WO2006090810A2 - Peptide vaccines for lung cancers expressing ttk, urlc10 or koc1 polypeptides - Google Patents

Abstract

The present invention provides peptides comprising the amino acid sequence of SEQ ID NO: 8, 67, 89, as well as peptides comprising the above-mentioned amino acid sequences in which 1, 2, or several amino acids are substituted, deleted, or added, and having cytotoxic T cell inducibility. The present invention also provides drugs for treating or preventing tumors comprising these peptides. The peptides of the present invention can also be used as vaccines.

Description

DESCRIPTION

PEPTIDE VACCINES FOR LUNG CANCERS EXPRESSING TTK, URLClO OR KOCl POLYPEPTIDES This application claims the benefit of U.S. Provisional Application Serial

^■No.60/656,857 filed February 25, 2005, the contents of which are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates to the field of biological science, more specifically to the field of cancer therapy. In particular, the present invention relates to novel peptides that are extremely effective as cancer vaccines, and drugs for treating and preventing tumors that contain these peptides.

Background Art

Lung cancer is one of the most commonly fatal human tumors. Many genetic alterations associated with the development and progression of lung cancer have been reported. Genetic changes can aid prognostic efforts and predictions of metastatic risk or response to certain treatments. (Mitsudomi T et ah, (2000) Clin Cancer Res 6: 4055- 63.; Niklinski et ah, (2001) Lung Cancer. 34 Suppl 2: S53-8.; Watine J. (2000) Bmj 320: 379-80.). Non-small cell lung cancer (NSCLC) is by far the most common form of lung cancer, accounting for nearly 80% of lung tumors (Society, A. C. Cancer Facts and

Figures 2001, 2001.). The overall 10-year survival rate remains as low as 10%, despite recent advances in multi-modality therapy, because the majority of NSCLCs are not diagnosed until advanced stages (Fry, W. A. et al, (1999) Cancer. 86: 1867-76.). Although chemotherapy regimens based on platinum are considered the reference standards for treatment of NSCLC, those drugs are able to extend survival of patients with advanced NSCLC only about six weeks (Non-small Cell Lung Cancer Collaborative Group, (1995) BMJ. 311: 899-909.). Numerous targeted therapies are being investigated for this disease, including tyrosine kinase inhibitors; however, to date promising results have been achieved in only a limited number of patients and some recipients suffer severe adverse reactions (Kris MG, et ah, (2002) Proc Am Soc Clin Oncol. 27: 292a(Al 166).).

It has been demonstrated that CD8⁺ cytotoxic T lymphocytes (CTLs) recognize - 9 -

epitope peptides derived from tumor-associated antigens (TAAs) presented on MHC class I molecules, and lyse the tumor cells. Since the discovery of the MAGE family as the first example of TAAs, many other TAAs have been discovered using immunological approaches (Boon T. (1993) Int J Cancer 54: 177-80.; Boon T. et al, (1996) J Exp Med 183: 725-9.; van der Bruggen P et al, (1991) Science 254: 1643-47.; Brichard V et al, (1993) J Exp Med 178: 489-95.; Kawakami Y et al, (1994) J Exp Med 180: 347-52.). Some of them are now in clinical development as targets of immunotherapy. TAAs discovered so far include MAGE (van der Bruggen P et al, (1991) Science 254: 1643-7.), gplOO (Kawakami Y et al, (1994) J Exp Med 180: 347-52.), SART (Shichijo S et al, (1998) J Exp Med 187:277-88.), and NY-ESO-I (Chen YT. et al, (1997) Proc.Natl.Acd. Sci.USA, 94: 1914-8.). On the other hand, certain gene products demonstrated to be somewhat specifically over-expressed in tumor cells have been shown to be recognized as targets inducing cellular immune responses. Such gene products include p53 (Umano Y et al, (2001) Br J Cancer, 84:1052-7.), HER2/neu (Tanaka H et al, (2001) Br J Cancer, 84: 94-9.), CEA (Nukaya I et al, (1999) Int. J. Cancer 80, 92-7.) and the like.

Despite significant progress in basic and clinical research concerning TAAs (Rosenberg SA et al, (1998) Nature Med, 4: 321-7.; Mukherji B. et al, (1995) Proc Natl Acad Sci USA, 92: 8078-82.: Hu X et al, (1996) Cancer Res, 56: 2479-83.), only a very limited number of candidate TAAs suitable for treatment of adenocarcinomas, such as lung cancer, are available. TAAs that are abundantly expressed in cancer cells, and whose expression is restricted to cancer cells, would be promising candidates as immunotherapeutic targets.

It has been repeatedly shown in ⁵ Cr-release assays that peptide-stimulated peripheral blood mononuclear cells (PBMCs) from certain healthy donors produce significant levels of IFN-Y in response to the peptide, but rarely exert cytotoxicity against tumor cells in an HLA- A24 or A0201 restricted manner (Kawano K et al, (2000) Cancer Res 60: 3550-8.; Nishizaka et al, (2000) Cancer Res 60: 4830-7.; Tamura et al, (2001) Jpn J Cancer Res 92: 762-7.). However, both HLA-A24 and HLA-A0201 are common HLA alleles in Japanese and Caucasian populations (Date Y et al, (1996) Tissue Antigens 47: 93-101.; Kondo A et al, (1995) J Immunol 155: 4307-12.; Kubo RT et al, (1994) J Immunol 152: 3913-24.; Imanishi et al, Proceeding of the eleventh International Histocompatibility Workshop and Conference Oxford University Press, Oxford, 1065 (1992); Williams F et al, (1997) Tissue Antigen 49: 129.). Thus, antigenic peptides of cancers presented by these HLA alleles may be especially useful for the treatment of cancers among Japanese and Caucasian patients. Further, it is known that the induction of low-affinity CTL in vitro usually results from the use of peptide at a high concentration, generating a high level of specific peptide/MHC complexes on antigen-presenting cells (APCs), which will effectively activate these CTL (Alexander- Miller et al, (1996) Proc Natl Acad Sci USA 93: 4102-7.).

Recent developments in cDNA microarray technologies have enabled the constructions of comprehensive profiles of gene expression of malignant cells as compared to normal cells (Okabe, H. et al, (2001) Cancer Res., 61, 2129-37.; Lin YM. et al, (2002) Oncogene, 21;4120-8.; Hasegawa S. et al, (2002) Cancer Res 62:7012-7.). This approach enables an understanding of the complex nature of cancer cells and the mechanisms of carcinogenesis and facilitates the identification of genes whose expression is deregulated in tumors (Bienz M. et al , (2000) Cell 703, 311 -320.).

Among the transcripts identified as commonly up-regulated in lung cancers, TTK (TTK Protein kinase; GenBank Accession No. NM_003318; SEQ ID Nos.l, 2), URLClO (cDNA for differentially expressed CO16 gene; GenBank Accession No. AB105187; SEQ ID Nos.3, 4) and KOCl (IGF II mRNA Binding Protein 3; GenBank Accession No. NM_006547; SEQ ID Nos.5, 6) are of particular interest to the present inventors, being specifically up-regulated in tumor cells of the lung cancer tissues in more than 80 % of the cases analyzed. In contrast, Northern blot analysis demonstrated that these gene products are not found in normal vital organs (See WO2004/031413, the entire contents of which are incorporated by reference herein). Thus, immunogenic peptides derived from TTK, URLClO and KOCl may find utility in killing tumor cells expressing those antigens. The present invention addresses these and other needs.

Summary of the Invention

Three genes, TTK (TTK Protein kinase), URLClO (cDNA for differentially expressed CO 16 gene) and KOCl (IGF II mRNA Binding Protein 3) have been identified as up-regulated in lung cancer. The genes were identified using gene expression profiling with a genome-wide cDNA microarray containing 23,040 genes. As discussed above, expression of TTK, URLClO and KOCl is specifically up-regulated in tumor cells in more than 80% of the patients with lung cancer yet absent in other normal vital organs.

The present invention is based, at least in part, on the identification of epitope peptides of the gene products of these genes (TTK, URLClO, and KOCT) which elicit cytotoxic T lymphocytes (CTLs) specific to the corresponding molecules. As discussed in detail below, Peripheral Blood Mononuclear Cells (PBMC) of healthy donor were stimulated using HLA-A*2402 binding candidate peptides derived from TTK, URLClO or KOCl. CTL clones were then established with specific cytotoxicity against the - A -

HLA- A24 positive target cells pulsed with each of the candidate peptides. Further analysis of the CTL clones showed the potent cytotoxic activity against, not only the peptide-pulsed target cells, but also tumor cells that endogenously express TTK, URLClO or KOCl . Furthermore, both a cold target inhibition assay and an antibody blocking assay revealed that CTL cell clones specifically recognized the MHC class I - peptide complex. These results demonstrate that these peptides are HLA- A24 restricted epitope peptides that can induce potent and specific immune responses against lung cancer cells expressing TTK, URLClO or KOCl.

