JP4628208B2 - Peptide vaccine for the treatment of gastric cancer or colorectal cancer expressing CXADRL1 or GCUD1 protein - Google Patents

Description

  The present invention relates to a novel peptide effective as a cancer vaccine, and a pharmaceutical composition for treating and / or preventing a tumor containing the peptide.

  Gastric cancer and colorectal cancer are the leading causes of cancer death worldwide. Despite recent advances in diagnostic and therapeutic strategies, the prognosis for patients with advanced cancer remains very poor. Molecular studies have revealed that changes in tumor suppressor genes or oncogenes are involved in carcinogenesis, but the exact mechanism remains unclear.

  The cDNA microarray technology has made it possible to obtain a comprehensive profile of gene expression in normal cells and malignant cells, and to compare gene expression in malignant cells and corresponding normal cells (Non-Patent Documents 1 to 4). This approach makes it possible to elucidate the complex nature of cancer cells and helps to elucidate the mechanism of carcinogenesis. The identification of genes that are deregulated in tumors can lead to a more accurate and error-free diagnosis of individual cancers and the development of new therapeutic targets (Non-Patent Document 5). In order to elucidate the underlying mechanisms of tumors from a genome-wide perspective, and to explore target molecules for the development of diagnostics and novel therapeutics, we have cDNA containing 23040 genes. Tumor cell expression profiles have been analyzed using microarrays (Non-Patent Documents 1 to 4).

  It is already easy to identify molecular targets for antitumor drugs by studies designed to elucidate the mechanisms of carcinogenesis. For example, a farnesyltransferase inhibitor (FTI), originally developed to inhibit the growth signaling pathways involved in Ras, was effective in the treatment of Ras-dependent tumors in animal models (Non-Patent Document 6). Clinical trials are being conducted in humans with anti-HER2 monoclonal antibody trastuzumab for the purpose of antagonizing anticancer drugs and proto-oncogene receptor HER2 / neu. Clinical efficacy and overall survival of breast cancer patients Has been achieved (Non-patent Document 7). The tyrosine kinase inhibitor STI-571, which selectively inactivates the bcr-abl fusion protein, is used in chronic myeloid leukemia where constitutive activation of the bcr-abl tyrosine kinase plays a crucial role in leukocyte transformation. Developed for therapeutic purposes. These types of drugs are designed for the purpose of suppressing the carcinogenic activity of specific gene products (Non-patent Document 8). Thus, gene products that are frequently up-regulated in cancer cells may serve as target candidates for developing new anticancer drugs.

  CD8 + cytotoxic T lymphocytes (CTL) have been shown to recognize tumor peptides from tumor associated antigens (TAA) presented on MHC class I molecules and lyse tumor cells. Since the discovery of the MAGE family as the first example of TAA, many other TAAs have been discovered using immunological approaches (Non-Patent Documents 9 to 13). Some of the discovered TAAs are currently in clinical development as targets for immunotherapy. TAA discovered so far includes MAGE (Non-Patent Document 11), gp100 (Non-Patent Document 13), SART (Non-Patent Document 14), and NY-ESO-1 (Non-Patent Document 15). On the other hand, gene products that have been shown to be specifically overexpressed in tumor cells have been shown to be recognized as targets that induce cellular immune responses. Such gene products include p53 (Non-patent document 16), HER2 / neu (Non-patent document 17), CEA (Non-patent document 18), and the like.

  Despite significant progress in basic and clinical research on TAA (Non-Patent Documents 19-21), the number of candidate TAAs for the treatment of adenocarcinoma including colorectal and gastric cancer is very limited . TAA, which is expressed in large amounts in cancer cells and at the same time is restricted to cancer cells, is considered to be a promising candidate for immunotherapy targets. In addition, the identification of new TAAs that elicit potent and specific anti-tumor immune responses would facilitate clinical use of peptide vaccination in various types of cancer (Non-Patent Documents 10-15, 22-29). .

