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  • A61K40/24—Antigen-presenting cells [APC]
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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  • C12N5/0634—Cells from the blood or the immune system
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  • A61K2039/515—Animal cells
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  • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase

Definitions

• the present invention relates to the field of biological science, more specifically to the field of cancer therapy.
• the present invention relates to novel peptides that are extremely effective as cancer vaccines, and drugs for treating and preventing tumors.
• CD8 positive CTLs recognize epitope peptides derived from the tumor-associated antigens (TAAs) found on major histocompatibility complex (MHC) class I molecules, and then kill the tumor cells.
• TAAs tumor-associated antigens
• MHC major histocompatibility complex
• MELK maternal embryonic leucine zipper kinase
• MELK is up-regulated in several cancer cells, for example lung, bladder, lymphoma and cervical cancer cells (See WO2004/031413, WO2007/013665, and WO2006/085684, the disclosures of which are incorporated by reference herein).
• Northern blot analysis on multiple human tissues and cancer cell lines demonstrated that MELK was over- expressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney) (WO2006/016525).
• siRNA suppression of MELK expression by siRNA was shown to significantly inhibit growth of human breast cancer cells. Accordingly, MELK is considered to be a suitable target for cancer immunotherapy and epitope peptides derived therefrom may be expected to serve as cancer immunotherapeutics effective in the treatment of a wide array of cancer types.
• the present invention is based in part on the discovery of suitable targets of immunotherapy. Because TAAs are generally perceived by the immune system as "self” and therefore often have no innate immunogenicity, the discovery of appropriate targets is of extreme importance. Recognizing that MELK has been identified as up- regulated in cancer tissues of breast cancer, bladder cancer, cervical cancer, cholangio- cellular carcinoma, chronic myeloid leukemia (CML), colorectal cancer, en- dometriosis, esophagus cancer, gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and small cell lung cancer (SCC), the present invention targets this maternal embryonic leucine zipper kinase (MELK) protein (SEQ ID NO: 94) encoded by the gene of GenBank Accession No.
• MML chronic myeloid leukemia
• SCCLC non-small cell lung cancer
• SCC renal carcinoma and small cell lung cancer
• NM_014791 SEQ ID NO: 93
• MELK gene products containing epitope peptides that elicit CTLs specific to the corresponding molecules were selected for study.
• Peripheral blood mononuclear cells (PBMCs) obtained from a healthy donor were stimulated using HLA-A*2402 or HLA-A*0201 binding candidate peptides derived from MELK.
• CTLs that specifically recognize HLA- A24 or HLA- A02 positive target cells pulsed with the respective candidate peptides were established, and HLA- A24 or HLA- A02 restricted epitope peptides that can induce potent and specific immune responses against MELK expressed on the surface of tumor cells were identified.
• the present invention contemplates modified peptides, having an amino acid sequence of SEQ ID NOs: 14, 21, 23, 27, 36, 46, 57, 60 or 62, wherein one, two or more amino acids are substituted, deleted, incorporated, and/or added, so long as the modified peptides retain the original CTL inducibility.
• the present peptides When administered to a subject, the present peptides are presented on the surface of antigen-expressing cells and then induce CTLs targeting the respective peptides. Therefore, it is an object of the present invention to provide antigen-presenting cells which present any of the present peptides, as well as methods for inducing antigen- presenting cells.
• An anti-tumor immune response is induced by the administration of the present MELK polypeptides or polynucleotide encoding the polypeptides, as well as exosomes and antigen-presenting cells which present the MELK polypeptides.
• the pharmaceutical agents of the present invention find use as vaccines.
• the CTLs of the invention also find use as vaccines against cancer.
• cancer examples include, but are not limited to breast cancer, bladder cancer, cervical cancer, cholangiocellular carcinomas, CML, colorectal cancer, endometriosis, esophagus cancer, gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and SCC.
• Figure 1 includes a series of photographs, (a) - (i) depicting the results of IFN- gamma ELISPOT assay on CTLs that were induced with peptides derived from MELK.
• Figure 2 includes a series of line graphs, (a) - (i), depicting the IFN-gamma production of CTL lines stimulated with SEQ ID NO: 14 (a), SEQ ID NO: 21 (b), SEQ ID NO: 23 (c), SEQ ID NO: 27 (d), SEQ ID NO: 36 (e), SEQ ID NO: 46 (f), SEQ ID NO: 57 (g), SEQ ID NO: 60 (h) and SEQ ID NO: 62 (i) with IFN-gamma ELISA assay.
• CTL lines established by stimulation with each peptide showed potent IFN- gamma production as compared with the control.
• "+" indicates that the target cells were pulsed with the appropriate peptide and "-" indicates that the target cells had not been pulsed with any peptides.
• FIG.3 Figure 3 depicts the IFN-gamma production of the CTL clone established by limiting dilution from the CTL line stimulated with SEQ ID NO: 27.
• the results depicted herein demonstrate that the CTL clone established by stimulation with SEQ ID NO: 27 showed potent IFN-gamma production as compared with the control.
• "+” indicates that the target cells were pulsed with SEQ ID NO: 27 and "-" indicates that the target cells had not been pulsed with any peptides.
• Figure 4 is composed of line graphs (a) and (b) depicting specific CTL activity of the CTL clones established with MELK-A02-9-138 (SEQ ID NO:36) (a) or MELK- A02-9-171 (SEQ ID NO: 57) (b) against target cells that exogenously express MELK and HLA-A*0201.
• the target cells were prepared by co-transfecting COS7 cells with the full length of both MELK and HLA-A*0201 molecule gene (-closed diamond-), and the control target cells were prepared by transfecting COS7 cells with HLA- A*0201 molecule gene and pulsing with an inappropriate peptide that is different from the peptides with which the CTL clones were established (-open triangle-), or transfecting COS7 cells with MELK gene (-open circle-).