Accordingly, the present invention provides methods for treating or preventing lung cancer in a subject comprising the step of administering to the subject the TTK,

URLClO and KOCl polypeptides of the invention. Anti-tumor immunity is induced by the administration of these polypeptides. Thus, the present invention provides methods for inducing anti-tumor immunity in a subject comprising the step of administering to the subject the TTK, URLClO and KOCl polypeptides, as well as pharmaceutical compositions for treating or preventing lung cancer comprising the TTK, URLClO and KOCl polypeptides.

These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of preferred embodiments, and not restrictive of the invention or other alternate embodiments of the invention.

Brief Description of the Drawings

Figure 1 is a graph showing that the CTL clone raised by TTK-567 has peptide-specific cytotoxicity. Specifically, the CTL clone showed high cytotoxic activity against target cells (A24LCL) pulsed with TTK-567, whereas it did not show significant cytotoxic activity against the same target cells (A24LCL) pulsed with no peptides.

Figure 2 is a graph showing that the CTL clone raised by URLC 10- 177 has peptide-specific cytotoxicity. Specifically, the CTL clone showed high cytotoxic activity against target cells (A24LCL) pulsed with URLC 10- 177, whereas it did not show significant cytotoxic activity against the same target cells (A24LCL) pulsed with no peptides.

Figure 3 is a graph showing that the CTL clone raised by KOCl-508 has peptide-specific cytotoxicity. Specifically, the CTL clone showed high cytotoxic activity against target cells (A24LCL) pulsed with KOCl-508, whereas it did not show significant cytotoxic activity against the same target cells (A24LCL) pulsed with no peptides. Figure 4 is a graph showing that the CTL clone raised by TTK-567 recognizes and lyses tumor cells endogenously expressing TTK in an HLA restricted fashion. Cytotoxic activity against TEl cells, which endogenously express TTK and HLA- A24, was tested using as effector cells the CTL clones raised by TTK-567. PC9 cells were used as the target cells that endogenously express TTK but do not express HLA- A24. The CTL clone showed high cytotoxic activity against TEl cells that express both TTK and HLA-A24. On the other hand, it did not show significant cytotoxic activity against PC9 cells that express TTK but not HLA- A24.

Figure 5 is a graph showing that the CTL clone raised by URLC 10- 177 recognizes and lyses tumor cells endogenously expressing URLClO in an HLA restricted fashion. Cytotoxic activity against TEl cells, which endogenously express URLClO and HLA- A24, was tested using as effector cells the CTL clone raised by URLC 10- 177. TE 13 cells were used as the target cells that endogenously express URLClO but do not express HLA-A24. The CTL clone showed high cytotoxic activity against TEl cells that express both URLCl 0 and HLA- A24. On the other hand, it did not show significant cytotoxic activity against TE 13 cells that express URLClO, but not HLA- A24.

Figure 6 is a graph showing that the CTL clone raised by KOCl-508 recognizes and lyses the tumor cells endogenously expressing KOCl in an HLA restricted fashion. Cytotoxic activity against TEl cells, which endogenously express KOCl and HLA- A24, was tested using as effector cells the CTL clone raised by KOCl- 508. PC9 cells were used as the target cells that endogenously express KOCl but do not express HLA-A24. The CTL clone showed high cytotoxic activity against TEl cells that express both KOCl and HLA- A24. On the other hand, it did not show significant cytotoxic activity against PC9 cells that express KOCl, but not HLA- A24.

Figure 7 is a graph showing that the CTL clone raised by TTK-567 specifically recognizes TTK-567 in an HLA-A24 restricted manner. TEl cells labeled by Na₂ ⁵¹Cr O₄ were prepared as hot target, while TTK-567 peptide-pulsed A24LCL cells were used as cold target (Inhibitor). The E/T ratio was fixed at 20. The cytotoxic activity of TTK-567 CTL clone against TEl cells was inhibited by the addition of A24LCL cells pulsed with the identical peptide. Figure 8 is a graph showing that the CTL clone raised by URLC 10- 177 specifically recognizes URLClO in an HLA- A24 restricted manner. TEl cells labeled by Na₂ ⁵¹Cr O₄ were prepared as hot target, while URLClO-177 peptide-pulsed A24LCL cells were used as cold target (Inhibitor). The E/T ratio was fixed at 20. The cytotoxic activity of URLC 10- 177 CTL clone against TEl was inhibited by the addition of A24LCL cells pulsed with the identical peptide.

Figure 9 is a graph showing that the CTL clone raised by KOCl-508 specifically recognizes KOCl in an HLA-A24 restricted manner. TEl cells labeled by Na₂ ⁵¹Cr O₄ were prepared as hot target, while KOCl-508 peptide-pulsed A24LCL cells were used as cold target (Inhibitor). The E/T ratio was fixed at 20. The cytotoxic activity of KOCl-508 CTL clone against TEl cells was inhibited by the addition of A24LCL cells pulsed with the identical peptide.

Figure 10 is a graph showing that the cytotoxic activity of the CTL clone raised by TTK-567 is specifically blocked by antibodies recognizing the T-cell surface antigens of HLA class I or CD8. The specificity of cytotoxicity of the CTL clone was confirmed by an antibody blocking assay. TEl cells were co-cultured with monoclonal antibodies respectively, and used as a target. The CTL activity was clearly blocked the addition of antibodies that recognize HLA Class I or CD8 and was marginally affected the addition of antibodies to HLA Class II or CD4; however, it was not inhibited at all by the addition of an isotype-matched control antibody.

Figure 11 is a graph showing that the cytotoxic activity of the CTL clone raised by URLC 10- 177 is specifically blocked by antibodies recognizing the T-cell surface antigens of HLA class I or CD8. The specificity of cytotoxicity of the CTL clone was confirmed by an antibody blocking assay. TEl cells were co-cultured with monoclonal antibodies respectively, and used as a target. The CTL activity was clearly blocked by the addition of antibodies that recognize HLA Class I or CD8 and was marginally affected by the addition of antibodies to HLA Class II or CD4; however, it was not inhibited at all by the addition of an isotype-matched control antibody.

Figure 12 is a graph showing that the cytotoxic activity of the CTL clone raised by KOCl-508 is specifically blocked by antibodies recognizing the T-cell surface antigens of HLA class I or CD8. The specificity of cytotoxicity of the CTL clone was confirmed by an antibody blocking assay. TEl cells were co-cultured with monoclonal antibodies respectively, and used as a target. The CTL activity was clearly blocked by the addition of antibodies that recognize HLA Class I or CD8 and was marginally affected by the addition of antibodies to HLA Class II or CD4; however, it was not inhibited at all by the addition of an isotype-matched control antibody.

Detailed Description of the Invention

The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Identification of new TAAs, particularly those that induce potent and specific anti-tumor immune responses, warrants further development of the clinical application of the peptide vaccination strategy in various types of cancer (Boon T et al, (1996) J Exp Med 183: 725-9.; van der Bruggen P et al, (1991) Science 254: 1643-7.; Brichard V et al, (1993) J Exp Med 178: 489-95.; Kawakami Y et al, (1994) J Exp Med 180: 347-52.; Shichijo S et al, (1998) J Exp Med 187:277-88.; Chen YT et al, (1997) Proc.NatlAcd. ScWSA, 94: 1914-8.; Harris CC, (1996) J Natl Cancer Inst 88:1442-5.; Butterfield LH et al, (1999) Cancer Res 59:3134-42.; Vissers JL et al, (1999) Cancer Res 59: 5554-9.; van der Burg SH et al, (1996) J. Immunol 156:3308-14.; Tanaka F et al, (1997) Cancer Res 57:4465-8.; Fujie T et al, (1999) Int J Cancer 80:169-72.; Kikuchi M et al, (1999) Int J Cancer 81 : 459-66.; Oiso M et al, (1999) Int J Cancer 81:387-94.). As noted above, TTK, URLCIO and KOCl were previously identified as over-expressed in lung cancer using cDNA microarray technologies. As discussed in WO2004/031413, TTK encodes an S_TKc domain. The protein encoded by the TTK gene phosphorylates proteins on serine, threonine and tyrosine, such phosphorylation likely associated with cell proliferation (Mills GB et al, (1992) J Biol Chem 267: 16000-6.; Schmandt R et al, (1994) J Immunol.;152(l):96-105.; Stucke VM et al, (2002) EMBO J.;21(7):1723-32.). KOCl encodes insulin-like growth factor 2 (IGF2) mRNA-binding protein 3 (IMP-3). IMP-3 protein contains 2 functional RNA recognition motifs (RRM) in addition to the 4 KH domains. The protein associates specifically with the 5-prime UTR of the human 6.0-kb insulin-like growth factor II (IGF2) leader-3 mRNA, suggesting a role for IMP-3 in the physiologic regulation of IGF2 production. (Nielsen, J. et al, (1999) Molec. Cell Biol. 19: 1262-1270) IMP-3 was also over-expressed in pancreatic cancers (Mueller- Pillasch, F. et al, (1997) Oncogene 14: 2729-2733).