Peripheral blood mononuclear cells (PBMC) from certain healthy donors, when subjected to peptide stimulation, produce significant levels of IFN-γ in response to the peptide, but HLA-A24 or HLA-A0201 in ⁵¹ Cr release assays It has been repeatedly reported that there is almost no cytotoxicity against tumor cells in a restrictive manner (Non-Patent Documents 30 to 32). However, both HLA-A24 and HLA-A0201 are HLA alleles common to Japanese people, and the same applies to Caucasians (Non-patent Documents 33 to 37). Therefore, the cancer antigen peptides presented by these HLAs may be particularly useful in the treatment of Japanese and Caucasian cancers. In addition, in vitro induction of low-affinity CTLs is usually a specific peptide / MHC complex that is thought to effectively activate these CTLs on the surface of antigen-presenting cells (APC) using peptides at high concentrations. It is known to occur by raising the body to a high level (38).
Okabe et al., Cancer Res 61: 2129-37 (2001) Kitahara et al., Cancer Res 61: 3544-9 (2001) Lin et al., Oncogene 21: 4120-8 (2002) Hasegawa et al., Cancer Res 62: 7012-7 (2002) Bienz and Clevers, Cell 103: 311-20 (2000) Sun J, et al., Oncogene 16: 1467-73 (1998) Molina MA, et al., Cancer Res 61: 4744-9 (2001); O'Dwyer ME and Druker BJ, Curr Opin Oncol 12: 594-7 (2000) Boon, Int J Cancer 54: 177-80 (1993) Boon and van der Bruggen, J Exp Med 183: 725-9 (1996) van der Bruggen et al., Science 254: 1643-7 (1991) Brichard et al., J Exp Med 178: 489-95 (1993) Kawakami et al., J Exp Med 180: 347-52 (1994) Shichijo et al., J Exp Med 187: 277-88 (1998) Chen et al., Proc Natl Acad Sci USA 94: 1914-8 (1997) Umano et al., Brit J Cancer 84: 1052-7 (2001) Tanaka et al., Brit J Cancer 84: 94-9 (2001) Nukaya et al., Int J Cancer 80: 92-7 (1999) Rosenberg et al., Nature Med 4: 321-7 (1998) Mukherji et al., Proc Natl Acad Sci USA 92: 8078-82 (1995) Hu et al., Cancer Res 56: 2479-83 (1996) Harris, J Natl Cancer Inst 88: 1442-55 (1996) Butterfield et al., Cancer Res 59: 3134-42 (1999) Vissers et al., Cancer Res 59: 5554-9 (1999) van der Burg et al., J Immunol 156: 3308-14 (1996) Tanaka et al., Cancer Res 57: 4465-8 (1997) Fujie et al., Int J Cancer 80: 169-72 (1999) Kikuchi et al., Int J Cancer 81: 459-66 (1999) Oiso et al., Int J Cancer 81: 387-94 (1999) Kawano et al., Cancer Res 60: 3550-8 (2000) Nishizaka et al., Cancer Res 60: 4830-7 (2000) Tamura et al., Jpn J Cancer Res 92: 762-7 (2001) Date et al., Tissue Antigens 47: 93-101 (1996) Kondo et al., J Immunol 155: 4307-12 (1995) Kubo et al., J Immunol 152: 3913-24 (1994) Imanishi et al., Proceeding of the eleventh International Hictocompatibility Workshop and Conference Oxford University Press, Oxford, 1065 (1992) Williams et al., Tissue Antigens 49: 129 (1997) Alexander-Miller et al., Proc Natl Acad Sci USA 93: 4102-7 (1996)

  The present invention has been made in view of such a situation, and the problem to be solved by the present invention is a cell targeting the gene CXADRL1 or GCUD1 whose expression is significantly increased in gastric cancer or colorectal cancer. It is to provide a novel peptide effective as a cancer vaccine capable of inducing cytotoxic T cell activity, and a pharmaceutical composition for treatment and / or prevention of a tumor containing the peptide.

  The present inventors pay attention to the possibility of cancer vaccine therapy targeting two genes CXADRL1 or GCUD1 whose expression is remarkably increased in gastric cancer or colorectal cancer, and can be used effectively as a vaccine. I tried to search. As a result, the inventors succeeded in finding an epitope peptide having an increased binding affinity for HLA by modification of the anchor residue, and completed the present invention.

That is, the present invention provides the following (1) to (18).
(1) The peptide according to any one of (a) and (b) below;
(A) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or 6, and (b) one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 3 or 6 are substituted, deleted, added, and / or A peptide having cytotoxic T cell-inducing activity, which is an inserted peptide, wherein the second amino acid from the N-terminus is leucine or isoleucine, and / or the C-terminal amino acid is leucine, isoleucine or valine,
(2) A pharmaceutical composition for treating and / or preventing a tumor, comprising at least one peptide according to (1),
(3) The pharmaceutical composition according to (2), wherein the tumor is gastric cancer or colorectal cancer,
(4) A vaccine for suppressing tumor growth and / or metastasis, comprising at least one peptide according to (1),
(5) The vaccine according to (4) for administration to a patient whose HLA antigen is HLA-A02,
(6) The vaccine according to (5), wherein the tumor is gastric cancer or colorectal cancer,
(7) An exosome presenting on the surface a complex containing at least one peptide according to (1) and an HLA antigen,
(8) The exosome according to (7), wherein the HLA antigen is HLA-A02,
(9) The exosome according to (8), wherein the HLA antigen is HLA-A0201,
(10) A method for producing an antigen-presenting cell having cytotoxic T cell-inducing activity, comprising a step of contacting at least one of the peptides according to (1) with a cell having an antigen-presenting ability;
(11) A method for producing an antigen-presenting cell having cytotoxic T cell-inducing activity, comprising introducing at least one polynucleotide encoding the peptide according to (1) into a cell having an antigen-presenting ability;
(12) An agent for inducing an antigen-presenting cell having cytotoxic T cell-inducing activity, comprising at least one of the peptides according to (1),
(13) A method for producing activated cytotoxic T cells, comprising a step of contacting at least one of the peptides according to (1) with a cell having an antigen-presenting ability,
(14) An inducer of activated cytotoxic T cells, comprising at least one peptide according to (1),
(15) an antigen-presenting cell obtained by presenting a complex of the HLA antigen and the peptide according to (1),
(16) The antigen-presenting cell according to (15), which is induced by the inducer according to (12),
(17) An isolated cytotoxic T cell induced by the peptide according to (1),
(18) The cytotoxic T cell according to (17), which is induced by the inducer according to (14).

