Medicament for preventing or treating benign tumors

Abstract

The present disclosure provides a prophylactic or therapeutic agent for a benign tumor. Provided is a prophylactic or therapeutic agent for a benign tumor, which comprises a WT1 peptide, an analog thereof, or a nucleic acid molecule encoding the same. In a specific embodiment, the present disclosure provides a prophylactic or therapeutic agent for a benign tumor, which comprises a WT1 peptide, an analog thereof, or a nucleic acid molecule encoding the same. In a preferred embodiment, the prophylactic or therapeutic agent for a benign tumor (e.g., familial adenomatous polyposis) according to the present disclosure comprises a killer-type and / or helper-type WT1 peptide. In a further preferred embodiment, the aforementioned WT1 peptide is a WT1 126 killer peptide and / or WT1 35 helper peptide. In another aspect, provided are WT1 peptide-specific CTLs, a method for inducing WT1-specific helper T cells, and a method for inducing dendritic cells presenting a WT1 peptide.

Description

Technical Field

[0001] This disclosure includes pharmaceuticals and methods for the prevention or treatment of benign tumors. In one specific example, this disclosure relates to compositions for the prevention or treatment of familial adenomatous polyposis. Background Technology

[0002] Benign tumors are tumors that do not show a pathological malignancy. They are generally considered distinct from malignant tumors and do not exhibit a tendency to metastasize or invasively. Most benign tumors are asymptomatic, but when they become large, they can cause symptoms by compressing other tissues or become malignant, requiring treatment or prevention. Therefore, there are few treatments and preventative measures available for benign tumors.

[0003] Familial adenomatous polyposis (APC) is a hereditary disease characterized by a heterozygous deletion of the tumor suppressor gene APC. Starting around age 20, homozygous deletion of the APC gene occurs in glandular cells in the large intestine where APC is heterozygous, and adenomas develop throughout the colon, eventually leading to colon cancer. Initially, the adenomas are removed endoscopically as a treatment. However, because the adenomas subsequently become too dense to be removed, and cancer develops from them, a total colectomy is usually performed. The prevalence is 100%, and it is a very serious disease that occurs around age 20 in all carriers. There are approximately 6,000 patients in Japan.

[0004] Aspirin is known to be effective in preventing the occurrence and / or inhibiting the progression of adenomas and adenocarcinomas in familial adenomatous polyposis, but its effect is weak and there is a risk of gastrointestinal bleeding as a side effect of aspirin.

[0005] The Wilms tumor gene WT1 was isolated as a gene involved in the tumorigenesis of Wilms tumor, a pediatric renal tumor (see Non-Patent Document 1). This gene encodes a zinc finger transcription factor involved in the regulation of cell proliferation and differentiation, as well as apoptosis and tissue development.

[0006] Existing technical documents

[0007] Non-patent documents

[0008] Non-patent document 1: Gessler, M et al., Nature, Vol. 343, pp. 774-778, 1990. Summary of the Invention

[0009] Problem-solving methods

[0010] Through diligent research, the inventors have discovered that the WT1 peptide vaccine effectively inhibits and / or delays the occurrence of adenomas in benign tumors (e.g., familial adenomatous polyposis) and inhibits and / or delays the onset of symptoms arising from adenomas. Furthermore, the WT1 peptide vaccine is effective in treating and preventing benign tumors, such as familial adenomatous polyposis, which is considered refractory because there is no fundamental cure other than complete resection. The inventors have discovered that adenomas in patients with benign tumors such as familial adenomatous polyposis express the WT1 cancer antigen, thus proposing that the WT1 cancer vaccine can effectively inhibit and / or delay the occurrence of adenomas in benign tumors such as familial adenomatous polyposis and inhibit and / or delay the onset of symptoms arising from adenomas, thereby ultimately completing this invention.

[0011] This disclosure is based on the surprising finding that cells in adenomas, benign tumors, express the WT1 protein. It is known in the art that the WT1 protein is highly expressed in cancer cells of malignant tumors, and that the usefulness of WT1 peptide vaccines in malignant tumors can be readily predicted. This disclosure is the first to reveal that cells in adenomas of familial adenomatous polyposis, a benign tumor, express the WT1 protein, and proposes the idea of ​​providing a treatment or preventative agent for benign tumors in general. Furthermore, since the effectiveness of WT1 peptide vaccines depends on the expression pattern (including expression level) of the WT1 protein, this cannot be reasonably predicted even if WT1 protein expression is found in adenomas. In this respect, this disclosure is the first to demonstrate the effectiveness of WT1 peptide vaccines against benign tumors and to discover effects that are unpredictable under the prior art.

[0012] Therefore, this disclosure provides:

[0013] (Term X1)

[0014] Compositions for the prevention or treatment of benign tumors, comprising WT1 peptide or an analogue thereof.

[0015] (Term X2)

[0016] The composition according to Clause X1, wherein the WT1 peptide or its analogues include killer peptides and / or helper peptides.

[0017] (Term X3)

[0018] The composition according to Clause X1 or Clause X2, wherein the WT1 peptide or its analogue comprises WT1 126 Killer peptides and / or WT1 35 Helper peptides.

[0019] (Clause X4)

[0020] The composition according to any one of clauses X1-X3, wherein the composition comprises a nucleic acid molecule encoding a WT1 peptide or an analogue thereof.

[0021] (Term X5)

[0022] The composition according to any one of clauses X1-X4, wherein the nucleic acid molecule comprises RNA and / or DNA.

[0023] (Term X6)

[0024] The composition according to any one of clauses X1-X5, wherein the composition further comprises an adjuvant.

[0025] (Term X7)

[0026] The composition according to any one of clauses X1-X6, wherein the adjuvant is Montanide® ISA51 adjuvant.

[0027] (Term X8)

[0028] The composition according to any one of clauses X1-X7, wherein a benign tumor expresses WT1.

[0029] (Term X9)

[0030] The composition according to any one of clauses X1-X8, wherein the benign tumor is selected from familial adenomatous polyposis, non-hereditary colorectal adenoma, intraductal papillary mucinous tumor, meningioma, schwannoma, epithelial adenoma of organ, papilloma, non-epithelial fibroid, lipoma, chondroma and hemangioma.

[0031] (Term X10)

[0032] The composition according to any one of clauses X1-X9, wherein the benign tumor is familial adenomatous polyposis.

[0033] (Term X11)

[0034] The composition is according to any one of the terms X1-X10, wherein the composition is given once a week.

[0035] (Term X12)

[0036] A method for inducing WT1-specific CTLs and / or WT1-specific helper T cells for the prevention or treatment of benign tumors, comprising culturing peripheral blood mononuclear cells from a subject requiring treatment for a benign tumor in the presence of a WT1 peptide or an analogue thereof according to any of the preceding clauses, or introducing a nucleic acid molecule encoding a WT1 peptide or an analogue thereof according to any of the preceding clauses into peripheral blood mononuclear cells from a subject requiring treatment for a benign tumor, and inducing WT1-specific CTLs and / or WT1-specific helper T cells from peripheral blood mononuclear cells.

[0037] (Term X13)

[0038] A method for inducing WT1-presenting dendritic cells for the prevention or treatment of benign tumors includes culturing immature dendritic cells from a subject requiring treatment of a benign tumor in the presence of a WT1 peptide or an analogue thereof according to any of the preceding clauses, or introducing a nucleic acid molecule encoding a WT1 peptide or an analogue thereof according to any of the preceding clauses into immature dendritic cells from a subject requiring treatment of a benign tumor, and inducing WT1-presenting dendritic cells.

[0039] (Term X13A)

[0040] The method of Clause X12 or 13 further includes one or more features pursuant to any one or more of Clauses X1-X11.

[0041] (Clause X14)

[0042] Compositions for inducing WT1-specific CTLs and / or WT1-specific helper T cells, comprising a WT1 peptide or an analogue thereof, or a nucleic acid molecule encoding a WT1 peptide or an analogue thereof, wherein the composition is used for the prevention or treatment of benign tumors.

[0043] (Term X15)

[0044] Compositions for inducing dendritic cells to present WT1, comprising a WT1 peptide or an analogue thereof, or a nucleic acid molecule encoding a WT1 peptide or an analogue thereof, wherein the composition is intended for the prevention or treatment of benign tumors.

[0045] (Term X15A)

[0046] The composition according to Clause X14 or Clause X15 further includes one or more features according to any one or more of Clauses X1-X13.

[0047] (Term X16)

[0048] Compositions for the prevention or treatment of benign tumors, comprising WT1-specific CTLs and / or WT1-specific helper T cells.

[0049] (Term X17)

[0050] A composition for the prevention or treatment of benign tumors, comprising dendritic cells that present WT1.

[0051] (Clause X17A)

[0052] The composition according to Clause X16 or Clause X17 further includes one or more features according to any one or more of Clauses X1-X13.

[0053] This disclosure also provides:

[0054] (Clause 1)

[0055] Compositions for the prevention or treatment of benign tumors, comprising WT1 peptide or an analogue thereof.

[0056] (Clause 2)

[0057] The composition according to Clause 1, wherein the WT1 peptide or its analogues include killer peptides and / or helper peptides.

[0058] (Clause 3)

[0059] The composition according to Clause 1 or 2, wherein the WT1 peptide or its analogue comprises WT1 126 Killer peptide, WT1 235 Killer peptides and / or WT1 35 Helper peptides, or peptides that differ from WT1 due to the deletion, substitution, and / or addition of one or more amino acids. 126 Killer peptide, WT1 235 Killer peptides and WT1 35 A peptide that has the amino acid sequence of any of the accessory peptides and possesses CTL-inducible activity.

[0060] (Clause 4)

[0061] The composition according to any one of clauses 1-3, wherein the WT1 peptide or its analogue is a peptide composed of an amino acid sequence selected from:

[0062] RMFPNAPYL (SEQ ID NO: 2),

[0063] RYFPNAPYL (SEQ ID NO: 46),

[0064] YMFPNAPYL (SEQ ID NO: 14),

[0065] CYTWNQMNL (SEQ ID NO: 45),

[0066] CMTWNQMNL (SEQ ID NO: 3),

[0067] C-CYTWNQMNL (SEQ ID NO: 47) (where CC indicates that C residues are linked together by disulfide bonds), and

[0068] C-CMTWNQMNL (SEQ ID NO: 48) (where CC indicates that C residues are linked together by disulfide bonds),

[0069] Or its pharmaceutically acceptable salt.

[0070] (Clause 5)

[0071] The composition according to any one of clauses 1-4, wherein the WT1 peptide or its analogue is a compound of formula (2):

[0072]

[0073] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0074] Or its pharmaceutically acceptable salt.

[0075] (Clause 6)

[0076] The composition according to any one of clauses 1-5, wherein the WT1 peptide or its analogue is a compound of formula (3):

[0077]

[0078] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0079] Or its pharmaceutically acceptable salt.

[0080] (Clause 7)

[0081] The composition according to any one of clauses 1-6, wherein the WT1 peptide or its analogue is a composition, further comprising at least one peptide consisting of an amino acid sequence selected from:

[0082] WAPVLDFAPPGASAYGSL (SEQ ID NO: 4),

[0083] CWAPVLDFAPPGASAYGSL (SEQ ID NO: 50) and

[0084] WAPVLDFAPPGASAYGSLC (SEQ ID NO: 51),

[0085] Or its pharmaceutically acceptable salt.

[0086] (Clause 8)

[0087] A composition according to any one of clauses 1-7, wherein the WT1 peptide or its analogue is a composition comprising a compound of formula (2):

[0088]

[0089] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0090] A peptide or a pharmaceutically acceptable salt thereof consisting of the amino acid sequence: WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4).

[0091] (Clause 9)

[0092] A composition according to any one of clauses 1-8, wherein the WT1 peptide or its analogue is a composition comprising a compound of formula (3):

[0093]

[0094] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0095] A peptide or a pharmaceutically acceptable salt thereof consisting of the amino acid sequence: WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4).

[0096] (Clause 10)

[0097] Compositions for the prevention or treatment of benign tumors, comprising nucleic acid molecules encoding WT1 peptide or analogues thereof.

[0098] (Clause 11)

[0099] The composition according to any one of Clause 10, wherein the nucleic acid molecule comprises RNA and / or DNA.

[0100] (Clause 12)

[0101] The composition according to any one of clauses 1-11, wherein the composition further comprises an adjuvant.

[0102] (Clause 13)

[0103] The composition according to any one of Clauses 1-12, wherein the adjuvant is Montanide® ISA51 adjuvant.

[0104] (Clause 14)

[0105] The composition according to any one of clauses 1-13, wherein a benign tumor expresses WT1.

[0106] (Clause 15)

[0107] The composition according to any one of Clauses 1-14, wherein the benign tumor is selected from familial adenomatous polyposis, non-hereditary colorectal adenoma, intraductal papillary mucinous tumor, meningioma, schwannoma, epithelial adenoma of organ, papilloma, non-epithelial fibroid, lipoma, chondroma and hemangioma.

[0108] (Clause 16)

[0109] The composition according to any one of clauses 1-15, wherein the benign tumor is familial adenomatous polyposis.

[0110] (Clause 17)

[0111] The composition according to any one of claims 1-16, wherein the composition is administered once a week.

[0112] (Clause 18)

[0113] A method for inducing WT1-specific CTLs and / or WT1-specific helper T cells for the prevention or treatment of benign tumors, comprising culturing peripheral blood mononuclear cells from a subject requiring treatment for a benign tumor in the presence of a WT1 peptide or an analogue thereof according to any of the preceding clauses, or introducing a nucleic acid molecule encoding a WT1 peptide or an analogue thereof according to any of the preceding clauses into peripheral blood mononuclear cells from a subject requiring treatment for a benign tumor, and inducing WT1-specific CTLs and / or WT1-specific helper T cells from peripheral blood mononuclear cells.

[0114] (Clause 19)

[0115] A method for inducing WT1-presenting dendritic cells for the prevention or treatment of benign tumors includes culturing immature dendritic cells from a subject requiring treatment of a benign tumor in the presence of a WT1 peptide or an analogue thereof according to any of the preceding clauses, or introducing a nucleic acid molecule encoding a WT1 peptide or an analogue thereof according to any of the preceding clauses into immature dendritic cells from a subject requiring treatment of a benign tumor, and inducing WT1-presenting dendritic cells.

[0116] (Clause 19A)

[0117] The method of Clause 18 or 19 further includes one or more features pursuant to any one or more of Clauses 1-17.

[0118] (Clause 20)

[0119] Compositions for inducing WT1-specific CTLs and / or WT1-specific helper T cells, comprising a WT1 peptide or an analogue thereof, or a nucleic acid molecule encoding a WT1 peptide or an analogue thereof, wherein the composition is used for the prevention or treatment of benign tumors.

[0120] (Clause 21)

[0121] Compositions for inducing dendritic cells to present WT1, comprising a WT1 peptide or an analogue thereof, or a nucleic acid molecule encoding a WT1 peptide or an analogue thereof, wherein the composition is intended for the prevention or treatment of benign tumors.

[0122] (Clause 21A)

[0123] The composition of Clause 20 or 21 further comprises one or more features according to any one or more of Clauses 1-19.

[0124] (Clause 22)

[0125] Compositions for the prevention or treatment of benign tumors, comprising WT1-specific CTLs and / or WT1-specific helper T cells.

[0126] (Clause 23)

[0127] A composition for the prevention or treatment of benign tumors, comprising dendritic cells that present WT1.

[0128] (Clause 23A)

[0129] The composition according to Clause 22 or 23 further comprises one or more features according to any one or more of Clauses 1-19.

[0130] (Term A1)

[0131] Methods for preventing or treating benign tumors in a subject include administering an effective amount of WT1 peptide or an analogue thereof to the subject.

[0132] (Clause A2)

[0133] According to the method of Clause A1, the WT1 peptide or its analogues include killer peptides and / or helper peptides.

[0134] (Clause A3)

[0135] According to the method of clause A1 or A2, wherein the WT1 peptide or its analogues include WT1 126 Killer peptide, WT1 235 Killer peptides and / or WT1 35 Helper peptides, or peptides that differ from WT1 due to the absence, substitution, and / or addition of one or more amino acids. 126 Killer peptide, WT1 235 Killer peptides and WT1 35 A peptide that has the amino acid sequence of any of the accessory peptides and possesses CTL-inducible activity.

[0136] (Clause A4)

[0137] According to the method of any one of clauses A1-A3, wherein the WT1 peptide or its analogue is a peptide composed of an amino acid sequence selected from:

[0138] RMFPNAPYL (SEQ ID NO: 2),

[0139] RYFPNAPYL (SEQ ID NO: 46),

[0140] YMFPNAPYL (SEQ ID NO: 14),

[0141] CYTWNQMNL (SEQ ID NO: 45),

[0142] CMTWNQMNL (SEQ ID NO: 3),

[0143] C-CYTWNQMNL (SEQ ID NO: 47) (where CC indicates that C residues are linked together by disulfide bonds), and

[0144] C-CMTWNQMNL (SEQ ID NO: 48) (where CC indicates that C residues are linked together by disulfide bonds),

[0145] Or its pharmaceutically acceptable salt.

[0146] (Clause A5)

[0147] The method according to any one of clauses A1-A4, wherein the WT1 peptide or its analogue is a compound of formula (2):

[0148]

[0149] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0150] Or its pharmaceutically acceptable salt.

[0151] (Clause A6)

[0152] The method according to any one of clauses A1-A5, wherein the WT1 peptide or its analogue is a compound of formula (3):

[0153]

[0154] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0155] Or its pharmaceutically acceptable salt.

[0156] (Clause A7)

[0157] The method according to any one of clauses A1-A6, wherein the WT1 peptide or its analogue is a composition further comprising at least one peptide consisting of an amino acid sequence selected from:

[0158] WAPVLDFAPPGASAYGSL (SEQ ID NO: 4),

[0159] CWAPVLDFAPPGASAYGSL (SEQ ID NO: 50) and

[0160] WAPVLDFAPPGASAYGSLC (SEQ ID NO: 51),

[0161] Or its pharmaceutically acceptable salt.

[0162] (Term A8)

[0163] The method according to any one of clauses A1-A7, wherein the WT1 peptide or its analogue is a composition comprising a compound of formula (2):

[0164]

[0165] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0166] A peptide or a pharmaceutically acceptable salt thereof consisting of the amino acid sequence: WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4).

[0167] (Clause A9)

[0168] The method according to any one of clauses A1-A8, wherein the WT1 peptide or its analogue is a composition comprising a compound of formula (3):

[0169]

[0170] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0171] A peptide or a pharmaceutically acceptable salt thereof consisting of the amino acid sequence: WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4).

[0172] (Clause A10)

[0173] Methods for preventing or treating benign tumors in a subject include administering to the subject an effective amount of a nucleic acid molecule encoding a WT1 peptide or an analogue thereof.

[0174] (Clause A11)

[0175] The method according to clause A10 includes nucleic acid molecules including RNA and / or DNA.

[0176] (Clause A12)

[0177] The method according to any one of clauses A1-A11, wherein the WT1 peptide or an analogue thereof, or a nucleic acid molecule encoding the WT1 peptide or an analogue thereof, is used in combination with an adjuvant.

[0178] (Clause A13)

[0179] The method is according to any one of clauses A1-A12, wherein the adjuvant is Montanide® ISA51 adjuvant.

[0180] (Clause A14)

[0181] The method is based on any one of clauses A1-A13, wherein a benign tumor expresses WT1.