Previous experiments demonstrated that TTK, URLClO and KOCl were over- expressed in lung cancer and show minimal expression in normal tissues. In addition, these genes were shown to have a significant function related to cell proliferation (See WO2004/031413).

In the present invention, peptides derived from TTK, URLClO or KOCl are shown to be TAA epitopes restricted by HLA- A24, an HLA allele commonly found in the Japanese and Caucasian populations. Specifically, using their binding affinities to HLA- A24, candidates of HLA-A24 binding peptides derived from TTK, URLClO or KOCl were identified. After the in vitro stimulation of T-cells by dendritic cells (DCs) loaded with these peptides, CTLs were successfully established using TTK-567 (SYRNEIAYL (SEQ ID No.8)), URLClO-177 (RYCNLEGPPI (SEQ ID No.67)) and KOCl-508 (KTVNELQNL (SEQ ID No.89)). These CTLs showed potent cytotoxic activity against the peptide-pulsed A24LCL cells. Furthermore, CTL clones derived from these cells also showed specific cytotoxicity against HLA- A24 positive lung carcinoma cell lines that endogenously over-express TTK, URLClO or KOCl. However, these CTL clones did not show cytotoxic activity against cell lines lacking expression of either HLA- A24 or target TAA. The specific cytotoxic activities of these CTL clones were significantly inhibited by the cold target. These results demonstrate that TTK, URLClO and KOCl axe useful as TAAs of lung cancer cells and that TTK-567. KOCl- 508 and URLClO-177 are epitope peptides of each TAA restricted by HLA-A24. Since these antigens are over-expressed in most lung cancers and are associated with tumor cell proliferation, they find utility as immunotherapeutic targets against lung cancers. Accordingly, the present invention further provides methods of treating or preventing lung cancer in a subject, said methods comprising the steps of administering an immunogenic peptide of less than about 40 amino acids, often less than about 20 amino acids, usually less than about 15 amino acids and comprising the amino acid sequence of SEQ ID NOs: 8, 67, or 89 to the subject in need thereof. Alternatively, the immunogenic peptide may comprise a sequence of SEQ ID NOs: 8, 67, or 89 in which 1, 2, or several amino acids are substituted, deleted or added, provided the resulting variant peptide retains the immunogenic activity (i.e., the ability to induce CTLs specific to lung cancer cells). The number of residues to be substituted, deleted, or added is generally 5 amino acids or less, preferably 4 amino acids or less, more preferably 3 amino acids or less, even more preferably one or two amino acids.

Variant peptides (i.e., peptides comprising an amino acid sequence modified by substituting, deleting, or adding one, two or several amino acid residues to an original amino acid sequence) have been known to retain the original biological activity (Mark DF et αl., (1984) Proc Natl Acad Sci USA 81: 5662-6.; Zoller MJ and Smith M, (1982) Nucleic Acids Res 10:6487-500.; Dalbadie-McFarland G et αl., (1982) Proc Natl Acad Sci USA 79: 6409-13.)- In the context of the present invention, the amino acid modification results in conservation of the properties of the original amino acid side- chain (a process known as conservative amino acid substitution). Examples of properties of amino acid side chains are hydrophobic amino acids (A₅ 1₅ L₅ M, F₅ P₅ W, Y₅ V), hydrophilic amino acids (R₅ D₅ N₅ C₅ E₅ Q₅ G₅ H₅ K₅ S₅ T)₅ and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G₅ A₅ V₅ L₅ 1₅ P); a hydroxyl group containing side-chain (S₅ T₅ Y); a sulfur atom containing side-chain (C₅ M); a carboxylic acid and amide containing side-chain (D₅ N₅ E₅ Q); a base containing side-chain (R₅ K₅ H); and an aromatic containing side-chain (H₅ F₅ Y₅ W). Note, the parenthetic letters indicate the one-letter codes of amino acids.

In preferred embodiments, the immunogenic peptide is a nonapeptide (9-mer) or a decapeptide (10-mer).

The present invention further provides a method of inducing anti-tumor immunity for lung cancer in a subject, said method comprising the steps of administering to the subject an immunogenic peptide of the invention, namely one comprising the amino acid sequence of SEQ ID NOs: 8, 67, or 89 or a variant thereof (i. e. , including 1 , 2, or several amino acid substitutions, deletions, or additions) to the subject in need thereof.

In the context of the present invention, the subject is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow.

In the present invention, the peptide can be administered to a subject via in vivo or ex vivo. Furthermore, the present invention also provides use of nonapeptide or decapeptide selected from peptides comprising the amino acid sequence of SEQ ID NOs: 8, 67, and 89 (and variants thereof) for manufacturing an immunogenic composition for treating or preventing lung cancer.

Homology analyses of TTK-567, KOCl-508 and URLClO-177 demonstrate that they do not have significant homology with the peptides derived from any known human gene products. Accordingly, the possibility of unknown or undesirable immune responses with immunotherapy against these molecules is significantly reduced. Regarding HLA antigens, the use of an A-24 type that is highly expressed among the Japanese population is favorable for obtaining effective results, and the use of subtypes, such as A-2402, is even more preferable. Typically, in the clinic, 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 this antigen, or having cytotoxic T cell (CTL) inducibility by antigen presentation. Furthermore, in order to obtain peptides showing high binding affinity and CTL inducibility, substitution, deletion, or addition of 1, 2, or several amino acids may be performed based on the amino acid sequence of the naturally occurring TTK, URLClO and KOCl partial peptide. Herein, the term "several" means refers to 5 or less, more preferably 3 or less. Furthermore, in addition to peptides that are naturally displayed, since the regularity of the sequences of peptides displayed by binding to HLA antigens is already known (Kubo RT, et al, (1994) J. Immunol., 152, 3913-24.; Rammensee HQ et al, (1995) Immunogenetics. 41:178-228.; Kondo A, et al, (1995) J. Immunol. 155:4307-12.), modifications based on such regularity can be performed on the immunogenic peptides of the invention. For example, peptides showing high HLA-24 binding affinity in which the second amino acid from the N terminus substituted with phenylalanine, tyrosine, methionine, or tryptophan may be favorably used. Likewise, peptides whose C-terminal amino acid is substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine may also be used favorably.

However, when 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 or allergic symptoms against specific substances may be induced. Therefore, it is preferable to avoid the situation wherein the immunogenic sequence matches the amino acid sequence of a known protein. This situation may be avoided by performing a homology search using available databases. If homology searches confirm that peptides in which 1, 2 or several different amino acids do not exist, then the danger that modifications of the above-mentioned amino acid sequence that, for example, increase the binding affinity with HLA antigens, and/or increase the CTL inducibility can be avoided.

Although peptides having high binding affinity to the HLA antigens as described above are expected to be highly effective as cancer vaccines, the candidate peptides, which are selected according to the presence of high binding affinity as an indicator, must be examined for the actual presence of CTL inducibility. CTL inducibility may be confirmed by inducing antigen-presenting cells carrying human MHC antigens (for example, B-lymphocytes, macrophages, and dendritic cells), or more specifically dendritic cells derived from human peripheral blood mononuclear leukocytes, and, after stimulation with the peptide of interest, mixing with CD8-positive cells and measuring the cytotoxic activity against the target cells. As the reaction system, transgenic animals produced to express a human HLA antigen (for example, those described in BenMohamed L, et al, (2000) Hum. Immunol.; 61(8):764-79 Related Articles, Books, Linkout.) may be used. For example, the target cells can be radio-labeled with ⁵¹Cr and such, and cytotoxic activity can be calculated from radioactivity released from the target cells. Alternatively, it can be examined by measuring IFN-Y produced and released by CTL in the presence of antigen-presenting cells that carry immobilized peptides, and visualizing the inhibition zone on the media using anti-IFN-Y monoclonal antibodies.

