myeloblast change rate) of the proportion of myeloblasts in a subject after administration of a pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof divided by the proportion of myeloblasts in the subject before administration of the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, which is 0% to 300% or less, 0% to 200% or less, 0% to 150% or less, 0% to 100% or less, or 0% to 50% or less may be used as an indicator that the subject is one for whom the pharmaceutical composition is expected to be effective. Here, the change rate of myeloblasts may be, for example, 0% or more or less than 300% or less, or may be, for example, 50%, 100%, 150%, 200%, or 250% or more or may be less than 50%, 100%, 150%, 200%, or 250% or less.

[0128] Alternatively, for example, the determination of being below or equal to the standard value may be based on the average rate of change in myeloblasts from one to several time points, or based on the maximum rate of change in myeloblasts from one to several time points. For example, the determination of being below or equal to the standard value may be based on the value remaining below or equal to the standard value for two or more time points. The above time points refer to the timing of measurement of the myeloblast rate after administration of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof. The timing of measurement of the myeloblast rate may be, for example, every 1 to 365 days, every 1 to 180 days, every 1 to 90 days, every 1 to 60 days, every 1 to 30 days, or every 1 to 4 weeks. The timing of measurement may be, for example, every day or more or less than 365 days, or may be every 180 days, 90 days, 60 days, 30 days, 4 weeks, 3 weeks, 2 weeks, 1 week, 3 days, or more than or more than every 180 days, 90 days, 60 days, 30 days, 4 weeks, 3 weeks, 2 weeks, 1 week, 3 days, or less than or equal to every 180 days, 90 days, 60 days, 30 days, 4 weeks, 3 weeks, 2 weeks, 1 week, or 3 days. Furthermore, for example, if the change rate in myeloblasts up to 100 days after treatment remains 150% or less at two or more time points, it can be determined that the change rate in myeloblasts is 150% or less.

[0129] (7) Gender differences The selection method of this embodiment can provide an indicator that the subject is one for whom the effect can be expected based on one or a combination of two or more selected from the group consisting of (1) to (6) above, and may further include a step of providing an indicator that the subject is one for whom the effect of the pharmaceutical composition can be expected if the subject is male (male if the subject is human).

[0130] (Cancer treatment or prevention method) The method for treating or preventing cancer of this embodiment includes the steps of selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer by the above-described selection method; and administering the pharmaceutical composition to the selected subject.

[0131] The method for treating or preventing cancer of this embodiment includes determining a subject to be administered a pharmaceutical composition for treating or preventing cancer by a selection method based on a combination of one or more selected from the group consisting of (1) to (6) above, and administering the pharmaceutical composition. The method for treating or preventing cancer of this embodiment also includes determining whether or not to administer a pharmaceutical composition for treating or preventing cancer to a subject by a selection method based on a combination of one or more selected from the group consisting of (1) to (6) above, and administering the pharmaceutical composition to the subject. Therefore, the method for treating or preventing cancer of this embodiment includes, for example, determining the presence or absence of a mutation in the TP53 gene and / or the BCOR gene as described in (1) above, and administering a pharmaceutical composition for treating or preventing cancer to a subject with a wild-type TP53 and / or BCOR gene, as described in (2) above. The method for treating or preventing cancer of this embodiment also includes, for example, determining the mRNA expression level of the WT1 gene as described in (2) above, and administering a pharmaceutical composition for treating or preventing cancer to a subject whose mRNA expression level of the WT1 gene is below or equal to the reference value. The method for treating or preventing cancer of this embodiment includes, for example, administering a pharmaceutical composition for treating or preventing cancer to a subject whose karyotype based on IPSS-R is other than Very Poor, as described in (3). The method for treating or preventing cancer of this embodiment includes, for example, detecting WT1 antigen peptide-specific CD8 T cells using a sample collected from a subject administered with the pharmaceutical composition, peptide, or a pharmaceutically acceptable salt thereof, as described in (4), and administering the pharmaceutical composition to a subject in which the number of WT1 antigen peptide-specific CD8 T cells has increased compared to a sample collected from the subject before administration. The method for treating or preventing cancer of this embodiment includes, for example, administering the pharmaceutical composition to a subject in which a delayed-type hypersensitivity reaction has been detected following multiple administrations of the pharmaceutical composition, peptide, or a pharmaceutically acceptable salt thereof, as described in (5).The method for treating or preventing cancer of this embodiment includes, for example, as described above in (6), administering the pharmaceutical composition to a subject for whom the ratio of myeloblasts after administration of the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof divided by the ratio of myeloblasts before administration of the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof is less than or equal to a reference value.

[0132] The dosage of the pharmaceutical composition of this embodiment in the formulation in the cancer treatment or prevention method of this embodiment can be adjusted appropriately depending on the disease to be treated, the age and body weight of the patient, etc., but may be 0.0001 mg to 1000 mg, 0.001 mg to 1000 mg, or 0.1 mg to 10 mg. For example, the dosage per administration may be 1.75 mg to 17.5 mg, 3.5 mg to 10.5 mg, or 10.5 mg. The dosage per dose is 0.0001 mg or more, 0.0005 mg or more, 0.001 mg or more, 0.005 mg or more, 0.01 mg or more, 0.05 mg or more, 0.1 mg or more, 0.25 mg or more, 0.5 mg or more, 0.75 mg or more, 1.0 mg or more, 1.25 mg or more, 1.5 mg or more, 1.75 mg or more, 2.0 mg or more, 2.25 mg or more, 2.5 mg or more, 2.75 mg or more, 3.0 mg or more, 3.25 mg or more, 3.5 mg or more, 3.75 mg or more, 4.0 mg or more, 4.25 mg or more, 5.5 mg or more, 5.75 mg or more, 6.0 mg or more, and may be 1000 mg or less, 750 mg or less, 500 mg or less, 250 mg or less, 125 mg or less, 100 mg or less, 50 mg or less, mg or less, 40 mg or less, 35 mg or less, 30 mg or less, 25 mg or less, 20 mg or less, 15 mg or less, 10 mg or less.

[0133] Examples of administration methods include intradermal, subcutaneous, intramuscular, intravenous, and transdermal administration. Intradermal and subcutaneous administration are preferred, as they efficiently induce CTLs. The number of administrations and the interval between administrations can be adjusted as appropriate depending on the disease to be treated or prevented and individual patient differences, but are usually multiple times, preferably once every few days to several months. For example, the drug may be administered once every day to six months, once every three days to three months, once every one to four weeks, once every two to four weeks, or once every six, five, four, three, two, or one week. Alternatively, the compound may be administered once every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months at the minimum, or once every 12 months, 10 months, 8 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, or 5 days at the maximum.

[0134] The timing of administration can be changed after a predetermined period of time, for example, after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. For example, the administration can be once every 2 weeks for the first 6 months, once every 2 weeks from 1 month to 5 months, and once every 2 to 4 weeks from 6 months onward. For example, the administration can be every 2 weeks for the first 6 months, and then every 2 to 4 weeks thereafter.

