RU2588442C2 - Helper peptide for cancer antigen - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and represents peptide which has amino acid sequence consisting of adjacent amino acids obtained from protein WT1, and induces WT1-specific T-helper cells by binding with class II MHC molecule. Such amino acid sequence is selected from group consisting of amino acid sequence shown in SEQ ID NO: 3; amino acid sequence shown in SEQ ID NO: 4; amino acid sequence shown in SEQ ID NO: 5. Invention also relates to expression vector containing polynucleotide coding peptide, peptide antibodies, pharmaceutical composition containing peptide, method of treating cancer by administering peptide to individual.

EFFECT: invention enables to obtain effective agent for induction of WT1-specific T-helper cells by binding with class II MHC molecule and use thereof for treating cancer.

17 cl, 18 dwg, 9 tbl, 5 ex

Description

2420-180885RU / 015

FIELD OF THE INVENTION

This invention relates to a WT1 helper peptide, a polynucleotide encoding the peptide, WT1-specific helper T cells induced by this peptide, a pharmaceutical composition for treating / preventing cancer containing them, and the like. This application claims priority with respect to Japanese Patent Application No. 2009-105286, filed April 23, 2009, and the description of Japanese Patent Application No. 2009-105286 is incorporated herein by reference.

BACKGROUND

The WT1 gene (Wilms tumor gene 1) is a gene identified as the etiological factor of the Wilms tumor, which is kidney cancer in children (non-patent documents 1 and 2), and is a transcription factor having the structure of “zinc fingers”. Initially, this WT1 gene was considered a cancer suppressor gene. However, a subsequent study showed that the above gene serves more as a cancer gene in tumors of hematopoietic organs and solid cancerous tumors (non-patent documents 3-6).

Since the WT1 gene is highly expressed in many malignant tumors, the product of the WT1 gene, which is an auto-protein without mutations, was tested for the presence or absence of immunogenicity in vivo. As a result, it was shown that the protein obtained from this WT1 gene, expressed highly in tumor cells, is fragmented by intracellular processing, and the resulting peptide forms a complex with the MHC class I molecule, which is exposed (represented) on the cell surface, and that it is cytotoxic T cells (hereinafter referred to as CTL) recognizing such a complex can be induced by vaccination with the WT1 peptide (non-patent documents 7-9). It was also shown that mice immunized with the WT1 peptide or WT1 cDNA reject highly implantable WT1 gene expressing tumor cells (Non-Patent Documents 7 and 10), but normal tissues endogenously expressing the WT1 gene are not damaged by CTL induced. Prior to this, it was assumed with great certainty that WT1-specific CTLs can be induced not only in mice, but also in humans, and that such CTLs have cytotoxic activity against tumor cells expressing a high degree of WT1 gene, but do not have cytotoxic activity against normal cells endogenously expressing this WT1 gene (non-patent documents 7 and 10-14).

On the other hand, it has been reported that the presence of helper T cells specific for a cancer antigen is important for the effective induction of CTL (Non-Patent Document 15). These helper T cells (CD-4 positive T cells) are induced, proliferated and activated by recognition of the complex of the MHC class II molecule with the antigenic peptide on antigen-presenting cells. Activated helper T cells produce cytokines such as IL-2, IL-4, IL-5, IL-6, or interferon (IFN) and stimulate the proliferation, differentiation and maturation of B cells and other subpopulations of T cells. Thus, it is believed that the binding of an antigenic peptide to a MHC class II molecule effectively activates CTL and other cells through the induction of helper T cells and enhances immune function (non-patent document 16). Prior to this, only the binding of the antigenic peptide to HLA-DRB1 * 0401 and HLA-DRB1 * 0405 of the MHC class II molecule with respect to WT1 (Non-Patent Document 17 and Patent Document 1) was reported, and antigenic peptides for other subtypes had to be found.

DOCUMENTS OF KNOWN TECHNOLOGY

Non-Patent Documents

Patent Document 1: International Publication No. WO 2005/045027

Non-Patent Documents:

Non-Patent Document 1: Daniel A. Harber et al., Cell. 1990 Jun 29; 61 (7): 1257-69.

Non-Patent Document 2: Call K.M. et al., Cell. 1990 Feb 9; 60 (3): 509-20.

Non-Patent Document 3: Menke A.L., Int Rev. Cetol. 1998; 181: 151-212. Review

Non-Patent Document 4: Yamagami T. Et al., Blood. 1996 Apr 1: 87 (7): 2878-84.

Non-Patent Document 5: Inoue K. et al., Blood. 1998 Apr 15; 91 (8): 2969-76.

Non-Patent Document 6: Tsuboi A. et al., Leuk Res. 1999 May; 23 (5): 499-505.

Non-Patent Document 7: Oka Y. et al., J. Immunol. 2000 Feb 15; 164 (4): 1873-80.

Non-Patent Document 8: Melief C.J. et al., Immunol. Rev. 1995 Jun; 145: 167-77.

Non-Patent Document 9: Ritz J, J. Clin. Oncol. 1994 Feb; 12 (2): 237-8.

Non-Patent Document 10: Tsuboi A. Et al., J. Clin. Immunol. 2000 May; 20 (3): 195-202.

Non-Patent Document 11: Oka Y. et al., J. Immunogenetics. 2000 Feb; 51 (2): 99-107.

Non-Patent Document 12: Ohminami H. et al., Blood. 2000 Jan 1; 95 (1): 286-93.

Non-Patent Document 13: Gao L. et al., Blood. 2000 Apr 1; 95 (7): 2198-203.

Non-Patent Document 14: Ohminami H. et al., Blood. 2000 Jan 1; 95 (1): 286-93.

Non-Patent Document 15: Cancer. Res. 62: 6438, 2002.

Non-Patent Document 16: J. Immunol. Immunother., 24: 195, 2001.

Non-Patent Document 17: Cancer. Immunol. Immunother. 51: 271, 2002.

DESCRIPTION OF THE INVENTION

PROBLEMS TO BE SOLVED BY THIS INVENTION

Thus, the goal to be achieved by this invention is to provide a peptide inducing WT1-specific helper T cells by binding to various MHC class II molecules, a polynucleotide encoding this peptide, WT1 helper T cells induced by this peptide, and a pharmaceutical composition for treating / preventing cancer containing them.

WAYS TO SOLVE THESE PROBLEMS

The authors of this invention have carried out intensive research to achieve this goal. As a result, they found that a peptide having a portion of the sequence of adjacent amino acids representing the WT1 protein functions as a cancer antigenic helper peptide, in other words, this peptide is exposed on antigen-presenting cells by binding to a MHC class II molecule and induces WT1-specific helper T cells, and showed that this peptide can be used in a pharmaceutical composition for the treatment / prevention of cancer.

Thus, this invention provides:

(1) A peptide that has an amino acid sequence consisting of contiguous amino acids derived from a WT1 protein and induces WT1-specific helper T cells by binding to a MHC class II molecule, where this amino acid sequence is selected from the group consisting of:

(a) the amino acid sequence depicted in SEQ ID NO: 3;

(b) the amino acid sequence depicted in SEQ ID NO: 4;

(c) the amino acid sequence depicted in SEQ ID NO: 5, and

(d) an amino acid sequence in which one or more amino acids are substituted, deleted or added to the amino acid sequences depicted in (a) to (c);

(2) The peptide according to (1), where this amino acid sequence is the amino acid sequence depicted in SEQ ID NO: 3;

(3) The peptide according to (1) or (2), where this MHC class II molecule is selected from the group consisting of DRB1 * 0101, DRB1 * 0405, DRB1 * 0802, DRB1 * 0803, DRB1 * 0901, DRB1 * 1201, DRB1 * 1403, DRB1 * 1501, DRB1 * 1502, DPB1 * 0201, DPB1 * 0202, DPB1 * 0402, DPB1 * 0501, DPB1 * 0901, DQB1 * 0301, DQB1 * 0302, DQB1 * 0401, DQB1 * 0501, DQB1 * 0601 , DQB1 * 0602 and DRB5 * 0102;

(4) The peptide according to (1) or (2), wherein the MHC class II molecule is selected from the group consisting of DRB1 * 0101, DRB1 * 0405, DRB1 * 1502, DPB1 * 0201, DPB1 * 0202 and DQB1 * 0601;

(5) A polynucleotide encoding a peptide according to any one of (1) to (4);

(6) An expression vector containing the polynucleotide according to (5);

(7) An anti-peptide antibody according to any one of (1) to (4) or a polynucleotide according to (5);

(8) A pharmaceutical composition for treating or preventing cancer, comprising a peptide according to any one of (1) to (4), a polynucleotide according to (5) or a vector according to (6);

(9) A method for treating or preventing cancer, which comprises administering an effective amount of a peptide according to any one of (1) to (4), a polynucleotide according to (5), or a vector according to (6) to a subject having an MHC class II molecule according to (3) or ( four);

(10) The use of a peptide according to any one of (1) to (4), a polynucleotide according to (5) or a vector according to (6) for the treatment or prevention of cancer;