• polypeptide polypeptide
• peptide protein
• protein protein
• amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly function to the naturally occurring amino acids.
• Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine).
• amino acid analog refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium).
• modified R group or modified backbones e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium.
• amino acid mimetic refers to chemical compounds that have different structures but similar functions to general amino acids.
• Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
• cancer refers to the cancers over-expressing the MELK gene, examples of which include, but are not limited to, breast cancer, bladder cancer, cervical cancer, cholangiocellular carcinoma, chronic myeloid leukemia (CML), colorectal cancer, endometriosis, esophagus cancer, gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and small cell lung cancer (SCC)s.
• CML chronic myeloid leukemia
• NSCLC non-small cell lung cancer
• SCC small cell lung cancer
• cytotoxic T lymphocyte refers to a sub-group of T lymphocytes that are capable of recognizing non-self cells (e.g., tumor cells, virus-infected cells) and inducing the death of such cells.
• non-self cells e.g., tumor cells, virus-infected cells
• the present invention provides non- apeptides (peptides consisting of nine amino acid residues) and decapeptides (peptides consisting of ten amino acid residues) corresponding to CTL-recognized epitopes of MELK.
• nonapeptides and decapeptides of the present invention include those peptides consisting of the amino acid sequence selected from among SEQ ID NOs: 14, 21, 23, 27, 36, 46, 57, 60 and 62.
• the nonapeptides and decapeptides of the present invention can be flanked with additional amino acid residues so long as the resulting peptide retains its CTL in- ducibility.
• Such peptides having CTL inducibility are typically less than about 40 amino acids, often less than about 20 amino acids, usually less than about 15 amino acids.
• the particular amino acid sequences flanking the nonapeptide and decapeptides of the present invention e.g., peptides consisting of the amino acid sequence selected from among SEQ ID NOs: 14, 21, 23, 27, 36, 46, 57, 60 and 62 is not limited and can be composed of any kind of amino acids so long as it does not impair the CTL inducibility of the original peptide.
• the present invention also provides peptides having CTL inducibility and including the amino acid sequence selected from among SEQ ID NOs: 14, 21, 23, 27, 36, 46, 57, 60 and 62.
• modified peptides i.e., peptides composed of an amino acid sequence in which one, two or several amino acid residues have been modified (i.e., substituted, deleted, added or inserted as compared to an original reference sequence) have been known to retain the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13).
• the peptides of the present invention may have both CTL inducibility and an amino acid sequence selected from among SEQ ID NOs: 14, 21, 23, 27, 36, 46, 57, 60 and 62 wherein one, two or even more amino acids are added, inserted, deleted and/or substituted.
• amino acid side chain characteristics that are desirable to conserve include, for example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side- chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W).
• the following eight groups each contain amino acids that are accepted in the art as conservative substitutions for one another:
• Such conservatively modified peptides are also considered to be peptides of the present invention.
• peptides of the present invention are not restricted thereto and can include non-conservative modifications, so long as the modified peptide retains the CTL inducibility of the original peptide.
• modified peptides should not exclude CTL inducible peptides of polymorphic variants, interspecies ho- mologues, and alleles of MELK.
• a small number for example, 1, 2 or several
• a small percentage of amino acids for example, 1, 2 or several
• the term “several” means 5 or fewer amino acids, for example, 4 or 3 or fewer.
• the percentage of amino acids to be modified is preferably 20% or less, more preferably 15% or less, even more preferably 10% or less or 1 to 5%.
• these peptides are expected to be highly useful for eliciting immunity in tumor patients against MELK on cancer cells, such as breast cancer, bladder cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophagus cancer, gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and SCC.
• cancer cells such as breast cancer, bladder cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophagus cancer, gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and SCC.
• peptides of the present invention When used in the context of immunotherapy, peptides of the present invention should be presented on the surface of a cell or exosome, preferably as a complex with an HLA antigen. Therefore, one is preferable to select peptides that not only induce CTLs but also possess high binding affinity to the HLA antigen. To that end, the peptides can be modified by substitution, insertion, deletion, and/or addition of the amino acid residues to yield a modified peptide having improved binding affinity.
• peptides possessing high HLA- A02 binding affinity have their second amino acid from the N- terminus substituted with leucine or methionine, and/or the amino acid at C-terminus substituted with valine or leucine.
• peptides having the amino acid sequences selected from among SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 57, SEQ ID NO: 60 and SEQ ID NO: 62 wherein the second amino acid from the N-terminus is substituted with leucine or methionine, and/or wherein the C-terminus is substituted with valine or leucine are encompassed by the present invention.
• Substitutions can be introduced not only at the terminal amino acids but also at the position of potential TCR recognition of peptides.
• amino acid substitutions in a peptide can be equal to or better than the original, for example CAPl, p53 (264 - 272 ) , Her-2/neu ( 369 . 377 ) or gpl00 (209 - 2 i 7 ) (Zaremba et al. Cancer Res. 57, 4570-4577, 1997, T. K. Hoffmann et al. J Immunol. (2002) Feb 1; 168(3): 1338-47., S. O. Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206 and S. O. Dionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-314).
• the present invention also contemplates the addition of one to two amino acids to the N and/or C-terminus of the described peptides.
• modified peptides having high HLA antigen binding affinity and retained CTL inducibility are also included in the present invention.
• the peptide sequence is identical to a portion of the amino acid sequence of an endogenous or exogenous protein having a different function, side effects such as autoimmune disorders and/or allergic symptoms against specific substances may be induced. Therefore, it is preferable to first perform homology searches using available databases to avoid situations in which the sequence of the peptide matches the amino acid sequence of another protein.
• the objective peptide can be modified in order to increase its binding affinity with HLA antigens, and/or increase its CTL in- ducibility without any danger of such side effects.
• CTL inducibility indicates the ability of the peptide to induce cytotoxic lymphocytes (CTLs) when presented on antigen-presenting cells.