[0135] The present embodiment also includes a pharmaceutical composition for use in a method for treating or preventing cancer. The pharmaceutical composition, the method for treating or preventing cancer, etc. are as described above.

[0136] This embodiment also includes a method for determining subjects to whom a pharmaceutical composition for treating or preventing cancer should be administered. The subjects can be determined based on one or a combination of two or more selected from the group consisting of (1) to (6) above. The method includes, for example, determining the presence or absence of mutations in the TP53 gene and / or the BCOR gene, as described in (1), and determining subjects with wild-type TP53 and / or wild-type BCOR as subjects to whom a pharmaceutical composition for treating or preventing cancer should be administered. The method also includes, for example, determining the mRNA expression level of the WT1 gene, as described in (2), and determining subjects whose mRNA expression level of the WT1 gene is below or equal to the reference value as subjects to whom a pharmaceutical composition for treating or preventing cancer should be administered. The method also includes, for example, determining subjects whose karyotype based on IPSS-R is other than Very Poor as subjects to whom a pharmaceutical composition for treating or preventing cancer should be administered, as described in (3). The above method also includes, for example, as described in (4), detecting WT1 antigen peptide-specific CD8 T cells in a sample collected from a subject administered with a pharmaceutical composition or a peptide or a pharmaceutically acceptable salt thereof, comparing the sample with a sample collected from the subject before administration, and determining a subject in which the WT1 antigen peptide-specific CD8 T cells have increased as a subject to administer the pharmaceutical composition. The above method also includes, for example, as described in (5), determining a subject in which a delayed-type hypersensitivity reaction has been detected after multiple administrations of the pharmaceutical composition or a peptide or a pharmaceutically acceptable salt thereof as a subject to administer the pharmaceutical composition. The above method also includes, for example, as described in (6), determining a subject in which the myeloblast percentage after administration of the pharmaceutical composition or a peptide or a pharmaceutically acceptable salt thereof divided by the myeloblast percentage before administration of the pharmaceutical composition or a peptide or a pharmaceutically acceptable salt thereof is less than or equal to a reference value as a subject to administer the pharmaceutical composition.

[0137] This embodiment also includes a method for determining whether to administer a pharmaceutical composition for treating or preventing cancer. The decision to administer a pharmaceutical composition for treating or preventing cancer can be based on one or a combination of two or more selected from the group consisting of (1) to (6) above. The method includes, for example, determining the presence or absence of mutations in the TP53 gene and / or BCOR gene using a sample collected from a subject, as described in (1), and deciding to administer a pharmaceutical composition for treating or preventing cancer to the subject if the TP53 gene is wild-type or BCOR wild-type, or deciding not to administer a pharmaceutical composition for treating or preventing cancer to the subject if the TP53 gene is mutant and / or BCOR mutant. The method also includes, for example, determining the mRNA expression level of the WT1 gene in the sample collected from the subject, as described in (2), and deciding to administer a pharmaceutical composition for treating or preventing cancer to the subject if the mRNA expression level of the WT1 gene is below or equal to a reference value, or deciding not to administer a pharmaceutical composition for treating or preventing cancer to the subject if the mRNA expression level of the WT1 gene is above or equal to the reference value. The above method also includes, for example, as described in (3), determining to administer a pharmaceutical composition for treating or preventing cancer to the subject if the subject's karyotype based on IPSS-R is other than Very Poor, and not to administer a pharmaceutical composition for treating or preventing cancer to the subject if the subject's karyotype based on IPSS-R is Very Poor.The above method also includes, for example, as described in (4), determining to detect WT1 antigenic peptide-specific CD8 T cells in a sample collected from the subject to which the pharmaceutical composition or the peptide or a pharmaceutically acceptable salt thereof has been administered, and administering the pharmaceutical composition to the subject if the number of WT1 antigenic peptide-specific CD8 T cells has increased compared to a sample collected from the subject before administration, and not to administer the pharmaceutical composition to the subject if the number of WT1 antigenic peptide-specific CD8 T cells has remained the same or decreased.Furthermore, the above method includes, for example, as described in (5), determining to administer the pharmaceutical composition to the subject when a delayed-type hypersensitivity reaction is detected in the subject after multiple administrations of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof, and determining not to administer the pharmaceutical composition to the subject when a delayed-type hypersensitivity reaction is not detected in the subject. Furthermore, the above method includes, for example, as described in (6), determining to administer the pharmaceutical composition to the subject when the myeloblast percentage after administration of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof divided by the myeloblast percentage before administration of the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof is less than or equal to a reference value, and determining not to administer the pharmaceutical composition to the subject when the value is equal to or greater than the reference value.

[0138] This embodiment also includes a method for screening subjects to be administered with a pharmaceutical composition for treating or preventing cancer. The screening can be performed based on one or a combination of two or more selected from the group consisting of (1) to (6) above. The method includes, for example, determining the presence or absence of mutations in the TP53 gene and / or the BCOR gene and selecting subjects with wild-type TP53 and / or wild-type BCOR, as described in (1). The method also includes, for example, determining the expression level of WT1 gene mRNA and selecting subjects whose expression level of WT1 gene mRNA is below or equal to the reference value, as described in (2). The method also includes, for example, selecting subjects whose karyotype based on IPSS-R is other than Very Poor, as described in (3). The method also includes, for example, detecting WT1 antigen peptide-specific CD8 T cells in a sample collected from a subject administered with a pharmaceutical composition, a peptide, or a pharmaceutically acceptable salt thereof, and selecting subjects whose WT1 antigen peptide-specific CD8 T cells have been increased compared to a sample collected from the subject before administration, as described in (4). The method also includes, for example, selecting a subject in whom a delayed-type hypersensitivity reaction has been detected after multiple administrations of the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof, as described above in (5).The method also includes, for example, selecting a subject in whom the myeloblast percentage after administration of the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof divided by the myeloblast percentage before administration of the pharmaceutical composition, peptide, or pharmaceutically acceptable salt thereof is less than or equal to a reference value, as described above in (6). [Example]

[0139] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. It's not that.