(11) Antigen-presenting cells that expose (represent) the peptide according to any one of (1) to (4) through a MHC class II molecule according to (3) or (4);

(12) A method for inducing antigen-presenting cells, which comprises culturing immature antigen-presenting cells in the presence of a peptide according to any one of (1) to (4) and inducing antigen-presenting cells that expose this peptide via an MHC class II molecule according to (3) or ( 4), from immature antigen-presenting cells;

(13) WT1-specific helper T cells that are induced by the peptide according to any one of (1) to (4);

(14) A method for inducing WT1-specific helper T cells, which comprises culturing peripheral blood mononuclear cells in the presence of a peptide according to any one of (1) to (4) and inducing WT1-specific helper T cells from these peripheral blood mononuclear cells;

(15) A kit for inducing WT1-specific helper T cells, comprising, as an essential ingredient, the peptide according to any one of (1) to (4);

(16) A kit for preventing or treating cancer, comprising, as an essential ingredient, the peptide according to any one of (1) to (4), the polynucleotide according to (5) or the vector according to (6);

(17) A method for determining the presence or amount of WT1-specific helper T cells in a subject having a MHC class II molecule according to (3) or (4), said method comprising the steps of:

(a) the reaction of the peptide according to any one of (1) to (4) with a sample obtained from this subject; and then

(b) determining the presence or amount of a cytokine contained in this sample.

EFFECTS OF THIS INVENTION

According to this invention, WT1 helper peptides can be prepared that bind to many types of MHC class II molecules, such as DRB1 * 0101, DRB1 * 0405, DRB1 * 0802, DRB1 * 0803, DRB1 * 0901, DRB1 * 1201, DRB1 * 1403, DRB1 * 1501, DRB1 * 1502, DPB1 * 0201, DPB1 * 0202, DPB1 * 0402, DPB1 * 0501, DPB1 * 0901, DQB1 * 0301, DQB1 * 0302, DQB1 * 0401, DQB1 * 0501, DQB1 * 0601, DQB1 * 0602 and DRB5 * 0102, a pharmaceutical composition for treating / preventing cancer, including them and the like. Thus, it becomes possible to induce WT1-specific helper T cells in vivo and in vitro in various subjects (in particular, most Japanese have the above molecules). Since WT1-specific helper T cells are induced by the present invention, it is also possible to activate T cells and B cells in cancer expressing WT1 to a high degree.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results obtained by measuring cell proliferation after stimulation of each peptide-specific T cell line, which was obtained by pulsed treatment with each of the three peptides (mWT1 ₃₅ , mWT1 ₈₆ and mWT1 ₂₉₄ ), with each peptide. In this figure, the “-“ symbol indicates the absence of peptide stimulation.

Figure 2 shows the results obtained by measuring cell proliferation after stimulation of each peptide-specific T cell line, which was obtained by pulsed processing with three peptides (mWT1 ₃₅ , mWT1 ₈₆ and mWT1 ₂₉₄ ), with each corresponding peptide in the presence of anti-MHC class I or II antibodies. In this figure, the “-“ symbol indicates the absence of peptide stimulation. The “imp / min” symbol in the ordinate indicates the number of pulses per minute.

Figure 3 shows the results obtained by measuring cell proliferation of each WT1-peptide-specific T cell line in response to C1498 cells, C1498 cells treated in a pulsed fashion with three peptides (mWT1 ₃₅ , mWT1 ₈₆ and mWT1 ₂₉₄ ), as well as C1498 cells having enhanced expression of the WT1 protein. The “imp / min” symbol in the ordinate indicates the number of pulses per minute.

Figure 4 shows the results obtained by measuring IFN-γ-producing ability in each peptide-specific T cell line obtained by pulsed treatment with three peptides (mWT1 ₃₅ , mWT1 ₈₆ and mWT1 ₂₉₄ ).

Figure 5 shows the results obtained by measuring the CTL cytotoxic activity of three peptides (mWT1 ₃₅ , mWT1 ₈₆ and mWT1 ₂₉₄ ). ● Shows the results of experiments using RMAS cells, pulsedly treated with the WT1 ₁₂₆ peptide (a restricted MHC class I peptide). ○ shows the results of experiments conducted using RMAS control cells.

6 shows a schematic representation of a time series during tumor implantation and immunization in an experiment with tumor implantation. Immunization with the mWT1 ₃₅ helper peptide was performed on the 7th, 14th and 21st days after subcutaneous implantation of WT1-expressing leukemia cells in mice and the autopsy was performed on the 29th day. The downward-pointing white arrows indicate the time points at which the control (PBS) was injected intradermally (IFA / 30 μl). The downward-pointing black arrows indicate the time points at which the mWT1 ₃₅ helper peptide (50 μM / IFA / 30 μl) was intradermally injected.

7 shows the size of tumors in mice immunized with the mWT1 ₃₅ helper peptide and the proportion of disease-free mouse populations. In mice immunized with the mWT1 helper peptide ₃₅ , 4 out of 10 mice had no disease. On the other hand, in mice immunized with control, in 9 mice there were no mice without disease.

Fig. 8 shows disease-free survival rate in mice immunized with mWT1 ₃₅ helper peptide.

Figure 9 shows the cytotoxic activity of CTL in mice immunized with the mWT1 ₃₅ helper peptide. ● indicates the results of experiments conducted using RMAS cells pulsed with peptide mWT1 ₁₂₆ (peptide-MHC I). ○ shows the results of experiments conducted using RMAS control cells. The number in parentheses indicates the size of the tumor (mm).

Figure 10 shows the cytotoxic activity of mWT1-specific CTLs in control mice. ● shows the results of experiments conducted using RMAS cells treated in a pulsed mode with the mWT1 ₁₂₆ peptide (MHC I peptide) ○ shows the results of experiments conducted using RMAS control cells. The number in parentheses indicates the size of the tumor (mm).

11 shows cytotoxic activity mWT1 ₁₂₆ specific CTL (left) and ₁₂₆ share WT1 tetramer-positive T cells (right) when administered ₃₅ WT1 peptide.

12 shows the results obtained by measuring cell proliferation by stimulation with the WT1 ₃₅ peptide in peripheral blood mononuclear cells of each healthy subject having MHC class I molecules.

13 shows the results obtained by measuring cell proliferation in the treatment of the Responder [PBMC derived from DRB1 * 0101 / 0405-, DPB1 * 0201 / 0402- and DQB1 * 0401/0501-positive healthy subject (healthy subject A)] Stimulator [PBMC derived from DRB1 * 0405 / 0901-, DPB1 * 0201 / 0501- and DQB1 * 0303/0401-positive subject (healthy subject B)]. The ordinate indicates the amount of ³ H-thymidine incorporated (cpm). The abscissa shows the types of various added antibodies (without antibody, anti-HLA-DR antibodies, anti-HLA-DP antibodies and anti-HLA-DQ antibodies).

Fig. 14 shows the results obtained by measuring cell proliferation in the treatment of the Responder [PBMC derived from DRB1 * 0101 / 0405-, DPB1 * 0201 / 0402- and DQB1 * 0401/0501-positive healthy subject (healthy subject A)] Stimulator [PBMC derived from DRB1 * 0405 / 0803-, DPB1 * 0202 / 0501- and DQB1 * 0401/0601-positive subject (healthy subject G)]. The ordinate indicates the amount of ³ H-thymidine incorporated (cpm). The abscissa shows the types of various added antibodies (without antibody, anti-HLA-DR antibodies, anti-HLA-DP antibodies and anti-HLA-DQ antibodies).

Fig. 15 shows the results obtained by measuring cell proliferation in the treatment of the Responder [PBMC derived from DRB1 * 0101 / 0405-, DPB1 * 0201 / 0402- and DQB1 * 0401/0501-positive healthy subject (healthy subject A)] Stimulator [PBMC derived from DRB1 * 0101 / 0803-, DPB1 * 0501 / - and DQB1 * 0501/0601-positive subject (healthy subject H)]. The ordinate indicates the amount of ³ H-thymidine incorporated (cpm). The abscissa shows the types of various added antibodies (without antibody, anti-HLA-DR antibodies, anti-HLA-DP antibodies and anti-HLA-DQ antibodies).

Fig.16 shows the results obtained by measuring IFN-γ-producing ability in the treatment of the Responder [PBMC obtained from DRB1 * 0405 / 0803-, DPB1 * 0202 / 0501- and DQB1 * 0401/0601-positive healthy subject (healthy subject G) ] Stimulator [l-cells containing the included gene DQB1 * 0601]. The ordinate shows the proportion of the amount of IFN-γ in T cells. The abscissa indicates the presence or absence of an IFN-γ (+ or -) pulse with the WT1 ₃₅ peptide _.