[0182] (Clause A15)

[0183] According to any one of the provisions A1-A14, the benign tumor is selected from familial adenomatous polyposis, non-hereditary colorectal adenoma, intraductal papillary mucinous tumor, meningioma, schwannoma, epithelial adenoma of organ, papilloma, non-epithelial fibroid, lipoma, chondroma and hemangioma.

[0184] (Clause A16)

[0185] According to any of the provisions A1-A15, the benign tumor is familial adenomatous polyposis.

[0186] (Clause A17)

[0187] The method according to any one of clauses A1-A16, wherein the WT1 peptide or its analogue, or a nucleic acid molecule encoding the WT1 peptide or its analogue, is administered once a week.

[0188] (Clause A18)

[0189] Methods for preventing or treating benign tumors in subjects, including

[0190] Peripheral blood mononuclear cells from subjects requiring treatment of benign tumors are cultured in the presence of the WT1 peptide or its analogues according to any of the foregoing clauses, or a nucleic acid molecule encoding the WT1 peptide or its analogues according to any of the foregoing clauses is introduced into peripheral blood mononuclear cells from subjects requiring treatment of benign tumors, thereby inducing WT1-specific cytotoxic T cells (CTLs) and / or WT1-specific helper T cells from the peripheral blood mononuclear cells.

[0191] Subjects were given WT1-specific CTLs and / or WT1-specific helper T cells prepared therefrom.

[0192] (Clause A19)

[0193] Methods for preventing or treating benign tumors in subjects, including

[0194] Immature dendritic cells from a subject requiring treatment of a benign tumor are cultured in the presence of the WT1 peptide or its analogue according to any of the foregoing clauses, or a nucleic acid molecule encoding the WT1 peptide or its analogue according to any of the foregoing clauses is introduced into immature dendritic cells from a subject requiring treatment of a benign tumor, thereby inducing WT1-presenting dendritic cells.

[0195] The subject was given dendritic cells that presented WT1 prepared therefrom.

[0196] (Clause A19A)

[0197] According to the method of Clause A18 or A19, it further includes one or more features according to any one or more of Clauses A1-A17.

[0198] (Clause A20)

[0199] Methods for preventing or treating benign tumors in a subject include administering to the subject an effective amount of WT1-specific CTLs and / or WT1-specific helper T cells induced by a WT1 peptide or its analogues or a nucleic acid molecule encoding a WT1 peptide or its analogues.

[0200] (Clause A21)

[0201] Methods for preventing or treating benign tumors in a subject include administering to the subject an effective amount of dendritic cells that present WT1 induced by a WT1 peptide or an analogue thereof or a nucleic acid molecule encoding a WT1 peptide or an analogue thereof.

[0202] (Clause A21A)

[0203] The method according to Clause A20 or A21 further includes one or more features according to any one or more of Clauses A1-A19.

[0204] (Clause A22)

[0205] Methods for preventing or treating benign tumors in a subject include administering an effective amount of WT1-specific CTLs and / or WT1-specific helper T cells to the subject.

[0206] (Clause A23)

[0207] Methods for preventing or treating benign tumors in a subject include administering an effective amount of dendritic cells that present with WT1 to the subject.

[0208] (Clause A23A)

[0209] The method according to Clause A22 or A23 may further include one or more features according to any one or more of Clauses A1-A19.

[0210] (Term B1)

[0211] WT1 peptide or its analogues are used for the prevention or treatment of benign tumors.

[0212] (Clause B2)

[0213] According to Clause B1, WT1 peptides or analogues thereof include killer peptides and / or helper peptides.

[0214] (Clause B3)

[0215] According to clauses B1 or B2, the WT1 peptide or its analogues include WT1. 126 Killer peptide, WT1 235 Killer peptides and / or WT1 35 Helper peptides, or peptides that differ from WT1 due to the absence, substitution, and / or addition of one or more amino acids. 126 Killer peptide, WT1 235 Killer peptides and WT1 35 A peptide that has the amino acid sequence of any of the accessory peptides and possesses CTL-inducible activity.

[0216] (Clause B4)

[0217] The WT1 peptide or analogue thereof according to any one of clauses B1-B3, wherein the WT1 peptide or analogue thereof is a peptide composed of an amino acid sequence selected from the following:

[0218] RMFPNAPYL (SEQ ID NO: 2),

[0219] RYFPNAPYL (SEQ ID NO: 46),

[0220] YMFPNAPYL (SEQ ID NO: 14),

[0221] CYTWNQMNL (SEQ ID NO: 45),

[0222] CMTWNQMNL (SEQ ID NO: 3),

[0223] C-CYTWNQMNL (SEQ ID NO: 47) (where CC indicates that C residues are linked together by disulfide bonds), and

[0224] C-CMTWNQMNL (SEQ ID NO: 48) (where CC indicates that C residues are linked together by disulfide bonds),

[0225] Or its pharmaceutically acceptable salt.

[0226] (Clause B5)

[0227] According to any one of clauses B1-B4, the WT1 peptide or its analogue is a compound of formula (2):

[0228]

[0229] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0230] Or its pharmaceutically acceptable salt.

[0231] (Clause B6)

[0232] The WT1 peptide or analogue thereof according to any one of clauses B1-B5, wherein the WT1 peptide or analogue thereof is a compound of formula (3):

[0233]

[0234] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0235] Or its pharmaceutically acceptable salt.

[0236] (Clause B7)

[0237] The WT1 peptide or analogue according to any one of clauses B1-B6, wherein the WT1 peptide or analogue is a composition further comprising at least one peptide consisting of an amino acid sequence selected from:

[0238] WAPVLDFAPPGASAYGSL (SEQ ID NO: 4),

[0239] CWAPVLDFAPPGASAYGSL (SEQ ID NO: 50) and

[0240] WAPVLDFAPPGASAYGSLC (SEQ ID NO: 51),

[0241] Or its pharmaceutically acceptable salt.

[0242] (Clause B8)

[0243] The WT1 peptide or analogue thereof according to any one of clauses B1-B7, wherein the WT1 peptide or analogue thereof is a composition comprising a compound of formula (2):

[0244]

[0245] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0246] A peptide or a pharmaceutically acceptable salt thereof consisting of the amino acid sequence: WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4).

[0247] (Clause B9)

[0248] The WT1 peptide or analogue thereof according to any one of clauses B1-B8, wherein the WT1 peptide or analogue thereof is a composition comprising a compound of formula (3):

[0249]

[0250] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0251] A peptide or a pharmaceutically acceptable salt thereof consisting of the amino acid sequence: WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4).

[0252] (Term B10)

[0253] Nucleic acid molecules encoding WT1 peptide or its analogues are used to prevent or treat benign tumors in subjects.

[0254] (Clause B11)

[0255] Nucleic acid molecules according to clause B10, wherein nucleic acid molecules include RNA and / or DNA.

[0256] (Clause B12)

[0257] The WT1 peptide or its analogue or nucleic acid molecule according to any of clauses B1-B11, used in combination with an adjuvant.

[0258] (Clause B13)

[0259] The WT1 peptide or its analogue or nucleic acid molecule according to any of clauses B1-B12, wherein the adjuvant is Montanide® ISA51 adjuvant.

[0260] (Clause B14)

[0261] The WT1 peptide or its analogue or nucleic acid molecule according to any one of clauses B1-B13, wherein a benign tumor expresses WT1.

[0262] (Clause B15)

[0263] The WT1 peptide or its analogues or nucleic acid molecules according to any one of clauses B1-B14, wherein the benign tumor is selected from familial adenomatous polyposis, non-hereditary colorectal adenoma, intraductal papillary mucinous tumor, meningioma, schwannoma, epithelial adenoma of organ, papilloma, non-epithelial fibroid, lipoma, chondroma and hemangioma.

[0264] (Clause B16)

[0265] According to any of clauses B1-B15, the WT1 peptide or its analogue or nucleic acid molecule, wherein the benign tumor is familial adenomatous polyposis.

[0266] (Clause B17)

[0267] According to any of the terms B1-B16, the WT1 peptide or its analogues or nucleic acid molecules will be administered once a week.

[0268] (Clause B18)

[0269] The WT1 peptide or its analogue, or the nucleic acid molecule according to any of the foregoing clauses, is used to induce WT1-specific CTLs and / or WT1-specific helper T cells for the prevention or treatment of benign tumors, wherein the WT1 peptide or its analogue is cultured together with peripheral blood mononuclear cells from a subject requiring treatment for benign tumors, or the nucleic acid molecule is introduced into peripheral blood mononuclear cells from a subject requiring treatment for benign tumors, thereby inducing WT1-specific CTLs and / or WT1-specific helper T cells.

[0270] (Clause B19)

[0271] The WT1 peptide or its analogue, or the nucleic acid molecule, according to any of the foregoing provisions, is used to induce WT1-presenting dendritic cells for the prevention or treatment of benign tumors, wherein the WT1 peptide or its analogue is cultured together with immature dendritic cells from a subject requiring treatment for benign tumors, or the nucleic acid molecule is introduced into immature dendritic cells from a subject requiring treatment for benign tumors, thereby inducing WT1-presenting dendritic cells.

[0272] (Clause B19A)

[0273] The WT1 peptide or its analogues or nucleic acid molecules according to Clause B18 or B19 further include one or more features according to any one or more of Clauses B1-B17.

[0274] (Clause B20)

[0275] WT1 peptide or analogues thereof or nucleic acid molecules encoding WT1 peptide or analogues thereof, for inducing WT1-specific CTLs and / or WT1-specific helper T cells, wherein the WT1 peptide or analogues thereof or nucleic acid molecules are used for the prevention or treatment of benign tumors.

[0276] (Clause B21)

[0277] WT1 peptide or analogues thereof, or nucleic acid molecules encoding WT1 peptide or analogues thereof, for inducing dendritic cells to present WT1, wherein the WT1 peptide or analogues thereof or nucleic acid molecules are used for the prevention or treatment of benign tumors.

[0278] (Clause B21A)

[0279] The WT1 peptide or its analogues or nucleic acid molecules according to Clause B20 or B21 further include one or more features according to any one or more of Clauses B1-B19.

[0280] (Clause B22)

[0281] WT1-specific CTLs and / or WT1-specific helper T cells are used to prevent or treat benign tumors in subjects.

[0282] (Clause B23)

[0283] Dendritic cells that present WT1 are used to prevent or treat benign tumors in subjects.

[0284] (Clause B23A)

[0285] The WT1-specific CTLs and / or WT1-specific helper T cells according to Clause B22 or the WT1-presenting dendritic cells according to Clause B23 further include one or more features according to any one or more of Clauses B1-B19.

[0286] (Clause C1)

[0287] Use of WT1 peptide or analogues in the preparation of compositions for the prevention or treatment of benign tumors.

[0288] (Clause C2)

[0289] According to the use of Clause C1, WT1 peptides or their analogues include killer peptides and / or helper peptides.

[0290] (Clause C3)

[0291] According to the purpose of clause C1 or C2, wherein the WT1 peptide or its analogues include WT1 126 Killer peptide, WT1235 Killer peptides and / or WT1 35 Helper peptides, or peptides that differ from WT1 due to the absence, substitution, and / or addition of one or more amino acids. 126 Killer peptide, WT1 235 Killer peptides and WT1 35 A peptide that has the amino acid sequence of any of the accessory peptides and possesses CTL-inducible activity.

[0292] (Clause C4)

[0293] According to the use of any of clauses C1-C3, the WT1 peptide or its analogues are peptides composed of amino acid sequences selected from the following:

[0294] RMFPNAPYL (SEQ ID NO: 2),

[0295] RYFPNAPYL (SEQ ID NO: 46),

[0296] YMFPNAPYL (SEQ ID NO: 14),

[0297] CYTWNQMNL (SEQ ID NO: 45),

[0298] CMTWNQMNL (SEQ ID NO: 3),

[0299] C-CYTWNQMNL (SEQ ID NO: 47) (where CC indicates that C residues are linked together by disulfide bonds), and

[0300] C-CMTWNQMNL (SEQ ID NO: 48) (where CC indicates that C residues are linked together by disulfide bonds),

[0301] Or its pharmaceutically acceptable salt.

[0302] (Clause C5)

[0303] According to the use of any one of clauses C1-C4, wherein the WT1 peptide or its analogue is a compound of formula (2):

[0304]

[0305] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0306] Or its pharmaceutically acceptable salt.

[0307] (Clause C6)

[0308] According to any of the provisions C1-C5, wherein the WT1 peptide or its analogue is a compound of formula (3):

[0309]

[0310] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0311] Or its pharmaceutically acceptable salt.

[0312] (Clause C7)

[0313] According to the use of any of clauses C1-C6, wherein the WT1 peptide or its analogue is a composition further comprising at least one peptide consisting of an amino acid sequence selected from:

[0314] WAPVLDFAPPGASAYGSL (SEQ ID NO: 4),

[0315] CWAPVLDFAPPGASAYGSL (SEQ ID NO: 50) and

[0316] WAPVLDFAPPGASAYGSLC (SEQ ID NO: 51),

[0317] Or its pharmaceutically acceptable salt.

[0318] (Clause C8)

[0319] According to any of the provisions C1-C7, wherein the WT1 peptide or its analogue is a composition comprising a compound of formula (2):

[0320]

[0321] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0322] A peptide or a pharmaceutically acceptable salt thereof consisting of the amino acid sequence: WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4).

[0323] (Clause C9)

[0324] According to any of the provisions C1-C8, wherein the WT1 peptide or its analogue is a composition comprising a compound of formula (3):

[0325]

[0326] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0327] A peptide or a pharmaceutically acceptable salt thereof consisting of the amino acid sequence: WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4).

[0328] (Clause C10)

[0329] Use of nucleic acid molecules encoding WT1 peptide or analogues in the preparation of compositions for the prevention or treatment of benign tumors.

[0330] (Clause C11)

[0331] According to the purpose of Clause C10, nucleic acid molecules include RNA and / or DNA.

[0332] (Clause C12)

[0333] According to any of the provisions C1-C11, the WT1 peptide or its analogue or nucleic acid molecule is used in combination with an adjuvant.

[0334] (Clause C13)

[0335] For use in any of clauses C1-C12, wherein the adjuvant is Montanide® ISA51 adjuvant.

[0336] (Clause C14)

[0337] According to the use of any of clauses C1-C13, in which benign tumors express WT1.

[0338] (Clause C15)

[0339] According to any of the provisions C1-C14, the benign tumor is selected from familial adenomatous polyposis, non-hereditary colorectal adenoma, intraductal papillary mucinous tumor, meningioma, schwannoma, epithelial adenoma of organ, papilloma, non-epithelial fibroid, lipoma, chondroma and hemangioma.

[0340] (Clause C16)

[0341] According to any of the provisions C1-C15, the benign tumor is familial adenomatous polyposis.

[0342] (Clause C17)

[0343] According to any of the provisions C1-C16, the WT1 peptide or its analogue or the nucleic acid molecule encoding the WT1 peptide or its analogue shall be administered once a week.

[0344] (Clause C18)

[0345] The use of the WT1 peptide or its analogues according to any of the foregoing clauses or the nucleic acid molecule according to any of the foregoing clauses for inducing WT1-specific CTLs and / or WT1-specific helper T cells for the prevention or treatment of benign tumors, wherein the WT1 peptide or its analogues are cultured together with peripheral blood mononuclear cells from a subject requiring treatment for benign tumors, or the nucleic acid molecule is introduced into peripheral blood mononuclear cells from a subject requiring treatment for benign tumors, thereby inducing WT1-specific CTLs and / or WT1-specific helper T cells.

[0346] (Clause C19)

[0347] The use of the WT1 peptide or analogue thereof, or the nucleic acid molecule thereof, according to any of the foregoing terms, for inducing WT1-presenting dendritic cells for the prevention or treatment of benign tumors, wherein the WT1 peptide or analogue thereof is cultured together with immature dendritic cells from a subject requiring treatment for a benign tumor, or the nucleic acid molecule is introduced into immature dendritic cells from a subject requiring treatment for a benign tumor, thereby inducing WT1-presenting dendritic cells.

[0348] (Clause C19A)

[0349] Depending on the purpose of Clause C18 or C19, it may further include one or more features pursuant to any one or more of Clauses C1-C17.

[0350] (Clause C20)

[0351] Use of WT1 peptide or analogues or nucleic acid molecules encoding WT1 peptide or analogues in the preparation of WT1-specific CTLs and / or WT1-specific helper T cells for the prevention or treatment of benign tumors.

[0352] (Clause C21)

[0353] Use of WT1 peptide or analogues or nucleic acid molecules encoding WT1 peptide or analogues in the preparation of WT1-presenting dendritic cells for the prevention or treatment of benign tumors.

[0354] (Clause C21A)

[0355] Depending on the purpose of Clause C20 or C21, it may further include one or more features pursuant to any one or more of Clauses C1-C19.

[0356] (Clause C22)

[0357] Use of WT1-specific CTLs and / or WT1-specific helper T cells in the preparation of compositions for the prevention or treatment of benign tumors.

[0358] (Clause C23)

[0359] Use of WT1-presenting dendritic cells in the preparation of compositions for the prevention or treatment of benign tumors.

[0360] (Clause C23A)

[0361] Depending on the purpose of Clause C22 or C23, it may further include one or more features pursuant to any one or more of Clauses C1-C19.

[0362] This disclosure anticipates that one or more of the features described above can be combined with any specific combination described herein. Other embodiments and advantages of this disclosure will become apparent to those skilled in the art upon reading and understanding the following detailed description.

[0363] Invention Effects

[0364] According to the present invention, prevention, delay, and treatment of benign tumors are achieved. In an exemplary embodiment, the present invention achieves prevention, delay, and treatment of familial adenomatous polyposis. In another aspect, the present invention achieves prevention, delay, and treatment of symptoms arising from adenomas of benign tumors (e.g., familial adenomatous polyposis).

[0365] Furthermore, aside from redness and swelling of the skin at the administration site, the WT1 peptide cancer vaccine of this disclosure has no serious side effects and is very safe. Therefore, the WT1 peptide cancer vaccine can be safely and easily administered to most patients with benign tumors and familial adenomatous polyposis, and from a patient quality of life perspective, avoids endoscopic resection and surgery. From the perspective of reducing the economic impact of medical expenses, it can be considered superior to conventional techniques. Brief description of the attached diagram

[0367] Figure 1 The images show micrographs illustrating the expression of the WT1 protein in adenomas from patients with human familial adenomatous polyposis. The image on the left is of adenoma tissue, and the image on the right is of normal glandular ducts. In both images, light staining indicates WT1 protein, and dark stained areas indicate cell nuclei.

[0368] Figure 2 These are photomicrographs, displayed on APC. Min / + Expression of WT1 protein in mice. Photographs at 5x, 10x, and 20x magnification are shown sequentially from top to bottom. A scale bar is shown in the lower left of each photograph, along with lengths of 500 μm, 250 μm, and 100 μm sequentially from top to bottom. The lower right photograph of each tissue shows a magnified view of the overall tissue image. In all photographs, dark staining indicates WT1 protein, and circled staining areas indicate cell nuclei.

[0369] Figure 3 These are photomicrographs, displayed on APC.Min / + Expression of WT1 protein in mice. Magnification is 40x, and a scale bar showing 100 μm length is displayed in the lower left of the image. Dark staining indicates WT1 protein, and circled staining areas indicate cell nuclei.