[0140] Example 1: Subjects in this Example and Effect of WT1 Peptide Cocktail Vaccine Unless otherwise specified, the following examples are based on the data of 47 patients with relapsed / refractory myelodysplastic syndrome (MDS) who provided informed consent and participated in phase 1 and 2 clinical trials of a W / O emulsion cocktail vaccine (see WO 2014 / 157692; hereinafter, also referred to as the WT1 peptide cocktail vaccine) containing the WT1 killer peptide conjugate shown in formula (3) below and the WT1 helper peptide represented by WAPVLDFAPPGASAYGSL (SEQ ID NO: 14). The breakdown of the HLA types of these patients is as follows: HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H ... * 02:01 or HLA-A * 02:06 12 cases of myelodysplastic syndrome (MDS) patients with HLA-A * 28 cases of 24:02-positive MDS patients, HLA-A * 02:01 positive and HLA-A * 24:02 5 cases of MDS patients positive for HLA-A * 02:06 Positive and HLA-A * Two cases of MDS patients were 24:02 positive. The cases in the Phase 1 study included seven high-risk patients (H) and five low-risk patients (L), while the cases in the Phase 2 study included 35 high-risk patients (H). This example focused on high-risk patients only, including 42 azacitidine-refractory high-risk patients (7 cases in the Phase 1 study and 35 cases in the Phase 2 study). The high-risk patients were classified as intermediate to very high in the risk classification of the revised IPSS (IPSS-R). The 42 azacitidine-refractory high-risk patients included 40 patients who were ineffective or no longer effective with azacitidine, and 2 patients who could not continue azacitidine treatment due to side effects. [ka] (wherein the bond between C and C represents a disulfide bond.)

[0141] In phase 1 and 2 clinical trials, the WT1 peptide cocktail vaccine was administered intradermally (ID) every 2 weeks for 6 months as a vaccine induction, followed by administration every 2 to 4 weeks until discontinuation. In phase 1, cohort 1 received a 3.5 mg dose (2 mg WT1 killer peptide conjugate + 1.5 mg WT1 helper peptide), and cohort 2 received a 10.5 mg dose (6 mg WT1 killer peptide conjugate + 4.5 mg WT1 helper peptide). In phase 2, the recommended dose was 10.5 mg (6 mg WT1 killer peptide conjugate + 4.5 mg WT1 helper peptide).

[0142] The median survival time (mOS) in this study was 8.6 months (90% confidence interval 6.8-11.1 months), which tended to be longer than the 5.6 months in historical data (95% confidence interval 5.0-7.2 months, Prebet et al., Journal of Clinical Oncology 29, no. 24, p3322-3327 (2011)).

[0143] Example 2: Effect of WT1 peptide vaccine and influence of karyotype A comparison of the test results of the WT1 peptide cocktail vaccine in Example 1 with the control (BSC in the Rigosertib study) in the ONTIME study of Rigosertib (a phase 3 clinical trial; Garcia-Manero et al., The Lancet Oncology 17, no. 4, p. 496-508 (2016) Table S1 "MDS cytogenetic prognosis") is shown in Figure 1. Taking into account differences from the patient population in the phase 3 study of Rigosertib, the comparison was performed on 33 cases, excluding two cases in which azacitidine did not respond due to side effects, six high-risk cases with a blast count of <5%, and one case with an unknown karyotype, from the 42 cases in Example 1.

[0144] The mOS by karyotype tended to be longer in the Good / Very good to Poor group (excluding Very Poor karyotype) compared with the BSC in the Rigosertib study, suggesting that the prolonged mOS may be due to the effect of the WT1 peptide cocktail vaccine.In the Very Poor karyotype group, the mOS in the WT1 peptide cocktail vaccine group was equivalent to the BSC in the Rigosertib study.

[0145] Example 3: Search for genetic biomarkers for selecting patients who are expected to benefit from treatment with a WT1 peptide vaccine Of the 42 high-risk cases described in Example 1, 29 cases for which informed consent was obtained were subjected to genetic testing related to myelodysplastic syndrome (MDS). Of the 29 cases, 28 cases were used for subsequent genetic analysis, excluding one case in which azacitidine was ineffective due to side effects. Bone marrow fluid was collected from the patients within 28 days prior to the start of administration of the WT1 peptide cocktail vaccine.

[0146] DNA extraction from bone marrow fluid and specimen quality assessment DNA was extracted from 0.5 mL of bone marrow fluid sample using the Wizard Genomic DNA purification Kit (Promega Corporation) according to the protocol (3A Isolating Genomic DNA from whole blood) included with the kit.

[0147] The quality of the extracted DNA was evaluated by agarose gel electrophoresis to confirm whether clear bands in the high molecular weight region could be detected. The fluorescence intensity was measured using a plate reader with the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies), and the concentration was calculated from a calibration curve.

[0148] Next-generation sequencing (NGS) assays Next-generation sequencing (NGS) assays were performed using the TruSight Myeloid Sequencing Panel (Illumina, Inc.), which targets mutations associated with myeloid malignancies, including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML). Genomic DNA extracted from bone marrow was analyzed for sequences targeted by the TruSight Myeloid Sequencing Panel. Target regions were amplified using the panel to generate libraries. Strand length distribution was confirmed by electrophoresis analysis using the Agilent DNA 1000 Kit (Agilent Technologies, Inc.), and quantification was performed using a real-time PCR system (Applied Biosystems, Inc.). The quantification results were corrected for the average library size to calculate library concentration. Nucleotide sequences were determined using MiSeq (Illumina, Inc.). The panel covers the 40 genes listed in Table 1 below.

[0149] [Table 1]

[0150] Bioinformatics analysis The base sequences determined by MiSeq (Illumina Inc.) were analyzed for gene mutations by bioinformatics analysis using MiSeq Reporter (Illumina Inc.).

[0151] result The results of gene mutation analysis are shown in Table 2. The thick horizontal lines in the table represent mOS, with overall survival (OS) increasing from top to bottom. Note that the IPSS-R karyotypes of patients with Good / Very good or Intermediate IPSS-R karyotypes are omitted.

[0152] [Table 2]

[0153] Patients with TP53 mutations or BCOR mutations tended to have a shorter OS. This suggests that the presence or absence of mutations in the TP53 and BCOR genes may be useful genetic markers for selecting patients who are likely to benefit from treatment with WT1 peptide vaccines. Furthermore, there was a tendency for very poor karyotypes to be more prevalent in the group with a short OS.

[0154] Example 4: Usefulness as a genetic marker for TP53 / BCOR gene mutations To verify the usefulness of the presence or absence of mutations in the TP53 and BCOR genes as genetic markers, we compared survival curves based on the presence or absence of the above mutations.

[0155] The patients described in Example 1 were divided into 17 patients with TP53 wild-type and BCOR wild-type disease and 11 patients with TP53 mutant or BCOR mutant disease, and the survival curves were compared. As shown in Figure 2, the median overall survival (mOS) of the TP53 wild-type and BCOR wild-type patients tended to be longer than the mOS of the TP53 or BCOR mutant patients.

[0156] Furthermore, we confirmed the WT1 antigen peptide-specific immune responses induced by the WT1 peptide cocktail vaccine in 17 TP53 wild-type and BCOR wild-type cases and 11 TP53 mutant or BCOR mutant cases using the HLA tetramer assay and delayed-type hypersensitivity reaction described below. Figure 3 shows the results of a comparison of overall survival (OS) and immune responses between TP53 wild-type and BCOR wild-type cases and TP53 mutant or BCOR mutant cases. The results shown in Figure 3 apply to 15 TP53 wild-type and BCOR wild-type cases and 9 TP53 mutant or BCOR mutant cases, excluding 4 cases in which the immune response could not be determined.

