WT1 antigenic polypeptide, and anti-tumor agent containing said polypeptide

Abstract

[Problem] To provide a polypeptide which enables stimulation of numerous T cells of different derivation, and to induce strong immunological response against numerous types of cancers. [Solution] Provided is a fused polypeptide that includes a helper epitope derived from the WT1 protein, and a killer epitope derived from the WT1 protein, wherein the polypeptide includes a total of 3 to 6 of the helper epitopes and the killer epitopes per molecule of the fused polypeptide.

Claims

1. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 47 or 48.

2. The polypeptide according to claim 1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 47.

3. The polypeptide according to claim 1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 48.

4. An anti-tumor agent comprising the polypeptide according to claim 1 as an active ingredient.

5. The anti-tumor agent according to claim 4, further comprising antigen-presenting cells.

6. A method for in vitro producing T cells that produce a cytokine against tumor cells expressing a WT1 protein, comprising (a) incubating in vitro the polypeptide according to claim 1 with mononuclear cells, and (b) incubating the mononuclear cells from step (a) with antigen presenting cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram showing a method of inducing, from peripheral blood mononuclear cells, T cells (antigen-specific T cells) to elicit a specific immunological response to a polypeptide derived from the WT1 protein, using said polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

(2) <Antigenic Polypeptide of the Invention Derived from WT1 Protein>

(3) As shown in Examples below, a long-chain polypeptide in which a total of 3 or more of helper epitopes derived from the WT1 protein and killer epitopes derived from the WT1 protein are bound is able to stimulate numerous CD4+ T cells and CD8+ T cells of different derivation and induce more numerous Th1 cells and CTLs capable of producing cytokines, as compared with a case of stimulating T cells with amixture of these epitopes . On the other hand, induction of such Th1 cells or CTLs was not enhanced even with a long-chain polypeptide in which one helper epitope derived from the WT1 protein is bound to one killer epitope from the WT1 protein.

(4) Accordingly, the antigenic polypeptide derived from the WT1 protein of the present invention is a fused polypeptide comprising a helper epitope derived from the WT1 protein and a killer epitope derived from the WT1 protein, wherein the fused polypeptide includes a total of 3 to 6 of the helper epitopes and the killer epitopes per molecule of the fused polypeptide.

(5) A polypeptide as used herein means a compound in which two or more amino acids are bound. In other words, it also includes so-called peptides, oligopeptides, and proteins. Further, the amino acid constituting the polypeptide may be a naturally occurring amino acid or a non-naturally occurring amino acid, or may be an analog (N-acylated compounds, O-acylated compounds, esters, acid amide compounds, alkylated compounds, etc. of amino acids). Also, the side chain of amino acids may be chemically modified (glycosylation, lipidation, acetylation,phosphorylation,ubiquitination, etc.). Furthermore, the amino-terminus and free amino groups of the polypeptide may be bonded to formyl group, acetyl group, t-butoxycarbonyl (t-Boc), etc., and the carbonyl-terminus and free carboxyl groups of the peptide of the present invention may be bonded to methyl group, ethyl group, t-butyl group, benzyl group, etc.

(6) The WT1 protein as used herein means a protein encoded by the Wilms' tumor gene WT1 that is highly expressed in leukemia and various solid cancers, and includes typically a protein consisting of the amino acid sequence as set forth in SEQ ID NO: 1 (protein encoded by the base sequence identified by RefSeq ID: NM_024426.4) when the WT1 protein is derived from human beings.

(7) Further, as the WT1 protein other than that having such a typical amino acid sequence, proteins in which an amino acid(s) is/are naturally mutated may also be present. Thus, for example, a human WT1 protein includes a protein consisting of the amino acid sequence as set forth in SEQ ID NO: 1, wherein one or more amino acids may be substituted, deleted, inserted, or added. The substitution, deletion, insertion, or addition for the amino acid sequence is generally carried out within 10 or less amino acids (e.g., within 5 or less amino acids, within 3 or less amino acids, or with 1 amino acid).

(8) In the present invention, the helper epitope, also called as Th epitope or CD4+ helper epitope, means an antigenic determinant having an activity of stimulating CD4+ T cells to induce the immunological response, an amino acid sequence constituting this antigenic determinant, or an antigenic polypeptide consisting of said amino acid sequence. There are no particular limitations on the length of the helper epitope in the present invention, and the helper epitope is usually 5 to 30 amino acids in length, preferably 7 to 25 amino acids in length, more preferably 9 to 22 amino acids in length, particularly preferably 16 amino acids in length.

(9) Note that immunological response in the present invention refers to a reaction performed specifically in response to exogenous substances (bacteria, viruses, etc. or fragments thereof) or endogenous substances (cancer cells, etc. or their fragments) which are recognized as antigens by cells responsible for immunity, such as T cells. For example, the immunological responses as a result of recognition of such antigens include production of cytokines such as IFN-gamma, etc., and eventually elimination of bacteria, viruses, and cancer cells induced by such cytokine production.

(10) The killer epitope in the present invention, also called as CTL epitope orcytotoxic T cell epitope, means an antigenic determinant having an activity of stimulating CD8+ T cells to induce the immunological response, an amino acid sequence constituting this antigenic determinant, ox: an antigenic polypeptide consisting of said amino acid sequence. There are no particular limitations on the length of the killer epitope in the present invention, and the killer epitope is usually 5 to 30 amino acids in length, preferably 6 to 25 amino acids in length, more preferably 8 to 20 amino acids in length, particularly preferably 9 amino acids in length.