As a result of examining the CTL inducibility of peptides as described above, it was discovered that peptides having high binding affinity to an HLA antigen did not necessarily have high inducibility. However, nonapeptides or decapeptides selected from peptides comprising the amino acid sequences indicated by SYRNEIAYL (SEQ ID NO: 8), RYCNLEGPPI (SEQ ID NO: 67), KTVNELQNL (SEQ ID NO: 89) showed particularly high CTL inducibility. As noted above, the present invention provides peptides having cytotoxic T cell inducibility, namely those comprising the amino acid sequence of SEQ ID NOS: 8, 67, or 89 or a variant thereof (i.e., those in which 1, 2, or several amino acids are substituted, deleted, or added). It is preferably that the amino acid sequence comprise 9 or 10 amino acids indicated in SEQ ID NOS: 8, 67, 89 or a variant thereof do not match an amino acid sequence associated with another endogenous protein. In particular, amino acid substitution to phenylalanine, tyrosine, methionine, or tryptophan at the second amino acid from the N terminus, and to phenylalanine, leucine, isoleucine, tryptophan, or methionine at the C-terminal amino acid, and amino acid addition of 1 to 2 amino acids at the N terminus and/or C terminus are favorable examples. Peptides of the present invention can be prepared using well known techniques.

For example, the peptides can be prepared synthetically, using either recombinant DNA technology or chemical synthesis. Peptides of the present invention may be synthesized individually or as longer polypeptides comprising two or more peptides. The peptides of the present invention are preferably isolated, i.e., substantially free of other naturally occurring host cell proteins and fragments thereof.

The peptides of the present invention may contain modifications, such as glycosylation, side chain oxidation, or phosphorylation; so long as the modifications do not destroy the biological activity of the peptides as described herein. Other modifications include incorporation of D-amino acids or other amino acid mimetics that can be used, for example, to increase the serum half life of the peptides.

The peptides of this invention can be prepared as a combination, which comprises two or more of peptides of the invention, for use as a cancer vaccine that may induce CTL in vivo. The peptides may be in a cocktail or may be conjugated to each other using standard techniques. For example, the peptides can be expressed as a single polypeptide sequence. The peptides in the combination may be the same or different. By administering the peptides of this invention, the peptides are presented at a high density on the HLA antigens of antigen-presenting cells, which, in turn, induces CTLs that specifically react toward the complex formed between the displayed peptide and the HLA antigen. Alternatively, antigen-presenting cells having immobilized the peptides of this invention on their cell surface, obtained by removing dendritic cells from the subjects, may be stimulated by the peptides of this invention. Re-administration of these cells to the respective subjects induces CTL, and, as a result, aggressiveness towards the target cells can be increased.

More specifically, the present invention provides drugs for treating tumors or preventing proliferation, metastasis, and such of tumors, which comprise one or more of peptides of this invention. The peptides of this invention find particular utility in the treatment of tumors, such as lung cancer.

The peptides of this invention can be administered to a subject directly, as a pharmaceutical composition that has been formulated by conventional formulation methods. In such cases, in addition to the peptides of this invention, carriers, excipients, and such that are ordinarily used for drugs can be included as appropriate, without particular limitations. The immunogenic compositions of this invention may be used for treatment and prevention of various tumors, including lung cancers.

The immunogenic compositions for treatment and/or prevention of tumors, which comprise as the active ingredient one ore more peptides of the present invention, can further include an adjuvant so that cellular immunity will be established effectively. Alternatively, they may be administered with other active ingredients, such as anti-tumor agents. Suitable formulations include granules. Suitable adjuvants are described in the literature (Johnson AG. (1994) Clin. Microbiol . Rev., 7:277-89.). Exemplary adjuvants include, but are not limited to, aluminum phosphate, aluminum hydroxide, and alum. Furthermore, liposome formulations, granular formulations in which the drug is bound to few-μm diameter beads, and formulations in which a lipid is bound to the peptide may be conveniently used. The method of administration may be oral, intradermal, subcutaneous, intravenous injection, or such, and may include systemic administration or local administration to the vicinity of the targeted tumor. The dose of the peptide(s) of this invention can be adjusted appropriately according to the disease to be treated, age of the patient, weight, method of administration, and such. Though the dosage is ordinarily 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, more preferably 0.1 mg to 10 mg, preferably administered once in a few days to few months, one skilled in the art can readily select the appropriate dose and method of administration, as, the selection and optimization of these parameters is well within routine skill.

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 described in detail in Published Japanese Translation of International Publication Nos. Hei 11-510507 and 2000-512161, and are preferably prepared using antigen-presenting cells obtained from subjects who are targets of treatment and/or prevention. The exosomes of this invention can be inoculated as cancer vaccines, similarly to the peptides of this invention.

The type of HLA antigens used must match that of the subject requiring treatment and/or prevention. For example, in the Japanese population, HLA- A24, particularly HLA-A2402, is often appropriate.

In some embodiments, the vaccine compositions of the present invention include a component which primes cytotoxic T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the ε-and α-amino groups of a lysine residue and then linked to an immunogenic peptide of the invention. The lipidated peptide can then be administered either directly, in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant. As another example of a lipid priming of CTL 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 K, et al., (1989) Nature 342:561-4.).

The immunogenic compositions of the present invention may also comprise nucleic acids encoding one or more of the immunogenic peptides disclosed here. See, e.g., Wolff JA et al, (1990) Science 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. Examples of DNA- based delivery technologies include "naked DNA", facilitated (bupivicaine, 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 immunogenic peptides of the invention can also be expressed by viral or bacterial vectors. Examples of suitable 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. Another suitable vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover CK, et al., (1991) Nature 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, are known in the art. See, e.g. , Shata MT, et al , (2000) MoI. Med. Today 6:66-71; Shedlock DJ and Weiner DB., et al, (2000) J. Leukoc. Biol. 68:793-806; and Hipp JD, et al. , (2000) In Vivo 14:571-85.

The present invention also provides methods of inducing antigen-presenting cells using one or more peptides of this invention. The antigen-presenting cells can be induced by inducing dendritic cells from the peripheral blood monocytes and then contacting (stimulating) them with one or more peptides of this invention in vitro, ex vivo or in vivo. When peptides of the present invention are administered to the subjects, antigen-presenting cells that have the peptides of this invention immobilized to them are induced in the body of the subject. Alternatively, after immobilizing the peptides of this invention to the antigen-presenting cells, the cells can be administered to the subject as a vaccine. For example, the ex vivo administration may comprise steps of: a: collecting antigen-presenting cells from a subject, and b: contacting the antigen-presenting cells of step a with a peptide of the present invention.

The antigen-presenting cells obtained by step b can be administered to the subject as a vaccine. This invention also provides a method for inducing antigen-presenting cells having a high level of cytotoxic T cell inducibility, in which the method comprises the step of transferring genes comprising polynucleotide(s) encoding one or more peptides of this invention to antigen-presenting cells in vitro. The introduced genes may be in the form of DNAs or RNAs. For the method of introduction, without particular limitations, various methods conventionally performed in this field, such as lipofection, electroporation, and calcium phosphate method may be suitably used. More specifically, transfection may be performed as described in Reeves ME, et al., (1996) Cancer Res., 56:5672-7.; Butterfield LH, et al., (1998) J. Immunol., 161:5607-13.; Boczkowski D, et al., (1996) J. Exp. Med., 184:465-72.; Published Japanese Translation of International Publication No. 2000-509281. By transferring the gene into antigen- presenting cells, 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 partial peptides.