[0157] TP53 wild-type and BCOR wild-type patients tended to have a longer overall survival period. Furthermore, many TP53 wild-type and BCOR wild-type patients had a positive immune response (14 / 15 cases). On the other hand, TP53 mutant and BCOR mutant patients tended to have a shorter overall survival period, regardless of whether the immune response was positive or negative.

[0158] This indicates that the presence or absence of mutations in the TP53 and BCOR genes is useful as a genetic marker for selecting patients who are expected to benefit from treatment with the WT1 peptide vaccine.

[0159] Example 5: Usefulness of WT1 mRNA as a genetic marker 1 Of the 42 high-risk patients in Example 1 who were refractory to azacitidine, WT1 mRNA expression was confirmed in 40 cases, excluding 2 cases in which azacitidine was ineffective due to side effects.

[0160] DNA extraction from peripheral blood and sample quality assessment Peripheral blood and bone marrow fluid were collected from patients within 28 days prior to the start of administration of the WT1 peptide cocktail vaccine. RNA was extracted and purified from 7 mL of whole blood or 0.5 mL of bone marrow fluid using the QIAcube (Qiagen) fully automated RNA purification system. Following the QIAcube user manual, reagents, tubes, and samples from the RNeasy Mini Kit (Qiagen) were loaded. RNA was extracted using the RNeasy Mini program, selected from the QIAcube Standard Programs stored in the QIAcube.

[0161] WT1 mRNA expression analysis WT1 mRNA expression analysis was performed using the WT1 mRNA Measurement Kit II "Otsuka" (Otsuka Pharmaceutical Co., Ltd.) Unless otherwise specified, the reagents used were those provided with the kit.

[0162] The concentration of extracted RNA was adjusted to 50 ng / μL by adding RNase-free water. A WT1 / GAPDH mixed RNA standard solution was prepared as standard solution 1. Standard solution 1 was diluted 10-fold with standard diluent to prepare standard solution 2. Repeated 10-fold dilutions were repeated to prepare standard solution 5. Each reaction mixture consisted of 10 μL of RT-PCR mix (R1) and 5 μL of metal ion solution. RT-PCR reactions were performed using a real-time PCR system (Applied Biosystems 7500 Fast Dx, Applied Biosystems) according to "3. Measurement Procedure" in the protocol provided with the kit. Measurement values ​​for WT1 mRNA and GSDPH mRNA in the samples were calculated using a standard curve constructed from standards 1–5.

[0163] According to "4. Method for calculating WT1 mRNA expression level" in the protocol attached to the kit, the WT1 mRNA expression level was calculated as follows. Specifically, the WT1 mRNA expression level was calculated by dividing the measured WT1 mRNA value by the measured GAPDH mRNA value (WT1 mRNA copy number per GAPDH mRNA copy), multiplying this value by the average GAPDH mRNA copy number per μg of RNA in healthy adults (GAPDH mRNA expression level), as shown in the following formula: 7 (copies / μg RNA) is the average GAPDH mRNA measurement per μg RNA in healthy adults.

number

[0164] Result(1) The results of WT1 mRNA expression analysis are shown in Figure 4. WT1 mRNA expression was detected in the peripheral blood of all 40 cases. The mOS of cases with WT1 mRNA expression levels of less than 10,000 copies / μg RNA tended to be longer than that of cases with WT1 mRNA expression levels of 10,000 copies / μg RNA or more.

[0165] Furthermore, WT1 antigen peptide-specific immune responses were confirmed by HLA tetramer assay and delayed-type hypersensitivity test (described below) in 27 cases with WT1 mRNA expression levels below 10,000 copies / μg RNA and 13 cases with WT1 mRNA expression levels of 10,000 copies / μg RNA or higher. Figure 5 shows the results of comparing overall survival (OS) and WT1 antigen peptide-specific immune responses with respect to WT1 mRNA expression levels. The results shown in Figure 5 apply to 25 cases with WT1 mRNA expression levels below 10,000 copies / μg RNA and 11 cases with WT1 mRNA expression levels of 10,000 copies / μg RNA or higher, excluding 4 cases in which the WT1 antigen peptide-specific immune responses could not be determined.

[0166] In the group of cases with WT1 mRNA expression levels below 10,000 copies / μg RNA, patients with WT1 antigen peptide-specific immune responses tended to have a longer OS. In contrast, in the group of cases with WT1 mRNA expression levels above 10,000 copies / μg RNA, no difference in OS was observed between patients with a positive or negative WT1 antigen peptide-specific immune response. Furthermore, we confirmed that WT1 mRNA expression levels in peripheral blood and bone marrow fluid correlated, with a similar trend observed in bone marrow fluid (Figure 16).

[0167] Result (2) The results of WT1 mRNA expression analysis are shown in Figure 6. WT1 mRNA expression was detected in the peripheral blood of all 40 cases. The mOS of cases with WT1 mRNA expression levels of less than 4000 copies / μg RNA tended to be longer than that of cases with WT1 mRNA expression levels of 4000 copies / μg RNA or more.

[0168] Furthermore, WT1 antigen peptide-specific immune responses were confirmed by HLA tetramer assay and delayed-type hypersensitivity test (described below) in 22 cases with WT1 mRNA expression levels below 4000 copies / μg RNA and 18 cases with WT1 mRNA expression levels above 4000 copies / μg RNA. Figure 7 shows the results of comparing overall survival (OS) and WT1 antigen peptide-specific immune responses with respect to WT1 mRNA expression levels. The results shown in Figure 7 apply to 21 cases with WT1 mRNA expression levels below 4000 copies / μg RNA and 15 cases with WT1 mRNA expression levels above 4000 copies / μg RNA, excluding 4 cases in which the WT1 antigen peptide-specific immune responses could not be determined.

[0169] In the group of cases in which WT1 mRNA expression levels were less than 4000 copies / μg RNA, cases in which WT1 antigen peptide-specific immune responses were observed tended to have a longer OS. On the other hand, in the group of cases in which WT1 mRNA expression levels were 4000 copies / μg RNA or higher, no difference in OS was observed between positive and negative WT1 antigen peptide-specific immune responses. Furthermore, we confirmed that WT1 mRNA expression levels in peripheral blood and bone marrow fluid correlated, and a similar trend was observed in bone marrow fluid (Figure 16).

[0170] These results demonstrate that the expression level of WT1 mRNA is useful as a genetic marker for selecting patients who are likely to benefit from treatment with a WT1 peptide vaccine. Furthermore, both 10,000 copies / μg RNA and 4,000 copies / μg RNA were useful reference values ​​for this marker.

[0171] Example 6: Detection of WT1 antigen peptide-specific immune response by HLA tetramer assay Blood samples were collected from patients before and after administration of the WT1 peptide cocktail vaccine, and the proportion of WT1 antigen peptide-specific CD8 T cells in lymphocytes or CD8-positive lymphocytes was measured by flow cytometry using a tetramer reagent.