• CTL inducibility includes the ability of the peptide to induce CTL activation, CTL proliferation, promote CTL lysis of target cells, and to increase CTL IFN-gamma production.
• CTL inducibility is accomplished by inducing antigen-presenting cells carrying human MHC antigens (for example, B -lymphocytes, macrophages, and dendritic cells (DCs)) by stimulating with the peptides, mixing the induced antigen- presenting cells with CD8-positive cells, and then measuring the IFN-gamma produced and released by CTL against the target cells.
• human MHC antigens for example, B -lymphocytes, macrophages, and dendritic cells (DCs)
• DCs dendritic cells
• transgenic animals that have been produced to express a human HLA antigen (for example, those described in BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000 Aug, 61(8): 764-79, Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DRl transgenic mice: dependence on HLA class II restricted T(H) response) can be used.
• the target cells can be radio-labeled with 51 Cr and such, and cytotoxic activity can be calculated from radioactivity released from the target cells.
• CTL inducibility can be assessed by measuring IFN-gamma produced and released by CTL in the presence of antigen-presenting cells (APCs) that carry immobilized peptides, and visualizing the inhibition zone on the media using anti-IFN-gamma monoclonal antibodies.
• APCs antigen-presenting cells
• the peptides of the present invention can also be linked to other substances, so long as the resulting linked peptide retains the requisite CTL inducibility of the original peptide.
• suitable substances include, but are not limited to: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc.
• the peptides can contain modifications such as glycosylation, side chain oxidation, or phosphorylation, etc., provided the modifications do not destroy the biological activity of the original peptide. These kinds of modifications can be performed to confer additional functions (e.g., targeting function, and delivery function) or to stabilize the polypeptide.
• polypeptides For example, to increase the in vivo stability of a polypeptide, it is known in the art to introduce D-amino acids, amino acid mimetics or unnatural amino acids; this concept can also be adapted to the present polypeptides.
• the stability of a polypeptide can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, can be used to test stability (see, e.g., Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).
• the peptides of the present invention may be linked to other peptides via spacers or linkers.
• other peptides include, but are not limited to, CTL inducible peptides derived from other TAAs.
• two or more peptides of the present invention may be linked via spacers or linkers.
• the peptides linked via spacers or linkers may be the same or different each other.
• Spacers or linkers are not specifically limited, but are preferably peptides, more preferably peptides having one or more cleavage sites which are capable of being cleaved by enzymes such as peptidases, proteases and proteasomes.
• linkers or spacers include, but are not limited to: AAY (P. M. Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (R. P. M. Sutmuller et al., J Immunol. 2000, 165: 7308-7315) or, one to several lysine residues (S. Ota et al., Can Res. 62, 1471-1476, K. S. Kawamura et al., J Immunol. 2002, 168: 5709-5715).
• the peptides of the present invention encompass those peptides linked to other peptides via spacers or linkers.
• the peptides of the present invention may be existed on the surface of a cell carrying human MHC antigens (e.g. antigen presenting cell) or an exosome as complexes in combination with MHC molecules and then induce CTLs.
• the cells and the exosomes can be prepared by well-known methods in the art, for example, the cells may be prepared by contacting with the peptides of the present invention, and the exosomes may be prepared by collecting an exosome-containing fraction from the cells contacted with the peptides of the present invention (see, e.g., Japanese Patent Application Kohyo Publications Nos. Hei 11-510507 and WO99/03499).
• the peptide of the present invention encompass those peptides existed on the surface of a cell or an exosome as complexes in combination with MHC molecules. [0043] Herein, the peptides of the present invention can also be described as "MELK peptide(s)" or "MELK polypeptide(s)”. [0044] III. Preparation of MELK peptides
• the peptides of the present invention can be prepared using well known techniques.
• the peptides can be prepared synthetically, using recombinant DNA technology or chemical synthesis.
• Peptide of the invention can be synthesized individually or as longer polypeptides composed of two or more peptides.
• the peptides can then be isolated i.e., purified or isolated so as to be substantially free of other naturally occurring host cell proteins and fragments thereof, or any other chemical substances.
• a peptide of the present invention can be obtained through chemical synthesis based on the selected amino acid sequence. Examples of conventional peptide synthesis methods that can be adapted to the synthesis include, but are not limited to:
• the present peptides can be obtained adapting any known genetic engineering methods for producing peptides (e.g., Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62).
• a suitable vector harboring a polynucleotide encoding the objective peptide in an expressible form e.g., downstream of a regulatory sequence corresponding to a promoter sequence
• the host cell is then cultured to produce the peptide of interest.
• the peptide can also be produced in vitro adopting an in vitro translation system.
• the present invention also provides a polynucleotide which encodes any of the aforementioned peptides of the present invention.
• polynucleotides derived from the natural occurring MELK gene (GenBank Accession No. NM_014791 (SEQ ID NO: 93)) as well as those having a conservatively modified nucleotide sequence thereof.
• conservatively modified nucleotide sequence refers to sequences which encode identical or essentially identical amino acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
• nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
• Every nucleic acid sequence herein which encodes a peptide also describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
• TGG which is ordinarily the only codon for tryptophan
• the polynucleotide of the present invention can be composed of DNA, RNA, and derivatives thereof.
• a DNA is suitably composed of bases such as A, T, C, and G, and T is replaced by U in an RNA.
• the polynucleotide of the present invention can encode multiple peptides of the present invention, with or without intervening amino acid sequences in between.
• the intervening amino acid sequence can provide a cleavage site (e.g., enzyme recognition sequence) of the polynucleotide or the translated peptides.
• the polynucleotide can include any additional sequences to the coding sequence encoding the peptide of the present invention.
• the polynucleotide can be a recombinant polynucleotide that includes regulatory sequences required for the expression of the peptide or can be an expression vector (plasmid) with marker genes and such.