The present invention further provides methods for inducing CTL using one or more peptides of this invention. When the peptides of this invention are administered to a subject, CTL are induced in the body of the subject, and the strength of the immune system targeting the lung cancer cells in the tumor tissues is thereby enhanced. Alternatively, the peptides of the present invention may be used in the context of an ex vivo therapeutic method, in which subject-derived antigen-presenting cells and CD8- positive cells or peripheral blood mononuclear leukocytes are contacted (stimulated) with one or more peptides of this invention in vitro, and, after inducing CTL, the cells are returned to the subject. For example, the method may comprise steps of: a: collecting antigen-presenting cells from a subject, b: contacting the antigen-presenting cells of step a with a peptide of the present invention, c: mixing the antigen-presenting cells of step b with CD ⁺ T cells and co- culturing so as to induce cytotoxic T-cells:, and d: collecting CD⁸⁺ T cells from the co-culture of step c. The CD⁸⁺ T cells having cytotoxic activity obtained by step d can be administered to the subject as a vaccine. The present invention further provides isolated cytotoxic T cells induced using the peptides of this invention. The cytotoxic T cells, induced by stimulation with an antigen-presenting cell presenting one or more peptides of this invention, are preferably derived from subjects who are the target of treatment and/or prevention, and can be administered alone or in combination with other drugs, including one or more peptides of this invention or exosomes having anti-tumor activity. The obtained cytotoxic T cells act specifically against target cells presenting the peptides of this invention, or preferably the same peptide(s) used for induction. The target cells may be cells that express TTK, URLClO and KOCl endogenously, or cells that are transfected with TTK, URLClO and KOCl genes. Cells that present the peptides of this invention on the cell surface, due to stimulation with these peptides, can also become targets of attack.

The present invention also provides antigen-presenting cells presenting complexes formed between HLA antigens and one or more peptides of this invention. The antigen-presenting cells, obtained through contact with the peptides of this invention or the nucleotides encoding such peptides, are preferably derived from subjects who are the target of treatment and/or prevention, and can be administered as vaccines, alone or in combination with other drugs, including the peptides, exosomes, or cytotoxic T cells of the present invention.

In the context of the present invention, the term "vaccine" (also referred to as an immunogenic composition) refers to a substance that induces anti-tumor immunity or suppresses lung cancer upon inoculation into animals. According to the present invention, polypeptides comprising the amino acid sequence of SEQ ID NO: 8, 67, or 89 were suggested to be HLA- A24 restricted epitope peptides that may induce potent and specific immune response against lung cancer cells expressing TTK, URLClO or KOCl. Thus, the present invention also encompasses a method of inducing anti-tumor immunity using polypeptides comprising the amino acid sequence of SEQ ID NO: 8, 67, or 89 or a variant thereof (i.e., including 1, 2, or several amino acid substitutions, deletions, or additions). In general, anti-tumor immunity includes immune responses such as follows: induction of cytotoxic lymphocytes against tumors comprising cells expressing TTK, URLClO₃ or KOCl, - induction of antibodies that recognize tumors comprising cells expressing

TTK, URLClO, or KOCl, and - induction of anti-tumor cytokine production.

Therefore, when a certain peptide induces any one of these immune responses upon inoculation into an animal, the peptide is decided to have anti-tumor immunity inducing effect. The induction of the anti-tumor immunity by a peptide can be detected by observing in vivo or in vitro the response of the immune system in the host against the peptide.

For example, a method for detecting the induction of cytotoxic T lymphocytes is well known. A foreign substance that enters the living body is presented to T cells and B cells by the action of antigen-presenting cells (APCs). T cells that respond to the antigen presented by APC in antigen specific manner differentiate into cytotoxic T cells (also referred to as cytotoxic T lymphocytes or CTLs) due to stimulation by the antigen, and then proliferate; this process is referred to herein as "activation" of T cells. Therefore, CTL induction by a certain peptide can be evaluated by presenting the peptide to a T cell by APC, and detecting the induction of CTL. Furthermore, APCs have the effect of activating CD4+ T cells, CD8+ T cells, macrophages, eosinophils and NK cells. Since CD4+ T cells are also important in anti-tumor immunity, the anti-tumor immunity inducing action of the peptide can be evaluated using the activation effect of these cells as indicators. A method for evaluating the inducing action of CTL using dendritic cells (DCs) as APC is well known in the art. DC is a representative APC having the strongest CTL inducing action among APCs. In this method, the test polypeptide is initially contacted with DC and then this DC is contacted with T cells. Detection of T cells having cytotoxic effects against the cells of interest after the contact with DC shows that the test polypeptide has an activity of inducing the cytotoxic T cells. Activity of CTL against tumors can be detected, for example, using the lysis of ⁵¹Cr-labeled tumor cells as the indicator. Alternatively, it is well known to evaluate the degree of tumor cell damage using ³H-thyrnidine uptake activity or LDH (lactose dehydrogenase)-release as the indicator.

Apart from DC, peripheral blood mononuclear cells (PBMCs) may also be used as the APC. The induction of CTL is reported to be enhanced by culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL has been shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

The test polypeptides confirmed to possess CTL inducing activity by these methods are polypeptides having DC activation effect and subsequent CTL inducing activity. Therefore, polypeptides that induce CTL against tumor cells are useful as vaccines against lung cancer. Furthermore, APC that have acquired the ability to induce CTL against lung cancer by contacting with the polypeptides are useful as vaccines against lung cancer. Furthermore, CTL that have acquired cytotoxicity due to presentation of the polypeptide antigens by APC can be also used as vaccines against lung cancer. Such therapeutic methods for lung cancer, using anti-tumor immunity due to APC and CTL, are referred to as cellular immunotherapy.

Generally, when using a polypeptide for cellular immunotherapy, efficiency of the CTL-induction can be increased by combining a plurality of polypeptides having different structures and contacting them with DC. Therefore, when stimulating DC with protein fragments, it is advantageous to use a mixture of multiple types of fragments.

The induction of anti-tumor immunity by a polypeptide can be further confirmed by observing the induction of antibody production against tumors. For example, when antibodies against a polypeptide are induced in a laboratory animal immunized with the polypeptide, and when growth, proliferation and/or metastasis of tumor cells is suppressed by those antibodies, the polypeptide is determined to induce anti-tumor immunity.

Anti-tumor immunity can be induced by administering a vaccine of this invention, and the induction of anti-tumor immunity enables treatment and prevention of lung cancer. Therapy against or prevention of the onset of lung cancer may include inhibition of the growth of lung cancer cells, involution of lung cancer cells and suppression of occurrence of lung cancer cells. Decrease in mortality of individuals having lung cancer, decrease of lung cancer markers in the blood, alleviation of detectable symptoms accompanying lung cancer and such are also included in the therapy or prevention of lung cancer. Such therapeutic and preventive effects are preferably statistically significant, for example, observed at a significance level of 5% or less, wherein the therapeutic or preventive effect of a vaccine against lung cancer is compared to a control without vaccine administration. For example, Student's t-test, the Mann- Whitney U-test or ANOVA may be used for determining statistical significance.

The above-mentioned peptide, having immunological activity, or a polynucleotide or vector encoding such a peptide, may be combined with an adjuvant. An adjuvant refers to a compound that enhances the immune response against the peptide when administered together (or successively) with the peptide having immunological activity. Examples of suitable adjuvants include cholera toxin, salmonella toxin, alum and such, but are not limited thereto. Furthermore, a vaccine of this invention may be combined appropriately with a pharmaceutically acceptable carrier. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid and such. Furthermore, the vaccine may contain as necessary, stabilizers, suspensions, preservatives, surfactants and such. The vaccine is administered systemically or locally. Vaccine administration may be performed by single administration or boosted by multiple administrations.

When using APC or CTL as the vaccine of this invention, lung cancer can be treated or prevented, for example, by the ex vivo method. More specifically, PBMCs of the subject receiving treatment or prevention are collected, contacted ex vivo with a peptide of the present invention. Following the induction of APC or CTL, the cells may be administered to the subject. APC can be also induced by introducing a vector encoding the peptide into PBMCs ex vivo. APC or CTL induced in vitro can be cloned prior to administration. By cloning and growing cells having high activity of damaging target cells, cellular immunotherapy can be performed more effectively. Furthermore, APC and CTL isolated in this manner may be used for cellular immunotherapy not only against individuals from whom the cells are derived, but also against similar types of diseases in other individuals.

Aspects of the present invention are described in the following examples, which are presented only to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. EXAMPLES The present invention is illustrated, but not restricted, by following Examples.

MATERIALS AND METHODS Cell lines A24LCL cells (HLA-A24/24) and EHM (HLA-A3/3), human B- lymphoblastoid cell lines, were generous gifts from Takara Shuzo Co, Ltd. (Otsu, Japan). The A24LCL celis were used for peptide-mediated cytotoxicity assays. Lung carcinoma cell lines TEl (HLA-A2402+), TEl 3 (HLA- A2402-) and PC9 (HLA- A2402-) were purchased from ATCC Expression levels of TTK, URLClO and KOCl in the lung carcinoma cell lines were determined by cDNA microarray and RT-PCR that revealed strong expression of all three genes in TEl, TTK and KOCl expression in PC9, and URLClO expression in TE 13 (data not shown).