(11) The antigenic polypeptides can be identified using a well-known technique (e.g., Paul WE editing, Fundamental Immunology, 3rd ed., pages 243-247, Raven Press, 1993). As a technique for identifying antigenic polypeptides, there is exemplified a screening method of polypeptides with use of the ability to react with antigen-specific antisera and/or T cell lines or clones as an index. For example, in an ELISA and/or T-cell reactivity assay, a portion that reacts with antisera and/or T cells in the polypeptide among the WT1 proteins can be selected as the antigenic polypeptide derived from the WT1 protein, using an index that the reactivity of the portion is substantially not less than the reactivity of the WT1 full length protein. Such screening can be carried out using the method well-known to those skilled in the art, as described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1998.

(12) The amino acid sequence constituting the epitope may also be inferred by an analysis using a computer program. The amino acid sequence of the helper epitopes can be inferred by computer programs, such as MHC-THREAD (http://www.csd.abdn.ac.uk/gjlk/MHC-Thread/), EpiPredict (http://www.epipredict.de/index.html), HLA-DR4 binding (http://www-dcs.nci.nih.gov/branches/surgery/sbprog.html), and ProPred (http://www.imtech.res.in/raghava/propred/). The amino acid sequence of the killer epitopes can he inferred by computer programs, such as BIMAS (http://bimas.dcrt.nih.gov/ molbio/hla_bind/), SVMHC (http://www.sbc.su.se/svmhc/), PREDEP (http://bioinfo.md.huji.ac.il/marg/Teppred/mhc-bind/), NetMHC (http://www.cbs.dtu.dk/services/NetMHC/), PREDICT (http://sdmc.krdl.org.sg:8080/predict/), and LpPep (http://reiner.bu.edu/zhiping/lppep.html). In addition, it is also possible to deduce the amino acid sequences of the killer epitope and the helper epitope by computer programs, such as SYFPEITHI (http://syfpeithi.bmi-heidelberg.com/Scripts/MHCServer,dll/EpiPredict.htm), and RankPep (http://www.mifoundation.org/Tools/rankpep.html).

(13) Whether or not a polypeptide consisting of the amino acid sequence inferred by these computer programs has an activity to stimulate CD4+ T cells or CD8+ T cells and to induce the immunological response, can be evaluated by subjecting such a polypeptide to in vitro stimulation assay using dendritic cells, peripheral blood cells or fibroblasts, as shown in Examples below. Further, evaluation can also be performed by subjecting such a polypeptide Lc in vivo stimulation assay using transgenic mice expressing HLA.

(14) The helper epitope derived from the WT1 protein or killer epitope derived from the WT1 protein in the present invention includes not only an antigenic polypeptide consisting of a continuous amino acid sequence of a portion of a native WT1 protein, but also a variant thereof. Such variants include those consisting of the amino acid sequence wherein one or more amino acids are substituted, deleted, inserted, or added in the continuous amino acid sequence of a portion of the WT1 protein. The substitution, deletion, insertion or addition for the amino acid sequence is generally performed within 10 or less amino acids (e.g., within 5 or less amino acids, preferably within 3 or less amino acids, more preferably with 1 amino acid), and a variant in the present invention consisting of the amino acid sequence wherein one amino acid is substituted in the continuous amino acid sequence of a portion of the WT1 protein, is particularly preferred.

(15) Further, as a suitable example of the helper epitope derived from the WT1 protein in the present invention, there are exemplified polypeptides consisting of the amino acid sequence of any one of SEQ ID NOs: 2 to 39 described in Table 1, and a more preferred example includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5. As a suitable example of the killer epitope derived from the WT1 protein in the present invention, there are exemplified polypeptides consisting of the amino acid sequence of any one of SEQ ID NOs: 40 to 45 described in Table 5, and a more preferred example includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 40 or 44.

(16) TABLE-US-00001 TABLE1 positionin humanWT1 protein(the aminoacid amino sequence HLA helper acid representedby re- SEQ epitope sequence SEQIDNo:1) striction IDNo: 1 PQQMGSDV 66-80 DRB1*0401 2 RDLNALL 2 NKRYFKLS 399-419 DRB1*0401 3 HLQWHSR 3 LSHLQMHS 405-415 DRB1*0401, 4 RKH DRB1*0405, DRB1*1501, DRB1*1502, DRP1*0901 4 KRYFKLSH 400-415 DRB1*0101, 5 LQMHSRKH DRB1*0301, DRB1*0401, DRB1*0701, DRP1*1101, DRP1*1501 5 RSDELVRH 495-513 DRB1*0101, 6 HNMHQRNM DRB1*0301, TKL DRB1*0401, DRB1*0701, DPB1*1101, DPB1*1501 6 PGCNKRYF 396-417 DRB1*0101, 7 KLSHLQMH DRB1*0301, SRKHTG DRB1*0401, DRB1*0701, DPB1*1101, DPB1*1501 7 SGQARMFP 190-208 DRB1*0101, 8 NAPYLPSC DRB1*0301, LES DRB1*0401, DRB1*0701, DPB1*1101, DPB1*1501 8 SGQAYMFP 190-208 DRB1*0101, 9 NAPYLPSC [Arginineat DRB1*0301, LES 5thposition DRB1*0401, issubstituted DRB1*0701, withTyrosine] DRP1*1101, DRP1*1501 9 LKGYAAGS 315-329 DRB1*0101, 10 SSSYKWT DRB1*0401, DRB1*0701, DR53

(17) TABLE-US-00002 TABLE2 positionin humanWT1 protein(the aminoacid amino sequence HLA helper acid representedby re- SEQ epitope sequence SEQIDNo:1) striction IDNo: 10 KLSHLQMH 404-423 DP5 11 SRKHTGEK PYQC 11 KLSHLQMH 404-422 DP5 12 SRKHTGEK PYQ 12 KLSHLQMH 404-421 DP5 13 SRKHTGEK PY 13 KLSHLQMH 404-420 DP5 14 SRKHTGEK P 14 KLSHLQMH 404-419 DP5 15 SRKHTGEK 15 KLSHLQMH 404-418 DP5 16 SRKHTGE 16 KLSHLQMH 404-417 DP5 17 SRKHTG 17 KLSHLQMH 404-416 DP5 18 SRKHT 18 KLSHLQMH 404-415 DP5 19 SRKH 19 KLSHLQMH 404-414 DP5 20 SRK