[0370] Figure 4 The WT1 peptide vaccine was administered to APC. Min / + The experimental administration protocol for mice. The horizontal axis above shows the APC. Min / + The mice's age in weeks, vaccination time, and euthanasia time for analysis are shown on the horizontal axis above. The numbers below indicate the mice's age in weeks, the short arrows above indicate the vaccination time, and the long arrows above indicate the euthanasia time and analysis time. The composition of the WT1 vaccine and control vaccine is shown below the horizontal axis.

[0371] Figure 5 This is a graph showing the suppression of adenoma development by administration of the WT1 peptide vaccine. The vertical axis of the graph indicates the number of adenomas per small intestine, and the scatter plot on the left indicates the APCs that received the WT1 peptide vaccine. Min / + The results for mice and the scatter plot on the right indicate APCs that received the control vaccine. Min / + Results for mice. The horizontal lines in each scatter plot show the mean, and the results of the significance test are shown above the plot.

[0372] Figure 6 This indicates that administration of the WT1 peptide vaccine increases WT1 tetramer levels. + CD3 + CD8 + A graph of T cells. The vertical axis of the graph shows CD3. + CD8 + H-2D in T cells b WT1 tetramer + CD3 + CD8 + The frequency of T cells, and the scatter plot on the left showing APCs receiving the WT1 peptide vaccine. Min / + The results for mice, and the scatter plot on the right showing APCs that received the control vaccine. Min / + Results for mice. The horizontal lines in each scatter plot show the mean, and the results of the significance test are shown above the plot.

[0373] Figure 7 This is a graph showing the regression analysis of the WT1 peptide vaccine-administered group and the control vaccine-administered group. The vertical axis indicates the CD3... + CD8 + H-2D in T cells b WT1 tetramer + CD3 + CD8+ The frequency of T cells and the horizontal axis indicating the number of adenomas per small intestine. Individual points are the number of adenomas and WT1 tetramers in the WT1 peptide vaccine-treated group and the control vaccine-treated group. + CD3 + CD8 + A plot of T cell frequencies. The straight line in the plot shows the regression line, along with the correlation coefficient (R), the functional expression of the regression line, and the coefficient of determination (R²). 2 They are displayed sequentially from top to bottom on the right.

[0374] Figure 8 The WT1 peptide vaccine was administered to APC. Min / + The experimental administration protocol for mice. The horizontal axis above shows the APC. Min / + The mice's age in weeks, vaccination time, and euthanasia time for analysis are shown on the horizontal axis above. The numbers below indicate the mice's age in weeks, the short arrows above indicate the vaccination time, and the long arrows above indicate the euthanasia time and analysis time. The composition of the WT1 vaccine and control vaccine is shown below the horizontal axis.

[0375] Figure 9 Micrographs showing the expression of WT1 protein in adenomas from patients with non-hereditary colorectal adenomas are displayed. The first image from the left is a photograph of normal glandular ducts, and the three images from the right are photographs of adenoma tissue obtained from three patients with non-hereditary colorectal adenomas. In all images, light staining indicates WT1 protein, and dark circles or oval-shaped stained areas indicate cell nuclei. The expression level of WT1 protein is shown below each image, with a - sign indicating low expression and a + sign indicating high expression.

[0376] Figure 10 The compound represented by equation (3) and WT1 are shown in this paper. 35 Mixture of accessory peptides to APC Min / + The experimental administration protocol for mice. The horizontal axis shows the APC. Min / + The mice's age in weeks, vaccination time, and euthanasia time for analysis are shown. On the horizontal axis, the numbers below indicate the mice's age in weeks, and the arrows above indicate the time of vaccination and the time of euthanasia for analysis.

[0377] Figure 11 This is shown by giving the compound represented by equation (3) and WT1. 35 A graph showing the inhibition of adenoma formation by a mixture of helper peptides. The vertical axis of the graph indicates the number of adenomas per small intestine, and the scatter plot on the left indicates APCs receiving the WT1 peptide vaccine. Min / + The results for mice and the scatter plot on the right indicate APCs that received the control vaccine.Min / + Results for mice. The horizontal lines in each scatter plot show the mean, and the results of the significance test are shown above the plot.

[0378] Figure 12 This is shown by giving the compound represented by equation (3) and WT1. 35 The mixture of accessory peptides increases WT1 tetramer. + CD3 + CD8 + A graph of T cells. The vertical axis of the graph indicates the CD3+ level. + CD8 + H-2D in T cells b WT1 tetramer + CD3 + CD8 + The frequency of T cells, and the scatter plot on the left showing the acceptance of the compound represented by equation (3) and WT1 in this paper. 35 APC of a mixture of accessory peptides Min / + The results for mice and the scatter plot on the right show APCs that received the control vaccine. Min / + Results for mice. The horizontal lines in each scatter plot show the mean, and the results of the significance test are shown above the plot.

[0379] Figure 13 This shows that the compound represented by equation (3) and WT1 are acceptable. 35 A graph showing the regression analysis of the group receiving the combination of helper peptides and the group receiving the control vaccine. The vertical axis of the graph indicates the CD3... + CD8 + H-2D in T cells b WT1 tetramer + CD3 + CD8 + The frequency of T cells, and the horizontal axis indicating the number of adenomas per small intestine. The points are based on the compounds represented by equation (3) and WT1. 35 The number of adenomas and WT1 tetramers in the group receiving the mixture of accessory peptides and the group receiving the control vaccine. + CD3 + CD8 + A plot of T cell frequencies. The straight line in the plot shows the regression line, along with the correlation coefficient (R), the function of the regression line, and the coefficient of determination (R²). 2 They are displayed sequentially from top to bottom on the right. Detailed Implementation

[0380] The present invention will be described below in its best mode. Throughout this specification, it should be understood that singular expressions include their plural forms unless otherwise stated. Therefore, it should be understood that singular articles (e.g., "a," "an," or "the" in English) also include their plural forms unless otherwise stated. It should also be understood that the terminology used herein is used in the sense commonly understood in the art unless otherwise stated. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, this specification (including definitions) shall prevail.

[0381] The specific terminology and / or basic technical content used in this specification are defined and / or described appropriately below.

[0382] (Terminology Definition)

[0383] In this specification, "WT1 (Wilson's Bladder Gene 1)," "WT1 protein," or "WT1 peptide" refers to a polypeptide comprising at least a portion (or all) of the nephroblastoma (WT1) gene product or an analogue thereof. Specifically, "WT1 protein" preferably refers to the human WT1 protein (SEQ ID NO: 1) typically composed of 449 amino acids, or a protein whose amino acid sequence differs from that of SEQ ID NO: 1 due to the deletion, substitution, and / or addition of one to several (preferably about two to six) amino acids. The amino acids to be inserted or substituted may be non-natural amino acids not included in the 20 amino acids encoded by the gene. "WT1 peptide" refers to a peptide consisting of a portion of the amino acid sequence constituting the WT1 protein. In this specification, the term "WT1" or "WT1 peptide" will include mutated WT1 unless otherwise specified. Furthermore, when "WT1" or "WT1 peptide" is mentioned in this specification, "WT1" refers to human WT1 unless otherwise specified.

[0384] The length of the WT1 peptide used in this disclosure is not particularly limited, but preferably, is from about 7 to about 30 amino acids. Preferred WT1 peptides have a specific sequence pattern (motif) as an antigenic peptide that binds to and is presented by HLA molecules, and have the ability to bind HLA molecules. The ability to bind HLA molecules can be studied by methods known in the art. Such methods include, for example, computer-based methods such as Rankpep, BIMAS, and SYFPEITHI, and competitive binding tests using known WT1 peptides capable of binding HLA molecules. WT1 peptides that can be used in this disclosure are described herein in the section "WT1 Peptides" and also in WO2016 / 093326, which is incorporated herein by reference.

[0385] The preferred WT1 peptide used in this disclosure is a peptide that activates killer T cells and / or helper T cells. Activation of killer T cells and / or helper T cells can be achieved by a single type of peptide or multiple types of peptides (each of which may be specialized to activate killer or helper T cells). Furthermore, the WT1 peptide used in the pharmaceutical compositions of this disclosure can be a single type of peptide or may include multiple types of peptides. Additionally, the WT1 peptide used in the medicaments or pharmaceutical compositions of this disclosure can be a killer WT1 peptide or a helper WT1 peptide, or a mixture of these peptides. The WT1 peptide of this disclosure can be a single peptide or a conjugate, mixture, or combination of multiple peptides. More preferably, the WT1 peptide may include a combination of killer WT1 peptides and helper WT1 peptides. Examples of such combinations include compounds or compositions described in WO2014 / 157692.

[0386] As used in this article, "benign tumor" is a tumor without pathological findings of malignancy and is understood as distinct from malignant tumors. Benign tumors are considered to show no tendency to metastasize or invasive. A diagnosis of benign tumor does not necessarily imply a good clinical prognosis. For example, a low-grade meningioma occurring in the brainstem is a benign tumor, but it is difficult to treat and is clinically malignant because it compresses the brainstem and shows a poor prognosis, thus usually requiring treatment or prevention. Benign tumors include, but are not limited to, familial adenomatous polyposis, non-hereditary colorectal adenomas, intraductal papillary mucinous tumors, meningiomas, schwannomas, organ epithelial adenomas, papillomas, non-epithelial fibroids, lipomas, chondromas, and hemangiomas.

[0387] As used herein, the terms "familial adenomatous polyposis," "familial colonic polyposis," "familial adenomatous colonic polyposis," and "FAP" are used interchangeably and refer to a genetic disease characterized by mutations in the tumor suppressor gene APC (adenomatous colonic polyposis) and the formation of numerous adenomas in the intestine, and which are classified as benign tumors. As used herein, familial adenomatous polyposis also includes diseases consistent with tumors in tissues outside the intestine, such as Gardner syndrome. Typical animal models of familial adenomatous polyposis include, but are not limited to, APC... Min / + Mice (Jackson Institute, Bar Harbor, Maine, USA).

[0388] As used herein, terms such as "killer" or "killer peptide" mean that the peptide has the ability to activate cytotoxic T cells (CTLs, killer T cells). Killer T cell activation means an increase in the cytotoxic activity of killer T cells and / or an increase in the number of killer T cells. Whether a peptide is a "killer" peptide is typically determined by the tests described below. That is, it can be determined by detecting CD8 on the surface of T cells via methods such as flow cytometry. Alternatively, it can be determined by methods such as...51 The cytotoxic activity against the target is determined by measuring Cr release assay or lactate dehydrogenase (LDH) assay. In a preferred embodiment, the WT1 peptide of this disclosure has this killer activity.

[0389] MHC is known as human leukocyte antigen (HLA) in humans. HLA corresponding to MHC class I molecules is classified into subtypes, such as HLA-A, B, Cw, F, and G. The term "MHC class I - restricted" preferably includes "HLA-A - restricted", "HLA-B - restricted", and "HLA-Cw - restricted".

[0390] For each HLA subtype, polymorphisms (alleles) are known. Examples of HLA-A polymorphisms include 27 or more types, such as HLA-A1, HLA-A0201, and HLA-A24; examples of HLA-B polymorphisms include 59 or more types, such as HLA-B7, HLA-B40, and HLA-B4403; and examples of HLA-Cw polymorphisms include 10 or more types, such as HLA-Cw0301, HLA-Cw0401, and HLA-Cw0602. Among these polymorphisms, HLA-A0201 and HLA-A24 are preferred.

[0391] The "WT1 peptide" in this disclosure is a partial peptide consisting of 7-30 consecutive amino acids from the amino acid sequence of human WT1 shown in SEQ ID NO: 1.

[0392] As used herein, the term "MHC Class I-restricted" refers to the ability of a peptide to bind MHC (major histocompatibility complex) Class I molecules and induce CTLs. An "MHC Class I-restricted WT1 peptide" is a peptide capable of binding to MHC Class I molecules, presenting as a complex, and inducing CTLs from precursor T cells that recognize the complex in vitro and / or in vivo; therefore, it is synonymous with "WT1 killer peptide." The number of amino acid residues in an "MHC Class I-restricted WT1 peptide" is 7-30, preferably 7-15, more preferably 8-12, even more preferably 8-11, and most preferably 8 or 9.

[0393] Examples of "MHC Class I-restricted WT1 peptides" include amino acid positions 2, 3, 4, 6, 7, 10, 17, 18, 20, 23, 24, 26, 29, 30, 32, 33, 37, 38, 39, 40, 47, 63, 64, 65, 70, 73, 80, 81, 82, 83, 84, 85, 86, 88, 92, 93, 96, 98, 99, 100, 101, 104, 107, 110, 118, 119, 120, 123, 125, 126, 128, 130, 136, 137, 138, 139, 141, 143 of the human WT1 shown in SEQ ID NO: 1. 144, 146, 152, 161, 163, 165, 168, 169, 174, 177, 179, 180, 185, 187, 191, 192, 194, 202, 204, 206, 207, 208, 209, 210, 211, 213, 217, 218, 219, 221, 222, 223, 225, 227, 228, 230, 232, 233, 235, 239, 240, 242, 243, 244, 250, 251, 252, 260, 261, 263, 269 270, 272, 273, 276, 278, 279, 280, 285, 286, 287, 289, 292, 293, 294, 295, 298, 299, 301, 302, 303, 306, 309, 312, 313, 315, 316, 317, 318, 319, 324, 325, 326, 327, 329, 332, 334, 337, 340, 343, 345, 347, 349, 351, 354, 356, 358, 362, 363, 364, 366, 368 Peptides of nine amino acid residues beginning at 371, 372, 373, 375, 379, 383, 384, 386, 387, 389, 390, 391, 394, 396, 401, 406, 408, 409, 410, 412, 415, 416, 417, 418, 419, 420, 423, 424, 425, 426, 427, 428, 429, 432, 433, 434, 436, 437, 439, 440, or 441, or peptides containing variants thereof (see WO2014 / 157692). WO2014 / 157692 is incorporated herein by reference in its entirety.

[0394] "MHC Class I-restricted WT1 peptide" is preferably a peptide comprising an amino acid sequence selected from the following:

[0395] RMFPNAPYL (SEQ ID NO: 2),

[0396] CMTWNQMNL (SEQ ID NO: 3),

[0397] ALLPAVPSL (SEQ ID NO: 52),

[0398] SLGEQQYSV (SEQ ID NO: 53), and

[0399] RVPGVAPTL (SEQ ID NO: 54)

[0400] Alternatively, it may be a peptide comprising an amino acid sequence that differs from the amino acid sequence selected from SEQ ID NO: 2, 3, 52, 53, and 54 through amino acid alterations and possessing CTL-inducible activity. More preferably, the peptide may be a peptide composed of an amino acid sequence selected from SEQ ID NO: 2, 3, 52, 53, and 54.

[0401] When "WT1 peptide or analogue thereof" in this disclosure is a killer peptide, a peptide containing an amino acid sequence that differs from a given amino acid sequence through amino acid alteration has CTL-inducible activity.

[0402] This disclosure includes peptides with altered amino acid sequences that differ from a given amino acid sequence and possess CTL-inducible activity, also referred to as "altered killer peptides." An altered killer peptide is a peptide composed of an amino acid sequence that differs from a given amino acid sequence due to the deletion, substitution, and / or addition of one to three amino acids and is capable of binding to MHC class I to induce CTLs. When the peptide consists of a 9-residue amino acid sequence, amino acid substitutions can be at position 1 (N-terminus), 2, 3, or 9. The number of amino acid additions (including insertions) is typically 1 to several, preferably 1-3, more preferably 1 or 2, and even more preferably 1. Preferred positions for amino acid additions include the C-terminus. The number of amino acid deletions is preferably 1. In such alterations, the amino acids used for addition or substitution can be non-natural amino acids different from the 20 amino acids encoded by a gene.

[0403] Examples of altered killer peptides include the following peptides:

[0404] As a killer peptide modified from RMFPNAPYL (SEQ ID NO: 2),

[0405] RYFPNAPYL (SEQ ID NO: 46) (WO2003 / 106682),

[0406] FMFPNAPYL (SEQ ID NO: 13),

[0407] RLFPNAPYL (SEQ ID NO: 18),

[0408] RMMPNAPYL (SEQ ID NO: 25),

[0409] RMFPNAPYV (SEQ ID NO: 28) or

[0410] YMFPNAPYL (SEQ ID NO: 14) (WO2009 / 072610);

[0411] As a modified killer peptide of CMTWNQMNL (SEQ ID NO: 3),

[0412] CYTWNQMNL (SEQ ID NO: 45) (WO2002 / 79253),

[0413] Xaa-Met-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 55)

[0414] (where Xaa is Ser or Ala) or

[0415] Xaa-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu(SEQ ID NO: 56)

[0416] (where Xaa is Ser, Ala, Abu, Arg, Lys, Orn, Cit, Leu, Phe or Asn) (WO2004 / 026897);

[0417] As a modified killer peptide of ALLPAVPSL (SEQ ID NO: 52),

[0418] AYLPAVPSL (SEQ ID NO: 57) (WO2003 / 106682);

[0419] As a modified killer peptide of SLGEQQYSV (SEQ ID NO: 53),

[0420] FLGEQQYSV (SEQ ID NO: 58),

[0421] SMGEQQYSV (SEQ ID NO: 59), or

[0422] SLMEQQYSV (SEQ ID NO: 60) (WO 2009 / 072610); and

[0423] As a killer peptide modified from RVPGVAPTL (SEQ ID NO: 54),

[0424] RYPGVAPTL (SEQ ID NO: 61) (WO2003 / 106682).

[0425] Examples of amino acid sequences of peptides that are not a partial peptide consisting of 8-35 consecutive amino acid residues in the amino acid sequence of human WT1 as shown in SEQ ID NO: 1 include the following amino acid sequences (see WO2007 / 063903).

[0426] C-CYTWNQMNL (SEQ ID NO: 47) (where CC indicates that C residues are linked together by disulfide bonds) or

[0427] C-CMTWNQMNL (SEQ ID NO: 48) (where CC means that C residues are linked together by disulfide bonds).

[0428] Examples of killer peptides modified in this disclosure also include multimeric peptides, such as dimers as shown below (see WO2014 / 157692):

[0429] Compounds of formula (2):

[0430]

[0431] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0432] Compounds of formula (3):

[0433]

[0434] (where CC in the formula means that C residues are linked together by disulfide bonds), or

[0435] Compounds of formula (4):

[0436]

[0437] (where CC in the formula means that C residues are linked together by disulfide bonds).

[0438] As used herein, terms such as "helper" or "helper peptide" mean that the peptide has the ability to activate helper T cells. Activation of helper T cells means an increased function of helper T cells in assisting events such as antibody production from B cells or activation of killer T cells, and / or an increase in the number of helper T cells. Whether a peptide is a "helper" peptide is typically determined by the tests described below. That is, it can be determined by detecting CD4 on the surface of T cells via methods such as flow cytometry. Alternatively, it can be determined by stimulating target cells with an antigen and examining whether antigen-specific cytokines such as IFN-α or IFN-γ are produced by immunostaining.

[0439] As used in this article, the term "MHC class II-restricted" refers to the ability of peptides to bind to MHC class II molecules and induce helper T cells.

[0440] HLA corresponding to MHC class II molecules is classified into subtypes, such as HLA-DR, DQ, and DP. The term "MHC class II - restricted" preferably includes "HLA-DR - restricted", "HLA-DQ - restricted", and "HLA-DP - restricted".

[0441] Therefore, "MHC class II-restricted WT1 peptide" is a peptide capable of binding to MHC class II molecules and inducing helper T cells in vitro and / or in vivo. The number of amino acid residues in "MHC class II-restricted WT1 peptide" is 7-30, preferably 14-30.