[0172] Administration and blood sampling Testing was performed on 42 high-risk patients who were refractory to azacitidine in Example 1. The breakdown of the HLA types of the 42 patients was as follows: HLA-A * 02:01 or HLA-A * 02:06 10 cases of myelodysplastic syndrome (MDS) patients with HLA-A * 24:02 25 cases of MDS patients positive for HLA-A * 02:01 positive and HLA-A * 24:02 5 cases of MDS patients positive for HLA-A * 02:06 Positive and HLA-A * Two 24:02-positive MDS patients were included. Peripheral blood samples were collected and assayed from the patients within 28 days before the start of administration of the pharmaceutical composition, 15 days after the second administration, 15 days after the sixth administration, 15 days after the twelfth administration, 15 days after the eighteenth administration, 15 days after every six subsequent administrations, and within 28 days after the final administration. Each administration consisted of intradermal administration of 1.75 mg, 3.5 mg, or 10.5 mg of the WT1 peptide cocktail vaccine.

[0173] HLA tetramer reagents Because WT1 antigen peptide-specific CD8 T cells are HLA-restricted, measurements were performed using tetramer reagents that matched the patient's HLA. * For 24:02-positive patients, fluorescent dye Phycoerythrin (PE)-labeled HLA-A was prepared using a peptide consisting of CYTWNQMNL (SEQ ID NO: 4) of the WT1 protein. * 24:02 tetramer (T-Select HLA-A * 24:02 WT1 (mutant) Tetramer-CYTWNQMNL PE-labeled (Medical and Biological Laboratories, Inc.) was used. Hereinafter, this reagent will also be referred to as "PE-labeled WT1 2402."

[0174] HLA-A * 02:01 or HLA-A *For 02:06-positive patients, fluorescent dye allophycocyanin (APC)-labeled HLA-A was prepared using a peptide consisting of RMFPNAPYL of the WT1 protein (SEQ ID NO: 2). * 02:01 Tetramer (T-Select HLA-A * 02:01 WT1 126-134 Tetramer-RMFPNAPYL-APC, Medical and Biological Laboratories Co., Ltd., HLA-A * 02:01 and HLA-A * 02:06) and a peptide consisting of VLDFAPPGA of the WT1 protein (SEQ ID NO: 9) labeled with the fluorescent dye Phycoerythrin (PE). * 02:01 tetramer (HLA-A * 02:01 Tetramer-VLDFAPPGA-PE, manufactured by Medical and Biological Laboratories Co., Ltd., HLA-A * 02:01 and HLA-A * The tetramer reagent was used (which can detect both WT1 0201 and WT1 02:06). Hereafter, the former reagent will be referred to as "APC-labeled WT1 0201" and the latter reagent will be referred to as "PE-labeled WT1 0201." Before use, the tetramer reagent was placed in a microtube and centrifuged at 1620 × g for 5 minutes at room temperature, and the supernatant was used.

[0175] staining HLA-A * Staining of patients with 24:02 was performed as follows. 2.5 mL of collected blood was dispensed into a sample tube, and the remaining blood was dispensed into a control tube. 5 μL of PE-labeled WT1 2402 was added to the sample tube. After incubation at room temperature for 10 minutes in the dark, 15 μL of FITC-labeled CD8 (Cytostat / Coulter Clone T8-FITC, Beckman Coulter, Inc.), 12.5 μL each of 7-AAD (7-AAD Staining Solution, Becton Dickinson Japan), PC5-labeled CD4 (IO Test CD4-PC5, Beckman Coulter, Inc.), and PC5-labeled CD19 (IO Test CD19-PC5, Beckman Coulter, Inc.) were added to the sample tube and mixed. After 15 minutes of incubation at room temperature, 26 mL of lysing solution (BD FACS™ Lysing solution (Becton Dickinson and Company, Japan) diluted 10-fold with purified water) was added to each tube, followed by stirring. After 10 minutes at room temperature, the tubes were centrifuged at 300 × g for 5 minutes. The supernatant was aspirated and, after stirring, 30 mL of azide PBS (PBS containing 1% sodium azide) was dispensed into each tube and centrifuged at 300 × g for 5 minutes. The supernatant was aspirated and then resuspended in 300 μL of CellFix solution (BD FACS Cell Fix, Becton Dickinson and Company, Japan) and transferred to a measurement tube.

[0176] HLA-A * 02:01 or HLA-A * Staining of patients with 02:06 was performed as follows. 2.5 mL of collected blood was dispensed into a sample tube, and the remaining blood into a control tube. 5 μL each of APC-labeled WT1 0201 and PE-labeled WT1 0201 was added to the sample tube. After 10 minutes of incubation at room temperature in the dark, 15 μL of FITC-labeled CD8, 12.5 μL each of 7-AAD, APC-H7-labeled CD3 (CD3 APC-H7, Becton Dickinson Japan), Pacific Blue-labeled CD4 (IOTest CD4-Pacific Blue, Beckman Coulter), and PE-Cy7-labeled CD19 (IOTest CD19-PC7, Beckman Coulter) were added to the sample tube and vortexed. After 15 minutes of incubation at room temperature, 26 mL of lysing solution was added and vortexed thoroughly. The tubes were left to stand for 10 minutes at room temperature and then centrifuged at 300 × g for 5 minutes. The supernatant was removed by aspirating, and after stirring, 30 mL of azide PBS was dispensed into each tube and centrifuged at 300 × g for 5 minutes. After removing the supernatant by aspirating, the cells were resuspended in 300 μL of CellFix preparation solution and transferred to measurement tubes.

[0177] HLA-A * 24:02 and HLA-A * 02:01 or HLA-A * Staining of patients with both 02:06 and HLA-A * Staining of patients with 24:02 and HLA-A * 02:01 or HLA-A * Both staining procedures were performed on patients with 02:06.

[0178] Flow cytometry analysis The proportion of WT1 antigen peptide-specific CD8 T cells in lymphocytes or CD8-positive lymphocytes was measured by flow cytometry.

[0179] The samples prepared above were measured using a flow cytometer, FACS Canto II (BD Biosciences). The flow cytometer generates scattered light (forward scattered light (FSC) reflecting size and side scattered light (SSC) reflecting internal structure) with an intensity that corresponds to the characteristics of the cells (size and internal structure), as well as fluorescence that depends on the amount of bound labeled antibody. The intensity of these parameters was measured for each cell in the sample. By graphing the distribution of each cell based on the obtained parameter intensities, it is possible to calculate the specific population. Based on the cell size and internal structure, the cells corresponded to lymphocytes, and were negative for anti-CD4 antibody, anti-CD19 antibody, and 7-AAD staining solution, and positive only for anti-CD8 antibody (HLA-A * 02:01 or HLA-A * 02:06) or both anti-CD3 and anti-CD8 antibodies positive (HLA-A * Lymphocyte fractions (targeting patients with 02:01 / 06) were extracted, and the tetramer-positive / lymphocyte fraction or tetramer-positive / CD8-positive cell fraction within the fraction was evaluated. The FITC-labeled CD8-positive and PE-labeled WT1 2402-positive cell population was gated at the 10 of the y-axis. 3 The gate for the FITC-labeled CD8-positive and PE-labeled WT1 0201-positive cell population was set at 3 x 10 on the y-axis (Gate CD8(+)tet(+)). 2 The gate for the FITC-labeled CD8-positive and APC-labeled WT1 0201-positive cell population was set at 3 x 10 on the y-axis (Gate CD8(+)tet(+)). 2 The gate was set based on this (Gate CD8(+)tet(+)).