(18) TABLE-US-00003 TABLE3 positionin humanWT1 protein(the aminoacid amino sequence HLA helper acid representedby re- SEQ epitope sequence SEQIDNo:1) striction IDNo: 20 LSHLQMHS 405-423 DP5 21 RKHTGEKP YQC 21 LSHLQMHS 405-422 DP5 22 RKHTGEKP YQ 22 LSHLQMHS 405-421 DP5 23 RKHTGEKP Y 23 LSHLQMHS 405-420 DP5 24 RKHTGEKP 24 LSHLQMHS 405-419 DP5 25 RKHTGEK 25 LSHLQMHS 405-418 DP5 26 RKHTGE 26 LSHLQMHS 405-417 DP5 27 RKHTG 27 LSHLQMHS 405-416 DP5 28 RKHT 28 LSHLQMHS 405-414 DP5 29 RK

(19) TABLE-US-00004 TABLE4 positionin humanWT1 protein(the aminoacid amino sequence HLA helper acid representedby re- SEQ epitope sequence SEQIDNo:1) striction IDNo: 29 SHLQMHSR 406-423 DP5 30 KHTGEKPY QC 30 SHLQMHSR 406-422 DP5 31 KHTGEKPY Q 31 SHLQMHSR 406-421 DP5 32 KHTGEKPY 32 SHLQMHSR 406-420 DP5 33 KHTGEKP 33 SHLQMHSR 406-419 DP5 34 KHTGEK 34 SHLQMHSR 406-418 DP5 35 KHTGE 35 SHLQMHSR 406-417 DP5 30 KHTG 30 SHLQMHSR 406-416 DP5 37 KHT 37 SHLQMHSR 406-415 DP5 33 KH 38 SHLQMHSR 406-414 DP5 39 K

(20) TABLE-US-00005 TABLE5 positionin humanWT1 protein(the aminoacid amino sequence HLA killer acid representedby re- SEQ epitope sequence SEQIDNo:1) striction IDNo: 1 RMFPNAPYL 194-202 A*0201 40 2 SLGEQQYSV 255-263 A*0201, 41 A*0206 3 YMFPNAPYL 194-202 A*0201 42 [Arginineat 1stposition issubstituted withTyrosine] 4 CMTWNQMNL 303-311 A*0201, 43 A*2402 5 CYTWNQMNL 303-311 A*2402 44 [Methionineat 2ndposition issubstituted withtyrosine] 6 RWPSCQKKF 485-493 A*2402 45

(21) It is to be noted that Patent Literatures 4 to 6 and Non-Patent Literatures 1 to 3 have demonstrated that the polypeptides listed in Tables 1 to 5 have antigenicity against CD4+ T cells or CD8+ T cells. Further, as described in Tables 1 to 5, the polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 9 (helper epitope 8) is a polypeptide wherein the 194th arginine residue in the native WT1 protein is substituted with a tyrosine residue The polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 42 (killer epitope 3) is a polypeptide wherein the 194th arginine residue in the native WT1protein is substituted with a tyrosine residue. The polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 44 (killer epitope 5) is a polypeptide wherein the 304th methionine residue in the native WT1 protein is substituted with a tyrosine residue.

(22) The fused polypeptide of the present invention is one wherein a total of 3 to 6 of the polypeptides including the helper epitopes and the killer epitopes per molecule are artificially bound. When the number of the helper epitope and the killer epitope is less than the lower limit, it becomes impossible to stimulate numerous T cells of different derivation. When the number of the helper epitope and the killer epitope exceeds the upper limit, it becomes difficult to prepare such polypeptides by chemical synthesis or the like. Further, although the chain length of the fused polypeptide of the present invention is not particularly limited, 30 to 100 amino acids are preferred.

(23) With reference to the number of epitopes (the helper epitope or the killer epitope) contained per molecule of the fused polypeptide according to the present invention, when one epitope contains other epitope, such other epitope shall not be counted as 1 (one). For example, with respect to the fused polypeptide of the present invention containing helper epitope 7 described in Table 1 (polypeptide consisting of the amino acid residues at positions 190-208 of the human WT1 protein), although killer epitopes 7 (polypeptide consisting of the amino acid residues at positions 194-202 of the human WT1 protein) is contained in the amino acids at positions 190-208 of the human WT1 protein as described in Table 5, the killer epitope 7 (one killer epitope) is not counted as the number of epitopes contained per molecule of the fused polypeptide. Further, for example, with respect to the fused polypeptide of the present invention containing helper epitope 6 (polypeptide consisting of the amino acids at positions 396-417 of the human WT1 protein) as described in Table 1, helper epitopes 2 to 4 (polypeptide consisting of the amino acid residues at positions 399-413 of the human WT1 protein, polypeptide consisting of the amino acid residues at positions 405-415 of the human WT1 protein, andpolypeptide consisting of the amino acid residues at positions 400-415 of the human WT1 protein, respectively) are included in the fused polypeptide, but the helper epitopes 2 to 4 (3 helper epitopes) are not counted as the number of epitopes contained per molecule of the fused polypeptide.

(24) In the fused polypeptide of the present invention, one or more epitopes of different sequence may be contained for each sequence, and a plurality of epitopes of the same sequence may be contained.

(25) Further, in the fusedpolypeptide of the present invention, no particular limitation is imposed on the arrangement of the helper epitope and the killer epitope, and two or more helper epitopes and killer epitope may be arranged consecutively without sandwiching the killer epitope and the helper epitope, respectively, or may be arranged alternately.