• such recombinant polynucleotides can be prepared by the manipulation of polynucleotides through conventional recombinant techniques using, for example, polymerases and endonucleases.
• a polynucleotide can be produced by insertion into an appropriate vector, which can be expressed when transfected into a competent cell.
• a polynucleotide can be amplified using PCR techniques or expression in suitable hosts (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989).
• a polynucleotide can be synthesized using the solid phase techniques, as described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.
• Vectors containing the polynucleotide of the present invention and host cells harboring the vectors are also included in the present invention.
• the present invention further provides intracellular vesicles called exosomes, which present complexes formed between the peptides of this invention and HLA antigens on their surface.
• Exosomes can be prepared, for example by using the methods detailed in Japanese Patent Application Kohyo Publications Nos. Hei 11-510507 and WO99/03499, and can be prepared using APCs obtained from patients who are subject to treatment and/or prevention.
• the exosomes of this invention can be inoculated as vaccines, in a fashion similar to the peptides of this invention.
• the type of HLA antigens contained in the complexes must match that of the subject requiring treatment and/or prevention.
• HLA- A24 particularly HLA-A2402
• the use of the A24 type or the A02 type that is highly expressed among the Japanese and Caucasian is favorable for obtaining effective results, and subtypes such as A2402 or A0201 also find use.
• the type of HLA antigen of the patient requiring treatment is investigated in advance, which enables the appropriate selection of peptides having high levels of binding affinity to the particular antigen, or having CTL inducibility by antigen presentation.
• substitution, insertion and/or addition of 1, 2, or several amino acids can be performed based on the amino acid sequence of the naturally occurring MELK partial peptide.
• the peptides having the amino acid sequences selected from among SEQ ID NOs: 14, 21, 23 and 27 are useful.
• the peptides having the sequences selected from among SEQ ID NO: 36, 46, 57, 60 and 62 are preferred.
• the present invention also provides isolated APCs that present complexes formed between HLA antigens and the peptides of this invention on its surface.
• the APCs that are obtained by contacting the peptides of this invention, or introducing the nucleotides encoding the peptides of this invention in an expressible form can be derived from patients who are subject to treatment and/or prevention, and can be administered as vaccines by themselves or in combination with other drugs including the peptides of this invention, exosomes, or cytotoxic T cells.
• the APCs are not limited to a particular kind of cells and include dendritic cells
• DCs Langerhans cells, macrophages, B cells, and activated T cells, which are known to present proteinaceous antigens on their cell surface so as to be recognized by lymphocytes. Since DC is a representative APC having the strongest CTL inducing action among APCs, DCs find use as the APCs of the present invention.
• an APC can be obtained by inducing DCs from peripheral blood monocytes and then contacting (stimulating) them with the peptides of this invention in vitro, ex vivo or in vivo.
• APCs that present the peptides of this invention are induced in the body of the subject.
• the phrase "inducing APC” includes contacting (stimulating) a cell with the peptides of this invention, or nucleotides encoding the peptides of this invention to present complexes formed between HLA antigens and the peptides of this invention on cell's surface.
• the APCs can be administered to the subject as a vaccine.
• the ex vivo administration can include the steps of: a: collecting APCs from a first subject, b: contacting with the APCs of step a, with the peptide and c: administering the peptide-loaded APCs to a second subject.
• the first subject and the second subject can be the same individual, or may be different individuals.
• use of the peptides of the present invention for manufacturing a pharmaceutical composition inducing antigen-presenting cells is provided.
• the present invention provides a method or process for manufacturing a pharmaceutical composition inducing antigen-presenting cells, wherein the method or the process includes the step of admixing or formulating the peptide of the present invention with a pharmaceutically acceptable carrier.
• the present invention also provides the peptides of the present invention for inducing antigen-presenting cells.
• the APCs obtained by step (b) can be administered to the subject as a vaccine.
• the APCs of the present invention have a high level of CTL inducibility.
• the high level is relative to the level of that by APCs contacted with no peptide or peptides which can not induce the CTL.
• Such APCs having a high level of CTL inducibility can be prepared by a method which includes the step of transferring genes containing polynucleotides that encode the peptides of this invention to APCs in vitro.
• the introduced genes can be in the form of DNAs or RNAs.
• Examples of methods for introduction include, without particular limitations, various methods conventionally performed in this field, such as lipofection, electroporation, and calcium phosphate method can be used. More specifically, it can be performed as described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; Published Japanese Translation of International Publication No. 2000-509281.
• the gene undergoes transcription, translation, and such in the cell, and then the obtained protein is processed by MHC Class I or Class II, and proceeds through a presentation pathway to present peptides.
• CTLs Cytotoxic T cells
• a cytotoxic T cell induced against any of the peptides of the present invention strengthens the immune response targeting tumor- associated endothelia in vivo and thus can be used as vaccines, in a fashion similar to the peptides per se.
• the present invention also provides isolated cytotoxic T cells that are specifically induced or activated by any of the present peptides.
• Such cytotoxic T cells can be obtained by (1) administering the peptide of the present invention to a subject and then collecting cytotoxic T cells from the subject, or (2) contacting (stimulating) subject-derived APCs and CD8-positive cells, or peripheral blood mononuclear leukocytes in vitro with the peptides of the present invention and then isolating cytotoxic T cells.
• the cytotoxic T cells which have been induced by stimulation with APCs that present the peptides of this invention, can be derived from patients who are subject to treatment and/or prevention, and can be administered by themselves or in combination with other drugs including the peptides of this invention or exosomes for the purpose of regulating effects.
• the obtained cytotoxic T cells act specifically against target cells presenting the peptides of this invention, or for example, the same peptides used for induction.
• the cytotoxic T cells can recognize (i.e., binding to) a complex formed between a HLA antigen and the peptide of the present invention on a target cell surface with the T cell receptor and then attack the target cell to induce the death of the target cell.