[0442] When "WT1 peptide or analogue thereof" in this disclosure is a helper peptide, a peptide comprising an amino acid sequence that differs from a given amino acid sequence through amino acid alteration has helper T cell-inducing activity.

[0443] This disclosure includes peptides with modified amino acid sequences that differ from a given amino acid sequence due to amino acid alterations and which possess helper T cell-inducing activity, also referred to as "modified helper peptides." Modified helper peptides consist of amino acid sequences that differ from a given amino acid sequence due to the deletion, substitution, and / or addition of one to three amino acids and are capable of binding to MHC class II to induce helper T cells. The number of amino acid additions (including insertions) is preferably 1-3. The number of amino acid deletions is preferably 1-5. In such modifications, the amino acids used for addition or substitution can be non-natural amino acids different from the 20 genetically encoded amino acids.

[0444] Examples of modified accessory peptides include the following peptides:

[0445] As an auxiliary peptide of SGQARMFPNAPYLPSCLES (SEQ ID NO: 69),

[0446] SGQAYMFPNAPYLPSCLES (SEQ ID NO: 70) (WO2004 / 063217),

[0447] SGQARMFPNAPYLPSC (SEQ ID NO: 71) or

[0448] SGQAYMFPNAPYLPSC (SEQ ID NO: 72); or

[0449] PGCNKRYFKLSHLQMHSRK (SEQ ID NO: 49),

[0450] PGCNKRYFKLSHLQMHSRKH (SEQ ID NO: 62),

[0451] CNKRYFKLSHLQMHSRK (SEQ ID NO: 64),

[0452] CNKRYFKLSHLQMHSRKH (SEQ ID NO: 65) or

[0453] CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 66); or

[0454] WAPVLDFAPPGASAYGSL (SEQ ID NO: 4),

[0455] CWAPVLDFAPPGASAYGSL (SEQ ID NO: 50) or

[0456] WAPVLDFAPPGASAYGSLC (SEQ ID NO: 51).

[0457] The "WT1 peptide or analogue thereof" disclosed herein may possess both killer and helper activities in a composition of a killer peptide and a helper peptide. Examples of possessing both killer and helper activities include the following:

[0458] A composition comprising compounds selected from the following

[0459] Compounds of formula (2):

[0460]

[0461] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0462] Compounds of formula (3):

[0463]

[0464] (where CC in the formula means that C residues are linked together by disulfide bonds), and

[0465] Compounds of formula (4):

[0466]

[0467] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds).

[0468] Or its pharmaceutically acceptable salt, and

[0469] Peptides composed of amino acid sequences selected from the following

[0470] CNKRYFKLSHLQMHSRK (SEQ ID NO: 63),

[0471] CNKRYFKLSHLQMHSRKH (SEQ ID NO: 64),

[0472] CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 65),

[0473] WAPVLDFAPPGASAYGSL (SEQ ID NO: 4),

[0474] CWAPVLDFAPPGASAYGSL (SEQ ID NO: 50) and

[0475] WAPVLDFAPPGASAYGSLC (SEQ ID NO: 51)

[0476] Or its pharmaceutically acceptable salt;

[0477] A composition comprising a compound of formula (2):

[0478]

[0479] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0480] A peptide consisting of the amino acid sequence of WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof; and

[0481] Composition comprising a compound of formula (3):

[0482]

[0483] (where CC in the formula means that C residues are linked together by disulfide bonds) or its pharmaceutically acceptable salts, and

[0484] A peptide consisting of the amino acid sequence of WAPLVLDFAPPGASAYGSL (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof.

[0485] As used herein, the term "adjuvant" refers to an auxiliary agent for a primary agent (e.g., the WT1 peptide of this disclosure). In this disclosure, an adjuvant is a substance that enhances or improves the immune response induced by the WT1 peptide in a therapeutic or prophylactic composition. In this disclosure, an adjuvant may be, for example, a precipitating adjuvant such as sodium hydroxide, aluminum hydroxide, calcium phosphate, aluminum phosphate, alum, Pepesu, or a carboxyvinyl polymer, or an oil adjuvant such as liquid paraffin, lanolin, Freund, Montanide ISA763AVG, Montanide ISA51, an incomplete Freund's adjuvant, or a complete Freund's adjuvant.

[0486] As used herein, the term "adenoma" refers to a polyp that carries a high risk of becoming cancerous. The term "polyp" is a general term for mushroom-shaped or wart-like growths that form on the inner wall of intestinal tissue such as the small intestine, large intestine, or rectum. Adenomas are proliferative polyps and are at high risk of becoming cancerous, such as colorectal cancer.

[0487] In adenomas, those occurring in the large intestine are collectively referred to as "colorectal adenomas." Colorectal adenomas include hereditary colorectal adenomas (e.g., familial adenomatous polyposis) and non-hereditary colorectal adenomas. "Non-hereditary colorectal adenomas" refer to diseases in which a large number of adenomas form in the intestine due to factors such as lifestyle, diet, alcohol consumption, smoking, and stress, and are not characterized by mutations in the APC gene and are classified as benign tumors.

[0488] As used herein, "treatment" means the cessation, preferably cure, of the progression of the disease, disorder, or symptom of interest in this disclosure. The effectiveness of treatment can be evaluated according to "New Guidelines to Evaluate the Response to Treatment in Solid Tumors" (RECIST).<http: / / www.jcog.jp / doctor / tool / C_150_0010.pdf> Or its latest version. The definition of the best response according to WHO or RECIST standards is as follows.

[0489] Table 1

[0490] Best response WHO product and changes RECIST longest diameter and variation CR Disappeared; confirmed in 4 weeks Disappeared; confirmed in 4 weeks PR 50% reduction; confirmed in 4 weeks 30% reduction; confirmed in 4 weeks SD Neither PR nor PD standards are met. Neither PR nor PD standards are met. PD 25% increase; no CR, PR, or SD were recorded prior to the increase in disease incidence. A 20% increase; no CR, PR, or SD were recorded prior to the increase in disease incidence.

[0491] WHO = World Health Organization, RECIST = Response Evaluation Criteria for Solid Tumors, CR = Complete Response, PR = Partial Response, SD = Stable Disease, PD = Progressive Disease

[0492] As used herein, the terms "composition for treatment" or "therapeutic composition (formulation)" broadly refer to any composition capable of treating a condition of interest (e.g., a disease such as familial adenomatous polyposis). In one embodiment of this disclosure, a "therapeutic composition" can be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition can be produced, for example, by mixing the active ingredient with the aforementioned carrier and using any method known in the art of pharmaceutical science. Furthermore, the therapeutic composition is not limited in its form of use, as long as it is intended for treatment, and can be a single active ingredient or a mixture of the active ingredient and any other ingredients. The form of the carrier is not particularly limited and can be, for example, a solid or a liquid (e.g., a buffer solution).

[0493] As used herein, “prevention” means by any means to prevent or at least delay the occurrence of a disease, disorder or symptom of interest in this disclosure before it occurs, or to prevent any dysfunction arising from the cause of the disease, disorder or symptom even if the cause of the disease, disorder or symptom occurs.

[0494] As used herein, the terms "composition for prevention" or "preventive composition (formulation)" broadly refer to any composition capable of preventing a condition of interest (e.g., a disease such as familial adenomatous polyposis).

[0495] As used herein, “improvement” means the complete or partial cessation or mitigation of the progression of a disease, disorder, or symptom of interest that has occurred in this disclosure.

[0496] As used herein, the term "subject" means the object intended for prevention or treatment of the present disclosure (e.g., an organism, such as a human, or a substance obtained from an organism, such as cells, blood, or serum).

[0497] As used herein, the term "peripheral blood mononuclear cell" or "PBMC" refers to a mononuclear or monocytic cell containing monocytes or lymphocytes isolated from peripheral blood. Peripheral blood mononuclear cells include various blood cells such as T cells, B cells, NK cells, monocytes, and dendritic cells. Upon stimulation, peripheral blood mononuclear cells can differentiate into cells such as cytotoxic T cells or helper T cells.

[0498] In this specification, for example, WT1-specific cytotoxic T cells and helper T cells are referred to as "WT1-specific cytotoxic T cells" and "WT1-specific helper T cells," respectively. WT1-specific cytotoxic T cells and / or WT1-specific helper T cells can be induced by using the WT1 peptide or its analogues described in this disclosure or the nucleic acid molecule encoding it.

[0499] As used herein, the term "immature dendritic cell" refers to a dendritic cell that, when sensitized by a peptide or substance such as a cell, is capable of presenting a peptide or antigen from said substance. Upon stimulation, immature dendritic cells can differentiate into cells that present specific peptides, such as dendritic cells.

[0500] In this specification, for example, dendritic cells that present WT1 are referred to as "WT1-presenting dendritic cells". WT1-presenting dendritic cells can be induced by using the WT1 peptide described in this disclosure or an analogue thereof or a nucleic acid molecule encoding it.

[0501] Methods for inducing or activating antigen-presenting cells, killer T cells, or helper T cells are known to those skilled in the art. In vivo, these cells can be induced or activated by administering the WT1 peptide to a subject. In vitro, in one example, killer T cells can be obtained by reacting a sample containing lymphocytes derived from a subject with a complex of the WT1 peptide and an HLA molecule. In another example, peripheral blood mononuclear cells derived from a subject can be cultured in the presence of the WT1 peptide, and WT1-specific CTLs can be induced from peripheral blood mononuclear cells. In a further example, antigen-presenting cells that present the WT1 peptide via an HLA molecule can be induced by culturing immature antigen-presenting cells derived from a subject in the presence of the WT1 peptide. Immature antigen-presenting cells refer to cells that are ready to mature into antigen-presenting cells, and immature dendritic cells are an example. In an optional example, the WT1 peptide can be added to antigen-presenting cells to activate helper T cells. The antigen-presenting cells, killer T cells, or helper T cells used in the medicaments or compositions of this disclosure can be those induced or activated using the WT1 peptide or its derivatives or nucleic acid molecules. The antigen-presenting cells, killer T cells, or helper T cells induced or activated therefrom may be given to the subject, preferably from whom these cells are obtained, to treat or prevent benign tumors (e.g., familial adenomatous polyposis).

[0502] As used herein, “analog,” “derivative,” “analog,” or “mutant” (e.g., “analog,” “derivative,” “analog,” or “mutant” of the WT1 peptide) is preferably, but not limited to, a molecule containing a region substantially homologous to the protein of interest (e.g., the WT1 peptide). In various embodiments, such molecules have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity with an amino acid sequence aligned to the same size as the amino acid sequence, or with an amino acid sequence aligned by a computer homology program known in the art, or the nucleic acid encoding such a molecule is capable of hybridizing with the sequence encoding the protein under (high) stringency, moderate stringency, or non-stringency conditions. They are obtained by altering the protein via amino acid substitution, deletion, or addition, and such derivatives still exhibit the biological function of the original protein, although not necessarily to the same degree. For example, the biological function of such proteins can be examined by suitable and available in vitro assays described herein or known in the art. In this specification, embodiments relating to polypeptides, fragments, or derivatives involving the present disclosure, as used herein, are described by the terms "functionally active" or "having functional activity," meaning that the polypeptide, fragment, or derivative has a structural, regulatory, or biochemical function, such as biological activity, of a protein.

[0503] In this disclosure, a fragment of WT1 peptide is a polypeptide containing a region of WT1 peptide, and it does not necessarily have all the biological functions of a natural WT1 peptide, provided that it functions for the purposes of this disclosure (e.g., a peptide vaccine).

[0504] As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural or non-natural, or may be modified amino acids. The term may also include those complexes assembled into multiple polypeptide chains. The term also includes polymers of natural or artificially modified amino acids. Such modifications include, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeled component). The definition also includes, for example, one or more analogues containing amino acids (e.g., containing non-natural amino acids), peptide-like compounds (e.g., peptide pseudomorphs), and other modified polypeptides known in the art. In this specification, "amino acid" is a general term for organic compounds having both amino and carboxyl groups. When a protein or enzyme according to an embodiment of this disclosure contains a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Furthermore, any amino acid in the amino acid sequence may form a salt or solvate. Additionally, any amino acid in the amino acid sequence may be L- or D-form. Even in such cases, a protein according to an embodiment of this disclosure is considered to contain the aforementioned "specific amino acid sequence". The amino acids contained in the protein undergo chemical modifications in vivo, including, for example, N-terminal modifications (e.g., acetylation and myristylation), C-terminal modifications (e.g., amidation and addition of glycosylphosphatidylinositol), and side-chain modifications (e.g., phosphorylation and glycosylation). The amino acids can be natural or non-natural, provided they satisfy the objectives of this disclosure.

[0505] As used herein, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. The term also includes "oligonucleotide derivatives" or "polynucleotide derivatives." "Oligonucleotide derivatives" or "polynucleotide derivatives" refers to oligonucleotides or polynucleotides containing nucleotides or having unusual bonds between nucleotides, and these terms are used interchangeably. Specifically, such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bond of the oligonucleotide is converted to a thiophosphate bond, oligonucleotide derivatives in which the phosphodiester bond of the oligonucleotide is converted to an N3'-P5' aminophosphate bond, oligonucleotide derivatives in which the ribose and phosphodiester bond of the oligonucleotide are converted to a peptide nucleic acid bond, oligonucleotide derivatives in which the uracil of the oligonucleotide is replaced by C-5 propynyluracil, oligonucleotide derivatives in which the uracil of the oligonucleotide is replaced by C-5 thiazolyluracil, oligonucleotide derivatives in which the cytosine of the oligonucleotide is replaced by C-5 propynylcytosine, oligonucleotide derivatives in which the cytosine of the oligonucleotide is replaced by phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose of the DNA is replaced by 2'-O-propylribose, and oligonucleotide derivatives in which the ribose of the oligonucleotide is replaced by 2'-methoxyethoxyribose. Unless otherwise specified, a given nucleic acid sequence is intended to include not only the explicitly specified sequence, but also variants and complementary sequences obtained through conserved modifications (e.g., degenerate codon substitutions). In particular, variants obtained through degenerate codon substitutions can be achieved by sequences that produce a third position of one or more selected (or all) codons replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). As used herein, the term "nucleic acid" is also used interchangeably with the terms gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotide" can be natural or non-natural.

[0506] As used in this article, the term "gene" refers to a factor that defines a hereditary characteristic and can refer to "polynucleotide," "oligonucleotide," and "nucleic acid."

[0507] In this specification, "homology" of genes means the degree of similarity between two or more gene sequences, and generally, having "homology" means having a high degree of similarity or identity. Therefore, the higher the homology between two genes, the greater the similarity or identity of these sequences. Whether two genes are homologous can be checked by direct sequence comparison, or, in the case of nucleic acids, by hybridization under strict conditions. When directly comparing two gene sequences, genes are homologous when the DNA sequences generally have at least 50% similarity, preferably at least 70%, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% similarity. Therefore, as used herein, "homologous" or "homologous gene product" refers to a protein of another species, preferably a mammal, more preferably a human, that performs the same biological function as the protein components of the complex further described herein. Such homologous products are sometimes also referred to as "orthologous gene products." It should be understood that such homologous products, homologous gene products, and orthologous gene products may also be used, provided they satisfy the objectives of this disclosure.

[0508] Amino acids are referred to herein by their commonly known three-letter or single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Nucleotides are also referred to by their known single-letter codes. In this specification, comparisons of similarity, identity, or homology of amino acid or nucleotide sequences are calculated using BLAST (a tool for sequence analysis) with default parameters. Identity searches can be performed using, for example, NCBI's BLAST 2.7.1 (released on October 19, 2017). The value of "identity" in this specification generally refers to the value obtained when aligning sequences using the aforementioned BLAST and under default conditions. However, if a higher value is obtained when parameters are changed, the highest value is set as the identity value. When evaluating identity across multiple regions, the highest value among them is set as its identity value. "Similarity" is a numerical value that considers similar amino acids in addition to identity.

[0509] In one embodiment of this disclosure, "several" can be, for example, 10, 8, 6, 5, 4, 3, or 2, or fewer than any of these values. It is known that polypeptides in which one or more amino acid residues have been deleted, added, or inserted, or replaced by different amino acids, retain their biological activity (Mark et al., Proc Natl Acad Sci USA. 1984 Sep; 81 (18):5662-5666., Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10(20): 6487-6500., Wang et al., Science 1984 Jun 29; 224 (4656): 1431-1433.). Such modified proteins can be prepared, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using a protein phage library. For site-directed mutagenesis, a KOD-Plus mutagenesis kit (TOYOBO CO., LTD.) can be used. From such mutant proteins with alterations, such as deletions, proteins that have the same activity as their wild-type counterparts can be selected using any of a variety of characterization methods, such as FACS analysis and ELISA.

[0510] In one embodiment of this disclosure, the numerical value "70% or more" indicating identity can be, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%, and can be within a range starting from any two of the mentioned values. The aforementioned "identity" is the proportion of the number of identical amino acids between two or more amino acid sequences calculated according to the known methods described above. Specifically, before calculating the proportion, the amino acid sequences to be compared are aligned, and if it is necessary to maximize the proportion of identical amino acids, vacancies are introduced into a portion of the amino acid sequence. The methods used for alignment, proportion calculation, and comparison, as well as the associated computer programs, are well known in the art (e.g., BLAST as described above). As used herein, "identity" and "similarity" can be expressed as values ​​measured by NCBI BLAST unless otherwise specified. When comparing amino acid sequences in BLAST, BLAST can be used with the algorithm's default settings. Measurement results are quantified as positive values ​​or identity.

[0511] As used herein, the term "polynucleotide hybridizing under stringent conditions" refers to a polynucleotide hybridizing under well-known stringent conditions commonly used in the art. Such polynucleotides can be obtained by methods such as colony hybridization, plaque hybridization, or DNA blotting using polynucleotides selected from the polynucleotides disclosed herein as probes. In particular, such polynucleotides can be identifiable polynucleotides that are hybridized with a filter membrane to which DNA derived from colonies or plaques is immobilized at 65°C in the presence of 0.7–1.0 M NaCl, followed by washing the filter membrane at 65°C with 0.1–2 times the amount of SSC (saline-sodium citrate) solution (1 times the amount of SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate). "Strict conditions" can be any of the following: (1) using low ionic strength and high temperature for washing (e.g., at 50°C with 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate), (2) using denaturing agents such as formamide during hybridization (e.g., at 42°C with 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% ficol / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5), and 750 mM sodium chloride and 75 mM sodium citrate), or (3) incubating overnight at 37°C in a solution containing 20% ​​formamide, 5 × SSC, 50 mM sodium phosphate (pH 7.6), 5 × Denhardt solution, 10% dextran sulfate and 20 mg / ml denatured cleaved salmon sperm DNA, followed by washing the filter membrane with 1 × SSC at approximately 37–50°C. The formamide concentration can be 50% or more. Washing times can last 5, 15, 30, 60, or 120 minutes or longer. Several factors, such as temperature and salt concentration, can be considered to affect the stringency of the hybridization reaction. For details, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of "highly stringent conditions" are 0.0015 M sodium chloride and 0.0015 M sodium citrate at 65–68 °C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42 °C.Hybridization can be performed according to the protocol described in Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Sequences containing only A or T are preferably excluded from hybridization under strict conditions. Moderately stringent conditions can be readily determined, for example, based on DNA length, by those skilled in the art, and see Sambrook et al., Molecular Cloning: A Laboratory Manual, No. 3, Vol. 1, 7.42-7.45, Cold Spring Harbor Laboratory Press, 2001. Regarding nitrocellulose membranes, this includes hybridization conditions such as a pre-washing solution of 5 × SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0) at about 40-50°C with 50% formamide and 2 × SSC-6 × SSC (or similar hybridization solutions, such as Stark solution in about 50% formamide at about 42°C), and washing conditions of 0.5 × SSC and 0.1% SDS at about 60°C. Therefore, polypeptides used in this disclosure also include polypeptides encoded by nucleic acid molecules that hybridize under high or moderate stringent conditions with nucleic acid molecules encoding polypeptides specifically described herein.