17 shows the results obtained by measuring cell proliferation in the treatment of the Responder [PBMC obtained from DRB1 * 1502 / 1502-, DPB1 * 0201 / 0901- and DQB1 * 0601/0601-positive healthy subject (healthy subject D)] Stimulator [PBMC obtained from the same healthy subject as in the case of the responder). The ordinate indicates the amount of ³ H-thymidine incorporated (cpm). The abscissa shows the types of various added antibodies (without antibody, anti-HLA-DR antibodies, anti-HLA-DP antibodies and anti-HLA-DQ antibodies).

Fig. 18 shows the results obtained by measuring IFN-γ-producing ability in the treatment of the Responder [PBMC obtained from DRB1 * 0101 / 1501-, DPB1 * 0201 / 0402- and DQB1 * 0501/0602-positive healthy subject (healthy subject I) ] Stimulant [PBMC obtained from the same healthy subject as in the case of the responder). The ordinate shows the proportion of the amount of IFN-γ in T cells. The abscissa indicates the presence or absence of an IFN-γ (+ or -) pulse with the WT1 ₃₅ peptide _.

MODE FOR CARRYING OUT THE INVENTION

In one aspect, the invention relates to a peptide having an amino acid sequence consisting of amino acids derived from mouse or human WT1 protein. The WT1 gene is highly expressed, for example, in tumors of hematopoietic organs such as leukemia; myelodysplastic syndrome, multiple myeloma and malignant lymphoma; solid cancers such as stomach cancer, intestinal cancer, lung cancer, breast cancer, germ cells, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer. Thus, the peptide of the invention is present in cancer cells expressing a large amount of the WT1 gene.

The peptide of this invention is a peptide that has an amino acid sequence consisting of adjacent amino acids derived from the human WT1 protein depicted in SEQ ID NO: 2, retains the ability to bind to MHC class II molecules, as shown below, and has the ability to induce WT1-specific helper T cells. There is no particular limitation on this amino acid sequence and the length of the peptide of the invention, as long as the peptide has the above features. However, a peptide that is too long is sensitive to the action of proteases, and a peptide that is too short cannot bind to a peptide adapted to deepen the groove. The length of the peptide of the invention is preferably 10-25 amino acids, more preferably 15-21 amino acids, even more preferably 16-20 amino acids, for example 17 amino acids, 18 amino acids or 19 amino acids. Specific examples of the peptide of the invention are peptides having the amino acid sequence shown in SEQ ID NO: 3; the amino acid sequence depicted in SEQ ID NO: 4 and the amino acid sequence depicted in SEQ ID NO: 5.

The peptide of the invention also includes variants of the aforementioned peptides. These options may contain, for example, a peptide selected from the group consisting of peptides having an amino acid sequence that has a substitution, deletion or addition of several amino acids, for example, 1-9, preferably 1-5, 1-4, 1-3, more preferably 1-2 amino acids, even more preferably one amino acid in one of the above amino acid sequences. Replacement of amino acids in peptides can be carried out in any positions and any types of amino acids. Conservative amino acid substitutions are preferred. For example, the Glu residue can be replaced by the Asp residue, the Phe residue by the Tyr residue, the Leu residue by the Ile residue, the Ala residue by the Ser residue, the His residue by the Arg residue. The addition or deletion of amino acids can preferably be carried out at the N-terminus and the C-terminus in the peptides, but can be carried out in an internal sequence. A preferred specific example of a peptide of the invention has the sequence of SEQ ID NO: 3. In this regard, all of the above peptides must retain the ability to bind to the MHC class II molecule and be able to induce WT1-specific helper T cells.

In this regard, the MHC class II molecule to which the peptide of this invention binds can belong to any subclass of HLA-DR, HLA-DQ and HLA-DP. Preferably, the MHC class II molecule is a molecule selected from the group consisting of DRB1 * 0101, DRB1 * 0405, DRB1 * 0802, DRB1 * 0803, DRB1 * 0901, DRB1 * 1201, DRB1 * 1403, DRB1 * 1501, DRB1 * 1502 , DPB1 * 0201, DPB1 * 0202, DPB1 * 0402, DPB1 * 0501, DPB1 * 0901, DQB1 * 0301, DQB1 * 0302, DQB1 * 0401, DQB1 * 0501, DQB1 * 0601, DQB1 * 0602 and DRB5 * 0102. More preferably, the MHC class II molecule is DRB1 * 0101, DRB1 * 0405, DRB1 * 1403, DRB1 * 1502, DPB1 * 0201, DPB1 * 0202, DPB1 * 0901, DQB1 * 0301, DQB1 * 0601 or DRB5 * 0102 and most preferably DRB1 * 0101, DRB1 * 0405, DRB1 * 1502, DPB1 * 0201, DPB1 * 0202 or DQB1 * 0601. In this description, a peptide that retains the ability to induce WT1-specific helper T cells is called he Pernod peptide WT1. In the examples described below, a peptide having an amino acid sequence as depicted in SEQ ID NO: 3, also referred to as WT1 peptide _{35 35} helper peptide or WT1 peptide, WT1 _35.

The peptide of this invention may also be a peptide having an amino acid sequence consisting of adjacent amino acids derived from the mouse WT1 protein depicted in SEQ ID NO: 1, and the above amino acid sequence may be a peptide (SEQ ID NO: 6), in which the amino acid residue at position 9 in the amino acid sequence depicted in SEQ ID NO: 4, replaced by leucine; or a peptide (SEQ ID NO: 7), in which the amino acid residue at position 11 in the amino acid sequence depicted in SEQ ID NO: 5 is replaced by serine. In addition, the peptide of the present invention may contain a peptide selected from the group consisting of peptides having an amino acid sequence that has the substitution, deletion or addition of several amino acids, for example, 1-9, preferably 1-5, 1-4, 1-3 , more preferably 1-2 amino acids, even more preferably one amino acid in the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7. In the examples described below, a peptide having an amino acid sequence as depicted in SEQ ID NO: 6, also referred to as peptide WT1 ₈₆ or the helper peptide of WT1 _86, and a peptide having the amino acid sequence depicted in SEQ ID NO: 7 peptide of WT1 _294, helper peptide WT1 ₂₉₄ .

The peptide of the invention may be derived from a WT1 protein and may consist of or contain the above sequence of contiguous amino acids. Thus, the peptide of the present invention can be, for example, a peptide consisting of the aforementioned amino acid sequence, or a WT1 protein containing the aforementioned amino acid sequence or part thereof. The peptide of the invention may also be a peptide obtained by modifying the above amino acid sequence. Amino acid residues in the above amino acid sequence can be modified in a known manner. Such a modification may be, for example, esterification, alkylation, halogenation, phosphorylation, sulfonation, amidation, etc. on a functional group in the side chain of the amino acid residue that is part of the peptide. You can also bind various substances with the N-terminus and / or C-terminus of a peptide containing the above amino acid sequence. For example, an amino acid, a peptide, an analog thereof, and the like may be associated with this peptide. If these substances are associated with the peptide of the present invention, they can be processed, for example, with an in vivo enzyme and the like, or using a method such as intracellular processing, so that ultimately they generate a peptide, consisting of the above amino acid sequence, which is presented on the cell surface as a complex with a MHC class II molecule, which is thereby capable of generating the induction effect of helper T cells. These substances can be substances that regulate the solubility of the peptide of this invention, substances that improve the stability of this peptide, for example, resistance to proteases, substances that enable specific delivery of the peptide of the invention, for example, to a specific tissue or specific organ, or substances that have an enhancing effect on the efficiency of absorption of antigen-presenting cells or other action. These substances can also be substances that increase the ability of CTL induction, for example, helper peptides, other than the peptide of the present invention.

The modification of the peptide of the present invention may be a modification of an amino group at the N-terminal amino acid or a carboxyl group at the C-terminal amino acid of this peptide. Modifying groups of the amino acid group on the N-terminal amino acid include, for example, one to three alkyl groups having 1-6 carbon atoms, phenyl groups, cycloalkyl groups and acyl groups. Specific examples of the acyl group include an alkanoyl group having 1-6 carbon atoms, an alkanoyl group having 1-6 carbon atoms substituted with a phenyl group, a carbonyl group substituted with a cycloalkyl group having 5-7 carbon atoms, an alkylsulfonyl group having 1 -6 carbon atoms, phenylsulfonyl group, alkoxycarbonyl group having 2-6 carbon atoms, alkoxycarbonyl group substituted by phenyl group, carbonyl group substituted by cycloalkoxy group having 5-7 carbon atoms, f enoxycarbonyl group and the like. Peptides having a modification of the carboxyl group at the C-terminal amino acid include, for example, esterified and amidated peptides. Specific examples of this ester include an alkyl ester having 1-6 carbon atoms, an alkyl ester having 0-6 carbon atoms substituted with a phenyl group, a cycloalkyl ester having 5-7 carbon atoms and the like, and specific examples of the amide include an amide, an amide substituted with one or two alkyl groups having 1-6 carbon atoms, an amide substituted with one or two alkyl groups having 0-6 carbon atoms substituted with a phenyl group, an amide forming 5- , 7 membered azocycloalkane, including ayuschy a nitrogen atom of an amide group, etc.