[0180] HLA-A *In 24:02 patients, if an event was observed in the gate CD8(+)tet(+) before administration of the WT1 peptide cocktail vaccine, the ratio of PE-labeled WT1 24:02-positive cells was compared using the number of strongly CD8-positive lymphocytes as the denominator before and after administration of the WT1 peptide cocktail vaccine.If no event was observed in the gate CD8(+)tet(+) before administration, the number of events in the gate CD8(+)tet(+) after administration was calculated.HLA-A * 02:01 or HLA-A * For 02:06 patients, if an event was detected in the gate CD8(+)tet(+) before administration of the WT1 peptide cocktail vaccine, the ratios of PE-labeled WT1 02:01- or 02:06-positive cells and APC-labeled WT1 02:01- or 02:06-positive cells were compared, with the number of strongly CD8-positive lymphocytes before and after administration of the WT1 peptide cocktail vaccine. If no event was detected in the gate CD8(+)tet(+) before administration, the number of events within the gate CD8(+)tet(+) after administration was calculated. The following criteria were used to determine whether the WT1 antigen peptide-specific immune response was positive or negative by HLA tetramer assay. If the response was positive or maintained at any time point after administration, the HLA tetramer assay was considered positive or maintained. If the response was negative at any time point after administration, the HLA tetramer assay was considered negative.

[0181] [Table 3]

[0182] result Based on the above criteria, all 47 cases were judged as positive by HLA tetramer assay in 30 cases (63.8%), maintained in 3 cases (6.4%), and negative in 13 cases (27.7%). In one case (2.1%), the HLA tetramer assay could not be determined.

[0183] Example 7: Detection of delayed hypersensitivity reaction (DTH reaction) Preparation of DTH chemical solution Of the WT1 peptide cocktail vaccines shown in Example 1, 20 mg of the WT1 killer peptide conjugate was mixed with 2 mL of water for injection, and 18 mg of the WT1 helper peptide was mixed with 1.8 mL of water for injection to prepare a WT1 killer peptide conjugate solution (10 mg / mL) and a WT1 helper peptide solution (10 mg / mL). Each solution was further diluted 10-fold (1 mg / mL) with lactated Ringer's solution. The DTH drug solution was used within 3 hours of preparation.

[0184] Inoculation and measurement Two DTH solutions (WT1 killer peptide conjugate solution and WT1 helper peptide solution) were intradermally injected in 100 μL (100 μg) into the patient's forearm, spaced at least 3 cm apart. As a negative control, 100 μL of lactated Ringer's solution (control solution) was intradermally injected into the ipsilateral forearm at least 3 cm away from the DTH solution injection site. Two days after administration, the diameter of the reddening lesion (long diameter, short diameter, and nature (double reddening, induration, ulcer, etc.)) was measured. The difference between the long diameter of the reddening lesion for the WT1 killer peptide conjugate solution or WT1 helper peptide solution and that for the control solution was calculated, and the DTH response was scored based on this difference according to the table below.

[0185] [Table 4]

[0186] DTH tests were performed before the start of WT1 peptide cocktail vaccine administration (within 28 days before administration), two days after the second administration, two days after the 12th administration, and after the final administration (within 28 days after administration). DTH responses were scored based on the above criteria. The highest score in each case after administration was used for subsequent analysis.

[0187] result Based on the above criteria, scores were determined for all 47 cases, with 11 cases (23.4%) receiving a score of 0, 4 cases (8.5%) receiving a score of + / -, 9 cases (19.1%) receiving a score of 1, 4 cases (8.5%) receiving a score of 2, 9 cases (19.1%) receiving a score of 3, 7 cases (14.9%) receiving a score that could not be determined, and 3 cases (6.4%) receiving a DTH test that could not be performed.

[0188] Example 8: Establishment of criteria for classifying WT1 antigen peptide-specific immune responses as positive or negative Regarding the WT1 antigen peptide-specific immune response induced by the WT1 peptide cocktail vaccine, the results of the HLA tetramer assay and the maximum score of the DTH test using the WT1 killer peptide conjugate were analyzed from both sides, and criteria for classifying the WT1 antigen peptide-specific immune response as positive or negative were established.

[0189] Since the objective of this Example was to comprehensively assess WT1 antigen peptide-specific immune responses, a total of 47 cases were analyzed, including 42 high-risk cases and 5 low-risk cases described in Example 1. However, of these, 1 case in which assessment by HLA tetramer assay was impossible in Example 6, and 10 cases in which DTH score determination was impossible and DTH test could not be performed in Example 7 were excluded from this analysis.

[0190] Many of the cases that were positive in the HLA tetramer assay also had a score of + / - or higher in the DTH test using the WT1 killer peptide conjugate (left panel of Figure 8). Furthermore, many of the cases that had a score of less than + / - in the DTH test using the WT1 killer peptide conjugate were negative in the HLA tetramer assay (right panel of Figure 8). Based on these results, the positions indicated by the arrows in the left and right panels of Figure 8 were set as the criteria for a positive WT1 antigen peptide-specific immune response.

[0191] That is, if the result of the HLA tetramer assay is positive or the result of the DTH test using a WT1 killer peptide conjugate is a score of + / - or higher (a difference of 2 mm or more from the control), the WT1 antigen peptide-specific immune response to the WT1 peptide cocktail vaccine can be determined to be positive. On the other hand, if the analysis result of the HLA tetramer assay is unchanged or negative and the result of the DTH test using a WT1 killer peptide conjugate is 0 (a difference of less than 2 mm from the control), the WT1 antigen peptide-specific immune response to the WT1 peptide cocktail vaccine can be determined to be negative.

[0192] When all 47 cases were classified according to the above criteria, 33 cases (70.2%) were judged to have a positive WT1 antigen peptide-specific immune reaction, and 9 cases (19.1%) were judged to have a negative WT1 antigen peptide-specific immune reaction. In 5 cases, the DTH test was not performed or the DTH test revealed internal bleeding, making the WT1 antigen peptide-specific immune reaction unknown (10.7%).