Candidate selection of peptide derived from TTK, URLClO and KOCl

9-mer and 10-mer peptides derived from TTK, URLClO or KOCl that bind to HLA- A24 molecule were predicted by binding prediction software "BIMAS"

(http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken parker comboform) (Parker KC, et al,

(1994) J Immunol.;152(l):163-75.; Kuzushima K, et al, (2001) Blood.;98(6): 1872-81.).

These peptides were synthesized by Mimotopes (San Diego, LA) according to the standard solid phase synthesis method and purified by reversed phase HPLC. The purity (>90%) and the identity of the peptides were determined by analytical HPLC and mass spectrometry analysis, respectively. Peptides were dissolved in dimethylsulfoxide

(DMSO) at 20 mg/ml and stored at -80 ⁰C.

In vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as antigen-presenting cells (APCs) to induce CTL responses against peptides presented on HLA. DCs were generated in vitro as described elsewhere (Nukaya I et al, (1999) Int. J. Cancer 80, 92-7., Tsai V et al. , (1997) J. Immunol 158:1796-802.). Briefly, peripheral blood mononuclear cells (PBMCs) isolated from a normal volunteer (HLA-A*2402) by Ficoll-Paque (Pharmacia) solution were separated by adherence to a plastic tissue culture flask (Becton Dickinson) so as to enrich them for the monocyte fraction. The monocyte- enriched population was cultured in the presence of 1000 U/ml of GM-CSF (provided by Kirin Brewery Company) and 1000 U/ml of IL-4 (Genzyme) in AIM-V (Invitrogen) containing 2% heat-inactivated autologous serum (AS). After 7 days in the culture, the cytokine-generated DCs were pulsed with 20 μg/ml of HLA-A24-binding peptides in the presence of 3 μg/ml of B2-microglobulin for 4 hrs at 20°C in AIM-V. These peptide- pulsed DCs were then irradiated (5500 rad) and mixed at a 1:20 ratio with autologous CD8⁺ T cells, obtained by positive selection with Dynabeads M-450 CD8 (Dynal) and DETACHa BEAD™ (Dynal). These cultures were set up in 48-well plates (Corning); each well contained 1.5x10⁴ peptide-pulsed DCs, 3x10⁵ CD8⁺ T cells and 10 ng/ml of IL- 7 (Genzyme) in 0.5 ml of AIM-V/2% AS. Three days later, these cultures were supplemented with IL-2 (CHIRON) to a final concentration of 20 IU/ml. On day 7 and 14, the T cells were further restimulated with the autologous peptide-pulsed DCs. The DCs were prepared each time by the same way described above. Cytotoxicity was tested against peptide-pulsed A24LCL cells after the 3rd round of peptide stimulation on day 21.

CTL Expansion Procedure

CTLs were expanded in culture using the method similar to the one described by Riddell SR₅ et al, (Walter EA et al, (1995) N Engl J Med 333:1038-44.;, Riddel et al, (1996) Nature Med. 2:216-23.). Atotal 5 x 10⁴ of CTLs were resuspended in 25 ml of AIM-V/5% AS with 25 x 10⁶ irradiated (3300 rad) PBMC and 5 x 10⁶ irradiated (8000 rad) EHM cells in the presence of 40 ng/ml of anti-CD3 monoclonal antibody (Pharmingen). One day after initiating the cultures, 120 IU/ml of IL-2 were added to the cultures. The cultures were fed with fresh AIM-V/5% AS containing 30 IU/ml of IL-2 on days 5, 8 and 11.

Establishment of CTL clones

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 7x10⁴ cells/well of allogenic PBMCs, IxIO⁴ cells/well of EHM, 30ng/ml of anti-CD3 antibody, and 125 U/ml of IL-2 in total of 150μl/well of AIM-V containing 5%AS. 50 μl /well of IL-2 was added to the medium 10 days later so that IL-2 became 125 U/ml in the final concentration. Cytotoxic activity of CTLs was tested on the 14th day, and CTL clones were expanded using the same method above.

Cytotoxicity Assay Target cells were labeled with 100 μCi OfNa₂ ⁵¹CrO₄ for 1 hr at 37°C in a CO₂ incubator (Perkin Elmer Life Sciences). Peptide-pulsed targets were prepared by incubating the cells with 20μg/ml of the peptide for 16 hrs at 37°C before labeling. Labeled target cells were rinsed and mixed with effector cells in a final volume of 0.2 ml in round-bottom microliter plates. The plates were centrifuged (4 minutes at 800 x g) to increase cell-to-cell contact and placed in a CO₂ incubator at 37°C. After 4 hrs of incubation, 0.1 ml of the supernatant was collected from each well and the radioactivity was determined with a gamma counter.

The percentage of specific cytotoxicity was determined by calculating the percentage of specific ⁵¹Cr-release by the following formula:

{(cpm of the test sample release - cpm of the spontaneous release) / (cpm of the maximum release - cpm of the spontaneous release)} x 100.

Spontaneous release was determined by incubating the target cells alone, in the absence of effector cells, and the maximum release was obtained by incubating the target cells with IN HCL. All measurements were done in duplicate, and the standard errors of the means were consistently below 10% of the value of the mean.

Antigen specificity was confirmed by the cold target inhibition assay, which utilized unlabeled A24LCL cells that were pulsed with or without peptide (20μg/ml for 16 hrs at 37°C) to compete for the recognition of ⁵¹Cr-labeled tumor cells. Blocking assay of cytotoxicity using the monoclonal antibodies (mAbs)

(mouse anti-MHC-class I mAb, anti-MHC-class II mAb, anti-CD8mAb, and anti- CD4mAb) was performed to confirm the HLA restriction manner. Anti-mouse IgGl , anti-mouse IgG2a mAbs were used as Isotype.

RESULTS Prediction ofHLA-A24 binding peptides derived from TTK URLClO or KOCl Table 1 shows the HLA-A*2402 binding peptides for TTK (GenBank Accession No. NM_003318; SEQ ID Nos.l, 2) in order of binding affinity. Table IA shows 9-mer peptides derived from TTK and Table IB shows 10-mer peptides derived from TTK. Table 2 shows the HLA-A*2402 binding peptides for URLClO (GenBank Accession No. AB105187; SEQ ID Nos.3, 4) in order of binding affinity. Table 2A shows 9-mer peptides derived from URLClO and Table 2B shows 10-mer peptides derived from URLCl 0. Table 3 shows the HLA- A*2402 binding peptides for KOCl (GenBank Accession No. NM_006547; SEQ ID Nos.5, 6) in order of binding affinity. Table 3 A shows 9-mer peptides derived from KOCl and Table 3B shows 10-mer peptides derived from KOCl .

Table IA HLA-A24 binding 9-mer peptides derived from TTK

Start Amino Acid SEQ Binding Start Amino Acid ^r Binding

Position Sequence ID No. Score Position Sequence Score

90 RYSQAIEAL 7 400 71 KLEKNSVPL 17 12 567 SYRNEIAYL 8 200 201 KNLSASTVL 18 12 N.D

549 IYAIKYVNL 9 200 467 RTPWKNDF 19 10.1 N.D

590 DYEITDQYI 10 90 102 KYGQNESFA 20 10 N.D

652 NFLIVDGML 11 42 19 KVRDIKNKF 21 8.9 N.D

141 KFAFVHISF 12 28 111 KQRISIPEL 22 8.8 N.D

214 SFSGSLGHL 13 20 75 NSVPLSDAL 23 8.6 N.D

28 KNEDLTDEL 14 19 605 GNTDLNSWL 24 8.6 N.D

111 RIQVRFAEL 15 15.8 596 QYIYMVMEC 25 8.3 N.D

108 SFARIQVRF 16 14 . 535 GSSKVFQVL 26 8.1 N.D

Start position indicates the number of amino acid from N-terminal of TTK. Binding score is derived from "BIMAS" described in Materials and Methods. N.D. indicates "not done"

Table IB HLA-A24 binding 10-mer peptides derived from TTK

Start Amino Acid SEQ ID Binding Start Amino Acid SEQ Binding

Position Sequence No. Score Position Sequence ID No. Score

810 KYVLGQLVGL 27 600 168 KAVERGAVPL 37 14.4

725 YYMTYGKTPF 28 150 232 RGQTTKARFL 38 12

598 ΓYMVMECGNI 29 75 185 RNLNLQKKQL 39 12

728 TYGKTPFQQI 30 72 111 KQRISIPELL 40 11.2

755 EFPDIPEKDL 31 36 573 AYLNKLQQHS 41 10.8

490 CFQQQQHQIL 32 36 373 EYQEPEVPES 42 9.9

143 AFVHISFAQF 33 18 74 KNSVPLSDAL 43 9.6

569 RNEIAYLNKL 34 15.8 ^' 315 KPSGNDSCEL 44 8.8

^' 359 KTESSLLAKL 35 15.8 61 NPEDWLSLLL 45 8.6

553 KYVNLEEADN 36 15 763 DLQDVLKCCL 46 8.6

Start position indicates the number of amino acid from N-terminal of TTK. Binding score is derived from "BIMAS" described in Materials and Methods.