Modification of the peptide of the present invention can also be carried out by linking amino acid residues to each other via a bond other than a peptide bond, such as a carbon-carbon bond and a carbon-sulfur bond. In addition, the peptide of the invention may contain one or more D-amino acids.

The aforementioned peptides, variant peptides and modified peptides according to this invention are only illustrative, and those skilled in the art can easily allow, obtain, evaluate and use other variations of the above peptides.

The peptide of the invention may be synthesized using a method routinely used in the art or a modified method thereof. Such a synthesis method is described, for example, in Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Basis and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1985; Development of Medicines (continuation), Vol. 14, Peptide Synthesis, Hirokawa Shoten Co., 1991, etc. The peptide of the present invention can be obtained using a genetic engineering method based on the information of the nucleotide sequence encoding the peptide of the present invention. This method of genetic engineering is well known to those skilled in the art. Such a method can be carried out in accordance with the method described in the literature [Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983); DNA Cloning, DM. Glover IRL PRESS (1985)], as described above, or by the method described below and other methods.

It can be determined whether the peptide of the invention or its candidate peptide binds to the aforementioned MHC class II molecule and induces helper T cells in a known manner, for example, such as the method described in Cancer Immunol. Immunother. 51: 271 (2002), or the method described in the examples of this description, and in other ways.

Since the peptide of the present invention activates helper T cells (CD4 positive T cells), this peptide induces and supports CTL differentiation and exhibits the effect of activating effector cells such as macrophages. Thus, the peptide of the invention can be used to effectively treat or prevent cancer.

In another aspect, the invention provides a polynucleotide encoding the aforementioned helper peptide WT1 (hereinafter referred to as the WT1 polynucleotide). The polynucleotide of the invention may be DNA or RNA. The base sequence of the polynucleotide of the present invention can be determined based on the amino acid sequence of the above WT1 helper peptide. This polynucleotide can be obtained, for example, by the method of synthesis of DNA or RNA, by PCR method, etc.

A polynucleotide of the invention includes a polynucleotide that hybridizes to a complementary sequence of a polynucleotide encoding a peptide of the invention, under stringent conditions, and encodes a peptide having activity comparable to that of the peptide of the invention. With regard to the term “hybridize under stringent conditions,” the hybridization used here can be carried out in accordance with the generally accepted method described, for example, in Molecular Cloning, 2 ^nd edition, Sambrook, J., Frisch, EF, Maniatis, T., Cold Spring Harbor Laboratory Press (1989) and the like. The term “stringent conditions” includes, for example, conditions under which a hybrid is formed in a solution containing 6 × SSC (10 × SSC is a solution containing 1.5 M NaCl and 0.15 M trisodium citrate) and 50% formamide, with 45 ° C and then washed with 2 × SSC at 50 ° C (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1.-6.3.6) and the like.

In yet another aspect, the invention relates to an expression vector comprising the aforementioned polynucleotide (hereinafter also referred to as the WT1 expression vector). Type of expression vectors, other sequences contained, along with the above polynucleotide sequence, and the like. may be selected appropriately depending on the type of hosts into which these expression vectors are introduced, the purpose of this administration, and the like. Examples of this expression vector include plasmids, phage vectors, viral vectors, and the like. In the case where the host are Escherichia coli cells, examples of this vector include plasmid vectors such as pUC118, pUC119, pBR322 and pCR3, as well as phage vectors such as λZAPII and λgt11. In the case where the host is yeast cells, examples of this vector include pYES2, pYEUra3 and the like. In the case where the host is insect cells, an example of this vector is pAcSGHisNT-A and the like. In the case where the host is animal cells, examples of this vector include plasmid vectors such as pKCR, pCDM8, pGL2, pcDNA3.1, pRc / RSV and pRc / CMV, viral vectors such as retroviral vector, adenovirus vector and adeno-associated viral vector. This vector may optionally contain factors such as an expression-induced promoter, a gene encoding a signal sequence, a marker gene for selection, and a terminator. For easy isolation and easy purification, a sequence expressed as a fusion protein with thioredoxin, His tag, GST (glutathione S transferase) and the like can be added to this vector. In this case, you can use a GST-fused protein vector (pGEX4T and others) having a suitable promoter (lac, tac, trc, trp, CMV, early SV40 promoter, etc.), functional in host cells, vector (pcDNA3 .1 / Myc-His, etc.) having a tag sequence such as Myc and His, as well as a vector (pET32a) expressing a thioredoxin fusion protein and His tag, and the like.

When an expression vector of the present invention is administered to a subject to obtain the WT1 helper peptide in vivo, WT1-specific helper T cells induced by this peptide produce various cytokines (e.g., IL-2, IL-4, IL-5, IL-6, or interferon (IFN), etc.) and stimulate the proliferation, differentiation and maturation of B cells and other T cells. Thus, tumor cells that have a MHC class II molecule and express highly WT1 can be damaged specifically using this WT1 expression vector of the present invention.

In another aspect, the invention provides an antibody against the aforementioned WT1 helper peptide or polynucleotide encoding the peptide (hereinafter referred to as the WT1 antibody). The antibody of the invention may be either a polyclonal antibody or a monoclonal antibody. A method for producing such an antibody is already known, and the antibody of the present invention can also be obtained in accordance with such a conventional method (Current protocols in Molecular Biology, Ausubel et al., (Ed.), 1987, John Wiley and Sons (pub.), Section 11.12-11.13, Antibodies; A Laboratory Manual, Lane, HD et al., (Ed.), Cold Spring Harber Laboratory Press (pub.), New York, 1989).

This invention relates to a pharmaceutical composition for treating or preventing cancer, comprising the aforementioned WT1 helper peptide, WT1 polynucleotide, or WT1 expression vector. The WT1 gene is highly expressed, for example, in tumors of hematopoietic organs such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma, as well as solid cancers such as stomach cancer, intestinal cancer, lung cancer, breast cancer, and germ cells , liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer, and therefore this pharmaceutical composition of the present invention can be used to treat or prevent cancer, express iruyuschego WT1 gene. When a pharmaceutical composition of the invention is administered to a subject having a MHC class II molecule, WT1-specific helper T cells induced by the WT1 helper peptide contained in this pharmaceutical composition produce various cytokines (e.g., IL-2, IL-4, IL-5 , IL-6 or interferon (IFN), etc.) and stimulate the proliferation, differentiation and maturation of b cells and other subpopulations of T cells. Thus, tumor cells that have a MHC class I molecule and express highly WT1 can be specifically damaged using the peptide of the invention.

The pharmaceutical composition of the present invention may contain, for example, a carrier, excipient and the like, along with the above WT1 helper peptide, WT1 polynucleotide or WT1 expression vector, as an effective component. The WT1 helper peptide contained in the pharmaceutical composition of the present invention induces WT1-specific helper T cells, and therefore the pharmaceutical composition of the present invention may contain a suitable adjuvant or may be administered with a suitable adjuvant to enhance induction efficiency. Examples of a preferred adjuvant include, but are not limited to, Freund's complete or incomplete adjuvant, aluminum hydroxide, and the like. The pharmaceutical composition of this invention may also contain a known antigenic cancer peptide other than the above WT1 helper peptide, for example, WT1 ₁₂₆ peptide inducing WT1-specific CTLs as an effective component (Oka et al., “Cancer immunotherapy targeting Wilms' tumor gene WT1 product ”, Journal of Immunology, 164: 1873-1880, 2000; and Oka et al.,“ Human cytotoxic T-lymphocyte responses specific for peptides of the wild-type Wilms' tumor gene (WT1) product ”, Immunogenetics, 51 : 99-107, 2000).

In addition, the pharmaceutical composition of the present invention can be administered in combination with a known cancer antigenic peptide. For example, a known cancer antigenic peptide, for example, the WT1 ₁₂₆ peptide, may be administered before or after administration of the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention has a feature that activates B cells or other T cells by inducing WT1-specific helper T cells, and therefore it is possible to further enhance the activity of CTLs induced by the introduction of a known cancer antigenic peptide and significantly increase therapeutic effects.

The route of administration of the pharmaceutical composition of this invention may be appropriately selected depending on conditions such as the type of disease, the condition of the subjects, and target sites. Examples of the method of administration include, but are not limited to, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, transnasal administration, oral administration and the like. The method of administration may also be therapy using lymphocytes or therapy using DC (dendritic cells). The amount of peptide contained in the pharmaceutical composition of the present invention, the form and frequency of administration of this pharmaceutical composition, etc. can be appropriately selected depending on conditions such as the type of disease, the condition of the subjects, and target sites. Typically, the amount of peptide administered per dose is 0.0001-1000 mg, and preferably 0.001-10000 mg.

In another aspect, the invention relates to a method for treating or preventing cancer, which comprises administering an effective amount of the above pharmaceutical composition to a subject having the aforementioned MHC class II molecule. Any carcinoma as long as it expresses the WT1 gene can be treated with carcinomas, and include, for example, tumors of hematopoietic organs such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma, as well as solid types of cancers such as stomach cancer, cancer bowel cancer, lung cancer, breast cancer, germ cells, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer.