[0512] The WT1 peptide of this disclosure is preferably "purified" or "isolated". As used herein, a "purified" substance or biological factor (e.g., nucleic acid or protein) is a substance or biological factor in which at least some factors associated with said substance or biological factor in nature have been removed. Therefore, purified biological factors generally have a higher purity than biological factors in their normal state (i.e., the biological factor is enriched). The term "purified" as used herein means that the biological factor is present at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight. The substance or biological factor used in this disclosure is preferably a "purified" substance. As used herein, an "isolated" substance or biological factor (e.g., nucleic acid or protein) is a substance or biological factor in which factors associated with said substance or biological factor in nature have been substantially removed. As used herein, the term "isolated" may have different meanings depending on its purpose and is therefore not necessarily expressed in terms of purity, but rather, if desired, means that the biological agent is present at a preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight. The substances used in this disclosure are preferably "isolated" substances or biological agents.

[0513] As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). The fragment length may be suitably varied depending on its purpose. For example, in the case of polypeptides, a lower limit of amino acid length of 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more, and any length expressed as an integer not specifically listed herein (e.g., 11) may also be a suitable lower limit. Similarly, in the case of polynucleotides, a lower limit of nucleotide length of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 or more, and any length expressed as an integer not specifically listed herein (e.g., 11) may also be a suitable lower limit. As used herein, it should be understood that such fragments fall within the scope of this disclosure, for example, when the full-length is used as a cancer vaccine and the fragment is also used.

[0514] As used herein, when referring to a gene or nucleic acid molecule or a related polypeptide, the term "biological function" means a specific function that a gene, nucleic acid molecule, or polypeptide may have in vivo or in vitro, and this may include, but is not limited to, activation of killer T cells or helper T cells. As used herein, a biological function may be performed by a corresponding "biological activity." As used herein, the term "biological activity" means the activity that a factor (e.g., a polynucleotide or protein) may have, and includes various activities that perform any of a variety of functions (e.g., activation of killer T cells or helper T cells). "Biological activity" can be an activity performed in vivo or an activity performed in vitro due to an event such as secretion. For example, if the factor is an enzyme, its biological activity includes its enzymatic activity. Such biological activity can be measured using techniques well known in the art. Therefore, "activity" can be any of a variety of measurable indicators that indicate or reveal (direct or indirect) binding; or affect any response (i.e., have a measurable effect in response to some exposure or stimulus), and includes the affinity of compounds that directly bind to polypeptides or polynucleotides of this disclosure, as well as the amount of upstream or downstream proteins or other similar functional measures following, for example, some stimulus or event.

[0515] As used herein, the term "expression" of a molecule such as a gene, polynucleotide, or polypeptide means that the molecule undergoes some in vivo process to present in another form. Preferably, the term "expression" means that a molecule such as a gene or polynucleotide is transcribed and translated into a polypeptide form, but it may also mean that the molecule is transcribed to produce mRNA. Thus, as used herein, "expression product" includes such polypeptides or proteins, or mRNA. More preferably, such polypeptide forms may be post-translational processed. For example, the expression level of WT1 can be determined by any method. In particular, the expression level of WT1 can be known by evaluating the amount of WT1 mRNA, the amount of WT1 protein, and the biological activity of the WT1 protein. The amount of WT1 mRNA or protein can be determined by methods detailed elsewhere herein or by other methods known in the art.

[0516] As used herein, the term "functional equivalent" refers to any entity having the same intended function as the original entity but with a different structure. Therefore, it should be understood that functional equivalents of the "WT1 peptide" of this disclosure include those that do not have the same sequence as SEQ ID NO: 1 but are mutants or variants (e.g., amino acid sequence variants) of it, which have the biological function of the WT1 peptide, and those that, upon action, can be converted to mutants or variants having the biological function of the WT1 peptide (including nucleic acids encoding the mutants or variants and vectors or cells containing such nucleic acids). It should be understood that functional equivalents of the WT1 peptide in this disclosure can be used in the same manner as the WT1 peptide, even if not specifically mentioned. Functional equivalents can be discovered by searching databases or other sources. As used herein, the term "search" means the discovery of different nucleic acid sequences having specific functions and / or properties by utilizing a nucleic acid sequence, through electronic, biological, or other methods. Electronic searches include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-2448 (1988)), the Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)) and the Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)). Biological searches include, but are not limited to, strict hybridization, microarrays of genomic DNA ligated to nylon membranes or glass plates (microarray assays), PCR, and in situ hybridization. In this specification, the genes intended for use in this disclosure should also include the corresponding genes identified by such electronic or biological searches.

[0517] Functional equivalents of this disclosure can be amino acid sequences that differ from the original amino acid sequence due to the insertion, substitution, deletion, or addition of one or more amino acids into the amino acid sequence. In this specification, the expression "one or more amino acids are inserted into the amino acid sequence, substituted or deleted, or added to one or two ends of the amino acid sequence" means the alteration of one or more naturally occurring amino acids, for example, substitution in the amino acid sequence performed by well-known techniques such as site-directed mutagenesis or by natural mutation. The altered amino acid sequence can be due to the insertion, substitution, and / or deletion, or addition of one or two amino acids into the amino acid sequence, particularly preferably 1-4, 1-3, 1-2, or 1 amino acid, into the amino acid sequence, or to the addition of one or two ends of the amino acid sequence, differing from the original amino acid sequence. The altered amino acid sequence is preferably due to a conserved substitution of one or more amino acids (preferably one or more, or 1, 2, 3, or 4 amino acids) that differs from the amino acid sequence of any of SEQ ID NO: 1-5. As used herein, the term "conserved substitution" refers to the substitution of one or more amino acid residues with different but chemically similar amino acid residues such that the function of the protein is substantially unchanged. For example, one hydrophobic residue can be replaced by another hydrophobic residue, and one polar residue can be replaced by another polar residue with the same charge. For each amino acid, functionally similar amino acids capable of such substitutions are known in the art. In particular, examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Furthermore, examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0518] (WT1 peptide)

[0519] In this disclosure, the WT1 peptide can be a peptide derived from the WT1 protein, which induces activation of cytotoxic T cells or helper T cells. Examples of WT1 peptides include WT1 killer peptides that induce activation of cytotoxic T cells and WT1 helper peptides that induce activation of helper T cells. Examples of WT1 killer peptides include peptides composed of 8-12 amino acids, preferably 8-9 amino acids, derived from the WT1 protein. Particularly preferably, the WT1 killer peptide is WT1… 126 Peptide: Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2), WT1 235Peptide: Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3) or a compound of formula (3):

[0520]

[0521] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds), which is WT1 126 Peptides and WT1 235m Peptide conjugates. Examples of WT1 accessory peptides include peptides consisting of 14-20 amino acids, preferably 16-18 amino acids, derived from the WT1 protein. Particularly preferred, the WT1 accessory peptide is WT1... 35 Peptide (Trp Ala Pro Val Leu Asp Phe Ala ProPro Gly Ala Ser Ala Tyr Gly Ser Leu; SEQ ID NO: 4) or WT1 332 The peptide (Lys Arg Tyr PheLys Leu Ser His Leu Gln Met His Ser Arg Lys His) (SEQ ID NO: 5). Variant peptides that differ from the WT1 peptide due to the deletion, substitution, or addition of one or more amino acids may also be used as the WT1 peptide of this disclosure. Examples of such variant peptides include WT1 126 Variant peptides, WT1 235 Variant peptides, WT1 35 Variant peptides and WT1 332 Variant peptides.

[0522] WT1 126 Preferred variant peptides are those in which amino acid residues 4-8, starting from the N-terminus, are modified from WT1. 126 The N-terminus of the peptide begins at position 4-8 (PNAPY) of the same peptide. WT1 126 Such variant peptides are preferably peptides composed of the amino acid sequences of any one of SEQ ID NO: 6-44:

[0523] WT1 126 P1G peptide (GMFPNAPYL; SEQ ID NO: 6)

[0524] WT1 126 P1A peptide (AMFPNAPYL; SEQ ID NO: 7)

[0525] WT1 126 P1V peptide (VMFPNAPYL; SEQ ID NO: 8)

[0526] WT1 126 P1L peptide (LMFPNAPYL; SEQ ID NO: 9)

[0527] WT1 126 P1I peptide (IMFPNAPYL; SEQ ID NO: 10)

[0528] WT1 126 P1M peptide (MMFPNAPYL; SEQ ID NO: 11)

[0529] WT1 126 P1W peptide (WMFPNAPYL; SEQ ID NO: 12)

[0530] WT1 126 P1F peptide (FMFPNAPYL; SEQ ID NO: 13)

[0531] WT1 126 P1Y peptide (YMFPNAPYL; SEQ ID NO: 14)

[0532] WT1 126 P2V peptide (RVFPNAPYL; SEQ ID NO: 15)

[0533] WT1 126 P2Q peptide (RQFPNAPYL; SEQ ID NO: 16)

[0534] WT1 126 P2A peptide (RAFPNAPYL; SEQ ID NO: 17)

[0535] WT1 126 P2L peptide (RLFPNAPYL; SEQ ID NO: 18)

[0536] WT1 126 P2I peptide (RIFPNAPYL; SEQ ID NO: 19)

[0537] WT1 126 P3I peptide (RMIPNAPYL; SEQ ID NO: 20)

[0538] WT1 126 P3L peptide (RMLPNAPYL; SEQ ID NO: 21)

[0539] WT1 126 P3G peptide (RMGPNAPYL; SEQ ID NO: 22)

[0540] WT1 126 P3A peptide (RMAPNAPYL; SEQ ID NO: 23)

[0541] WT1 126 P3V peptide (RMVPNAPYL; SEQ ID NO: 24)

[0542] WT1 126 P3M peptide (RMMPNAPYL; SEQ ID NO: 25)

[0543] WT1 126 P3P peptide (RMPPNAPYL; SEQ ID NO: 26)

[0544] WT1 126 P3W peptide (RMWPNAPYL; SEQ ID NO: 27)

[0545] WT1 126 P9V peptide (RMFPNAPYV; SEQ ID NO: 28)

[0546] WT1 126 P9A peptide (RMFPNAPYA; SEQ ID NO: 29)

[0547] WT1 126 P9I peptide (RMFPNAPYI; SEQ ID NO: 30)

[0548] WT1 126 P9M peptide (RMFPNAPYM; SEQ ID NO: 31)

[0549] WT1 126 P1D peptide (DMFPNAPYL; SEQ ID NO: 32)

[0550] WT1 126 P1E peptide (EMFPNAPYL; SEQ ID NO: 33)

[0551] WT1 126 P1H peptide (HMFPNAPYL; SEQ ID NO: 34)

[0552] WT1 126 P1K peptide (KMFPNAPYL; SEQ ID NO: 35)

[0553] WT1 126P1N peptide (NMFPNAPYL; SEQ ID NO: 36)

[0554] WT1 126 P1P peptide (PMFPNAPYL; SEQ ID NO: 37)

[0555] WT1 126 P1Q peptide (QMFPNAPYL; SEQ ID NO: 38)

[0556] WT1 126 P1S peptide (SMFPNAPYL; SEQ ID NO: 39)

[0557] WT1 126 P1T peptide (TMFPNAPYL; SEQ ID NO: 40)

[0558] WT1 126 P2I&P9I peptide (RIFPNAPYI; SEQ ID NO: 41)

[0559] WT1 126 P2I & P9V peptides (RIFPNAPYV; SEQ ID NO: 42)

[0560] WT1 126 P2L & P9I peptides (RLFPNAPYI; SEQ ID NO: 43) and

[0561] WT1 126 P2L&P9V peptide (RLFPNAPYV; SEQ ID NO: 44).

[0562] WT1 235 Examples of peptide variants include peptides containing WT1. 235 : Cys Met Thr Trp Asn Gln Met AsnLeu (SEQ ID NO: 3) or WT1 235m Cys Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO:45) or peptides thereof, dimers comprising the amino acid sequence of SEQ ID NO: 3 or 45 or peptides thereof, cystine forms comprising the amino acid sequence of SEQ ID NO: 3 or 45 or peptides thereof, and amino acid sequences or peptides thereof that differ from the above amino acid sequences due to substitution, deletion, or addition of one or more amino acids in the above amino acid sequences, but not limited thereto. In WT1 235 Among peptide variants, WT1 235mPeptides are particularly preferred because they are -Restricted WT1 peptide, while It is the most abundant HLA type among Japanese people, and it has more HLA types than wild-type WT1. 235 Peptides offer enhanced therapeutic efficacy and excellent water solubility.

[0563] Examples of these variant peptides include the following.

[0564] Cys-Cys Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 47) (where CC indicates that C residues are linked together by disulfide bonds) or Cys-Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 48) (where CC indicates that C residues are linked together by disulfide bonds) are particularly excellent in terms of physicochemical properties and stability because the thiol groups of the N-terminal cysteine ​​residues are modified (see WO2007 / 063903).

[0565] From the perspective of covering a wide range of HLA subtypes, the variant peptide of WT1 peptide in this embodiment preferably contains multiple peptides corresponding to different HLA subtypes. For example, it preferably contains both a peptide corresponding to the HLA-A2 subtype or a pharmaceutically acceptable salt thereof and a peptide corresponding to the HLA-A24 subtype or a pharmaceutically acceptable salt thereof. In such cases, for example, the pharmaceutical composition may comprise a compound of formula (1):

[0566]

[0567] [where X] a and Y a Independently representing a single bond, tumor antigen peptide A represents a peptide composed of an amino acid sequence selected from the following: RMFPNAPYL (SEQ ID NO: 2), ALLPAVPSL (SEQ ID NO: 52), SLGEQQYSV (SEQ ID NO: 53), RVPGVAPTL (SEQ ID NO: 7), YMFPNAPYL (SEQ ID NO: 14), and VLDFAPPGA (SEQ ID NO: 68), and the carbonyl group of the C-terminal amino acid of tumor antigen peptide A is bound to the hydroxyl group in formula (2).

[0568] R 1 It is hydrogen or tumor antigen peptide B, and

[0569] Tumor antigen peptide B represents a peptide having an amino acid sequence different from that of tumor antigen peptide A and consisting of amino acid sequences selected from the following: CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL (SEQ ID NO: 45), and the thioether group of the cysteine ​​residue of tumor antigen peptide B is combined with the thioether group in formula (1).

[0570] Or its pharmaceutically acceptable salt.

[0571] The compound represented by the above formula (1) has excellent stability against oxidants in solution (due to the formation of disulfide bonds by cysteine ​​residues) and stable quality as a raw material for pharmaceutical products.

[0572] Furthermore, when the variant peptide of the WT1 peptide in this embodiment comprises a compound represented by formula (1) (a conjugate of the WT1 killer peptide), the conjugate is degraded in vivo by reductive cleavage of the disulfide bond between N-terminal cysteine ​​residues induced by ERAP1, resulting in two epitopes corresponding to different HLA subtypes. The in vivo generation of conjugates corresponding to multiple epitopes of different HLA subtypes, such as those represented by formula (1), can broadly address different HLA subtypes that vary according to the subject and can cover large populations with a single conjugate, thus effectively inducing CTLs in subjects (see WO 2014 / 157692, which is incorporated herein by reference in its entirety).

[0573] In this embodiment, "tumor antigen peptide A" is an MHC class I-restricted WT1 peptide composed of 7-30 amino acid residues. In tumor antigen peptide A of formula (1), the N-terminal amino acid is bound to Ya in formula (1), and the carbonyl group of the C-terminal amino acid is bound to the hydroxyl group in formula (1).

[0574] The compound represented by formula (1) can be

[0575] Compounds of formula (2):

[0576]

[0577] (where CC in the formula means that C residues are linked together by disulfide bonds);

[0578] Compounds of formula (3):

[0579]

[0580] (Where CC is shown in the formula, it means that C residues are linked together by disulfide bonds); or

[0581] Compounds of formula (4):

[0582]

[0583] (where CC in the formula means that C residues are linked together by disulfide bonds).

[0584] WT1 35 Variant peptides can be any peptide as long as they have an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 4 due to the substitution, deletion or addition of one or more amino acids.

[0585] WT1 332 Variant peptides can be any peptide, as long as they have an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 5 due to the substitution, deletion, or addition of one or more amino acids. In a preferred embodiment, WT1 332 Variant peptides are preferably peptides whose amino acid sequences differ from the amino acid sequence of SEQ ID NO: 5 due to the substitution of amino acid residues at positions 3, 6, 8, and / or 11 by amino acid residues selected from the following.

[0586] Position 3: Phenylalanine, Tryptophan, Valine, Isoleucine, Leucine, Methionine

[0587] Position 6: Valine, Isoleucine, Methionine, Aspartic Acid, Glutamic Acid

[0588] Position 8: Asparagine, serine, threonine, glutamine, lysine, aspartic acid

[0589] Position 11: Aspartic acid, glutamic acid and glutamine.

[0590] Among them, WT1 126 The peptide variant is preferably WT1. 126 P1F peptide (SEQ ID NO: 13), WT1 126 P2L peptide (SEQ ID NO: 18), WT1 126 P3M peptide (SEQ ID NO: 25) or WT1 126 P9V peptide (SEQ ID NO: 28), more preferably WT1 126 P2L peptide, WT1 126 P3M peptide or WT1 126 P9V peptide, and even more preferably WT1 126 P9V peptide.

[0591] The WT1 peptide in the preventive or therapeutic compositions of this disclosure is preferably WT1. 126 Peptide, WT1 126 P1F peptide, WT1 126 P2L peptide, WT1126 P3M peptide or WT1 126 P9V peptide. More preferably, WT1 peptide is WT1. 126 Peptide, WT1 126 P2L peptide, WT1 126 P3M peptide or WT1 126 P9V peptide, or even more preferably WT1 126 peptide or WT1 126 P9V peptide, and particularly preferably WT1 126 Peptide.

[0592] WT1 peptide can also be a derivative of WT1 peptide. WT1 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 Examples of peptide derivatives include any of a variety of substances linked to one or both of the N- and C-termini of an amino acid sequence consisting of 9, 16, or 18 consecutive amino acids. For example, amino acids, peptides, or their analogues can be linked. Without being bound by theory, when such a substance is linked to WT1... 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 When a peptide or a variant thereof is processed, for example by an in vivo enzyme or by a method such as intracellular processing, it ultimately provides a peptide consisting of 9, 16, or 18 amino acids, which can be presented on the cell surface to induce a WT1-specific CTL response.