[0193] Example 9: Analysis of WT1 antigen peptide-specific immune responses and clinical effects Of the 42 high-risk azacitidine ineffective patients described in Example 1, 28 cases were determined to have a positive WT1 antigen peptide-specific immune response and 8 cases were determined to have a negative WT1 antigen peptide-specific immune response according to the criteria in Example 8. A total of 36 cases were analyzed for their relationship with clinical efficacy as follows: 2 cases in which azacitidine was ineffective due to side effects, and 4 cases in which the WT1 antigen peptide-specific immune response could not be determined because the DTH test was not performed or internal bleeding was observed in the DTH test.

[0194] Myeloblast changes WT1 antigen peptide-specific immune responses and changes in bone marrow blasts were analyzed. If the change in the percentage of bone marrow blasts relative to the pre-treatment value remained below 150% for two or more time points up to approximately 3 months after the first dose (administrations were given once every two weeks, with bone marrow aspirate collected approximately 15 days after the second dose and / or approximately 15 days after the sixth dose), the blasts were judged to have stabilized. If the change in the percentage of bone marrow blasts relative to the pre-treatment value exceeded 150%, the blasts were judged not to have stabilized (worsened). As shown in Figure 9, many cases in which the WT1 antigen peptide-specific immune responses were positive exhibited long-term blast stability.

[0195] AML Transition Period The time to progression to acute myeloid leukemia (AML) was compared between patients with and without a WT1 antigen peptide-specific immune response. As shown in Figure 10, the time to progression to AML tended to be longer in patients with a WT1 antigen peptide-specific immune response.

[0196] survival curve Survival curves were compared between patients with positive and negative WT1 antigen peptide-specific immune responses. As shown in Figure 11, the mOS of the 28 cases determined to be positive for the WT1 antigen peptide-specific immune response tended to be longer than the mOS of the 8 cases determined to be negative for the WT1 antigen peptide-specific immune response.

[0197] Stabilization of myeloblasts and survival curves As shown in Figure 9, of the eight cases in which the WT1 antigen peptide-specific immune response was determined to be negative, none showed stabilization of myeloblasts. On the other hand, of the 28 cases in which the immune response was determined to be positive, stabilization of myeloblasts was observed in 15 cases (53.6%). Cases in which the WT1 antigen peptide-specific immune response was positive and in which myeloblast stabilization was observed tended to have the longest mOS.

[0198] Karyotype and survival curve The 35 cases, excluding one case with an unknown karyotype, were classified by IPSS-R karyotype, and the WT1 antigen peptide-specific immune responses and survival curves were compared. As shown in Figure 12, in the Good / Intermediate / Poor groups, cases determined to be positive for WT1 antigen peptide-specific immune responses tended to have a longer mOS than cases determined to be negative for WT1 antigen peptide-specific immune responses. Similarly, in the Very Poor karyotype group, which has a poor prognosis, cases determined to be positive for WT1 antigen peptide-specific immune responses tended to have a longer mOS than cases determined to be negative for WT1 antigen peptide-specific immune responses.

[0199] result Compared with patients with negative WT1 antigen peptide-specific immune responses, patients with positive WT1 antigen peptide-specific immune responses showed more cases with stabilized myeloblasts and a longer AML transition period. Furthermore, mOS tended to be longer. It was suggested that the induction of WT1 antigen peptide-specific immune responses may stabilize myeloblasts, thereby extending the AML transition period and overall survival. A similar trend toward a longer mOS was observed when stratified by karyotypes with favorable and unfavorable prognosis.

[0200] Example 10: Analysis by gender Of the 42 high-risk azacitidine-refractory patients in Example 1, 2 cases in which azacitidine was ineffective due to side effects were excluded, and for 40 cases, the median survival time for each gender difference was compared with the median survival time of the historical data and the BSC in the Rigosertib trial.

[0201] As shown in Table 5, more men were found to have positive WT1 antigen peptide-specific immune responses. Comparisons with historical data (Prebet et al., Journal of Clinical Oncology 29, no. 24, 3322-3327) and the control (BSC from the Rigosertib study) from the ONTIME study of Rigosertib (Garcia-Manero et al., The Lancet Oncology 17, no. 4, pp. 496-508 (2016)) showed that the WT1 peptide cocktail vaccine extended OS in men more than in women, suggesting its potential for greater efficacy, as shown in Figure 13.

[0202] [Table 5]

[0203] Example 11: Usefulness of WT1 mRNA as a Gene Marker 2 Of the 42 high-risk patients in Example 1 who were refractory to azacitidine, WT1 mRNA expression was confirmed in 40 cases, excluding 2 cases in which azacitidine was ineffective due to side effects.

[0204] DNA extraction from peripheral blood and sample quality assessment Peripheral blood and bone marrow fluid were collected from patients within 28 days prior to the start of administration of the WT1 peptide cocktail vaccine. RNA was extracted and purified from 7 mL of whole blood or 0.5 mL of bone marrow fluid using the QIAcube (Qiagen) fully automated RNA purification system. Following the QIAcube user manual, reagents, tubes, and samples from the RNeasy Mini Kit (Qiagen) were loaded. RNA was extracted using the RNeasy Mini program, selected from the QIAcube Standard Programs stored in the QIAcube.

[0205] WT1 mRNA expression analysis WT1 mRNA expression analysis was performed using the WT1 mRNA Measurement Kit II "Otsuka" (Otsuka Pharmaceutical Co., Ltd.) Unless otherwise specified, the reagents used were those provided with the kit.

[0206] The concentration of extracted RNA was adjusted to 50 ng / μL by adding RNase-free water. A WT1 / GAPDH mixed RNA standard solution was prepared as standard solution 1. Standard solution 1 was diluted 10-fold with standard diluent to prepare standard solution 2. Repeated 10-fold dilutions were repeated to prepare standard solution 5. Each reaction mixture consisted of 10 μL of RT-PCR mix (R1) and 5 μL of metal ion solution. RT-PCR reactions were performed using a real-time PCR system (Applied Biosystems 7500 Fast Dx, Applied Biosystems) according to "3. Measurement Procedure" in the protocol provided with the kit. Measurement values ​​for WT1 mRNA and GSDPH mRNA in the samples were calculated using a standard curve constructed from standards 1–5.

[0207] According to "4. Method for calculating WT1 mRNA expression level" in the protocol attached to the kit, the WT1 mRNA expression level was calculated as follows. Specifically, the WT1 mRNA expression level was calculated by dividing the measured WT1 mRNA value by the measured GAPDH mRNA value (WT1 mRNA copy number per GAPDH mRNA copy), multiplying this value by the average GAPDH mRNA copy number per μg of RNA in healthy adults (GAPDH mRNA expression level), as shown in the following formula: 7 (copies / μg RNA) is the average GAPDH mRNA measurement per μg RNA in healthy adults.

number

[0208] The results of WT1 mRNA expression analysis are shown in Figure 14. WT1 mRNA expression was observed in the peripheral blood of all 40 cases. The mOS of cases with WT1 mRNA expression levels of 10,000 copies / μg RNA or less tended to be longer than the mOS of cases with WT1 mRNA expression levels higher than 10,000 copies / μg RNA.