(26) The fused polypeptide of the present invention includes preferably a total of 3 of the helper epitope and the killer epitope per molecule, more preferably one helper epitope and two killer epitopes, even more preferably a polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 5, a polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 40, and a polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 44.

(27) In addition, as shown in Examples below, from the viewpoint capable of stimulating more numerous CD4+ T cells and CD8+ T cells of different derivation to induce more numerous Th1 cells and CTLs producing cytokines antigen-specifically, the fused polypeptides of the present invention are more preferably those wherein the killer epitopes are bound to both sides of the helper epitope, and particularly preferably those wherein the polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 40 is bound to the amino terminal of the polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 5, and the polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 44 is bound to the carboxyl terminal of the polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 5.

(28) In the fused polypeptide of the present invention, the bonding between epitopes maybe made directly or indirectly. Such indirect bonding includes, for example, a bonding via a linker polypeptide. The length of such a linker polypeptide is generally 1 to 100 amino acids, preferably 1 to 50 amino acids, more preferably 1 to 30 amino acids, even more preferably 2 to 10 amino acids (e.g., 5 amino acids), particularly preferably a bonding via a linker polypeptide comprising 5 glycine residues.

(29) As long as the fused polypeptide of the present invention include a total of 3 to 6 of the helper epitopes and the killer epitopes per molecule, a His tag that is widely used as a useful tag for separation and purification of proteins, a marker protein such as GFP, or a labeled compound with biotin or the like may be added.

(30) It is possible to produce the fused polypeptide of the present invention as a recombinant protein by applying various known gene recombination techniques to DNA encoding such fused polypeptide. Typically, the fused polypeptide of the present invention may be produced by synthesizing DNA encoding the fused polypeptide of the present invention with use of an appropriate DNA synthesizer; constructing an expression vector expressing the fused polypeptide of the present invention by appropriately selecting or combining various methods that have been introduced in reference books in the art, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989, and the like.); and transforming a suitable host cell such as Escherichia coli, etc. with this expression vector; thereby to produce the desired polypeptide. At this time, as mentioned earlier, various operations used in the production of recombinant polypeptides, such as addition of a His tag or the like may be added.

(31) Moreover, the fused polypeptides of the present invention can also be prepared chemical synthetically, using amino acids modified with a variety of protecting groups as a raw material. Methods for synthesizing fused polypeptides organochemically without using a gene or a host cell are widely known to those skilled in the art. For example, Jikken Kagaku Koza (Encyclopedia of Experimental Chemistry, 5th Ed.) 16, Chemical Synthesis of Organic Compounds IV (Saburo Aimoto et al., Chemical Society of Japan) and the like introduce various chemical synthesis methods of polypeptides. The polypeptides of the present invention can also be synthesized using any of these methods. Further, the polypeptide of the present invention can also be synthesized using commercially available equipment that is generally called as a peptide synthesizer.

(32) <Anti-Tumor Agent of the Invention>

(33) As shown in Examples below, the fused polypeptide described above can stimulate numerous T cells of different derivation to strongly induce their immunological responses. Accordingly, it is also possible to provide an anti-tumor agent comprising the fused polypeptide of the present invention as an active ingredient.

(34) The anti-tumor agent in the present invention means a drug that is able to inhibit the growth of tumor cells and/or induce the death of tumor cells by stimulating T cells to induce their immunological responses. That is, the anti-tumor agent of the present invention includes not only a drug capable of stimulating T cells in a subject to activate the immunological response by administering it to the subject (a drug for active immunotherapy), but also a drug containing T cells and the like that are stimulated outside of a subject activate the immunological response (a drug for passive immunotherapy).

(35) The target tumor to be treated or prevented by the anti-tumor agent of the present invention is not limited as long as the WT1 protein is expressed therein, and includes, for example, leukemias (acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, etc.) and solid cancers (nephroblastoma (Wilms' tumor), kidney cancer, breast cancer, lung cancer, thyroid cancer, stomach cancer, colon cancer, prostate cancer, ovarian cancer, melanoma, etc.).

(36) Other embodiment of the anti-tumor agent of the present invention include an anti-tumor agent containing, as active ingredients, the fused polypeptide of the present invention and T cells producing cytokines to tumor cells expressing the WT1 protein, said T cells being produced by using the fused polypeptide.

(37) Such T cells producing cytokines to tumor cells expressing the WT1 protein can be produced, for example, by the following method.

(38) The method comprises the step of incubating the fused polypeptide of the present invention and mononuclear cells, and the step of incubating the incubated mononuclear cells and antigen-presenting cells.

(39) The term mononuclear cells as used herein refers to lymphocytes and monocytes, and includes, for example, embodiments such as peripheral blood mononuclear cells (PBMCs, peripheral blood monocytes), umbilical cord blood mononuclear cells, tumor tissue infiltrating lymphocytes, and the like. Note that the tumor tissue infiltrating lymphocytes means lymphocytes isolated from tumor tissue excised from a subject with cancer, or lymphocytes isolated from pleural fluid or abdominal dropsy collected from the subject.

(40) The incubation of the fused polypeptide of the present invention and mononuclear cells may be performed, for example, by adding the fused polypeptide of the present invention into a medium in which mononuclear cells are cultured. Further, the incubation may be carried out by culturing mononuclear cells in a plate to which the fused polypeptides of the present invention is immobilized. The conditions during such incubation are not limited as long as the conditions are suitable for the maintenance of mononuclear cells, and include, for example, the conditions under which culture is performed in a medium used to maintain the mononuclear cells g , AIM-V medium supplemented with serum) at 37 C. with 5% CO.sub.2 for 15 minutes to 48 hours.