[0593] The WT1 peptide or its analogue in this embodiment may further comprise a WT1 accessory peptide. When the WT1 peptide or its analogue in this embodiment further comprises a peptide containing an amino acid sequence selected from CNKRYFKLSHLQMHSRK (SEQ ID NO: 63), CNKRYFKLSHLQMHSRKH (SEQ ID NO: 64), CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 65), WAPPLDFAPPGASAYGSL (SEQ ID NO: 4), CWAPVLDFAPPGASAYGSL (SEQ ID NO: 66), and WAPPLDFAPPGASAYGSLC (SEQ ID NO: 51), it may further comprise a peptide containing another amino acid sequence selected from the above group and / or any other WT1 accessory peptide.

[0594] WT1 peptides can be prepared using methods commonly used in the art. For example, peptides can be synthesized using methods described in the literature, such as Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide synthesis, Maruzen Co., LTD., 1975; Basics and Experiments of Peptide Synthesis, Maruzen Co., LTD., 1985; or Development of Pharmaceutical Product subsequent vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991.

[0595] Such screening of WT1 peptides and variant peptides can preferably be carried out by stimulating PBMCs (peripheral blood mononuclear cells) of a number of patients with benign tumors (e.g., familial adenomatous polyposis) with each candidate peptide at a time, and selecting candidate peptides that show a good response in IFN-γ assays (because this is a simple method).

[0596] In this disclosure, polynucleotides, such as DNA or RNA, encoding the aforementioned WT1 protein or WT1 peptide can also be used as active ingredients in prophylactic or therapeutic compositions. That is, cancer immunity can be generated in vivo by inserting a polynucleotide encoding the WT1 protein or WT1 peptide into a suitable vector, preferably an expression vector, and then administering the vector to an animal (including a human). Examples of polynucleotides include DNA and RNA, and the polynucleotide is preferably DNA or RNA. The nucleotide sequence of the polynucleotide can be determined based on the amino acid sequence of the WT1 protein or WT1 peptide that is immunogenic to patients with benign tumors (e.g., familial adenomatous polyposis). The polynucleotide can be generated by known methods for DNA or RNA synthesis or, for example, by using PCR. Prophylactic or therapeutic compositions comprising DNA encoding the WT1 protein or WT1 peptide are also an aspect of this disclosure. The WT1 protein or WT1 peptide is preferably the WT1 peptide, more preferably WT1 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 Peptides, or their variant peptides, or combinations thereof, or even more preferably WT1 126 Peptide, WT1 235 peptide or WT1 35Peptides, or their variant peptides, or combinations thereof, and most preferably, compounds of formula (3) (WT1) 126 Peptides and WT1 235 peptide conjugates) and WT1 35 Combinations of peptides. DNA can be inserted into any expression vector. RNA itself can also be used as the active ingredient in the composition, even without insertion into a vector.

[0597] The prophylactic or therapeutic compositions of this disclosure may contain an adjuvant. The adjuvant can be any substance, provided that when administered with or even separately from the antigen, it nonspecifically enhances the immune response to the WT1 protein or WT1 peptide used as the antigen. Examples of adjuvants include precipitating adjuvants and oil-based adjuvants. Precipitating adjuvants include, for example, sodium hydroxide, aluminum hydroxide, calcium phosphate, aluminum phosphate, alum, Pepesu, and carboxyvinyl polymers. Oil-based adjuvants are preferably adjuvants in which the oil can encapsulate an aqueous antigen solution to form micelles, and specific examples include liquid paraffin, lanolin, Freund, Montanide ISA763AVG, Montanide ISA51, incomplete Freund's adjuvant, and complete Freund's adjuvant. Adjuvants may also be mixtures of two or more. Preferably, the adjuvant is an oil-based adjuvant. The amount of adjuvant in the prophylactic or therapeutic compositions of this disclosure is not particularly limited, provided that it is an amount that nonspecifically enhances the immune response to the antigen, and can be suitably selected depending on the type of adjuvant and other factors.

[0598] The prophylactic or therapeutic compositions of this disclosure can be administered orally or parenterally, such as intraperitoneally, subcutaneously, intradermally, intramuscularly, intravenously, or intranasally. Parenterial administration includes transdermal absorption of the active ingredient WT1 protein or WT1 peptide by applying the prophylactic or therapeutic composition to the skin or by adding the prophylactic or therapeutic composition to a patch and attaching it to the skin. The prophylactic or therapeutic compositions of this disclosure can also be administered by other methods of administration, including inhalation. Preferably, the prophylactic or therapeutic compositions are administered via parenterial administration. More preferably, the prophylactic or therapeutic compositions are administered via intradermal or subcutaneous administration. Preferred examples of body parts for intradermal or subcutaneous administration include the upper arm.

[0599] The preventive or therapeutic compositions of this disclosure may be in any of a variety of formulations, such as solid or liquid formulations, depending on the route of administration. For example, the preventive or therapeutic compositions may be oral solid or liquid formulations for oral administration or injectable formulations for parenteral administration.

[0600] Examples of oral solid dosage forms for oral administration include tablets, pills, capsules, powders, and granules.

[0601] Solid dosage forms for internal use can be prepared according to conventional methods and used as is with WT1 protein or WT1 peptide, or mixed with or granulated with additives (e.g., by stirred granulation, fluidized bed granulation, dry granulation, or rolling stirred fluidized bed granulation). For example, capsules can be prepared by filling capsules, and tablets can be prepared by compression. One or more additives can be suitably blended. Examples of additives include, for example, excipients such as lactose, mannitol, glucose, microcrystalline cellulose, corn starch; binders such as hydroxypropyl cellulose, polyvinylpyrrolidone, magnesium aluminum metasilicate; dispersants such as corn starch; disintegrants such as calcium carboxymethyl cellulose; lubricants such as magnesium stearate; solubilizers such as glutamic acid and aspartic acid; stabilizers; cellulose such as hydroxypropyl cellulose, hydroxypropyl methylcellulose and methylcellulose; and water-soluble polymers, including synthetic polymers such as polyethylene glycol, polyvinylpyrrolidone and polyvinyl alcohol; and sweeteners such as white sugar, powdered sugar, sucrose, fructose, glucose, lactose, reduced maltose starch syrup, powdered reduced maltose starch syrup, glucose fructose syrup, fructose glucose syrup, honey, sorbitol, maltitol, mannitol, xylitol, erythritol, aspartame, saccharin or sodium saccharin.

[0602] If desired, granules or tablets may be coated, for example, with a coating agent, and the coating may consist of two or more layers. Examples of coating agents include sugar, gelatin, hydroxypropyl cellulose, and hydroxypropyl methyl phthalate. Capsules may be prepared by suitably selecting any of the above-mentioned excipients and uniformly mixing WT1 protein or WT1 peptide with the selected excipients, and filling the mixture or granules (with or without a suitable coating) prepared from the mixture into capsules, or encapsulating them in a capsule matrix that has increased plasticity due to the addition of plasticizers such as glycerin or sorbitol to a suitable capsule matrix (e.g., gelatin). If desired, colorants or preservatives (sulfur dioxide; parabens, such as methylparaben, ethylparaben, and propylparaben) may be added to the capsule matrix. Capsules include hard capsules or soft capsules.

[0603] Examples of oral liquid formulations include liquids, suspensions, emulsions, syrups, pre-dissolved formulations such as dry syrups, and elixirs. For such oral liquid formulations, the WT1 protein or WT1 peptide is dissolved, suspended, or emulsified in a diluent commonly used in oral liquid formulations. Examples of diluents include purified water, ethanol, or mixtures thereof. The liquid formulation may also contain any of the following: wetting agents, suspending agents, emulsifiers, sweeteners, flavoring agents, fragrances, preservatives, and buffers. Dry syrups can be prepared, for example, by mixing WT1 protein or WT1 peptide with white sugar, powdered sugar, sucrose, fructose, glucose, or lactose. Dry syrups can be granulated according to conventional methods.

[0604] Dosage forms for parenteral administration include injections, ointments, gels, creams, patches, aerosols, and sprays, but injections are preferred. For example, injections are preferably prepared using WT1 protein or WT1 peptide and conventional carriers.

[0605] Injectable preparations for parenteral administration can be aqueous or oil-based. Aqueous preparations can be prepared, for example, by mixing a solution of WT1 protein or WT1 peptide with a pharmaceutically acceptable additive obtained by appropriate addition according to known methods to an aqueous solvent (e.g., water for injection or purified water), filtering the mixture with a sterile filter, and then filling the sterile mixture into a sterile container. Examples of pharmaceutically acceptable additives include, for example, the adjuvants described above; isotonic agents, such as sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, glucose, and propylene glycol; buffers, such as phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, and ε-aminocaproic acid buffer; preservatives, such as methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax; thickeners, such as hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, and polyethylene glycol; stabilizers, such as sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, and butylated hydroxytoluene; and pH adjusters, such as hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid. Injectable formulations may contain any suitable solubilizer, such as alcohols like ethanol; polyols like propylene glycol and polyethylene glycol; and nonionic surfactants such as polysorbate 80, polyoxyethylene hydrogenated castor oil 50, lysophosphatidylcholine, and pululanic polyols. Injectable formulations may further contain any protein, such as bovine serum albumin and keyhole cyanobacterium; and polysaccharides, such as glycosaminoglycans. To prepare oil-based injections, oil-based solvents such as sesame oil or soybean oil may be used, and solubilizers such as benzyl benzoate or benzyl alcohol may be added. Prepared injectable solutions are typically packaged in suitable containers, such as ampoules or vials. Liquid formulations, such as those for injection, may be stored frozen or after water removal by lyophilization. Lyophilized formulations are reconstituted with distilled water for injection before use.

[0606] Furthermore, the preventive or therapeutic compositions of this disclosure may be in a form in which, if desired, the WT1 protein or WT1 peptide is mixed in liposomes with polysaccharides and / or any other components to be incorporated into the cancer vaccine composition.

[0607] The dosage of the preventive or therapeutic compositions disclosed herein varies depending on the WT1 protein or WT1 peptide or DNA to be used and factors such as the patient's age and weight or the disease to be treated, and in the presence of WT1 peptides such as WT1126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 In the case of a peptide vaccine composition, for example, the amount of WT1 peptide is preferably from about 0.1 μg to 1 mg / kg body weight / day. The dose of WT1 peptide is typically from 0.0001 mg to 1000 mg, preferably from 0.01 mg to 1000 mg, more preferably from 0.1 mg to 10 mg, and this dose is preferably administered every few days to every few months.

[0608] Furthermore, the prophylactic or therapeutic compositions of this disclosure can be administered as follows: PBMCs are collected from peripheral blood of a patient with a benign tumor (e.g., familial adenomatous polyposis), dendritic cells are obtained from the peripheral blood, and peptides such as WT1, which are included as active ingredients in the prophylactic or therapeutic composition, are administered. 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 Pulsing cells are generated from peptides or polynucleotides such as DNA or RNA, and the cells are returned to the patient via subcutaneous administration or any other method of administration. Conditions for using reagents such as WT1 peptide-pulsing dendritic cells are not particularly limited, provided that the effects of this disclosure are demonstrated, and any commonly used conditions may be employed.

[0609] When a nucleic acid molecule encoding the WT1 protein or WT1 peptide is used in a prophylactic or therapeutic composition, it is preferred to administer the prophylactic or therapeutic composition such that the nucleic acid molecule is introduced into dendritic cells of a patient with a benign tumor (e.g., familial adenomatous polyposis). The nucleic acid molecule can be introduced into the dendritic cells of a patient with a benign tumor (e.g., familial adenomatous polyposis) by, for example, obtaining dendritic cells from a patient with a benign tumor (e.g., familial adenomatous polyposis) as described above, and by introducing the nucleic acid molecule into the dendritic cells using an electrical pulse. Since the WT1 protein or WT1 peptide expressed from the nucleic acid molecule introduced into the dendritic cells is presented on the surface of the dendritic cells, returning the dendritic cells pulsed with the nucleic acid molecule to the patient with a benign tumor (e.g., familial adenomatous polyposis) can rapidly induce cancer immunity in vivo. This method of treating or preventing cancer by introducing a nucleic acid molecule encoding the WT1 protein or WT1 peptide into the dendritic cells of a subject is one of the preferred embodiments of this disclosure. In the above embodiments, the nucleic acid molecule can be DNA or RNA, and preferably RNA.

[0610] Another aspect of this disclosure relates to methods for inducing WT1-specific CTLs and / or WT1-specific helper T cells, comprising culturing peripheral blood mononuclear cells derived from a subject in the presence of WT1 protein or WT1 peptide, or introducing a nucleic acid molecule encoding WT1 protein or WT1 peptide into such peripheral blood mononuclear cells, thereby inducing WT1-specific CTLs and / or WT1-specific helper T cells from the peripheral blood mononuclear cells. The subject from whom the peripheral blood mononuclear cells are derived is not particularly limited. The WT1 protein or WT1 peptide may be WT1 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 Peptide, or a variant peptide thereof, and preferably WT1 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 Peptides. For example, when peripheral blood mononuclear cells derived from a subject are in WT1 126 peptide or WT1 235 WT1-specific CTLs were induced from CTL precursor cells in peripheral blood mononuclear cells when cultured in the presence of peptides. 35 peptide or WT1 332 WT1-specific helper T cells are induced from helper T cell precursor cells in peripheral blood mononuclear cells when cultured in the presence of the peptide. Culture conditions for peripheral blood mononuclear cells derived from the subject are not particularly limited, and the cells can be cultured under any commonly used conditions. The resulting CTLs and helper T cells recognize WT1. 126 Peptide, WT1 235 Peptide, WT1 35 Peptides and WT1 332 Peptides. Therefore, WT1-specific CTLs and / or WT1-specific helper T cells induced by this disclosure can be specifically used to destroy tumor cells that highly express WT1 and to treat and / or prevent targeted benign tumors (e.g., familial adenomatous polyposis). The method of administering WT1-specific CTLs and / or WT1-specific helper T cells to a subject is not particularly limited, and the cells can be administered, for example, in the same manner as the prophylactic or therapeutic compositions described above.

[0611] Another aspect of this disclosure relates to a kit for inducing WT1-specific CTLs and / or WT1-specific helper T cells, comprising WT1 protein or WT1 peptide as a basic component. Preferably, the kit is used in the method described above for inducing WT1-specific CTLs and / or WT1-specific helper T cells from a subject. In addition to WT1 protein or WT1 peptide, such a kit may also contain any other components, such as tools, adjuvants, or reaction vessels for obtaining peripheral blood mononuclear cells. Using this kit, it is possible to effectively induce cells that recognize cancer antigens such as WT1. 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 WT1-specific CTLs and / or WT1-specific helper T cells of peptides.

[0612] In yet another embodiment, this disclosure relates to a method for inducing dendritic cells to present WT1 protein or WT1 peptide, comprising culturing immature dendritic cells derived from a subject in the presence of WT1 protein or WT1 peptide, or introducing a nucleic acid molecule encoding WT1 protein or WT1 peptide into such immature dendritic cells, thereby inducing dendritic cells to present WT1 protein or WT1 peptide from the immature dendritic cells. WT1 protein or WT1 peptide may be WT1 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 Peptide or its variant peptides, and preferably WT1 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 Peptide. Nucleic acid molecules encoding WT1 protein or WT1 peptide can be those encoding WT1. 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 Nucleic acid molecules of peptides or their variant peptides, preferably encoding WT1. 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332 The peptide is a nucleic acid molecule. The nucleic acid molecule can be DNA or RNA, and is preferably RNA. The subject from which the immature dendritic cells are derived is not particularly limited. Since immature dendritic cells are contained in cells such as peripheral blood mononuclear cells, such cells can be found in WT1. 126 Peptide, WT1 235 Peptide, WT1 35 peptide or WT1 332The dendritic cells are cultured in the presence of peptides. When the resulting dendritic cells are administered to a subject, the aforementioned WT1-specific CTLs and / or WT1-specific helper T cells are effectively induced, thereby treating and / or preventing benign tumors (e.g., familial adenomatous polyposis) in the subject. The method of administering the dendritic cells to the subject is not particularly limited, and the cells can be administered, for example, in the same manner as the aforementioned prophylactic or therapeutic compositions.

[0613] (Preferred Implementation Scheme)

[0614] Preferred embodiments of this disclosure are described below. It should be understood that the embodiments provided below are provided for a better understanding of this disclosure, and the scope of this disclosure should not be limited to the following description. Therefore, it will be apparent to those skilled in the art that suitable modifications can be made within the scope of this disclosure based on the description in this specification. It should also be understood that the following embodiments of this disclosure can be used alone or in combination.

[0615] Furthermore, all the following embodiments are general or specific examples. In the following embodiments, numerical values, shapes, materials, components, steps, sequence of steps, or other descriptions are examples and are not intended to limit the scope of the claims. Additionally, in the components of the following embodiments, components not described in the independent claims indicating the broadest concept are described as optional components.

[0616] (Preventive or therapeutic composition)

[0617] In one aspect, this disclosure provides compositions (preventive or therapeutic compositions) for the prevention or treatment of benign tumors, comprising the WT1 peptide or an analogue thereof. WT1 has been shown to be highly expressed in cancer cells of malignant tumors, but its expression in benign tumors is unknown. This disclosure is based on the surprising discovery that the WT1 protein is expressed in cells of benign tumors and provides a novel therapeutic approach for benign tumors.

[0618] In one embodiment, the benign tumor of this disclosure expresses WT1. Without being bound by theory, the WT1 peptide of this disclosure serves as a cancer antigen and enhances the cytotoxic activity of CTLs and / or the activity of helper T cells, thereby inducing cytotoxicity against benign tumor cells, and due to its activity, the WT1 peptide vaccine of this disclosure has a therapeutic effect against benign tumors expressing WT1. The expression of the WT1 protein is not limited to specific benign tumors but is observed in a variety of hereditary and non-hereditary benign tumors. Therefore, the WT1 protein can be expressed not only in hereditary benign tumors but also in acquired benign tumors, and it should be understood that this type of benign tumor can be treated or prevented using the techniques of this disclosure. In one embodiment, the benign tumor is selected from familial adenomatous polyposis, non-hereditary colorectal adenoma, intraductal papillary mucinous tumor, meningioma, schwannoma, organ epithelial adenoma, papilloma, non-epithelial fibroid, lipoma, chondroma, and hemangioma.

[0619] In one embodiment, this disclosure provides a composition for the prevention or treatment of familial adenomatous polyposis comprising a WT1 peptide or an analogue thereof. While WT1 peptides or analogues have been shown to be effective in treating angiogenesis, it is surprising that WT1 peptides or analogues are effective in treating or preventing familial adenomatous polyposis, which is difficult to treat and previously had virtually no cure except for excision. This disclosure provides a novel treatment option for familial adenomatous polyposis where excision is virtually the only treatment, and since the treatment or prevention of this disclosure does not require excision, it can therefore be used to promote improvements in quality of life (QOL).

[0620] In one embodiment, the WT1 peptide of this disclosure may be a single type of peptide or may comprise multiple types of peptides. Furthermore, the WT1 peptide used in the pharmaceutical or composition of this disclosure may be a killer WT1 peptide or a helper WT1 peptide, or may be a mixture of these peptides. More preferably, the WT1 peptide comprises both a killer WT1 peptide and a helper WT1 peptide. Dimers of WT1 peptides may be used in this disclosure. Dimers of WT1 peptides can be obtained by forming a disulfide bond between two WT1 peptides, each WT1 peptide having a cysteine ​​residue. The WT1 peptide used in the pharmaceutical composition of this disclosure may be a single type of peptide or may comprise multiple types of peptides.