• the target cells can be cells that endogenously express MELK, or cells that are transfected with the MELK gene; and cells that present a peptide of this invention on the cell surface due to stimulation by the peptide can also serve as targets of activated CTL attack.
• T cell receptor TCR
• the present invention also provides a composition containing nucleic acids sequence encoding polypeptides that are capable of forming a subunit of a T cell receptor (TCR), and methods of using the same.
• TCR subunits have the ability to form TCRs that confer specificity to T cells against tumor cells presenting MELK.
• the nucleic acid sequence of alpha- and beta- chains of the TCR expressed in the CTL induced with a peptide of the present invention can be identified (WO2007/032255 and Morgan et al., J Immunol, 171, 3288 (2003)).
• the derivative TCRs can bind to the MELK peptide displaying on the target cells with high avidity, and optionally mediate efficient killing of target cells presenting the MELK peptide in vivo and in vitro.
• the nucleic acids sequence encoding the TCR subunits can be incorporated into suitable vectors e.g. retroviral vectors. These vectors are well known in the art.
• the nucleic acids or the vectors containing them usefully can be transferred into a T cell, for example, a T cell from a patient.
• the invention provides an off- the-shelf composition allowing rapid modification of a patient's own T cells (or those of another mammal) to rapidly and easily produce modified T cells having excellent cancer cell killing properties.
• the present invention provides CTLs which are prepared by transduction with the nucleic acids encoding the TCR subunits polypeptides that bind to the MELK peptide e.g. SEQ ID NOs: 14, 21, 23, 27, 36, 46, 57, 60 and 62 in the context of HLA- A24 or HLA- A02.
• the transduced CTLs are capable of homing to cancer cells in vivo, and can be expanded by well known culturing methods in vitro (e.g., Kawakami et al., J Immunol., 142, 3452-3461 (1989)).
• the T cells of the invention can be used to form an immunogenic composition useful in treating or the prevention of cancer in a patient in need of therapy or protection (WO2006/031221).
• Prevention and prophylaxis include any activity which reduces the burden of mortality or morbidity from disease. Prevention and prophylaxis can occur "at primary, secondary and tertiary prevention levels.” While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease- related complications. Alternatively, prevention and prophylaxis include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
• Treating and/or the prophylaxis of cancer or tumor, and/or the prevention of postoperative recurrence thereof includes any of the following steps, such as surgical removal of cancer cells, inhibition of the growth of cancerous cells, involution or regression of a tumor, induction of remission and suppression of occurrence of cancer, tumor regression, and reduction or inhibition of metastasis.
• Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer.
• reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis includes 10%, 20%, 30% or more reduction, or stable disease.
• the peptides of this invention or polynucleotides encoding such peptides can be used for the treatment and/or for the prophylaxis of cancer or tumor, and/or prevention of postoperative recurrence thereof.
• the present invention provides a pharmaceutical agent or composition for treating and/or for the prophylaxis of cancer or tumor, and/or prevention of postoperative recurrence thereof, which includes one or more of the peptides of this invention, or polynucleotides encoding the peptides as an active ingredient.
• the present peptides can be expressed on the surface of any of the foregoing exosomes or cells, such as APCs for the use as pharmaceutical agents or compositions.
• the aforementioned cytotoxic T cells which target any of the peptides of the present invention can also be used as the active ingredient of the present pharmaceutical agents or compositions.
• the phrase "targeting a peptide” refers to recognizing (i.e., binding to) a complex formed between a HLA antigen and a peptide on a target cell surface with the T cell receptor, and then attacking the target cell to induce the death of the target cell.
• the present invention also provides the use of an active ingredient selected from among:
• cytotoxic T cell of the present invention in manufacturing a pharmaceutical composition or agent for treating cancer or tumor.
• the present invention further provides an active ingredient selected from among:
• a cytotoxic T cells of the present invention for use in for treating cancer or tumor.
• the present invention further provides a method or process for manufacturing a pharmaceutical composition or agent for treating cancer or tumor, wherein the method or process includes the step of formulating a pharmaceutically or physiologically acceptable carrier with an active ingredient selected from among:
• the present invention also provides a method or process for manufacturing a pharmaceutical composition or agent for treating cancer or tumor, wherein the method or process includes the step of admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is selected from among:
• the pharmaceutical composition or agent of the present invention may be used for either or both the prophylaxis of cancer or tumor and prevention of postoperative recurrence thereof.
• the present pharmaceutical agents or compositions find use as a vaccine.
• the phrase "vaccine” also referred to as an “immunogenic composition” refers to a substance that has the function to induce antitumor immunity upon inoculation into animals.
• the pharmaceutical agents or compositions of the present invention can be used to treat and/or prevent cancers or tumors, and/or prevention of postoperative recurrence thereof in subjects or patients including human and any other mammal including, but not limited to, mouse, rat, guinea-pig, rabbit, cat, dog, sheep, goat, pig, cattle, horse, monkey, baboon, and chimpanzee, particularly a commercially important animal or a domesticated animal.
• polypeptides having an amino acid sequence selected from among SEQ ID NOs: 14, 21, 23 and 27 or polypeptides having an amino acid sequence selected from among SEQ ID NOs: 36, 46, 57, 60 and 62 have been found to be HLA- A24 or HLA- A02 restricted epitope peptides or candidates, respectively, that can induce potent and specific immune response. Therefore, the present pharmaceutical agents or compositions which include any of these polypeptides having the amino acid sequences selected from among of SEQ ID NOs: 14, 21, 23 and 27 are particularly suited for the administration to subjects whose HLA antigen is HLA- A24.
• the present pharmaceutical agents or compositions which contain any of these polypeptides having the amino acid sequences of SEQ ID NOs: 36, 46, 57, 60 and 62 are particularly suited for the administration to subjects whose HLA antigen is HLA- A02.