(41) The antigen-presenting cells to be incubated with the mononuclear cells incubated in the above manner may be any cells as long as the cells express, on their surface, HLA capable of binding to the fused polypeptides of the present invention which was used in the incubation, and include, for example, dendritic cells, B cells, and macrophages. In addition, the antigen-presenting cells according to the present invention may be a cell expressing on its surface the HLApulsed with the fused polypeptide of the present invention. Furthermore, such antigen-presenting cells may be those obtained by X-ray irradiation or mitomycin treatment, etc., for losing proliferation potency prior to incubation with mononuclear cells.

(42) The conditions for incubation of such antigen-presenting cells with mononuclear cells are not limited as long as they are suitable for the maintenance of mononuclear cells and antigen-presenting cells, and include, for example, culture in a medium (e.g., AIM-V medium) used to maintain the mononuclear cells and antigen-presenting cells at 37 C. with 5% CO.sub.2 for 1 to 7 days. In addition, at the time of such incubation, IL-2, IL-7, IL-15, PHA, anti-CD3 antibody, IFN-, IL-12, anti-IL-4 antibody or a combination thereof may be added to the medium from the viewpoint of stimulating T cells. Further, from the viewpoint of enhancing the immunological response by the production of various cytokines, Toll-like receptor (TLR) agonists such as picibanil (OK-432) and CpG DNA may be added to the medium. Furthermore, incubation of the antigen-presenting cells with mononuclear cells may be repeated a plurality of times in order to secure the number of T cells necessary for passive immunotherapy.

(43) As shown in Examples below, T cells prepared in the above manner contain CD4+ T cells (antigen-specific CD4+ T cells) producing cytokines specifically to the fused polypeptide of the present invention, and CD8+ T cells (antigen-specific CD8+ T cells) producing cytokines specifically to the fused polypeptide of the present invention. As a method of purifying such T cells, for example, T cells can be separated using a carrier to which the fused polypeptide of the present invention is immobilized. Furthermore, such T cells can also be purified using a cytokine to be secreted as an index. That is, since antigen-specific CD4+ T cells produce cytokines, such as IFN-, IL-2, IL-4, T cells can be purified by flow cytometry, affinity chromatography, or magnetic beads purification method, using antibodies against these cytokines.

(44) On the other hand, since antigen-specific CD8+ T cells produce cytokines such as IFN- and TNF-, T cells can be purified by flow cytometry, affinity chromatography, and magnetic beads purification method, using antibodies against these cytokines.

(45) Further, such T cells can be purified by flow cytometry, affinity chromatography, and magnetic beads purification method, using antibodies against proteins that are expressed on the cell surface of each T cell. The protein expressed on the cell surface of antigen-specific CD4+ T cells include CD29, CD45RA, CD45RO, and the like, and the protein expressed on the cell surface of antigen-specific CD8+ T cells include CD107a, CD107b, CD63, CD69, and the like.

(46) Further, as described above, the fused polypeptides of the present invention and antigen-presenting cells are useful for inducing mononuclear cells to antigen-specific T cells. Accordingly, as a drug for treating or preventing a cancer by passive immunotherapy, it is also possible to provide an anti-tumor agent comprising the fused polypeptide of the present invention and antigen-presenting cells as active ingredients.

(47) Further, T cells and antigen-presenting cells contained in the anti-tumor agent of the present invention, as well as mononuclear cells and antigen-presenting cells used in the production of said T cells may be each independently those derived from the subject receiving the anti-tumor agent of the present invention (autologous), those that are allogeneic to said subject, or those that are allogeneic to said subject and in agreement with the type of HLA class of said subject.

(48) The anti-tumor agent of the present invention may contain other pharmaceutically active ingredients or various excipients that are commonly used for formulation of pharmaceuticals in a range that does not impair the effect of the fused polypeptides of the present invention, T cells or dendritic cells. In particular, the anti-tumor agent of the present invention is preferably in the form of a buffer solution or a liquid medium that can stably maintain the fused polypeptide of the present invention, T cells and dendritic cells.

(49) Non-limiting examples of the buffer solution may include a neutral buffered saline or a phosphate buffered saline. Further, the buffer solution may include, for example, saccharides (e.g., glucose, mannose, sucrose, dextran, mannitol, etc.), proteins, amino acids, antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide, KLH, QS21, complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum phosphate, BCG, alum, TLR agonists (e.g., CpG DNA, etc.)), tonicity adjusting agents, suspending agents, thickening agents and/or preservatives, etc.

(50) Also in the anti-tumor agent of the present invention, the fused polypeptide of the present invention and T cells, as well as the fused polypeptide of the present invention and dendritic cells are each preferably in the form of a mixture thereof. However, the fused polypeptide of the present invention and T cells or dendritic cells may be in the form of a so-called kit wherein the fused polypeptide and T cells or dendritic cells are separately stored and can be administered in admixture when used.

(51) The anti-tumor agent of the present invention may be used in combination with a known pharmaceutical composition for use in the treatment or prevention of cancer. Further, the anti-tumor agent of the present invention can be used for target animals including human beings as a subject. There are no particular limitations on the animals other than human beings, and such animal can include various livestock species, poultry, pets, experimental animals, and the like as a subject. The subject to which the anti-tumor agent of the present invention is administered is not particularly limited and may include not only those suffering from tumors in which WT1 protein is expressed, but also those that are not suffering from tumors. For example, from the viewpoint of prevention of cancer recurrence, the subject may include those who have received cancer therapy by surgery, chemotherapy, radiation therapy, and the like.

(52) There are no particular limitations on the dosage form of the anti-tumor agent of the present invention, and the dosage form includes, for example, a subcutaneous injection, an intravenous injection, an intradermal injection, an intramuscular injection, and a local injection into the tumor tissue site. When administering the anti-tumor agent of the present invention, the dosage is appropriately selected, depending on the age, body weight, symptom, and health condition of the subject. For example, the dosage of the anti-tumor agent of the present: invention (weight in terms of active ingredient) differs depending on the type of active ingredients. For example, when the active ingredient is a polypeptide, the dosage is usually from 0.01 to 10 mg, and when the active ingredient is a cell, the dosage is usually 10.sup.6 to 10.sup.12 cells.