Table 2A HLA-A24 binding 9-mer peptides derived from URLClO

Start

Amino Acid SEQ Bindin Start Amino Acid SEQ Bindin

Positio Sequence ID No. g Score Position Sequence ID No. g Score n

154 KPEEKRFLL 47 14.4 193 EYAGSMGES 57 5.5 N.D

48 RADPPWAPL 48 9.6 168 FFYLKCCKI 58 5.5 N.D

205 LWLAILLLL 49 8.4 128 AAVKIFPRF 59 5 N.D

57 GTMALLALL 50 7.2 58 TMALLALLL 60 4.8 N.D 203 GGLWLAILL 51 7.2 152 RPKPEEKRF 61 4.8 N.D

62 LALLLWAL 52 7.2 197 SMGESCGGL 62 4.8 N.D

WAPLGTMA

53 53 6 173 CCKIRYCNL 63 4 N.D L

214 ASIAAGLSL 54 6 28 DPGRGARRL 64 4 N.D

54 APLGTMALL 55 6 31 RGARRLRRF 65 4 N.D

212 LLASIAAGL 56 5.6 NJD 202 CGGLWLAIL 66 4 N.D

Start position indicates the number of amino acid from N-terminal of URLClO. Binding score is derived from "BIMAS" described in Materials and Methods. N.D. indicates "not done"

Table 2B HLA-A24 binding 10-mer peptides derived from URLClO

Start Amino Acid SEQ Binding Start Amino Acid SEQ Binding Position Sequence ID No. Score Position Sequence IDNo. Score

177 RYCNLEGPPI 67 100 196 GSMGESCGGL 77 6 N.D

159 RFLLEEPMPF 68 30 204 GLWLAILLLL 78 5.6 N.D

152 RPKPEEKRFL 69 9.6 123 PYCVIAAVKI 79 5.5 N.D

211 LLLASIAAGL 70 8.4 193 EYAGSMGESC 80 5 N.D

172 KCCKIRYCNL 71 8 61 LLALLLVVAL 81 4.8 N.S

169 FYLKCCKIRY 72 7.5 202 CGGLWLAILL 82 4.8 N.D

57 GTMALLALLL 73 7.2 56 LGTMALLALL 83 4.8 N.D

53 WAPLGTMALL 74 6 70 LPRVWTDANL 84 4 N.D

203 GGLWLAILLL 75 6 N.D 201 SCGGLWLAIL 85 4 N.D

198 MGESCGGLWL 76 6 N.D 213 LASIAAGLSL 86 4 N.D

Start position indicates the number of amino acid from N-terminal of URLClO. Binding score is derived from "BIMAS" described as Materials and Methods. N.D. indicates "not done". N.S. indicates "not synthesized"

Table 3A HLA-A24 binding 9-mer peptides derived from KOCl

Start Amino Acid SEQ Binding Start Amino Acid SEQ Binding

Position Sequence ID No. Score Position Sequence ID No. Score

350 SYENDIASM 87 37.5 4 LYIGNLSEN 97 8.25

141 GFQLENFTL 88 30 423 KQGQHIKQL 98 8 N.D

508 KTVNELQNL 89 14.4 561 KQHQQQKAL 99 8 N.D

26 KIPVSGPFL 90 12 310 ITISPLQEL 100 7.9 N.D

192 KPCDLPLRL 91 11.5 470 IYGKIKEEN 101 7.7 N.D

433 RFAGASIKI 92 11 356 ASMNLQAHL 102 7.2 N.D 505 KGGKTVNEL 93 10.6 93 QWEVLDSLL 103 7.2 N.D

190 KQKPCDLPL 94 9.6 43 DCPDESWAL 104 7.2 N.D

152 AYIPDEMAA 95 9 92 LQWEVLDSL 105 6.7 N.D

320 LYNPERTIT 96 9 55 EALSGKIEL 106 6.6 N.D

Start position indicates the number of amino acid from N-teπninal of KOCl. Binding score is derived from "BIMAS" described in Materials and Methods. N.D. indicates "not done"

Table 3B HLA-A24 binding 10-mer peptides derived from KOCl

Start Amino Acid SEQ ID Binding Start Amino Acid SEQ Binding

Position Sequence No. Score Position Sequence ID No. Score

470 IYGKIKEENF 107 100 91 HLQWEVLDSL 117 8.4 N.D

272 KFTEEIPLKI 108 18.5 359 NLQAHLIPGL 118 7.2 N.D

290 RLIGKEGRNL 109 12 364 LEPGLNLNAL 119 7.2 N.D

309 KITISPLQEL 110 10.6 165 LQQPRGRRGL 120 7.2 N.D

350 SYENDIASMN 111 10.5 273 FTEEIPLKIL 121 7.2 N.D

192 KPCDLPLRLL 112 9.6 406 ETVHLFIPAL 122 6 N.D

320 LYNPERTITV 113 9 N.D 138 KLNGFQLENF 123 6 N.D

4 LYIGNLSENA 114 9 N.D 9 LSENAAPSDL 124 6 N.D

548 VAQRKIQEIL 115 8.4 N.D 88 IPPHLQWEVL 125 6 N.D

83 LQIRNIPPHL 116 8.4 N.D 127 SSKDQARQAL 126 5.7 N.D Start position indicates the number of amino acid from N-terminal of KOC 1. Binding score is derived from "BIMAS" described in Materials and Methods. N.D. indicates "not done"

Stimulation of the T cells using the predicted peptides

CTLs for those peptides derived from TTK, URLClO or KOCl were generated in the manner described in "Materials and Methods" section above. Resulting CTLs showing detectable cytotoxic activity were expanded, and those CTL clones that demonstrated higher cytotoxic activities against the peptide-pulsed target as compared to the activities against target without peptide pulse were established.

The CTL clones stimulated by the HLA-A24 binding peptides TTK-567 (SYRNEIAYL (SEQ ID No.8)) (Figure I)₅ URLClO-177 (RYCNLEGPPI (SEQ ID

No.67)) (Figure 2) or by KOCl-508 (KTVNELQNL (SEQ ID No.89)) (Figure 3) showed potent cytotoxic activity against the peptide-pulsed target without showing any significant cytotoxic activity against targets not pulsed with any peptide. Cytotoxic activity against lunε cancer cell lines endogenousϊy expressing TTK URLClO or KOCl

The established CTL clones described above were examined for their ability to recognize and kill tumor cells endogenously expressing TTK, URLClO or KOCl. Cytotoxic activity against TEl cells, which endogenously express TTK and HLA-A24, was tested using as effector cells the CTL clone raised by TTK-567. PC9 cells were used as the target cells that endogenously express TTK but not HLA- A24. The CTL clone showed high cytotoxic activity against the TEl cells that express both TTK and HLA- A24. On the other hand, it did not show significant cytotoxic activity against the PC9 cells that express TTK but not HLA- A24 (Figure 4). Cytotoxic activity against TEl cells, which endogenously express URLClO and HLA- A24, was tested using as effector cells the CTL clone raised by URLClO-177. TE13 cells were used as the target cells that endogenously express URLClO but not HLA- A24. The CTL clone showed high cytotoxic activity against the TEl cells that express both URLClO and HLA-A24. On the other hand, it did not show significant cytotoxic activity against theTE13 cells that express URLClO but not HLA- A24 (Figure 5). Cytotoxic activity against TEl cells, which endogenously express KOCl and HLA-A24, was tested using as effector cells the CTL clone raised by KOCl-508. PC9 cells were used as the target cells that endogenously express KOCl but not HLA- A24. The CTL clone showed high cytotoxic activity against the TEl cells that express both KOCl, and HLA- A24. On the other hand, it did not show significant cytotoxic activity against the PC9 cells that express KOCl but not HLA-A24 (Figure 6).