In another aspect, the invention relates to the use of the above WT1 helper peptide, WT1 polynucleotide, or WT1 expression vector for the treatment or prevention of cancer.

In yet another aspect, the invention relates to the use of a WT1 helper peptide for the manufacture of a pharmaceutical composition for the treatment or prevention of cancer.

In yet another aspect, the invention relates to the use of a WT1 polynucleotide or WT1 expression vector for the preparation of a pharmaceutical composition comprising the above WT1 polynucleotide or WT1 expression vector.

In another aspect, the invention relates to cells comprising the aforementioned WT1 helper peptide, WT1 polynucleotide, or WT1 expression vector. These cells of the present invention can be obtained, for example, by transformation of host cells, such as Escherichia coli cells, yeast cells, insect cells and animal cells using the above expression vector. Transformation of host cells with an expression vector can be carried out using various methods, selected appropriately. The peptide of this invention can be obtained by culturing the transformed cells and extracting and purifying the WT1 helper peptide produced by them.

In yet another aspect, the invention relates to antigen-presenting cells (e.g., dendritic cells, B lymphocytes, macrophages, etc.) that exhibit (present) the above-described helper peptide WT1 via a MHC class II molecule. Antigen-presenting cells of the present invention are induced by the above WT1 helper peptide. WT1-specific helper T cells are effectively induced using the antigen-presenting cells of the present invention.

In yet another aspect, the present invention relates to a method for inducing antigen presenting cells that expose the WT1 helper peptide via an MHC class II molecule, the method comprising culturing immature antigen presenting cells in the presence of a WT1 helper peptide and inducing antigen presenting cells that exhibit ( represent) the above-described helper peptide WT1 through an MHC class II molecule from these immature antigen-presenting cells. As used herein, immature antigen presenting cells are called cells that can become antigen presenting cells, such as, for example, dendritic cells, B lymphocytes and macrophages, after maturation. The subjects from which immature antigen-presenting cells are derived can be any subjects as long as they have a MHC class II molecule. Since immature antigen-presenting cells are contained, for example, in peripheral blood mononuclear cells and the like, such cells can be cultured in the presence of the WT1 helper peptide described above.

In another aspect, the invention relates to a method for treating or preventing cancer, comprising administering antigen-presenting cells that expose (represent) the WT1 helper peptide via the above-described MHC class II molecule, to a subject having the same molecule as the above-described MHC class II molecule. The route of administration of antigen-presenting cells may be appropriately selected depending on conditions such as the type of disease, the condition of the subjects, and target sites. Examples of this method include, but are not limited to, intravenous administration, intradermal administration, subcutaneous administration, intramuscular administration, transnasal administration, oral administration and the like.

In yet another aspect, the invention relates to a method for preventing or treating cancer by inducing antigen-presenting cells that expose (present) the above-described WT1 helper peptide via an MHC class II molecule, the method comprising the steps of:

(a) reaction of the sample with a nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 2) of a WT1 protein, or a nucleic acid having a partial sequence thereof, or the above WT1 helper peptide;

(b) obtaining antigen-presenting cells that expose (represent) the WT1 helper peptide contained in this sample by means of the aforementioned MHC class II molecule; and

(c) administering antigen presenting cells to a subject having the same molecule as the aforementioned MHC class II molecule.

Samples in the above method can be any sample, as long as they have the ability to contain lymphocytes or dendritic cells and include, for example, samples obtained from a subject, such as blood, cell culture solutions, and the like. The reaction in the above method can be carried out using a conventional method and preferably using electroporation. Obtaining antigen-presenting cells can be carried out using a method known to those skilled in the art. The culture conditions of the cells in the sample at each stage can be determined by appropriately qualified specialists in this field. The method of administration of these antigen-presenting cells may be the method described above.

In a further aspect, the invention relates to WT1-specific helper T cells induced by the above WT1 helper peptide. Helper T cells of the present invention are induced, proliferate and activated upon recognition of the complex of the WT1 helper peptide with the MHC class II molecule. These activated WT1-specific helper T cells produce cytokines such as IL-2, IL-4, IL-5, IL-6, or interferon (IFN) and stimulate the proliferation, differentiation and maturation of B cells and other T-subpopulations cells. Thus, tumor cells that have a MHC class I molecule and express highly WT1 can be specifically damaged using the helper T cells of the present invention.

In another aspect, the present invention relates to a method for inducing WT1-specific helper T cells, comprising culturing peripheral blood mononuclear cells in the presence of a WT1 helper peptide and inducing WT1-specific helper T cells from peripheral blood mononuclear cells. The subjects from which peripheral blood mononuclear cells are obtained can be any subjects as long as they have the above class MHC molecule II. By culturing peripheral blood mononuclear cells in the presence of a WT1 helper peptide, WT1-specific helper T cells are induced from progenitor helper T cells in peripheral blood mononuclear cells. Tumors of hematopoietic organs and solid tumors in a subject can be treated or prevented by administering the WT1-specific helper T cells obtained according to this invention to a subject having the above class MHC class II molecule. In this regard, peripheral blood mononuclear cells in the present invention include immature antigen-presenting cells (e.g., dendritic cell precursor cells, B lymphocytes, macrophages, etc.). Since immature antigen-presenting cells are contained, for example, in peripheral blood mononuclear cells and the like, such cells can be cultured in the presence of the WT1 helper peptide described above.

In yet another aspect, the invention provides a kit for inducing WT1-specific helper T cells, comprising the above-described WT1 helper peptide as an essential ingredient. Preferably, this kit is used in the above method of inducing WT1-specific helper T cells. This kit of the present invention may contain, for example, agents for producing peripheral blood mononuclear cells, an adjuvant, a reaction vessel, and other components, along with the WT1 helper peptide described above. Usually, this kit is accompanied by instructions. WT1-specific helper T cells can be effectively induced using the kit of this invention.

In another aspect, the invention relates to a method for treating or preventing cancer, comprising administering WT1-specific helper T cells to a subject having a MHC class II molecule. The method of administering WT1-specific helper T cells may be appropriately selected depending on conditions such as the type of disease, the condition of the subjects, and target sites. Examples of the route of administration include, but are not limited to, intravenous administration, intradermal administration, subcutaneous administration, intramuscular administration, transnasal administration, oral administration and the like.

In addition, the present invention relates to a kit for the prevention or treatment of cancer, containing the above-described helper peptide WT1, polynucleotide WT1 or expressing vector WT1 as an essential ingredient. This kit is a kit characterized by the induction of antigen-presenting cells that expose (represent) the WT1 helper peptide via the above-described MHC class II molecule. The kit of the invention may also contain, for example, sample preparation agents, a reaction vessel, and other components, along with the main ingredient described above. Usually, this kit is accompanied by instructions. Antigen-presenting cells that expose the WT1 helper peptide via the above-described MHC class II molecule can be efficiently obtained using this kit of the present invention and used to treat or prevent cancer by administering them.

In another aspect, this invention relates to a method for determining the presence or amount of WT1-specific helper T cells in a subject having the above class MHC class II molecule, comprising the steps of:

(a) reaction of a complex of the aforementioned helper peptide WT1 with the aforementioned MHC class II molecule with a sample obtained from this subject; and then

(b) determining the presence or quantity of helper T cells recognizing the complex contained in this sample.

Samples obtained from subjects can be any samples as long as they have the ability to contain lymphocytes, and they include, for example, body fluids such as blood, lymphatic fluid, tissues, and the like. The complex of WT1-specific helper T cells with the MHC class II molecule can be, for example, in the form of a tetramer, a pentamer and the like, for example, using a method known to those skilled in the art, such as a method using biotin-streptavidin . The presence or quantity of helper T cells recognizing such a complex can be determined by a method known to those skilled in the art. In this aspect of the invention, the above complex may be labeled. As a label, known labels such as a fluorescent label and a radioactive label can be used. By labeling, the presence or quantity of helper T cells can be simply and quickly determined. Using the method of this aspect of the present invention, diagnosis and prognosis and the like become possible. cancer.

Thus, the invention also provides a composition comprising a complex of a WT1 helper peptide with the aforementioned MHC class II molecule for determining the presence or amount of WT1-specific helper T cells in a subject having the aforementioned MHC class II molecule.

In addition, the present invention provides a kit comprising a complex of a WT1 helper peptide with the aforementioned MHC class II molecule for determining the presence or amount of WT1-specific helper T cells in a subject having the aforementioned MHC class II molecule.

In another aspect, the invention relates to a method for determining the presence or quantity of WT1-specific helper T cells in a subject having the above class MHC class II molecule, comprising the steps of:

(a) reacting the aforementioned helper peptide WT1 with a sample obtained from this subject; and

(b) determining the presence or amount of a cytokine contained in this sample.