• pharmaceutical agents which include polynucleotides encoding any of these polypeptides are particularly suited for the administration to subjects whose HLA antigen is HLA- A02.
• Cancers or tumors to be treated by the pharmaceutical agents or compositions of the present invention are not limited and include all kinds of cancers or tumors wherein MELK is involved, including for example, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophagus cancer, gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and SCC.
• the present pharmaceutical agents or compositions can contain in addition to the aforementioned active ingredients, other peptides which have the ability to induce CTLs against cancerous cells, other polynucleotides encoding the other peptides, other cells that present the other peptides, or such.
• the other peptides that have the ability to induce CTLs against cancerous cells are exemplified by cancer specific antigens (e.g., identified TAAs), but are not limited thereto.
• the pharmaceutical agents or compositions of the present invention can optionally include other therapeutic substances as an active ingredient, so long as the substance does not inhibit the antitumoral effect of the active ingredient, e.g., any of the present peptides.
• formulations can include anti-inflammatory agents, pain killers, chemotherapeutics, and the like.
• the medicaments of the present invention can also be administered sequentially or concurrently with the one or more other pharmacologic agents.
• the amounts of medicament and pharmacologic agent depend, for example, on what type of pharmacologic agent(s) is/are used, the disease being treated, and the scheduling and routes of administration.
• compositions of this invention can include other agents conventional in the art having regard to the type of formulation in question.
• the present pharmaceutical agents or compositions can be included in articles of manufacture and kits containing materials useful for treating the pathological conditions of the disease to be treated, e.g, cancer.
• the article of manufacture can include a container of any of the present pharmaceutical agents or compositions with a label. Suitable containers include bottles, vials, and test tubes. The containers can be formed from a variety of materials, such as glass or plastic.
• the label on the container should indicate the agent is used for treating or prevention of one or more conditions of the disease.
• the label can also indicate directions for administration and so on.
• kits including a pharmaceutical agent or composition of the present invention can optionally further include a second container housing a pharmaceutically-acceptable diluent. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
• the pharmaceutical agents or compositions can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient.
• the pack can, for example, include metal or plastic foil, such as a blister pack.
• the pack or dispenser device can be accompanied by instructions for administration.
• the peptides of this invention can be administered directly as a pharmaceutical agent or composition, or if necessary, that has been formulated by conventional formulation methods.
• carriers, ex- cipients, and such that are ordinarily used for drugs can be included as appropriate without particular limitations. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid and such.
• the pharmaceutical agents or compositions can contain as necessary, stabilizers, suspensions, preservatives, surfactants and such.
• the pharmaceutical agents or compositions of this invention can be used for anticancer purposes.
• the peptides of this invention can be prepared as a combination, composed of two or more of peptides of the present invention, to induce CTLs in vivo.
• the peptide combination can take the form of a cocktail or can be conjugated to each other using standard techniques.
• the peptides can be chemically linked or expressed as a single fusion polypeptide sequence.
• the peptides in the combination can be the same or different.
• APCs that present any of the peptides of this invention on their cell surface which may be obtained by stimulating APCs (e.g., DCs) derived from a subject with the peptides of this invention, may be administered to the subject, and as a result, CTLs are induced in the subject and aggressiveness towards the cancer cells, such as bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophagus cancer, gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and SCC cells, can be increased.
• APCs e.g., DCs
• CTLs are induced in the subject and aggressiveness towards the cancer cells, such as bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophagus cancer, gastric cancer, liver cancer, NSC
• the pharmaceutical agents or compositions for the treatment and/or prevention of cancer or tumor which include a peptide of this invention as the active ingredient, can also include an adjuvant known to effectively cellular immunity.
• the pharmaceutical agents or compositions can be administered with other active ingredients or administered by formulation into granules.
• An adjuvant refers to a compound that enhances the immune response against the protein when administered together (or successively) with the protein having immunological activity.
• Adjuvants contemplated herein include those described in the literature (Clin Microbiol Rev 1994, 7: 277-89). Examples of suitable adjuvants include aluminum phosphate, aluminum hydroxide, alum, cholera toxin, salmonella toxin, and such, but are not limited thereto.
• liposome formulations may be conveniently used.
• granular formulations in which the peptide is bound to few-micrometers diameter beads, and formulations in which a lipid is bound to the peptide may be conveniently used.
• the pharmaceutical agents or compositions of the present invention may further include a component which primes CTLs.
• Lipids have been identified as agents capable of priming CTLs in vivo against viral antigens.
• palmitic acid residues can be attached to the epsilon- and alpha-amino groups of a lysine residue and then linked to a peptide of the present invention.
• the lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant.
• lipid priming of CTLs responses E.
• coli lipoproteins such as tripalmitoyl-S-glycerylcysteinlyseryl- serine (P3CSS) can be used to prime CTL when covalently attached to an appropriate peptide (see, e.g., Deres et al., Nature 1989, 342: 561-4).
• P3CSS tripalmitoyl-S-glycerylcysteinlyseryl- serine
• the method of administration can be oral, intradermal, subcutaneous, intravenous injection, or such, and systemic administration or local administration to the vicinity of the targeted sites.
• the administration can be performed by single administration or boosted by multiple administrations.
• the dose of the peptides of this invention can be adjusted appropriately according to the disease to be treated, age of the patient, weight, method of administration, and such, and is ordinarily 0.001 mg to 1000 mg, for example, 0.001 mg to 1000 mg, for example, 0.1 mg to 10 mg, and can be administered once in a few days to few months.
• One skilled in the art can appropriately select a suitable dose.
• the pharmaceutical agents or compositions of the present invention can also contain nucleic acids encoding the peptides disclosed herein in an expressible form.
• the phrase "in an expressible form” means that the polynucleotide, when introduced into a cell, will be expressed in vivo as a polypeptide that induces anti-tumor immunity.