(53) In this way, the present invention also provides a method for treating or preventing a cancer in a subject, characterized by administering the anti-tumor agent of the present invention to the subject.

(54) A product of the anti tumor agent of the present invention or an instruction thereof may be labelled to indicate that it can be used to treat or prevent a cancer. Herein, the phrase a product or an instruction thereof is labelled means that the body, container, or package of the product is labelled, or that an instruction thereof, a package insert, an advertisement, or other prints disclosing information on the product are labelled. The label indicating the use for treating or preventing a cancer may include information on a mechanism of suppressing the growth of tumor cells and/or induce the death of tumor cells by administering the fused polypeptide of the present invention; the fused polypeptide and T cells, produced by using the fused polypeptide, producing cytokines to tumor cells expressing WT1 protein; or the fused polypeptide and dendritic cells.

EXAMPLES

(55) Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, and the present invention is not limited to the following Examples.

(56) <Preparation of Dendritic Cells>

(57) From the blood that was donated from 6 healthy human volunteers, human peripheral blood mononuclear cells (PBMC) were separated using a Ficoll-Paque (manufactured by Amersham Bioscience Inc.). The separated PBMC was cultured at 37 C. for 1 hour in a cell culture flask (manufactured by ED Biosciences) in a CO.sub.2 incubator using a serum-free AIM-V medium, thereby to remove non-adherent cells. Then, adherent cells remaining on the flask were started to culture in the AIM-V medium in the presence of a recombinant GM-CSF (10 ng/ml, manufactured by Wako Company) and a recombinant IL-4 (10 ng/ml, manufactured by Wako Company). After 3 days from the start of culture, the medium was replaced by an AIM-V medium supplemented with GM-CSF and IL-4, and dendritic cells (DC) were recovered using trypsin after 7 days from the start of culture. These DCs were used as antigen-presenting cells (APC) in the induction of antigen-specific T cells or in the analysis of antigen-specific cytokine (IFN-) production from T cells.

(58) <Polypeptide Derived from WT1 Protein>

(59) The six polypeptides derived from WT1 protein shown in Table 6 were designed and chemically synthesized by PEPTIDE INSTITUTE, INC. to obtain polypeptides each having 95% or more purity.

(60) TABLE-US-00006 Polypeptidederived SEQ fromWT1protein aminoacidsequence IDNo: helperepitope4(HE4) KRYFKLSHLQMHSRKH 5 killerepitope1(KE1) RMFPNAPYL 40 killerepitope5(KF5) CYTWNQMNL 44 H/K-HELP1 KRYFKLSHLQMHSRKHGGG 46 GGRMFPNAPYL H/K-HELP2 KRYFKLSHLQMHSRKHGGG 47 GGRMFPNAPYLGGGGGCYT WNQMNL H/K-HELP3 RMFPNAPYLGGGGGKRYFK 48 LSHLQMHSRKHGGGGGCYT WNQMNL

(61) It is to be noted that helper/killer-hybridepitope longpeptide (H/K-HELP) 1 is a fused polypeptide wherein helper epitope (HE) 4 is bound to the N-terminus of killer epitope (KE) 1, wherein a linker polypeptide consisting of 5 glycine residues interposed therebetween. H/K-HELP 2 is a fused polypeptide wherein HE 4, KE 1, and KE 5 are bound in the order from the N-terminus, wherein a linker polypeptide consisting of 5 glycine residues interposed therebetween, H/K-HELP 3 is a fused polypeptide wherein KE 1, HE 4, and KE 5 are bound in the order from N-terminus, wherein a linker polypeptide consisting of 5 glycine residues interposed therebetween.

(62) Also, addition of PBMC to the culture medium described later was carried out by the following four embodiments.

(63) Mix (Comparative Example 1): a mixture of HE 4, KE 1, and KE 5 (each 5 M) was added to the medium.

(64) H/K-HELP 1 (Comparative Example 2): 5 M of H/K-HELP 1 was added to the medium.

(65) H/K-HELP 2 (Example 1): 5 M of H/K-HELP 2 was added to the medium.

(66) H/K-HELP 3 (Example 2): 5 M of H/K-HELP 3 was added to the medium.

(67) <Induction of Antigen-Specific Th Cells (CD4-Positive T Cells) and CTL (CD8-Positive T Cells)>

(68) The foregoing PBMCs separated from healthy human volunteers were seeded to a density of 210.sup.6 cells/well in a 24-well plate (manufactured by BD Biosciences). Then, culturing the cells were started in an AIM-V medium (1 ml/well) supplemented with each polypeptide (5 M) derived from the WT1 protein described above inducing antigen-specific T cells, and serum at 37 C. in a CO.sub.2 incubator.

(69) After 7 days from the start of culture, the above DC (autologous DC) was added to PBMC having the same derivation. Further, the medium was replaced with a serum-free AIM-V medium (manufactured by Life Technologies Inc.) supplemented with OK-432 (0.1 KE/ml, trade name: picibanil, manufactured by Chugai Pharmaceutical Co., Ltd.), and culture was performed for 2 hours. It should be noted that picibanil (OK-432) is a formulation obtained by treating an attenuated natural mutant strain (Su strain) of Group A Streptococcus with penicillin, and it is known that various cytokines (immune factor) are produced with use of picibanil to elicit an immune enhancing effect.