[0209] Furthermore, WT1 antigen peptide-specific immune responses were confirmed by HLA tetramer assay and delayed-type hypersensitivity test (described below) in 30 cases with WT1 mRNA expression levels of 10,000 copies / μg RNA or less and 10 cases with WT1 mRNA expression levels of more than 10,000 copies / μg RNA. The results of comparing survival time (OS) and WT1 antigen peptide-specific immune responses with respect to WT1 mRNA expression levels are shown in Figure 15. The results shown in Figure 15 apply to 28 cases with WT1 mRNA expression levels of 10,000 copies / μg RNA or less and 8 cases with WT1 mRNA expression levels of more than 10,000 copies / μg RNA, excluding 4 cases in which the WT1 antigen peptide-specific immune responses could not be determined.

[0210] These results suggest that WT1 mRNA expression levels are useful as genetic markers for selecting patients who are likely to benefit from treatment with WT1 peptide vaccines, and that a reference level of 10,000 copies / μg RNA is useful for this marker.

[0211] In the group of cases with WT1 mRNA expression levels below 10,000 copies / μg RNA, patients with WT1 antigen peptide-specific immune responses tended to have a longer OS. In contrast, in the group of cases with WT1 mRNA expression levels above 10,000 copies / μg RNA, no difference in OS was observed between positive and negative WT1 antigen peptide-specific immune responses. Furthermore, we confirmed that WT1 mRNA expression levels in peripheral blood and bone marrow fluid correlated, with a similar trend observed in bone marrow fluid (Figure 16).

Claims

1. A method for selecting a subject who is expected to benefit from a pharmaceutical composition for treating or preventing cancer, comprising: Determining the expression level of WT1 mRNA using a sample collected from the subject; and providing an indication that the subject is one who can be expected to benefit from the pharmaceutical composition when the expression level of WT1 mRNA is less than or equal to 10,000 copies / μg RNA; The pharmaceutical composition contains RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), VLDFAPPGA (SEQ ID NO: 9), CMTWNQMNL (SEQ ID NO: 3), CYTWNQMNL (SEQ ID NO: 4), CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKR A method comprising administering to a subject a peptide comprising an amino acid sequence selected from the group consisting of YFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7) and VLDFAPPGA (SEQ ID NO: 9), or a pharmaceutically acceptable salt thereof.

3. The method of claim 1 or 2, wherein the pharmaceutical composition comprises a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.

4. the pharmaceutical composition comprising a peptide comprising an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7) and VLDFAPPGA (SEQ ID NO: 9); The method of any one of claims 1 to 3, comprising a peptide comprising an amino acid sequence selected from the group consisting of CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition comprises a compound of formula (I): 【Chemistry 1】 [In the formula, X ａ and Y ａ represents a single bond, and tumor antigen peptide A has the following amino acid sequence: The present invention relates to a peptide having an amino acid sequence selected from the group consisting of RMFPNAPYL (SEQ ID NO: 2), YMFPNAPYL (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 5), SLGEQQYSV (SEQ ID NO: 6), RVPGVAPTL (SEQ ID NO: 7), and VLDFAPPGA (SEQ ID NO: 9), wherein the amino group of the N-terminal amino acid of the tumor antigen peptide A is Y in formula (1). ａ and the carbonyl group of the C-terminal amino acid of tumor antigen peptide A binds to the hydroxyl group in formula (1), R １ represents a hydrogen atom or tumor antigen peptide B, Tumor antigen peptide B has a different sequence from tumor antigen peptide A and has the following amino acid sequence: CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 4), and the thioether group of the cysteine ​​residue of tumor antigen peptide B is bonded to the thioether group in formula (1). The method according to any one of claims 1 to 4, comprising a compound represented by the formula:

6. The compound represented by formula (1) is a compound represented by formula (2): 【Chemistry 2】 (In the formula, the bond between C and C represents a disulfide bond.) The method according to claim 5, wherein the compound is a compound represented by the formula:

7. The compound represented by formula (1) may be a compound represented by formula (3): 【Transformation 3】 6. The method according to claim 5, wherein the compound is a compound represented by the formula: (wherein the bond between C and C represents a disulfide bond) or a pharmaceutically acceptable salt thereof.

8. The method of any one of claims 1 to 7, wherein the pharmaceutical composition further comprises a peptide comprising an amino acid sequence selected from the group consisting of CNKRYFKLSHLQMHSRK (SEQ ID NO: 11), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 12), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 13), WAPVLDFAPPGASAYGSL (SEQ ID NO: 14), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 15) and WAPVLDFAPPGASAYGSLC (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof.

9. The method of any one of claims 1 to 8, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

10. The method according to any one of claims 1 to 9, further comprising the step of providing an indication that, if the subject is of wild-type TP53, the subject is one who can be expected to benefit from the pharmaceutical composition.

11. The method according to any one of claims 1 to 10, further comprising the step of providing an indication that, if the subject is of the BCOR wild-type, the subject is one who can be expected to benefit from the pharmaceutical composition.

12. The method according to any one of claims 1 to 11, wherein when the mRNA expression level of the WT1 gene is less than or equal to 4000, an indication is provided that the subject is a subject who can be expected to benefit from the pharmaceutical composition.

13. Detecting WT1 antigen peptide-specific CD8 T cells using a sample collected from the subject to which the pharmaceutical composition or peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 has been administered; and The method according to any one of claims 1 to 12, further comprising the step of providing an indication that the subject is one who can be expected to benefit from the pharmaceutical composition when the number of WT1 antigen peptide-specific CD8 T cells is increased compared to a sample collected from the subject before administration.

14. The method of claim 13, wherein the step of detecting WT1 antigen peptide-specific CD8 T cells is carried out by reacting a complex of WT1 peptide and an HLA molecule with the sample and examining the presence or number of WT1 antigen peptide-specific CD8 T cells that recognize the complex contained in the sample.

15. The method according to claim 14, wherein the complex of the WT1 peptide and the HLA molecule is in the form of a tetramer.

16. 16. The method of claim 14 or 15, wherein the HLA molecule is HLA-matched to the subject.

17. The method according to any one of claims 13 to 16, wherein the step of detecting WT1 antigen peptide-specific CD8 T cells comprises analysis by flow cytometry.

18. The method according to any one of claims 1 to 17, further comprising, when a delayed-type hypersensitivity reaction is detected in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 has been administered multiple times, providing an indication that the subject is a subject who can be expected to benefit from the pharmaceutical composition.

19. The method according to claim 18, further comprising the step of comparing a reaction at a site in a subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 has been administered with a reaction at a site in the subject to which the pharmaceutical composition or peptide or pharmaceutically acceptable salt thereof has not been administered, and providing an indication that the subject is one in which the effect of the pharmaceutical composition can be expected, if the difference between the reaction at the administered site and the reaction at the non-administered site is equal to or greater than a reference value.

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