[0621] Whether a WT1 peptide exerts a therapeutic or preventative effect in a subject depends on whether the WT1 peptide corresponds to the subject's HLA type. Since suitable HLA types for many WT1 peptides are currently known, the WT1 peptide used in this disclosure can be selected based on the subject's HLA type. Furthermore, multiple types of WT1 peptides can be used in the pharmaceutical compositions of this disclosure to cover a wide range of subjects.

[0622] In a preferred embodiment, the WT1 peptide used in this disclosure is WT1. 126 Killer peptide, WT1 235 Killer peptide, WT1 35 Helper peptides and / or WT1 332 Helper peptide. Preferably, the WT1 peptide comprises peptides selected from WT1. 126 Peptides and WT1 235 Killer peptides and peptides selected from WT1 35 Peptides and WT1 332 Helper peptides of peptides. Not wishing to be bound by theory, it has been found in this disclosure that WT1 peptides are more effective when both killer and helper peptides are present than when used alone.

[0623] On the other hand, the WT1 peptide of this disclosure includes compounds of formula (3) and WT1 35 Both of the accessory peptides. To the best of the applicant's knowledge, no compound of formula (3) and WT1 are known. 35 Combinations of helper peptides have shown preventative and / or therapeutic efficacy against benign tumors.

[0624] On the other hand, this disclosure provides compositions for the prevention or treatment of benign tumors, comprising nucleic acid molecules encoding WT1 peptide or analogues thereof.

[0625] In some embodiments, this disclosure provides compositions for the prevention or treatment of familial adenomatous polyposis comprising a nucleic acid molecule encoding a WT1 peptide or an analogue thereof. In this case, the active ingredient in the pharmaceutical composition of this disclosure may be, but is not limited to, a polynucleotide encoding a WT1 peptide. The nucleotide sequence of the polynucleotide may be determined based on the amino acid sequence of the WT1 peptide. The polynucleotide may be produced, for example, by known methods used for DNA or RNA synthesis, such as chemical synthesis or PCR.

[0626] In one embodiment, the preventive or therapeutic composition of this disclosure comprises DNA encoding a WT1 peptide or an analogue thereof.

[0627] In another embodiment, the preventive or therapeutic composition of this disclosure comprises RNA encoding a WT1 peptide or an analogue thereof.

[0628] In another embodiment, the preventive or therapeutic composition of this disclosure comprises RNA and DNA encoding a WT1 peptide or an analogue thereof.

[0629] In one embodiment, the preventive or therapeutic composition of this disclosure contains an adjuvant in addition to the components described above.

[0630] In a preferred embodiment, the adjuvant used in this disclosure includes Montanide® ISA51 adjuvant.

[0631] In one embodiment, the preventive or therapeutic composition of this disclosure is administered once a week.

[0632] The drugs or compositions disclosed herein may be used in combination with agents for the treatment and / or prevention of benign tumors (e.g., familial adenomatous polyposis).

[0633] The route of administration of the drugs or compositions disclosed herein is not particularly limited, and examples of preferred routes of administration include intradermal administration, subcutaneous administration, transdermal administration, and transmucosal administration (e.g., eye drops, nasal drops, sublingual administration).

[0634] The dosage form of the medicine or composition disclosed herein is not particularly limited and may be, for example, a dosage form such as an injection, eye drops, nasal drops, lotion, cream, patch, sublingual tablet, or lozenge. These dosage forms can be prepared and administered by methods well known to those skilled in the art.

[0635] When using the pharmaceuticals or compositions of this disclosure, the dosage of the WT1 peptide may vary appropriately depending on factors such as the type of WT1 peptide, route of administration, dosage form, disease type, disease severity, and the subject's health condition. Generally, the dosage of the WT1 peptide is 0.1 μg / kg to 1 mg / kg / adult / day. Similarly, the type, route of administration, and dosage form of the WT1 peptide may be varied appropriately. In addition to pharmaceutically acceptable carriers or excipients, the pharmaceuticals or compositions of this disclosure may also contain suitable adjuvants, such as aluminum hydroxide. Alternatively, the pharmaceuticals or compositions of this disclosure may contain the WT1 peptide encapsulated in liposomes.

[0636] In this implementation, for example, when the WT1 peptide is a compound of formula (2) or formula (3), the WT1 peptide may be administered intradermally to adults at 3.5 mg / day every 2 weeks for 1-5 times during the introduction phase, and intradermally to adults at 3.5 mg / day every 1 or 2 months for 3-6 times during the maintenance phase.

[0637] Dosage ranges may be 1.0 mg or more, 2.5 mg or more, 5.0 mg or more, 10 mg or more, 15 mg or more, 20 mg or more, or 25 mg or more, or 100 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 25 mg or less, 20 mg or less, 15 mg or less, 10 mg or less, 5.0 mg or less, or 2.5 mg or less for adults daily. Specifically, dosages may be, for example, 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg, 7.5 mg, 8.0 mg, 8.5 mg, 9.0 mg, 9.5 mg, or 10 mg daily for adults.

[0638] The interval for granting may be appropriately selected from 1 week to 1 year, and may be, for example, 1 day or longer, 1 week or longer, 2 weeks or longer, 3 weeks or longer, 1 month or longer, 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, or 6 months or longer, or 1 year or less, 9 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, 2 weeks or less, or 1 week or less. In particular, the interval for granting may be, for example, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 1 year.

[0639] The number of times given can be appropriately selected from 1 to 100 times, and can be, 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, or 10 or more, or 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In particular, the number of times given can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times.

[0640] On the other hand, this disclosure provides WT1 peptides or analogues thereof, or nucleic acid molecules encoding WT1 peptides or analogues thereof, for the treatment and / or prevention of benign tumors (e.g., familial adenomatous polyposis).

[0641] In another aspect, this disclosure relates to the use of WT1 peptide or its analogues or nucleic acid molecules encoding WT1 peptide or its analogues for the treatment and / or prevention of benign tumors (e.g., familial adenomatous polyposis) or for the preparation of pharmaceutical compositions for the treatment and / or prevention of benign tumors (e.g., familial adenomatous polyposis). This disclosure further provides a method for treating and / or preventing benign tumors (e.g., familial adenomatous polyposis), comprising the step of administering an effective amount of WT1 peptide or its analogues or nucleic acid molecules encoding WT1 peptide or its analogues to a subject in need. In a further embodiment, this disclosure relates to the use of WT1 peptide or its analogues or nucleic acid molecules encoding WT1 peptide or its analogues for the preparation of medicaments for the treatment and / or prevention of benign tumors (e.g., familial adenomatous polyposis). In yet another aspect, this disclosure relates to a method for treating and / or preventing benign tumors (e.g., familial adenomatous polyposis), characterized by administering WT1 peptide or its analogues or nucleic acid molecules encoding WT1 peptide or its analogues to a subject in need. All descriptions herein, including the foregoing, also apply to these aspects.

[0642] (Methods for inducing immune cells)

[0643] On the other hand, this disclosure provides a method for inducing WT1-specific cytotoxic T cells (CTLs) and / or WT1-specific helper T cells for the prevention or treatment of benign tumors (e.g., familial adenomatous polyposis), characterized by comprising the steps of: culturing peripheral blood mononuclear cells derived from a subject requiring treatment for a benign tumor (e.g., familial adenomatous polyposis) in the presence of any WT1 peptide or analogue described herein, or introducing a nucleic acid molecule encoding any WT1 peptide or analogue described herein into such peripheral blood mononuclear cells, thereby inducing WT1-specific CTLs and / or WT1-specific helper T cells from the peripheral blood mononuclear cells. Those skilled in the art will understand that any descriptions given in the section "Prophylactic or Therapeutic Compositions" also apply to this induction method.

[0644] On the other hand, this disclosure provides a method for inducing WT1-presenting dendritic cells for the prevention or treatment of benign tumors (e.g., familial adenomatous polyposis), characterized by comprising the steps of: culturing immature dendritic cells derived from a subject requiring treatment for a benign tumor (e.g., familial adenomatous polyposis) in the presence of any WT1 peptide or analogue described herein, or introducing a nucleic acid molecule encoding any WT1 peptide or analogue described herein into such immature dendritic cells, thereby inducing WT1-presenting dendritic cells. Those skilled in the art will understand that any description given in the section "Prophylactic or Therapeutic Compositions" also applies to this induction method.

[0645] On the other hand, this disclosure relates to compositions comprising a WT1 peptide or an analogue thereof or a nucleic acid molecule encoding a WT1 peptide or an analogue thereof, for inducing WT1-specific cytotoxic T cells and / or WT1-specific helper T cells, wherein the composition is used for the prevention or treatment of benign tumors (e.g., familial adenomatous polyposis). Those skilled in the art will understand that any descriptions given in the section "Prophylactic or Therapeutic Compositions" also apply to such compositions.

[0646] In another aspect, this disclosure relates to compositions comprising a WT1 peptide or an analogue thereof or a nucleic acid molecule encoding a WT1 peptide or an analogue thereof, for inducing WT1-presenting dendritic cells, wherein the composition is used for the prevention or treatment of benign tumors (e.g., familial adenomatous polyposis). Those skilled in the art will understand that any descriptions given in the section "Prophylactic or Therapeutic Compositions" also apply to such compositions.

[0647] In another aspect, this disclosure provides compositions for the prevention or treatment of benign tumors (e.g., familial adenomatous polyposis) comprising WT1-specific cytotoxic T cells and / or WT1-specific helper T cells. Those skilled in the art will understand that any descriptions given in the section "Prophylactic or Therapeutic Compositions" also apply to such compositions.

[0648] In another aspect, this disclosure provides compositions for the prevention or treatment of benign tumors (e.g., familial adenomatous polyposis) comprising dendritic cells presenting WT1. Those skilled in the art will understand that any descriptions given in the section "Prophylactic or Therapeutic Compositions" also apply to such compositions.

[0649] (Preparation method)

[0650] The peptides, derivatives, or nucleic acid molecules of this disclosure can be prepared using methods commonly used in the art. These methods can be appropriately modified based on the knowledge of those skilled in immunological, molecular biological, biochemical, and microbiological approaches. In particular, the peptides, derivatives, or nucleic acid molecules of this disclosure can be designed based on the amino acid sequence of the natural WT1 protein (e.g., SEQ ID NO: 1) or the nucleic acid sequence of the natural WT1 protein, and can be prepared using systems employing microorganisms, such as expression systems. The starting materials and intermediates used in the preparation methods can be purchased as commercial products or obtained by methods described in publicly known documents or from publicly known compounds according to publicly known methods. Analogs of these starting materials and intermediates can be used, provided they do not interfere with the preparation process.

[0651] (General Technology)

[0652] The immunological, molecular biological, biochemical, and microbiological techniques used in this article are well-known and commonly used in the field, and are described in the references, such as Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its third edition (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods fromCurrentProtocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press; and Experimental Medicine, a separate volume, "Experimental methods for gene transfer & expression analysis," Yodosha, 1997. The relevant portions (or all) of each reference are incorporated herein by reference.

[0653] (Note)

[0654] In this specification, the term "or" is used when "at least one" of the substances listed herein may be used. In this specification, when specified as "within a range of two values," the range also includes both specific values.

[0655] References cited in this article, such as scientific literature, patents, and patent applications, are incorporated into this article as a whole, to the same extent as the content of each reference is specifically described in this article.

[0656] For ease of understanding, this disclosure has been described above with reference to preferred embodiments. This disclosure will be described below with reference to embodiments, but the above description and the following embodiments are for illustrative purposes only and not for limiting the scope of this disclosure. Therefore, the scope of this disclosure is not limited to the embodiments or examples specifically described in this specification, but is defined only by the scope of the claims. Example

[0657] Examples are described below. If necessary, the biological treatments used in the following examples conform to the standards set forth by Osaka University or relevant regulatory authorities. The reagents used in the examples are described below, but equivalents from other manufacturers (e.g., Sigma-Aldrich, FUJIFILM Wako Pure Chemical, Nacalai Tesque, R&D Systems, USCNLife Science INC, Thermo Fisher Scientific, Kanto Chemical, Funakoshi, Tokyo Chemical Industry, Merck) may also be used.

[0658] (Mouse)

[0659] The following examples use Apc Min / + (C57BL / 6J) mice (Jackson Laboratory, Bar Harbor, Maine, USA). Mice were fed in accordance with Osaka University's animal experiment regulations at the Specific Microorganism-Free (SPF) containment facility of the Department of Laboratory Animal Science, Osaka University School of Medicine.

[0660] (Reagents)

[0661] The following examples use MHC Class I (H-2D) b )-binding peptide WT1 126-134 (RMPNAPYL, 9a.a) and MHC class II (H-2I-A) b )-binding peptide WT1 35-52 (WAPVLDFAPPGASAYGSL, 18a.a.), purchased from SIGMA Genosys (Ishikari, Japan). Compounds of formula (3) and WT1 35-52 Raw materials used in the mixture of accessory peptides were supplied by Sumitomo Dainippon Pharma Co., Ltd. (Osaka, Japan). The peptides were stored at −20°C until use. The peptides were dissolved in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄) and stored at −20°C. Freund's incomplete adjuvant (IFA) and Montanide ISA 51 were purchased from Seppic SA (Orsay, France).

[0662] (Example 1: Expression of WT1 protein in adenomas of familial adenomatous polyposis)

[0663] This example demonstrates that the WT1 protein is expressed in adenomas in patients with familial adenomatous polyposis.

[0664] (Experimental Methods)

[0665] Small and large intestine sections from patients with familial adenomatous polyposis were fixed in formalin and embedded in paraffin, with the paraffin blocks cut to a thickness of 3 μm. To reduce endogenous peroxidase reactions, the paraffin sections were treated with 3% H2O2 solution, then deparaffinized in xylene, and after stepwise rehydration with ethanol, for antigen retrieval, heated in a 70°C oil bath for 25 minutes and a 110°C oil bath for 25 minutes in 10 mM Tris EDTA buffer (10 mM Tris, 1 mM EDTA, pH 9.0), followed by cooling at room temperature for 3 hours. The treated sections were reacted overnight at 4°C with anti-WT1 6FH2 mouse monoclonal antibody (Dako Cytomation, Inc., Carpinteria, CA) diluted 100-fold with PBST (PBS + 0.05% Tween20®). Then, the sections were treated with Dako REAL Envision HRP RABBIT / MOUSE (Dako Cytomation) at 37°C for 30 minutes. WT1 protein was visualized using the Dako REAL Envision Detection System Peroxidase / DAB+,Rabbit / Mouse (Dako Cytomation) according to the manufacturer's manual. The sections were then stained with hematoxylin.

[0666] (result)

[0667] The results of Example 1 are shown in Table 2 and Figure 1 Table 2 shows the expression levels of WT1 protein in adenomas, normal glandular ducts, and fibroblasts from samples derived from each patient. Table 2 confirms that the expression level of WT1 protein in adenomas is significantly higher than that in normal glandular ducts. Figure 1 In the images, light staining indicates WT1 protein, and dark staining areas indicate cell nuclei. WT1 protein expression was observed in adenomas but not in normal glandular ducts.

[0668] Table 2

[0669]

[0670] (Example 2: In APC) Min / + Expression of WT1 protein in mice

[0671] This example demonstrates the role of WT1 protein in APC. Min / + It is expressed in small intestinal adenomas of mice, which are models of familial adenomatous polyposis.

[0672] (Experimental Methods)

[0673] 20-week-old APC Min / +The small and large intestines of (C57BL / 6J) mice were fixed in formalin and embedded in paraffin, with the paraffin blocks cut into 3 μm thick sections. Paraffin sections were deparaffinized with xylene and, after stepwise rehydration with ethanol, microwaved for 15 minutes in 10 mM citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for antigen retrieval. The treated sections were reacted overnight at 4°C with anti-WT1 6FH2 mouse monoclonal antibody (Dako Cytomation, Inc., Carpinteria, CA) diluted 100-fold with PBS containing goat serum. The sections were then reacted at 37°C for 30 minutes with biotinylated anti-mouse IgG antibody (Vector Labs., Burlingame, CA) diluted 100-fold with PBS containing goat serum. To reduce endogenous peroxidase activity, sections were treated with 3% H2O2 solution and the WT1 protein was visualized using the Vectastain ABC kit (Vector Labs) according to the manufacturer's instructions. The sections were then stained with hematoxylin.

[0674] (result)

[0675] The results of Example 2 are shown in Figure 2 and 3 . Figure 2 The photomicrographs at 5x, 10x, and 20x magnification are displayed sequentially from top to bottom. Figure 3 Displaying photomicrographs at 40x magnification. Figure 2 and 3 In the image, dark staining indicates WT1 protein, and circle staining indicates the cell nucleus. WT1 protein expression is observed in APCs. Min / + It was observed in small intestinal adenomas in mice, which are a model of familial adenomatous polyposis.

[0676] (Example 3: Administration of WT1 peptide vaccine to mice with familial adenomatous polyposis)

[0677] This example demonstrates the administration of a WT1 peptide vaccine to APC. Min / + Administration protocol for experiments on mice.

[0678] (Experimental Methods)

[0679] The administration regimen for the WT1 peptide vaccine was shown in Figure 4 100 μg WT1 126-134 Killer peptide and 45 μg WT1 35-52A mixture of helper peptides or PBS is emulsified with IFA Montanide ISA51 adjuvant to obtain the WT1 peptide vaccine or control vaccine. The WT1 peptide vaccine or control vaccine is administered intradermally to APCs aged 4 to 5 weeks. Min / + Lateral flank of mice. Immunization began at 5 weeks of age and was administered 8 times weekly. Immunized mice were euthanized 10 days after the last immunization for further analysis. Administration of the WT1 peptide vaccine did not cause organ damage, as shown in Table 3 below.

[0680] Table 3

[0681]

[0682] (Example 4: Prevention and treatment of adenomas by administering WT1 peptide vaccine)

[0683] This example demonstrates the administration of a WT1 peptide vaccine to APC. Min / + Mice were used to prevent and treat adenomas.

[0684] (Experimental Methods)

[0685] Mice were obtained using the administration protocol described in Example 3. Whole intestines were collected from euthanized mice 10 days after the last immunization. The small intestine was divided into 8–10 segments, and the large intestine into the cecum and ascending colon. Each segment was longitudinally cut, washed with PBS, sandwiched between filter paper, and fixed in 4% paraformaldehyde / phosphate buffer at 4°C for 24 hours or longer. The fixed intestinal tissue was stained with 1% methylene blue solution and then examined under a stereomicroscope to count the number of adenomas (polyps). The difference in the number of adenomas (polyps) between the WT1 peptide vaccine-administered group and the control vaccine-administered group was statistically analyzed using a Student's t-test.

[0686] (result)

[0687] The results of Example 4 are shown in Figure 5 The horizontal axis of the graph shows the group receiving the WT1 vaccine on the left and the group receiving the control vaccine on the right, while the vertical axis shows the number of adenomas per small intestine. The mean number of adenomas per small intestine was approximately 29 in the WT1 peptide vaccine-administered group and approximately 34 in the control vaccine-administered group, indicating that administration of the WT1 peptide vaccine significantly inhibited the occurrence of adenomas and also treated them.

[0688] (Example 5: Increasing WT1 tetramer by administering WT1 peptide vaccine) + CD3 + CD8 + T cells)

[0689] This example demonstrates the administration of a WT1 peptide vaccine to APC. Min / + Increased WT1 tetramer in mice+ CD3 + CD8 + T cells.