Samples obtained from subjects can be any samples as long as they have the ability to contain lymphocytes and include, for example, peripheral blood mononuclear cells, blood, body fluids, tissues, and others, and preferably peripheral blood mononuclear cells. The reaction in the aforementioned step (a) can be carried out by reacting the aforementioned helper peptide WT1 in the aforementioned sample obtained from a subject using a conventional method. The culture conditions of the cells in the sample at each stage can be determined by properly qualified specialists in this field. The presence or amount of a cytokine contained in a sample can be measured by a method known to those skilled in the art. This cytokine may be a cytokine capable of being induced by helper T cells, such as interferon-γ and interleukin-10. In this aspect of the invention, the above cytokine may be labeled. As labels, known labels such as a fluorescent label and a radioactive label can be used. Using this presence or amount of the above cytokine as an indicator, it becomes possible to quickly and easily determine the presence or amount of WT1-specific helper T cells.

In a further aspect, the invention relates to a method for producing WT1-specific helper T cells using a complex of a WT1 helper peptide with a MHC class II molecule, comprising the steps of:

(a) reaction of the sample with this complex; and

(b) obtaining helper T cells that are contained in this sample and recognize this complex.

The complex of the WT1 helper peptide with the MHC class II molecule is such as the complex described above. Samples can be any samples, as long as they have the ability to contain lymphocytes and include, for example, samples obtained from a subject, such as blood, tissue culture solutions, etc. Obtaining helper T cells recognizing this complex can be carried out, for example, using a method known to those skilled in the art, such as FACS and MACS. The resulting WT1-specific helper T cells can be cultured and used to treat and prevent various cancers.

Thus, the present invention also relates to WT1-specific helper T cells, which can be obtained by a method for producing WT1-specific helper T cells using a complex of the WT1 helper peptide with the aforementioned MHC class II molecule.

In addition, this invention relates to a kit for producing WT1-specific helper T cells, comprising a complex of the WT1 helper peptide with the aforementioned MHC class II molecule.

In yet another aspect, the invention provides a method for diagnosing cancer, which comprises using the aforementioned WT1-specific helper T cells, the above antigen-presenting cells that expose (represent) the WT1 helper peptide via a MHC class II molecule, or the aforementioned antibody. Preferably, WT1-specific helper T cells are used for the cancer diagnostic method of the present invention. For example, the above helper T cells, antigen-presenting cells or antibody can be incubated with a sample obtained from a subject having a MHC class II molecule, or administered to a subject having a MHC class II molecule, and then, for example, location, region, number can be determined etc. these helper T cells, antigen presenting cells or antibodies for diagnosing cancer. The above helper T cells, antigen presenting cells or antibody may be labeled. By labeling, a method for diagnosing the cancer of the present invention can be effectively carried out.

In yet another aspect, the invention provides a kit for diagnosing cancer comprising the aforementioned WT1-specific helper T cells, antigen-presenting cells that expose (represent) a WT1 helper peptide via the aforementioned MHC class II molecule, or an antibody against a WT1 helper peptide or antibody against a polynucleotide encoding this peptide as an essential ingredient.

The invention will be specifically described and described in detail below by way of examples, but should not be construed as limiting the invention.

Example 1

Selection of candidate WT1 peptides that bind to MHC class II molecules

To search for peptide sequences that bind to MHC class II molecules, the method shown by Rammensee et al. (Rammensee et al, Immunogenetics 41: 178-228, 1995). Specifically, the selection was carried out using the programs described in the right-hand column in the tables together with the law of Rammensee et al. According to this method, WT1 ₃₅ peptides narrowed to the peptide sequences shown in Tables 1 and 2, WT1 ₈₆ peptides to the peptide sequences shown in Tables 3 and 4 and WT1 ₂₉₄ peptides to the peptide sequences shown in Tables 5 and 6. Left side column in tables 1-6 indicates “suitability” as a candidate peptide sequence. The higher the number “O”, the higher the suitability of the law Rammensee et al. The absence of any label indicates poor suitability. The group of amino acids in brackets of the column “candidate peptide sequences that bind to MHC class II molecules” in tables 1-6 shows that one amino acid can be selected from the group of amino acids listed in these brackets. For example, the description of [FLM] means one amino acid selected from the group of amino acids F, L and M. The description [VYI (AL)] also means one amino acid selected from the group of amino acids V, Y and I, or one amino acid from the group of amino acids A and L. “x” indicates that this amino acid can be any amino acid. The column on the right side shows the “program name” of the programs used for the list of candidate peptide sequences.

Then, the WT1 candidate peptides were visually selected from Tables 1-6, the peptides shown in the following Table 7 were identified as preferred candidate peptides for MHC class II molecules and the actual functions of these peptides were analyzed as described below.

Table 7 Identification of peptide candidates for MHC molecules
class II mouse WT1 ₃₅ WAPVLDFAPPGASAYGSL (SEQ ID NO: 3) 18-mer MW 1819.01 WT1 ₈₆ EQCLSAFTLHFSGQFTG (SEQ ID NO: 6) 17-mer MW 1944.01 WT1 ₂₉₄ FRGIQDVRRVSGVAPTLVR (SEQ ID NO: 7) 19-mer MW 2126.48

Obtaining WT1-peptide-specific cell lines and measuring the ability of cell proliferation

First, the above WT1 peptides were emulsified with incomplete Freund's adjuvant (Montanide ISA 51) and mice were inoculated intradermally with each WT1 peptide in an amount corresponding to 100 μg / mouse. Immunization was carried out 3 times at intervals of one week, the spleen was removed after week 1 of the final immunization and spleen cells were obtained. These spleen cells were stimulated 3 times at 10-day intervals using spleen cells from non-immunized mice that were pulsed with the same WT1 peptide that was used to immunize each mouse and irradiated as a stimulant. Then, the 4th stimulation was performed using spleen cells of non-immunized mice, which were pulsed with each peptide (WT1 ₃₅ , WT1 ₈₆ or WT1 ₂₉₄ peptide), as shown in Table 7, and the proliferation reaction was irradiated as the response to each stimulant was measured in a ³ H inclusion experiment. The OVA peptide (ovalbumin), not related to the WT1 peptides, was used as a control. As a result, mouse spleen cells immunized with the WT1 ₃₅ peptide, WT1 ₈₆ peptide or WT1 ₂₉₄ peptide, in each case, responded to a stimulator acting in a pulsed fashion with the WT1 ₃₅ peptide, WT1 ₈₆ peptide or WT1 ₂₉₄ peptide, and proliferated (Fig. 1A -1C).

As described above, spleen cells were stimulated in vitro 3 times at 10-day intervals using spleen cells from non-immunized mice that were pulsed with each WT1 peptide and irradiated. When conducting the 4th stimulation using spleen cells from non-immunized mice, which were pulsed with each peptide described above and irradiated as a stimulant, the proliferation reaction, MHC class I antibody (D ^b antibody) or MHC antibody were measured class II (A ^b antibody) was added to the culture solution and ³ H inclusion was measured. As a result, the proliferation reaction in response to a stimulant delivered in a pulsed manner with each of the WT1 ₃₅ peptide, WT1 ₈₆ peptide and WT1 ₂₉₄ peptide was suppressed by the addition of MHC class II antibody (FIGS. 2A-2C).

As described above, spleen cells were stimulated in vitro 3 times at 10-day intervals using spleen cells from non-immunized mice that were pulsed with each WT1 peptide and irradiated. Then, the proliferation reaction was measured by ³ H inclusion using irradiated C1498 cells not expressing WT1 protein, C1498 cells treated in a pulsed manner with each of the above WT1 peptides, or C1498 cells expressing WT1 protein due to the introduction of the WT1 gene as a stimulant. As a result, this proliferation reaction was obtained in response to C1498 cells treated in a pulsed fashion with the same WT1 peptide that was used in immunization in vivo, and C1498 cells expressing WT1 protein due to the introduction of the WT1 gene (Fig. 3). This revealed that the WT1 ₃₅ peptide, WT1 ₈₆ peptide, and WT1 ₂₉₄ peptide were produced by the intracellular process of the endogenous WT1 protein and were exposed (presented) on a MHC class II molecule. Based on the above facts, it was shown that these three WT1 peptides are WT1 peptides restricted to MHC class II.

Measurement of IFN-γ-producing activity

As described above, spleen cells were stimulated in vitro 3 times at 10-day intervals using spleen cells from non-immunized mice that were pulsed with each WT1 peptide and irradiated. The concentrations of IFN-γ and IL-4 in the culture supernatant were then measured using an ELISA kit (BIOSOURCE Immunoassay Kit, Invitrogen). As a result, spleen cells of two separate mice responded to spleen cells of non-immunized mice, which were pulsed with each WT1 peptide and irradiated, and produced interferon-γ, but little interleukin-4 (Fig. 4). This revealed that these three types of WT1 peptides induce the Th1 type of WT1-specific helper T cells.