(70) Then, after 2 hours of culture, the cells were treated for 50 minutes with addition of mitomycin C (MMC, manufactured by Kyowa Hakko Kirin Co., Ltd.). Furthermore, co-culture was performed in an AIM-V medium containing autologous DC and 5% human AB serum in which T cells are inactivated (Hokkaido Red Cross Center) in the presence of each polypeptide (5 M) for inducing the antigen-specific T cells, thereby to re-stimulate the T cells. Two days later, a recombinant IL-2 (manufactured by Shionogi Pharmaceutical Institute Co., Ltd.) was added thereto to a final concentration of 10 U/ml. After 14 days from the start of co-culture, the autologous DC was pulsed with each polypeptide (5 M) for inducing the antigen-specific T cells for 2 hours, and then treated with MMC. Such MMC treated cells as APC were used for re-stimulating T cells. In addition, on or after 14 days from the start of co-culture, culture was performed at the concentration of 20 U/ml of recombinant IL-2. After that, T cells on day 21 of the co-culture were collected by centrifugation or the like and subjected to an analysis of antigen-specific reactivity shown below.

(71) <Analysis of Antigen-Specific Cytokine-Production of T-Cells by Intracellular Staining (ICS)>

(72) T cells that were induced as described above (T cells on day 21 of the co-culture) and autologous DCs were seeded in the same well of a 96-well plate (manufactured by BD Biosciences) to a density of 510.sup.4 cells/well and 510.sup.3 cells/well, respectively. Then, the cells were cultured in the presence of each polypeptide (HE 4, KE 1, KE 5, H/K-HELP 1, H/K-HELP 2 or H/K-HELP 3) (5 M) for the analysis of antigen-specific reactivity. In addition, a group without addition of such polypeptide was prepared as a control group.

(73) The intracellular cytokine staining was carried out in a conventional manner by first adding brefeldin A (manufactured by Sigma-Aldrich) at the initiation of culture to inhibit intracellular protein transport, thereby to accumulate cytokines intracellularly. Then, the cells cultured for 15 to 20 hours were stained with an anti-CD4 antibody and an anti-cytokine antibody in order to detect CD4+ T cells, and stained with an anti-CD8 antibody and an anti-cytokine antibody in order to detect CD8+ T cells, and analyzed by flow cytometry. Incidentally, FITC-labeled mouse anti-human CD4 mAb, PerCP-Cy7-labeled mouse anti-human IFN- mAb, and APC-labeled mouse anti-human CD8 mAb were used as an anti-CD4 antibody, an anti-cytokine antibody and an anti-CD8 antibody, respectively. Further, FACS Canto (trademark) II was used in the measurement, and FACS Diva (trademark) 6.1 was used in the analysis (both manufactured by BD Biosciences).

(74) Then, the ratio of antigen-specific IFN--producing cells in CD4-positive T cells (Th cells) was measured to select the highest ratio in the measured values in the presence of each polypeptide for analyzing the antigen-specific reactivity. Then, when this ratio is 2.5 times or more than the ratio in the control group (polypeptide non-addition group), and the difference in the ratio is 2% or more, such a case was evaluated as a case where T cells of which cytokine production was induced by the polypeptide for inducing antigen-specific T cells (the antigen-specific T cells) were induced. The ratio of antigen specific IFN--producing cells in the CD8-positive T cells (CTL) was also measured, and the induction of antigen-specific T cells was evaluated in the same manner as in the CD4-positive T cells described above. The results obtained are shown in Table 7. The symbol + in Table 7 indicates that the induction of antigen-specific T cells was observed, and the symbol indicates that the induction of antigen-specific T cells was not observed. The induction efficiency (%) shows the proportion of kinds of PBMCs that could be induced to the antigen-specific T cells, to 6 kinds of PBMCs in different donors that were subjected. Further, the best peptide indicates a polypeptide to induce antigen-specific T cells, wherein the ratio of antigen-specific IFN--producing cells was the highest in each PBMC (the difference in the ratio of antigen-specific IFN--producing cells between a polypeptide-addition group and a polypeptide non-addition group became largest). Furthermore, the best peptide (%) indicates the percentage of kinds of PBMCs in which the peptide has been evaluated as the best peptide in 6 kinds of PBMCs.

(75) TABLE-US-00007 TABLE 7 polypeptide used for the induction of antigen-specific T cell mix H/K-HELP1 peripheral blood induced T (comparative (comparative H/K-HELP2 H/K-HELP3 mononuclear cell cell example 1) example 2) (Example 1) (Example 2) best peptide donor 1 CD4 + + + H/K-HELP3 CD8 + H/K-HELP2 donor 2 CD4 + H/K-HELP2 CD8 donor 3 CD4 + + H/K-HELP2 CD8 + + + H/K-HELP3 donor 4 CD4 + H/K-HELP3 CD8 + H/K-HELP3 donor 5 CD4 + + + + H/K-HELP3 CD8 + + H/K-HELP3 donor 6 CD4 CD8 + H/K-HELP2 induction CD4 33.33 33.33 50.00 66.67 efficiency (%) CD8 16.67 16.67 50.00 50.00 best peptide (%) CD4 0.00 0.00 33.33 50.00 CD8 0.00 0.00 33.33 50.00 CD4 + CD8 0.00 0.00 33.33 50.00

(76) As is apparent from the results shown in Table 7, in the case of using the fused polypeptide of the present invention (Examples 1 to 2), the fused polypeptide could stimulate more numerous CD4-positive T cells and CD8-positive T cells of different derivation than in the case where epitopes making up the fused polypeptide of the present invention were used by mixing (Comparative Example 1), thereby to induce Th1 cells and CTLs producing cytokines (IFN-) (see CD4 and CD8 of induction efficiency (%) in Table 7). Further, in the case of using the fused polypeptide of the present invention, wherein killer epitopes are bound to both sides of a helper epitope (Example 2), more numerous CD4-positive T cells of different derivation are stimulated, as compared to Example 1, revealing that Th1 cells producing cytokines (IFN-) can be induced (see CD4 of induction efficiency (%) in Table 7). On the other hand, when the fused polypeptide wherein one killer peptide and one helper epitope are bound is used (Comparative Example 2), the activities of inducing antigen-specific T cells in CD4-positive T cells and CD8-positive T cells were not different from those of Comparative Example 1 (see CD4 and CD8 of induction efficiency (%) in Table 7).