The above-described CTL clones showed potent cytotoxic activity against the TEl lung cancer cell line, a cell line that expresses TTK, URLClO and KOCl, and HLA- A24. On the other hand, the CTL clones against TTK-567 or KOCl-508 showed no cytotoxic activity against the PC9 lung cancer cell line, a cell line that expresses TTK and KOCl but not HLA-A24; likewise, the CTL clone raised against URLClO-177 did not show a cytotoxic activity against the TE 13 lung cancer cell line, a cell line that expresses URLClO but not HLA-A24. These CTL clones also show no cytotoxic activity against A24LCL cells, cell that express HLA-A24, but do not express TTK, URLClO or KOCl (Figure I₅ 2 and 3). These results clearly demonstrate that TTK-567, URLClO-177 and KOCl-508 were naturally expressed to the tumor cell surface with HLA- A24 molecule and recognized CTL.

Cold target inhibition assay Cold target inhibition assay was performed to confirm the specificity of the CTL clones as described in "Materials and Methods" section above. TEl cells labeled by Na₂ ⁵¹Cr O₄ were prepared as hot target, while TTK-567 peptide-pulsed A24LCL cells were used as cold target (Inhibitor). The cytotoxic activity of the TTK-567 CTL clone against TEl cells was specifically inhibited by the addition of A24LCL cells pulsed with the identical peptide (Figure 7). Regarding URLClO, TEl cells labeled by Na₂ ⁵¹Cr O₄ were prepared as hot target, while URLC 10- 177 peptide-pulsed A24LCL cells were used as cold target (Inhibitor). The cytotoxic activity of the URLClO-177 CTL clone against TEl was specifically inhibited by the addition of A24LCL cells pulsed with the identical peptide (Figure 8). As above, TEl cells labeled by Na₂ ⁵¹Cr O₄ were prepared as hot target, while KOCl-508 peptide-pulsed A24LCL cells were used as cold target

(Inhibitor). The E/T ratio was fixed at 20. The cytotoxic activity of KOC 1 -508 CTL clone against TEl cells was specifically inhibited by the addition of A24LCL cells pulsed with the identical peptide (Figure 9). Specific cytotoxicity against target TEl cells was significantly inhibited when peptide-pulsed cold target was added in the assay at various ratios but not inhibited at all by the addition of cold target. These results were indicated as a percentage of specific lysis inhibition at the E/T ratio of 20.

Blocking of CTL activity by antibodies that bind to T-cell surface antigens

To see whether the observed killing activity is mediated by the cytotoxic T- cells, effects of antibodies on the killing activities were investigated, in which antibodies recognizing T-cell surface antigens related to the function of CTL were used. CTL activities were clearly blocked by the addition of antibodies that recognize HLA Class I and CD 8 but are affected little by the addition of antibodies to HLA Class II or CD4, as TTK-567 CTL clone shown in Figure 10, URLC 10- 177 CTL clone in Figure 11 and KOCl-508 CTL clone in Figure 12. These results show that the cytotoxic activities of CTL clones against the lung carcinoma cells are the HLA class I restricted and CD8 mediated cytotoxic activity.

Homology analysis of the antigen peptides

The CTL clones established against TTK-567, URLC 10- 177 or KOCl-508 showed potent cytotoxic activity. Thus, it is possible that the sequence of TTK-567, URLClO-177 or KOCl-508 is homologous to the peptides derived from other molecules, which are known to sensitize human immune system. To exclude this possibility, homology analysis was performed with the peptide sequences as queries using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) (Altschul SF, et ah, (1997) Nucleic Acids Res.;25(17):3389-402.; Altschul SF, et al, (1990) J MoI Biol.;215(3):403-10.) and revealed no sequence with significant homology. These results indicate that the sequences of TTK-567, URLClO-177 and KOCl-508 are unique and there is little possibility that the peptides would raise unintended immunologic responses to any unrelated molecule.

DISCUSSION Identification of new TAAs, particularly those that induce potent and specific anti-tumor immune responses, warrants further development of the clinical application of ^■ peptide vaccination strategies in various types of cancer (Boon T. et al, (1996) J Exp Med 183: 725-9.; van der Bruggen P et al, (1991) Science 254: 1643-7.; Brichard V et al, (1993) J Exp Med 178: 489-95.; Kawakami Y et al, (1994) J Exp Med 180: 347-52.; Shichijo S et al, (1998) J Exp Med 187:277-88.; Chen Y.T. et al, (1997) Proc.Natl.Acd. Sci. USA₅ 94: 1914-8.; Harris CC, (1996) J Natl Cancer Inst 88:1442-5.; Butterfield LH et al, (1999) Cancer Res 59:3134-42.; Vissers JL et al, (1999) Cancer Res 59: 5554-9.; van der Burg SH et al, (1996) J. Immunol 156:3308-14.; Tanaka F et al, (1997) Cancer Res 57:4465-8.; Fujie T et al, (1999) Int J Cancer 80:169-72.; Kikuchi M et al, (1999) Int J Cancer 81 : 459-66.; Oiso M et al, (1999) Int J Cancer 81:387-94.). cDNA microarray technologies can disclose comprehensive profiles of gene expression of malignant cells (Okabe H. et al, (2001) Cancer Res., 61, 2129-37.; Lin Y- M. et al, (2002) Oncogene, 21;4120-8.; Hasegawa S. et al, (2002) Cancer Res 62:7012- 7.) and, find utility in the identification of potential TAAs. Among the transcripts that are up-regulated in lung cancers, three novel human genes, termed TTK, URLClO and KOCl, respectively, were identified using these technologies.

As demonstrated above, TTK, URLClO and KOCl are over-expressed in lung cancer and show minimal expression in normal tissues. In addition, these genes have been shown to have a significant function related to cell proliferation (See WO2004/031413). Thus, peptides derived from TTK, URLCl 0 and KOCl can serve as TAA epitopes, which, in turn, can be used to induce significant and specific immune responses against cancer cells.

Thus, as TTK, URLClO and KOCl are novel TAAs, cancer vaccines using these epitope peptides may be useful as immunotherapeutics against lung carcinoma or other cancer expressing these molecules.

INDUSTRIAL APPLICABILITY

The present invention identifies new TAAs, particularly those which induce potent and specific anti-rumor immune responses. Such TAAs warrants further development of the clinical application of peptide vaccination strategy in lung cancer.

All patents, patent applications, and publications cited herein are incorporated by reference.

While the invention has been described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

Claims

I . An isolated peptide of less than about 15 amino acids selected from the group consisting of peptides comprising the amino acid sequences of SEQ ID NO: 8, 67, and 89. 2. A peptide having cytotoxic T cell inducibility, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 67, and 89, wherein 1,

2, or several amino acids are substituted, deleted, or added.

3. The peptide of claim 2, wherein the second amino acid from the N-terminus is phenylalanine, tyrosine, methionine, or tryptophan.

4. The peptide of claim 2, wherein the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan, or methionine.

5. A pharmaceutical composition for treating or preventing lung cancer, said composition comprising one or more peptides of claim 1 or 2.

6. An exosome that presents on its surface a complex comprising a peptide of claim 1 or 2 and an HLA antigen.

7. The exosome of claim 6, wherein the HLA antigen is HLA- A24.

8. The exosome of claim 7, wherein the HLA antigen is HLA-A2402.

9. A method of inducing antigen-presenting cells having a high cytotoxic T cell inducibility comprising the step of contacting an antigen-presenting cell with a peptide of claim 1 or 2.

10. A method of inducing cytotoxic T cells by contacting a T cell with a peptide of claim 1 or 2.

I 1. The method of inducing antigen-presenting cells having high cytotoxic T cell inducibility, said method comprising the step of transferring a gene comprising a polynucleotide encoding a peptide of claim 1 or 2 to an antigen-presenting cell.

12. An isolated cytotoxic T cell is induced by contacting a T cell with a peptide of claim 1 or 2.

13. An antigen-presenting cell, which comprises a complex formed between an HLA antigen and a peptide of claim 1 or 2.

14. The antigen-presenting cell of claim 13, induced by the method of claim 9.

15. A vaccine for inhibiting proliferation of lung cancer cells, wherein the vaccine comprises a peptide of claim 1 or 2 as the active ingredient.

16. The vaccine of claim 15, formulated for administration to a subject whose HLA antigen is HLA-A24.

^■ 17. The vaccine of claim 15, said vaccine capable of suppressing the growth of lung cancer cells.

18. A method of treating or preventing lung cancer in a subject comprising administering to said subject a vaccine comprising a peptide of claim 1 or 2, an immunologically active fragment thereof, or a polynucleotide encoding said peptide or immunologically active fragment.