Example 2

Measurement of WT1-specific cytotoxic T cells (CTL)

Mice were immunized 3 times with one WT1 ₁₂₆ peptide (MHC class I), WT1 ₁₂₆ peptide (MHC class I) + WT1 ₃₅ peptide (MHC class II), WT1 ₁₂₆ peptide (MHC class I) + WTl ₈₆ peptide (MHC class II) or peptide WT1 ₁₂₆ MHC class I) + peptide WT1 ₂₉₄ (MHC class II) and received spleen cells of these mice. Then these spleen cells were stimulated once in vitro using the WT1 ₁₂₆ peptide (MHC class I) and on the 6th day cytotoxic activity was measured using the RMAS cells treated in a pulsed mode with the WT1 ₁₂₆ peptide (MHC class I) as the target. RMAS cells not pulsed with the WT1 ₁₂₆ peptide (MHC class I) were used as a control target cell. As a result, mouse spleen cells immunized with WT1 ₁₂₆ peptide (MHC class I) + WT1 helper peptide (MHC class II) induced WT1-specific cytotoxic T cells more strongly compared to mouse spleen cells immunized with WT1 ₁₂₆ (MHC class only) I) (Fig. 5). This demonstrated that these three peptides (MHC class II) are WT1-specific helper peptides.

Example 3

Tumor Implantation Experiment

WT1-expressing C1498 leukemic cells were implanted subcutaneously in mice at a ratio of 2.5 × 10 ⁵ cells per mouse and 50 μg / mouse of the helper peptide WT1 ₃₅ was administered intradermally with incomplete Freund's adjuvant once a week, only 3 times, control, instead of helper WT1 ₃₅ peptide was injected intradermally with physiological saline along with Freund's incomplete adjuvant. The size of the subcutaneous tumor was measured over time and the survival rate with no disease was calculated until day 29 after subcutaneous implantation. As a result, the tumor grew in all mice of the control group, while this proliferation is completely inhibited tumor in 4 out of 10 mice immunized with WT1 helper peptide ₃₅ (MHC class II) groups (Figure 7). A significant difference (p <0.05) was also observed between the immunized helper peptide WT1 ₃₅ group and the control group (Fig. 8). This has demonstrated that the WT1 ₃₅ helper peptide (MHC class II) is a WT1 peptide having the ability to induce tumor immunization in vivo.

Then the mice were opened on the 29th day after the start of the above experiment, the spleen was excised and the WT1-specific immune response was analyzed using spleen cells. Briefly, the spleen was dissected by opening the mice of the group immunized with the WT1 ₃₅ helper peptide (MHC class II) and control group and spleen cells were obtained. These spleen cells were stimulated once with the WT1 ₁₂₆ peptide (MHC class I), and on the 6th day after stimulation, the cytotoxic activity of the spleen cells was measured using RMAS cells treated with pulsed WT1 ₁₂₆ peptide (MHC class I) as the target cell . As a control, the cytotoxic activity of spleen cells was measured using RMAS cells as a target cell. As a result, WT1-specific cytotoxic T cells induced in all 4 mice immunized with WT1 helper peptide ₃₅ (MHC class II) groups (Figure 9). On the other hand, WT1-specific cytotoxic T cells were very weakly induced in 3 mice of the control group (Fig. 10). WT1-specific cytotoxic T cells were not induced in one mouse. It was also clear that the induction of WT1-specific cytotoxic T cells was lower compared to the group immunized with the WT1 ₃₅ helper peptide (MHC class II) (FIGS. 9 and 10). This shows that WT1-specific helper T cells were induced by the introduction of the WT1 ₃₅ class II helper peptide and, through the action of WT1-specific helper T cells, WT1-specific cytotoxic T cells induced by an immune response to the WT1 protein expressed by implanted tumor cells into highly amplified in vivo. Thus, these results demonstrated the applicability of the helper peptide WT1 ₃₅ .

Then, specific cytolysis was analyzed in mice of the above group and control group immunized with the WT1 ₃₅ helper peptide (MHC class II). Briefly, the degree of cytolysis (%) obtained by subtracting the degree of cytolysis (%) when the target cells were RMAS cells from the cytolysis degree (%) when the target cells were RMAS cells pulsedly treated with WT1 ₁₂₆ peptide (MHC class I ), in the above experiments was used as specific cytolysis (%) (Fig. 11, left). Obtained, as described above, spleen cells and fluorescently labeled tetramer WT1 (H-2Db WT1 Tetramer-REMPNAPYL-PE) were incubated at 4 ° C for 20 minutes, washed, then stained with fluorescently-labeled CD3 and CD8 antibodies, again washed and analyzed using FACS. CD3-positive, CD8-positive and WT1-tetramer-positive cells served as WT1-specific cytotoxic T cells (Fig. 11, right). As a result, significantly higher WT1-specific cytotoxic T cells (p <0.05) were induced in the spleen cells of mice immunized with the WT1 ₃₅ helper peptide (MHC class II) group compared to the spleen cells of control mice (Fig. 11).

Example 4

Measurement of the ability to proliferate WT1-specific cytotoxic T cells (CTL) in humans

Peripheral blood mononuclear cells were obtained from 6 healthy subjects having a subclass of DRB1, DPB1, DQB1 or DRB5 molecules shown in FIG. 12. Helper peptide WT1 _{35 was} added to the peripheral blood mononuclear cells and these cells were cultured for one week. Then, peripheral blood mononuclear cells were stimulated 4 times overall at intervals of one week using identical peripheral blood mononuclear cells obtained from the subject, which were pulsed with the WT1 ₃₅ helper peptide and irradiated as a stimulant, and ³ H inclusion was measured on 6th day. In all 6 subjects, peripheral blood mononuclear cells responded to the WT1 ₃₅ helper peptide and proliferated (FIG. 12). This showed that the WT1 ₃₅ helper peptide has the function of binding to the aforementioned class II HLA molecules and inducing a proliferation reaction. In this regard, the WT1 ₈₆ peptide and mouse WT1 ₂₉₄ peptide are different from the WT1 ₈₆ peptide (SEQ ID NO: 4) and the WT1 ₂₉₄ peptide (SEQ ID NO: 5) of a person in one amino acid in the positions enclosed in squares, as shown in the table 8.

Table 8 Sequence differences between mouse and human peptides WT1 ₃₅ , WT1 ₈₆ and WT1 ₂₉₄ mWT1 ₃₅ Mouse WAPVLDFAPPGASAYGSL (SEQ ID NO: 3) 18-mer hWT1 ₃₅ Person WAPVLDFAPPGASAYGSL (SEQ ID NO: 3) mWT1 ₈₆ Mouse EQCLSAFTLHFGQFTG (SEQ ID NO: 6) 17-mer hWT1 ₈₆ Person EQCLSAFTVHFGQFTG (SEQ ID NO: 4) mWT1 ₂₉₄ Mouse FRGIQDVRRVSGVAPTLVR (SEQ ID NO: 7) 19-mer hWT1 ₂₉₄ Person FRGIQDVRRVPGVAPTLVR (SEQ ID NO: 5)

Example 5

HLA Class II Restriction of the WT1 Peptide ₃₅

To determine the restriction of the class II HLA molecules of the WT1 ₃₅ peptide, an additional experiment was performed using a method well known to those skilled in the art, briefly described here. First, peripheral blood mononuclear cells (PBMCs) obtained from a healthy subject [DRB1 * 0101 / 0405-, DPB1 * 0201 / 0402- and DQB1 * 0401/0501-positive subject (hereinafter referred to as healthy subject A)] were stimulated 5 times peptide WT1 ₃₅ to obtain a Responder. Then peripheral blood mononuclear cells (PBMCs), obtained from another healthy subject, differing in type HLA class II [DRB1 * 0405 / 0901-, DPB1 * 0201 / 0501- and DQB1 * 0303/0401-positive healthy subject (hereinafter referred to as healthy subject B)], was treated in a pulsed mode with WT1 ₃₅ peptide to obtain a Stimulator, and cell proliferation was measured [amount of ³ H-thymidine incorporated (cpm)]. This measurement was carried out under conditions without the addition of an antibody, with the addition of anti-HLA-DR antibody (+ a-DR), with the addition of anti-HLA-DQ antibody (+ a-DQ). The usual type II HLA class II, which is positive in both the Responder and the Stimulator, shows the restriction of the WT1 ₃₅ peptide. As a result of these experiments, it was shown that the WT1 ₃₅ peptide is DRB1 * 0405-restricted, since proliferation was suppressed by the addition of anti-DR antibody, and DRB1 * 0405 was common in healthy subjects A and B, as shown in FIG. .13.

Then, the experiment was carried out under the same conditions as the conditions of the above experiment, except that as PBMCs obtained from a healthy subject, different from healthy subject A [DRB1 * 0405 / 0803-, DPB1 * 0202 / 0501- and A DQB1 * 0401/0601-positive subject called a healthy subject G)] was used as a Stimulator. As a result, it was shown that the WT1 ₃₅ peptide is DRB1 * 0405-, DPB1 * 0201- and DPB1 * 0202-restricted, since proliferation was suppressed by the addition of anti-HLA-DR antibody or anti-HLA-DP antibody and DRB1 * 0405, DPB1 * 0201 and DPB1 * 0202 were common in healthy subject A and healthy subject G (DPB1 * 0201 and DPB1 * 0202 have a high analogy and are cross-reactive and therefore considered to be a common molecule), as shown in FIG. fourteen.