(77) In addition, as shown in Table 7, in the case of using the fused polypeptides of the present invention (Examples 1 to 2), the induction efficiencies of cytokine-producing Th1 cells and CTLs are higher than those in Comparative Examples 1 and 2, i.e., it was revealed that immunological responses were more strongly induced (see best peptide in Table 7). Further, in the case of using the fused polypeptides of the present invention, wherein the killer epitopes are bound to both sides of the helper epitope (Example 2), it was revealed that, as compared to Example 1, more numerous CD4-positive T cells and CD8-positive T cells of different derivation could be stimulated to induce Th1 cells and CTLs producing cytokine (IFN-) (see best peptide (%) in Table 7).

INDUSTRIAL APPLICABILITY

(78) As described above, the present invention makes it possible to stimulate numerous T cells of different derivation, resulting in inducing strong immunological responses against many types of cancers.

(79) Thus, since the fused polypeptide of the present invention and the fused polypeptide and dendritic cells can stimulate T cells in a subject to strongly activate immunological responses by administration to the subject, the fused polypeptide and the fused polypeptide and dendritic cells are useful as a drug for active immunotherapy against cancers. Further, antigen-specific T cells induced by the fused polypeptide, and antigen-presenting cells pulsed with the fused polypeptide are useful as a drug or an anti-tumor agent for passive immunotherapy. These cells are preferably used together with the fused polypeptide.

SEQUENCE LISTING FREE TEXT

(80) SEQ ID NO: 1 <223> human WT1 protein SEQ ID NO: 2 <223> helper epitope 1 (HE1) SEQ ID NO: 3 <223> helper epitope 2 (HE2) SEQ ID NO: 4 <223:> helper epitope 3 (HE3) SEQ ID NO: <223> helper epitope 4 (HE4) SEQ ID NO: 6 <223> helper epitope 5 (HE5) SEQ ID NO: <223> helper epitope 6 (HE6) SEQ ID NO: 8 <223> helper epitope 7 (HE7) SEQ ID NO: 9 <223> helper epitope 8 (HE8) consisting of the amino acid sequence at positions 180-208 of the human WT1 protein wherein the 194th arginine is substituted with tyrosine. SEQ ID NO: 19 <223> helper epitope 9 (HE9) SEQ ID NO: 11 <223> helper epitope 10 (HE10) SEQ ID NO: 12 <223> helper epitope 11 (HE11) SEQ ID NO: 13 <223> helper epitope 12 (HE12) SEQ ID NO: 14 <223> helper epitope 13 (HE13) SEQ ID NO: 15 <223> helper epitope 14 (HE14) SEQ ID NO: 16 <223> helper epitope 15 (HE15) SEQ ID NO: 17 <223> helper epitope 16 (HE16) SEQ ID NO: 18 <223> helper epitope 17 (HE17) SEQ ID NO: 19 <223> helper epitope 18 (HE18) SEQ ID NO: 20 <223> helper epitope 19 (HE19) SEQ ID NO: 21 <223> helper epitope 20 (HE20) SEQ ID NO: 22 <223> helper epitope 21 (HE21) SEQ ID NO: 23 <223> helper epitope 22 (HE22) SEQ ID NO: 24 <223> helper epitope 23 (HE23) SEQ ID NO: 25 <223> helper epitope 24 (HE24) SEQ ID NO: 26 <223> helper epitope 25 (HE25) SEQ ID NO: 27 <223> helper epitope 26 (HE26) SEQ ID NO: 28 <223> helper epitope 27 (HE27) SEQ ID NO: 29 <223> helper epitope 28 (HE28) SEQ ID NO: 30 <223> helper epitope 29 (HE29) SEQ ID NO: 31 <223> helper epitope 30 (HE30) SEQ ID NO: 32 <223> helper epitope 31 (HE31) SEQ ID NO: 33 <223> helper epitope 32 (HE32) SEQ ID NO: 34 <223> helper epitope 33 (HE33) SEQ ID NO: 35 <223> helper epitope 34 (HE34) SEQ ID NO: 36 <223> helper epitope 35 (HE35) SEQ ID NO: 37 <223> helper epitope 36 (HE36) SEQ ID NO: 38 <223> helper epitope 37 (HE37) SEQ ID NO: 39 <223> helper epitope 38 (HE38) SEQ ID NO: 40 <223> killer epitope 1 (KE1) SEQ ID NO: 41 <223> killer epitope (KE2) SEQ ID NO: 42 <223> killer epitope 3 (KE3) consisting of the amino acid sequence at positions 194-202 of the human WT1 protein wherein the 194th arginine is substituted with tyrosine. SEQ ID NO: 43 <223> killer epitope 4 (KE4) SEQ ID NO: 44 <223> killer epitope 5 (KE5) consisting of the amino acid sequence at positions 303-311 of the human WT1 protein wherein the 304th methionine is substituted with tyrosine SEQ ID NO: 45 <223> killer epitope 6 (KE6) SEQ ID NO: 46 <223> fused polypeptide wherein HE4, linker consisting of five glycine residues and KE1 are bound in the order from N-terminus. SEQ ID NO: 47 <223> fusedpolypeptide wherein HE4, linker consisting of five glycine residues, KE1, linker polypeptide consisting of five glycine residues and KE5 are bound in the order from N-terminus. SEQ ID NO: 48 <223> fusedpolypeptide wherein KE1, linker consisting of five glycine residues, HE4, linker polypeptide consisting of five glycine residues and KE5 are bound in the order from N-terminus.