[0690] (Experimental Methods)

[0691] Mice were used according to the administration protocol described in Example 3. Spleen cells were collected from euthanized mice 10 days after the last immunization. After erythrocyte lysis, the cells were treated with H-2D. b WT1 tetramer (MBL, Aichi, Japan) staining was performed, and further stained with anti-mouse CD3 antibody (17A2, BioLegend, San Diego, CA) and anti-mouse CD8 antibody (KT15, MBL, Aichi, Japan). The stained cells were analyzed by FACSCanto, and CD3 levels were measured. + CD8 + H-2D in T cells b WT1 tetramer + CD3 + CD8 + T cell frequency. H-2D differences between the WT1 peptide vaccine-administered group and the control vaccine-administered group. b WT1 tetramer + CD3 + CD8 + The frequency differences of T cells were statistically analyzed using the Student t-test.

[0692] (result)

[0693] The results of Example 5 are shown in Figure 6 The horizontal axis of the graph shows the group receiving the WT1 peptide vaccine on the left and the group receiving the control vaccine on the right, and the vertical axis shows CD3. + CD8 + H-2D in cells b Frequency of WT1 tetramer. H-2D in the WT1 vaccine-administered group. b WT1 tetramer + CD3 + CD8 + The mean frequency of T cells was approximately 0.6%, compared to approximately 0.2% in the control vaccine-administered group, indicating that administration of the WT1 vaccine increased WT1 tetramer levels. + CD3 + CD8 + T cells.

[0694] (Example 6: Statistical Analysis)

[0695] This example shows the number of adenomas in Example 4 and the WT1 tetramer in Example 5. + CD3+ CD8 + Correlation was observed among T cells.

[0696] (Experimental Methods)

[0697] The number of adenomas per small intestine obtained from Example 4 and the CD3 count obtained from Example 5 + CD8 + H-2D in T cells b WT1 tetramer + CD3 + CD8 + The frequency of T cells was plotted on a scatter plot using Microsoft Excel®. Furthermore, the correlations of these results were statistically analyzed in Excel using the Pearson correlation coefficient.

[0698] (result)

[0699] The results from Example 6 showed a weak correlation between the number of adenomas (polyps) and the frequency of WT1 tetramers in the WT1 peptide vaccine-administered group and the control vaccine-administered group.

[0700] (Example 7: Single Drug Example)

[0701] This example demonstrates the difference in APC between the group receiving both the killer WT1 peptide vaccine and the adjuvant WT1 peptide vaccine and the group receiving either the killer WT1 peptide vaccine or the adjuvant WT1 peptide vaccine alone. Min / + Comparison of the effects of preventing and treating adenomas in mice.

[0702] (Experimental Methods)

[0703] The administration regimen for the WT1 peptide vaccine was shown in Figure 8 100 μg WT1 126-134 Killer peptide and 45 μg WT1 35-52 A mixture of accessory peptides, and 100 μg of WT1 alone. 126-134 Killer peptide, 45 μg WT1 alone 35-52 The helper peptide and PBS were emulsified with IFAmontanide ISA51 adjuvant. A mixture of killer peptide and helper peptide, killer peptide alone, helper peptide alone, or PBS-only control vaccine was administered intradermally to APCs up to 5 weeks of age. Min / + The flank of the mouse. Immunization began at 5 weeks of age and was administered 8 times at a frequency of once a week. The immunized mice were euthanized 10 days after the last immunization for further analysis.

[0704] (Example 8: WT1 expression in other benign tumors)

[0705] This example shows that WT1 is also expressed in non-hereditary colorectal adenomas.

[0706] (Experimental Methods)

[0707] Adenoma tissue was excised from three patients with non-hereditary colorectal adenomas and stained with WT1 protein and cell nuclei using the same method as in Example 1.

[0708] (result)

[0709] The results of Example 8 are shown in Figure 9 .exist Figure 9 In the middle, the first image from the left shows a microscopic image of normal glandular ducts, and the three images on the right show microscopic images of adenoma tissue from non-hereditary colorectal adenomas. Figure 9 In the images, light staining indicates WT1 protein, and dark circles or oval-shaped staining areas indicate the cell nucleus. WT1 protein expression was observed in adenomas but not in normal glandular ducts.

[0710] (Example 9: Giving the compound of formula (3) and WT1) 35-52 (A mixture of accessory peptides in a mouse model of familial adenomatous polyposis)

[0711] This embodiment shows the use of the compound of formula (3) and WT1. 35-52 Mixture of accessory peptides to APC Min / + Administration protocol for experiments on mice.

[0712] (Experimental Methods)

[0713] The administration regimen for the WT1 peptide vaccine was shown in Figure 10 The WT1 peptide vaccine was dissolved in PBS, and the solution containing the WT1 peptide vaccine or PBS alone was emulsified with IFA Montanide ISA51 adjuvant to obtain the WT1 peptide vaccine or control vaccine. The WT1 peptide vaccine or control vaccine was administered intradermally to APCs aged 4 to 5 weeks. Min / + The flank of the mouse. Immunization began at 4-5 weeks of age and was administered 6 times at a weekly frequency. Immunized mice were euthanized 7 days after the last immunization for further analysis.

[0714] (Example 10: By giving the compound of formula (3) and WT1) 35-52 (A mixture of co-peptides for the prevention and treatment of adenomas)

[0715] This embodiment shows the use of the compound of formula (3) and WT1. 35-52 Mixture of accessory peptides to APC Min / + Prevention and treatment of adenoma in mice. The compound of formula (3) and WT1 were administered. 35-52The mixture of accessory peptides did not cause organ damage, as shown in Table 4 below.

[0716] Table 4

[0717]

[0718] (Experimental Methods)

[0719] Mice were obtained according to the administration protocol described in Example 9. Organs were removed and weighed from euthanized mice 7 days after the last immunization. The small intestine was divided into 8-10 sections. Each section was longitudinally cut, washed with PBS, sandwiched between filter paper, and fixed in 4% paraformaldehyde / phosphate buffer at 4°C for 24 hours or longer. The fixed intestinal tissue was stained with 1% methylene blue solution and then observed under a stereomicroscope to count the number of adenomas (polyps). The difference in the number of adenomas (polyps) between the WT1 peptide vaccine-administered group and the control vaccine-administered group was statistically analyzed by Student's t-test.

[0720] (result)

[0721] The results of Example 10 are shown in Figure 11 The horizontal axis of the graph shows the group receiving the WT1 peptide vaccine on the left and the group receiving the control vaccine on the right, while the vertical axis shows the number of adenomas per small intestine. The mean number of adenomas per small intestine in the WT1 peptide vaccine-treated group was approximately 37.2, while in the control vaccine-treated group it was approximately 43.7. Therefore, administration of the WT1 peptide vaccine showed inhibition of adenoma development and also treated the adenomas.

[0722] (Example 11: By giving the compound of formula (3) and WT1) 35-52 The mixture of accessory peptides increases WT1 tetramer. + CD3 + CD8 + T cells)

[0723] This embodiment shows the use of the compound of formula (3) and WT1. 35-52 Mixture of accessory peptides to APC Min / + Increased WT1 tetramer in mice + CD3 + CD8 + T cells.

[0724] (Experimental Methods)

[0725] Mice were obtained according to the administration protocol described in Example 9. Spleen cells were collected from euthanized mice 7 days after the last immunization. After erythrocyte lysis, the cells were treated with H-2D. bWT1 tetramer (MBL, Aichi, Japan) was stained, and further stained with anti-mouse CD3 antibody (17A2, BioLegend, San Diego, CA), anti-mouse CD8 antibody (KT15, MBL, Aichi, Japan), and 7-AAD Viability Staining Solution (Bioscience, San Diego, CA). The stained cells were analyzed by FACSCanto to remove 7-AAD. + Dead cell debris and CD3 measurement + CD8 + H-2D in T cells b WT1 tetramer + CD3 + CD8 + T cell frequency. H-2D levels were observed between the WT1 peptide vaccine-administered group and the control vaccine-administered group. b WT1 tetramer + CD3 + CD8 + The frequency differences of T cells were statistically analyzed using the Student t-test.

[0726] (result)

[0727] The results of Example 11 are shown in Figure 12 The horizontal axis of the graph shows the group receiving the WT1 peptide vaccine on the left and the group receiving the control vaccine on the right, and the vertical axis shows CD3. + CD8 + H-2D in cells b Frequency of WT1 tetramer. CD3 in the WT1 vaccine-administered group. + CD8 + H-2D in cells b WT1 tetramer + CD3 + CD8 + The mean frequency of T cells was approximately 0.13%, compared to approximately 0.04% in the control vaccine-administered group, indicating that administration of the WT1 peptide vaccine increases WT1 tetramer levels. + CD3 + CD8 + T cells.

[0728] (Example 12: Statistical Analysis)

[0729] This example shows the number of adenomas in Example 10 and the WT1 tetramer in Example 11. + CD3 + CD8 + Correlation was observed among T cells.

[0730] (Experimental Methods)

[0731] The number of adenomas per small intestine obtained from Example 10 and the CD3 count obtained from Example 11 + CD8 + H-2D in T cells b WT1 tetramer + CD3 + CD8 + The frequency of T cells was plotted on a scatter plot using Microsoft Excel®. Furthermore, the correlations of these results were statistically analyzed in Excel using the Pearson correlation coefficient.

[0732] (result)

[0733] The results from Example 12 showed a high correlation between the number of adenomas (polyps) and the frequency of WT1 tetramers in the WT1 peptide vaccine-administered group and the control vaccine-administered group, confirming that WT1-specific immunity has a strong effect on tumor suppression.

[0734] (Note)

[0735] Although preferred embodiments of this disclosure have been described above, it should be understood that the scope of this disclosure should be interpreted solely by the claims. It should be understood that all patents, patent applications, and other documents cited herein are incorporated herein by reference in their entirety to the same extent as the contents specifically described herein. This application is accompanied by a priority claim to Japanese Patent Application No. 2018-190461, filed with the Japan Patent Office on October 5, 2018, and it should be understood that all prior applications are incorporated herein by reference.

[0736] Industrial applicability

[0737] This disclosure can be used to prevent, delay, and treat benign tumors (e.g., familial adenomatous polyposis) and to prevent, delay, and treat symptoms arising from adenomas of benign tumors (e.g., familial adenomatous polyposis). The WT1 peptide vaccine of this disclosure is useful because it allows us to avoid surgery, such as endoscopic resection, and it is very safe with no serious side effects.

[0738] Sequence List Free Text

[0739] Full-length amino acid sequence of human WT1 protein (SEQ ID NO: 1)

[0740] SEQ ID NO: 2 people WT1 126 amino acid sequence of peptide

[0741] SEQ ID NO: 3 people WT1 235 amino acid sequence of peptide

[0742] SEQ ID NO: 4 people WT1 35 amino acid sequence of peptide

[0743] SEQ ID NO: 5 people WT1 332 amino acid sequence of peptide

[0744] SEQ ID NO: 6 people WT1 126 The amino acid sequence of P1G peptide

[0745] SEQ ID NO: 7 people WT1 126 The amino acid sequence of P1A peptide

[0746] SEQ ID NO: 8 people WT1 126 The amino acid sequence of the P1V peptide

[0747] SEQ ID NO: 9 people WT1 126 The amino acid sequence of P1L peptide

[0748] SEQ ID NO: 10 people WT1 126 The amino acid sequence of P1I peptide

[0749] SEQ ID NO: 11 people WT1 126 The amino acid sequence of P1M peptide

[0750] SEQ ID NO: 12 people WT1 126 The amino acid sequence of P1W peptide

[0751] SEQ ID NO: 13 people WT1 126 The amino acid sequence of the P1F peptide

[0752] SEQ ID NO: 14 people WT1 126 The amino acid sequence of P1Y peptide

[0753] SEQ ID NO: 15 people WT1 126 The amino acid sequence of the P2V peptide

[0754] SEQ ID NO: 16 people WT1 126 The amino acid sequence of the P2Q peptide

[0755] SEQ ID NO: 17 people WT1 126 The amino acid sequence of the P2A peptide

[0756] SEQ ID NO: 18 people WT1 126 The amino acid sequence of P2L peptide

[0757] SEQ ID NO: 19 people WT1 126 The amino acid sequence of the P2I peptide

[0758] SEQ ID NO: 20 people WT1 126 The amino acid sequence of P3I peptide

[0759] SEQ ID NO: 21 people WT1 126 The amino acid sequence of P3L peptide

[0760] SEQ ID NO: 22 people WT1 126 The amino acid sequence of the P3G peptide

[0761] SEQ ID NO: 23 people WT1 126 The amino acid sequence of the P3A peptide

[0762] SEQ ID NO: 24 people WT1 126 The amino acid sequence of the P3V peptide

[0763] SEQ ID NO: 25 people WT1 126 The amino acid sequence of P3M peptide

[0764] SEQ ID NO: 26 people WT1 126 The amino acid sequence of P3P peptide

[0765] SEQ ID NO: 27 people WT1 126 The amino acid sequence of P3W peptide

[0766] SEQ ID NO: 28 people WT1 126 The amino acid sequence of the P9V peptide

[0767] SEQ ID NO: 29 people WT1 126 The amino acid sequence of the P9A peptide

[0768] SEQ ID NO: 30 people WT1 126 The amino acid sequence of P9I peptide

[0769] SEQ ID NO: 31 people WT1 126 The amino acid sequence of the P9M peptide

[0770] SEQ ID NO: 32 people WT1 126 The amino acid sequence of P1D peptide

[0771] SEQ ID NO: 33 people WT1 126 The amino acid sequence of P1E peptide

[0772] SEQ ID NO: 34 people WT1 126 The amino acid sequence of P1H peptide

[0773] SEQ ID NO: 35 people WT1 126 The amino acid sequence of P1K peptide

[0774] SEQ ID NO: 36 people WT1 126 The amino acid sequence of P1N peptide

[0775] SEQ ID NO: 37 people WT1 126 The amino acid sequence of P1P peptide

[0776] SEQ ID NO: 38 people WT1 126 The amino acid sequence of the P1Q peptide

[0777] SEQ ID NO: 39 people WT1 126 The amino acid sequence of the P1S peptide

[0778] SEQ ID NO: 40 people WT1 126 The amino acid sequence of P1T peptide

[0779] SEQ ID NO: 41 people WT1 126 amino acid sequences of P2I & P9I peptides

[0780] SEQ ID NO: 42 people WT1 126 amino acid sequence of P2I&P9V peptide

[0781] SEQ ID NO: 43 people WT1 126 amino acid sequence of P2L&P9I peptide

[0782] SEQ ID NO: 44 people WT1 126 The amino acid sequence of P2L&P9V peptides

[0783] SEQ ID NO: 45 people WT1 235m amino acid sequence of peptide

[0784] SEQ ID NO: 46 people WT1 126 amino acid sequences of peptide variants

[0785] SEQ ID NO: 47 people WT1 235amino acid sequences of peptide variants

[0786] SEQ ID NO: 48 people WT1 235 amino acid sequences of peptide variants

[0787] SEQ ID NO: 49 people WT1 122 amino acid sequences of peptide variants

[0788] SEQ ID NO: 50 people WT1 35 amino acid sequences of peptide variants

[0789] SEQ ID NO: 51 people WT1 35 amino acid sequences of peptide variants

[0790] SEQ ID NO: 52 people WT1 10 amino acid sequence of peptide

[0791] SEQ ID NO: 53 people WT1 187 amino acid sequence of peptide

[0792] SEQ ID NO: 54 people WT1 126 amino acid sequences of peptide variants

[0793] SEQ ID NO: 55 people WT1 235 amino acid sequences of peptide variants

[0794] SEQ ID NO: 56 people WT1 235 amino acid sequences of peptide variants

[0795] SEQ ID NO: 57 people WT1 235 amino acid sequences of peptide variants

[0796] SEQ ID NO: 58 people WT1 10 amino acid sequences of peptide variants

[0797] SEQ ID NO: 59 people WT1 187 amino acid sequences of peptide variants

[0798] SEQ ID NO: 60 people WT1 187 amino acid sequences of peptide variants

[0799] SEQ ID NO: 61 people WT1 126 amino acid sequences of peptide variants

[0800] SEQ ID NO: 62 people WT1 122amino acid sequences of peptide variants

[0801] SEQ ID NO: 63 people WT1 35 amino acid sequences of peptide variants

[0802] SEQ ID NO: 64 people WT1 35 amino acid sequences of peptide variants

[0803] SEQ ID NO: 65 people WT1 35 amino acid sequences of peptide variants

[0804] SEQ ID NO: 66 people WT1 35 amino acid sequences of peptide variants

[0805] SEQ ID NO: 67 people WT1 35 amino acid sequences of peptide variants

[0806] SEQ ID NO: 68 people WT1 37 amino acid sequences of peptide variants

[0807] SEQ ID NO: 69 people WT1 122 amino acid sequences of peptide variants

[0808] SEQ ID NO: 70 people WT1 122 amino acid sequences of peptide variants

[0809] SEQ ID NO: 71 people WT1 122 amino acid sequences of peptide variants

[0810] SEQ ID NO: 72 people WT1 122 The amino acid sequence of a peptide variant.

Claims

1. Use of the WT1 peptide in the preparation of a composition for treating benign tumors, wherein the benign tumor is familial adenomatous polyposis, and wherein the WT1 peptide is a composition. The composition comprises a peptide as shown in the amino acid sequence of RMFPNAPYL or a pharmaceutically acceptable salt thereof, and The amino acid sequence of WAP VLDF APPGASAY GSL represents the peptide or its pharmaceutically acceptable salt.

2. Use of the WT1 peptide in the preparation of a composition for treating benign tumors, wherein the benign tumor is familial adenomatous polyposis, and wherein the WT1 peptide is a composition. The composition comprises a compound of formula (3): Or its pharmaceutically acceptable salt, and The amino acid sequence of WAP VLDF APPGASAY GSL represents the peptide or its pharmaceutically acceptable salt. In equation (3), CC means that C residues are linked together by disulfide bonds.

3. Use of the nucleic acid molecule encoding the WT1 peptide of claim 1 or 2 in the preparation of a composition for treating benign tumors, wherein the benign tumor is familial adenomatous polyposis.

4. The use according to claim 3, wherein the nucleic acid molecule comprises RNA and / or DNA.

5. The use according to any one of claims 1-4, wherein the composition further comprises an adjuvant.

6. The use according to any one of claims 1-4, wherein the benign tumor expresses WT1.

7. The use according to any one of claims 1-4, wherein the composition is administered once a week.

8. The use according to any one of claims 1-4, wherein the composition is used in a method of inducing WT1-specific CTLs and / or WT1-specific helper T cells to treat benign tumors, the method comprising culturing peripheral blood mononuclear cells from a subject requiring treatment for benign tumors in the presence of the WT1 peptide according to claim 1 or 2, or introducing a nucleic acid molecule according to claim 3 or 4 into peripheral blood mononuclear cells from a subject requiring treatment for benign tumors, and inducing WT1-specific CTLs and / or WT1-specific helper T cells from the peripheral blood mononuclear cells, wherein the benign tumor is familial adenomatous polyposis.

9. The use according to any one of claims 1-4, wherein the composition is used in a method of inducing dendritic cells presenting WT1 to treat a benign tumor, the method comprising culturing immature dendritic cells from a subject requiring treatment of a benign tumor in the presence of the WT1 peptide according to claim 1 or 2, or introducing a nucleic acid molecule according to claim 3 or 4 into immature dendritic cells from a subject requiring treatment of a benign tumor, and inducing dendritic cells presenting WT1, wherein the benign tumor is familial adenomatous polyposis.

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