The experiment was then conducted under the same conditions as the conditions in the above experiment, except that PBMCs obtained from a healthy subject other than healthy subject A [DRB1 * 0101 / 0803-, DPB1 * 0501 / - and DQB1 * 0501 / 0601-positive subject (called healthy subject H)] was used as a Stimulator. As a result, it was shown that the WT1 ₃₅ peptide is DRB1 * 0101-restricted because proliferation was suppressed by adding anti-HLA-DR antibody and DRB1 * 0101 was common in healthy subject A and healthy subject H, as shown in FIG. fifteen.

In addition, PBMCs obtained from healthy subject G were used as the Responder and L cells having the introduced DQB1 * 0601 gene were used as a Stimulator to determine the restriction of the WT1 ₃₅ peptide. The difference in the amount of IFN-γ produced in the presence or absence of a pulse with the WT1 peptide of ₃₅ L cells was measured. The proportion of intracellular production of IFN-γ was measured using the FACS method, which is a method well known to those skilled in the art. As a result, it was shown that the WT1 ₃₅ peptide is DQB1 * 0601-restricted because this Responder was activated by a pulse with the WT1 ₃₅ peptide on L cells, as shown in FIG. 16.

Then, an experiment was performed as described above using PBMCs obtained from the same healthy subject as the Responder and Stimulator. The types of HLA class II molecules that healthy subjects used in this experiment possessed are summarized in table 9 below.

Table 9 Types of HLA Class II Molecules Available to Healthy Subjects Used in This Experiment No. of a healthy subject DRB1 DPB1 DQB1 BUT * 0101/0405 * 0201/0402 * 0401/0501 AT * 0405/0901 * 0201/0501 * 0303/0401 FROM * 0802/1201 * 0201/0501 * 0301/0302 D * 1502/1502 * 0201/0901 * 0601/0601 E * 0405/0901 * 0202/0501 * 0303/0401 F * 1403/1502 * 0201/0901 * 0301/0601 G * 0405/0803 * 0202/0501 * 0401/0601 H * 0101/0803 * 0501 / - * 0501/0601 I * 0101/1501 * 0201/0402 * 0501/0602

As a result, it was found that the addition of an anti-DR antibody or anti-DP antibody during this experiment using PBMCs obtained from healthy subjects AE reduced the amount of ³ H-thymidine incorporated (cpm) and, therefore, suppression of proliferation. Adding only anti-DR antibodies using PBMCs obtained from healthy subject F also led to suppression of proliferation. In addition, the addition of only anti-HLA-DP antibodies using PBMCs obtained from healthy subject G resulted in suppression of proliferation. Through an experiment using healthy subject A, it was shown that the WT1 ₃₅ peptide is DRB1 * 0101 or 0405-restricted and DPB1 * 0201 or 0402-restricted. Through an experiment using healthy subject C, it was shown that the WT1 ₃₅ peptide is DRB1 * 0802- or 1201-restricted and DPB1 * 0201- or 0501-restricted. Through an experiment using a healthy subject D, it was shown that the WT1 ₃₅ peptide is DRB1 * 1502-restricted because DRB1 * 1502 is homozygous (Fig. 17). In addition, it was shown that the peptide WT1 ₃₅ is DPB1 * 0201 - or 0901-restricted. Through an experiment using healthy subject E, it was shown that the WT1 ₃₅ peptide is DRB1 * 0405 or 0901-restricted and DPB1 * 0202 or 0501-restricted. Through an experiment using a healthy subject F, it was shown that the WT1 ₃₅ peptide is DRB1 * 1403- or 1502-restricted. Through an experiment using a healthy subject G, it was shown that the WT1 ₃₅ peptide is DRB1 * 0202- or 0501-restricted.

The difference in the amount of IFN-γ produced in the presence or absence of an impulse with WT1 ₃₅ peptide was also measured using PBMCs obtained from healthy subject I as a Responder and Stimulator. The proportion of intracellular production of IFN-γ was measured using FACS, a method well known to those skilled in the art. As a result, the proportion of IFN-γ was significantly increased by the pulse of the WT1 ₃₅ peptide (Fig. 18). This shows that the WT1 ₃₅ peptide is restricted to any of DRB1 * 0101, DRB1 * 1501, DPB1 * 0201, DPB1 * 0402, DQB1 * 0501, and DQB1 * 0602.

INDUSTRIAL APPLICABILITY

The present invention provides a WT1 peptide that is restricted to many types of MHC class II molecules, a polynucleotide encoding the peptide, a pharmaceutical composition containing them, and the like. Thus, they can be used in the field of pharmaceutical substances, for example, in the field of development and production of prophylactic or therapeutic drugs for various tumors of hematopoietic organs and solid tumors that highly express the WT1 gene.

Claims (17)

1. A peptide that has an amino acid sequence consisting of contiguous amino acids derived from a WT1 protein and induces WT1-specific helper T cells by binding to a MHC class II molecule, where this amino acid sequence is selected from the group consisting of:
(a) the amino acid sequence depicted in SEQ ID NO: 3;
(b) the amino acid sequence depicted in SEQ ID NO: 4;
(c) the amino acid sequence depicted in SEQ ID NO: 5, and
(d) an amino acid sequence in which one amino acid is substituted in the amino acid sequences depicted in (a) to (c). 2. The peptide according to claim 1, where this amino acid sequence is the amino acid sequence depicted in SEQ ID NO: 3. 3. The peptide according to claim 1 or 2, where this MHC class II molecule is selected from the group consisting of DRB1 * 0101, DRB1 * 0405, DRB1 * 0802, DRB1 * 0803, DRB1 * 0901, DRB1 * 1201, DRB1 * 1403, DRB1 * 1501, DRB1 * 1502, DPB1 * 0201, DPB1 * 0202, DPB1 * 0402, DPB1 * 0501, DPB1 * 0901, DQB1 * 0301, DQB1 * 0302, DQB1 * 0401, DQB1 * 0501, DQB1 * 0601, DQB1 * 0602 and DRB5 * 0102. 4. The peptide according to claim 1 or 2, where this MHC class II molecule is selected from the group consisting of DRB1 * 0101, DRB1 * 0405, DRB1 * 1502, DPB1 * 0201, DPB1 * 0202 and DQB1 * 0601. 5. A polynucleotide encoding a peptide according to claim 1 or 2, wherein the peptide induces WT1-specific helper T cells by binding to a MHC class II molecule. 6. An expression vector containing a polynucleotide according to claim 5. 7. The anti-peptide antibody according to any one of paragraphs. 1-4, where the antibody is obtained by immunizing an animal with a peptide according to any one of claims. 1-4, and where the antibody specifically binds to the peptide according to any one of paragraphs. 1-4. 8. A pharmaceutical composition for treating cancer, comprising an effective amount of a peptide according to any one of claims. 1-4 and a suitable adjuvant. 9. A method of treating cancer, which comprises administering an effective amount of a peptide according to any one of claims. 1-4 to a subject having a MHC class II molecule according to claim 3 or 4. 10. The use of the peptide according to any one of paragraphs. 1-4 for the treatment of cancer. 11. An isolated antigen-presenting cell that exhibits (represents) a peptide according to any one of claims. 1-4 through a MHC class II molecule according to claim 3 or 4. 12. A method of inducing antigen presenting cells, which comprises culturing immature antigen presenting cells in the presence of a peptide according to any one of claims. 1-4 and the induction of antigen-presenting cells that expose this peptide via the MHC class II molecule of claim 3 or 4 from immature antigen-presenting cells. 13. WT1-specific helper T cell, which is induced in vitro by the peptide according to any one of paragraphs. 1-4 with the MHC class II molecule, where the specified cell specifically recognizes the peptide complex according to any one of paragraphs. 1-4 and MHC class II molecules. 14. A method of inducing WT1-specific helper T cells, which comprises culturing peripheral blood mononuclear cells in the presence of a peptide according to any one of claims. 1-4 and the induction of WT1-specific helper T cells from these peripheral blood mononuclear cells. 15. A kit for the induction of WT1-specific helper T cells, containing, as an essential ingredient, the peptide according to any one of claims. 1-4 and a suitable adjuvant. 16. The use of a pharmaceutical composition for the treatment of cancer expressing the WT1 gene containing, as an essential ingredient, the peptide according to any one of claims. 1-4 and a suitable adjuvant, where the cancer is selected from the group consisting of hematopoietic organ tumors such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma, and solid cancers such as stomach cancer, intestinal cancer, lung cancer, breast cancer glands, germ cells, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer. 17. A method for determining the presence or quantity of WT1-specific helper T cells in a subject having a MHC class II molecule of claim 3 or 4, said method comprising the steps of:
(a) the reaction of the peptide according to any one of paragraphs. 1-4 with a sample obtained from this subject;
(b) determining the presence or amount of a cytokine contained in this sample; and then
(c) using the presence or amount of a cytokine as an indicator.

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