• the nucleic acid sequence of the polynucleotide of interest includes regulatory elements necessary for expression of the polynucleotide.
• the polynucleotide(s) can be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.
• DNA-based delivery technologies include "naked DNA”, facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
• the peptides of the present invention can also be expressed by viral or bacterial vectors.
• expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the peptide. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response.
• Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848. Examples of another vector include BCG (Bacille Calmette Guerin).
• BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
• a wide variety of other vectors useful for therapeutic administration or immunization e.g., adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent. See, e.g., Shata et al., MoI Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo 2000, 14: 571-85.
• Delivery of a polynucleotide into a subject can be either direct, in which case the subject is directly exposed to a polynucleotide-carrying vector, or indirect, in which case, cells are first transformed with the polynucleotide of interest in vitro, then the cells are transplanted into the subject.
• two approaches are known, respectively, as in vivo and ex vivo gene therapies.
• the method of administration can be oral, intradermal, subcutaneous, intravenous injection, or such, and systemic administration or local administration to the vicinity of the targeted sites finds use.
• the administration can be performed by single administration or boosted by multiple administrations.
• the dose of the polynucleotide in the suitable carrier or cells transformed with the polynucleotide encoding the peptides of this invention can be adjusted appropriately according to the disease to be treated, age of the patient, weight, method of administration, and such, and is ordinarily 0.001 mg to 1000 mg, for example, 0.001 mg to 1000 mg, for example, 0.1 mg to 10 mg, and can be administered once every a few days to once every few months.
• One skilled in the art can appropriately select the suitable dose.
• the peptides of the present invention and polynucleotides encoding such peptides can be used for inducing APCs and CTLs.
• the exosomes and APCs of the present invention can be also used for inducing CTLs.
• the peptides, polynucleotides, exosomes and APCs can be used in combination with any other compounds so long as the compounds do not inhibit their CTL inducibility.
• any of the aforementioned pharmaceutical agents or compositions of the present invention can be used for inducing CTLs, and in addition thereto, those including the peptides and polynucleotides can be also be used for inducing APCs as discussed below.
• the present invention provides methods of inducing APCs using the peptides of this invention or polynucleotides encoding the peptides.
• the induction of APCs can be performed as described above in section "VI. Antigen-presenting cells”.
• This invention also provides a method for inducing APCs having a high level of CTL inducibility, the induction of which has been also mentioned under the item of "VI. Antigen-presenting cells", supra.
• the methods for inducing APCs include at least one step selected from among: a: contacting APCs with the peptides of the present invention, and b: introducing the polypeptides of the present invention in an expressible form into APCs.
• Such methods for inducing APCs are preferably performed in vitro or ex vivo.
• APCs to be induced may be obtained from a subject to be treated or others whose HLA antigens are the same as the subject.
• the present invention provides methods for inducing CTLs using the peptides of this invention, polynucleotides encoding the peptides, or exosomes or APCs presenting the peptides.
• the present invention also provides methods for inducing CTLs using a polynucleotide encoding a polypeptide that is capable of forming a T cell receptor (TCR) subunit recognizing (i.e., binding to) a complex of the peptides of the present invention and HLA antigens on a cell surface.
• TCR T cell receptor
• the methods for inducing CTLs include at least one step selected from among: a: contacting a CD8-positive T cell with an antigen-presenting cell and/or an exosome that presents on its surface a complex of an HLA antigen and a peptide of the present invention, and b: introducing a polynucleotide encoding a polypeptide that is capable of forming a TCR subunit recognizing a complex of a peptide of the present invention and an HLA antigen into a CD8 positive T cell.
• the peptides of the present invention are administered to a subject, CTLs are induced in the body of the subject, and the strength of the immune response targeting the tumor-associated endothelia is enhanced.
• the peptides and polynucleotides encoding the peptides can be used for an ex vivo therapeutic method, in which subject-derived APCs and CD8-positive cells, or peripheral blood mononuclear leukocytes are contacted (stimulated) with the peptides of this invention in vitro, and after inducing CTLs, the activated CTL cells are returned to the subject.
• the method can include steps of: a: collecting APCs from subject, b: contacting with the APCs of step a, with the peptide, c: mixing the APCs of step b with CD 8+ T cells, and co-culturing for inducing CTLs, and d: collecting CD 8+ T cells from the co-culture of step c.
• the present invention provides a method or process for manufacturing a pharmaceutical agent or composition inducing CTLs, wherein the method includes the step of admixing or formulating the peptide of the present invention with a pharmaceutically acceptable carrier. Further, the present invention also provides the peptide of the present invention for inducing CTLs.
• the CD 8+ T cells having cytotoxic activity obtained by step d can be administered to the subject as a vaccine.
• the APCs to be mixed with the CD 8+ T cells in above step c can also be prepared by transferring genes coding for the present peptides into the APCs as detailed above in section "VI. Antigen-presenting cells"; but are not limited thereto. Accordingly, any APCs or exosomes which effectively presents the present peptides to the T cells can be used for the present method.
• A24 lymphoblastoid cell line was established by transformation with Epstein-bar virus into HLA- A24 positive human B lymphocyte.
• T2 HLA- A2
• human B -lymphoblastoid cell line and COS7 were purchased from ATCC.
• DCs Monocyte-derived dendritic cells
• APCs antigen-presenting cells
• CTL cytotoxic T lymphocyte
• HLA human leukocyte antigen
• DCs were generated in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003 JuI 15, 63(14): 4112-8).
• PBMCs peripheral blood mononuclear cells isolated from a normal volunteer (HLA- A*2402 or HLA-A*0201 positive) by Ficoll-Plaque (Pharmacia) solution were separated by adherence to a plastic tissue culture dish (Becton Dickinson) so as to enrich them as the monocyte fraction.
• the monocyte-enriched population was cultured in the presence of 1000 U/ml of granulocyte-macrophage colony- stimulating factor (GM-CSF) (R&D System) and 1000 U/ml of interleukin (IL)-4 (R&D System) in AIM-V Medium (Invitrogen) containing 2% heat-inactivated autologous serum (AS). After 7 days of culture, the cytokine-induced DCs were pulsed with 20 microgram/ml of each of the synthesized peptides in the presence of 3 microgram/ml of beta2-microglobulin for 3 hrs at 37 degrees C in AIM-V Medium.
• GM-CSF granulocyte-macrophage colony- stimulating factor
• IL interleukin-4
• AS heat-inactivated autologous serum
• the generated cells appeared to express DC-associated molecules, such as CD80, CD83, CD86 and HLA class II, on their cell surfaces (data not shown).
• DC-associated molecules such as CD80, CD83, CD86 and HLA class II
• MMC Mitomycin C
• CD8 Positive Isolation Kit CD8 Positive Isolation Kit
• These cultures were set up in 48-well plates (Corning); each well contained 1.5 x 10 4 peptide-pulsed DCs, 3 x 10 5 CD8+ T cells and 10 ng/ml of IL-7 (R&D System) in 0.5 ml of AIM-V/2% AS medium.
• CTLs were expanded in culture using the method similar to the one described by Riddell et al. (Walter EA et al., N Engl J Med 1995 Oct 19, 333(16): 1038-44; Riddell SR et al., Nat Med 1996 Feb, 2(2): 216-23).
• a total of 5 x 10 4 CTLs were suspended in 25 ml of AIM-V/5% AS medium with 2 kinds of human B-lymphoblastoid cell lines, inactivated by MMC, in the presence of 40 ng/ml of anti-CD3 monoclonal antibody (Pharmingen).
• 120 IU/ml of IL-2 were added to the cultures.
• the dilutions were made to have 0.3, 1, and 3 CTLs/well in 96 round-bottomed micro titer plate (Nalge Nunc International). CTLs were cultured with 1 x 10 4 cells/ well of 2 kinds of human B-lymphoblastoid cell lines, 30ng/ml of anti-CD3 antibody, and 125 U/ml of IL-2 in a total of 150 microliter/well of AIM-V Medium containing 5%AS. 50 microliter/well of IL-2 were added to the medium 10 days later so to reach a final concentration of 125 U/ml IL-2.
• IFN-gamma enzyme-linked im- munospot (ELISPOT) assay IFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed A24LCL or T2 (1 x 10 4 /well) was prepared as stimulator cells. Cultured cells in 48 wells were used as responder cells. IFN-gamma ELISPOT assay and IFN-gamma ELISA assay were performed under manufacture procedure. [0110] Results
• MELK GeneBank Accession No. NM_014791; SEQ ID No.93
• MELK expression was validly elevated in 29 out of 29 bladder cancer, 31 out of 34 breast cancer, 14 out of 15 cervical cancer, 11 out of 11 cholangiocellular carcinoma, 10 out of 13 CML, 12 out of 15 colorectal cancer, 2 out of 2 endometriosis, 19 out of 42 esophagus cancer, 5 out of 6 gastric cancer, 4 out of 4 liver cancer, 11 out of 11 NSCLC, 13 out of 14 lymphoma, 14 out of 18 osteosarcoma, 3 out of 6 ovarian cancer, 2 out of 2 pancreatic cancer, 18 out of 21 prostate cancer, 5 out of 6 renal carcinoma and 15 out of 15 small cell lung cancer as compared with corresponding normal tissue. (Table 1).
• Table 2 shows the HLA- A24 and HLA- A2 binding peptides of MELK in order of highest binding affinity. A total of 34 peptides with potential HLA-A24 binding ability were selected and examined to determine the epitope peptides (Table 2a), and a total of 58 peptides having potential HLA- A2 binding ability were similarly selected and examined to determine the epitope peptides (Table 2b and c).
• Start position indicates the number of amino acid residue from the N-terminal of MELK. Binding score is derived from "BIMAS”.
• the cells in the positive well number #5 stimulated with SEQ ID NO: 14, #3 with SEQ ID NO: 21, #4 with SEQ ID NO: 23, #4 with SEQ ID NO: 27, #5 with SEQ ID NO: 36, #3 with SEQ ID NO: 46, #1 with SEQ ID NO: 57, #2 with SEQ ID NO: 60 and #2 with SEQ ID NO: 62 were expanded and established CTL lines.
• CTL activity of those CTL lines was determined by IFN- gamma ELISA assay ( Figure 2a-i). All CTL lines demonstrated potent IFN-gamma production against the target cells pulsed with corresponding peptide as compared to target cells without peptide pulse.
• CTL clones were established by limiting dilution from CTL lines as described in "Materials and Methods", and IFN-gamma production from CTL clones against target cells pulsed peptide were determined by IFN-gamma ELISA assay. Potent IFN-gamma productions were determined from CTL clones stimulated with SEQ ID NO: 27 in Figure 3.
• the established CTL lines raised against these peptides were examined for their ability to recognize target cells presenting the candidate peptide endogenously processed from MELK with HLA-A*0201 molecule.
• Specific CTL activity against COS7 cells which transfected with both the full length of MELK and HLA-A*0201 molecule gene was tested using the CTL lines raised by corresponding peptide as the effecter cells.
• COS7 cells transfected with either full length of MELK genes or HLA- A* 0201 were prepared as control.
• the present invention describes new TAAs, particularly those derived from MELK which induce potent and specific anti-tumor immune responses and have applicability to a wide array of cancer types.
• TAAs warrant further development as peptide vaccines against diseases associated with MELK, e.g. cancers such as breast cancer, bladder cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophagus cancer, gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and SCC.
• diseases associated with MELK e.g. cancers such as breast cancer, bladder cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophagus cancer, gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and SCC.

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