  According to the present invention, a novel peptide effective as a cancer vaccine capable of inducing cytotoxic T cell activity targeting the gene CXADRL1 or GCUD1 whose expression is remarkably increased in gastric cancer or colorectal cancer, And a pharmaceutical composition for the treatment and / or prevention of tumors containing the same. Tumor associated antigen (TAA), which is expressed in large amounts in cancer cells and at the same time is restricted to cancer cells, is a promising candidate for immunotherapy targets. In addition, the identification of new TAAs that elicit strong and specific anti-tumor immune responses would facilitate clinical use of peptide vaccination in various types of cancer.

  The inventors of the present invention have found that the anchor-modified polypeptide of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (derived from CXADRL1) is more frequently and more abundant than the wild-type peptide. Clarified that it triggers. We have identified a specific partial TCR repertoire in which the anchor-modified polypeptide increases the binding affinity for the HLA-A0201 molecule and further recognizes a naturally processed wild-type epitope peptide presented by tumor cells. Has been found to activate. Therefore, the present invention provides a polypeptide in which an anchor residue is modified in a polypeptide derived from CXADRL1. Examples of these anchor-modified polypeptides include a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 (derived from CXADRL1).

  The inventors have also found that the anchor-modified polypeptide of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 (derived from GCUD1) is also more frequently and more abundant than the wild-type peptide. It was revealed that it induces CTL. We have identified a specific partial TCR repertoire in which the anchor-modified polypeptide increases the binding affinity for the HLA-A0201 molecule and further recognizes a naturally processed wild-type epitope peptide presented by tumor cells. Has been found to activate. Therefore, the present invention provides a polypeptide in which an anchor residue is modified in a GCUD1-derived polypeptide. An example of such an anchor-modified polypeptide is the amino acid sequence set forth in SEQ ID NO: 6 (derived from GCUD1).

  In the present invention, the “anchor residue” means an amino acid residue of an epitope that binds to the HLA class I peptide binding groove but does not contact the TCR. Epitope peptide positions 2 and 9 are known as anchor residues, and in the HLA-A02 antigen motif, leucine (Leu) and isoleucine (Ile) at position 2 have peptide binding affinity to the HLA-A * 0201 molecule. It has been reported to be the optimal anchor residue that enhances sex (Smith et al., Mol Immunol: 35 1033-43 (1998)). Similarly, valine at position 9 (Val) is also known as the optimal anchor residue.

  The present invention also provides a peptide in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 3 or 6, wherein the second amino acid from the N-terminal is Provided is a peptide having cytotoxic T cell-inducing activity, wherein leucine or isoleucine and / or the C-terminal amino acid is leucine, isoleucine or valine. Examples of such peptides include peptides having the amino acid sequence set forth in SEQ ID NO: 4 or 5.

  When modifying amino acid residues, it is desirable to mutate to another amino acid that preserves the properties of the amino acid side chain. For example, amino acid side chain properties include 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), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M) having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), and aromatics Amino acids having side chains (H, F, Y, W) can be mentioned (the parentheses represent single letter symbols of amino acids). Amino acid substitutions within each of these groups are referred to as conservative substitutions. It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by other amino acids of one or more amino acid residues to a certain amino acid sequence maintains its biological activity. (Mark, DF et al., Proc Natl Acad Sci USA (1984) 81: 5662-6; Zoller, MJ and Smith, M., Nucleic Acids Res. (1982) 10: 6487-500; Wang, A. et al., Science (1984) 224: 1431-3; Dalbadie-McFarland, G. et al., Proc Natl Acad Sci USA (1982) 79: 6409-13). The number of amino acids to be mutated is not particularly limited, but is usually 7 or less, preferably 5 or less, and more preferably 3 (for example, 2 or less). Amino acid sequence identity can be determined, for example, by the algorithm BLAST (Proc Natl Acad Sci USA (1993) 90: 5873-7) by Karlin and Altschul.

  The peptide of the present invention can be obtained by synthesizing a peptide from an arbitrary position based on the obtained amino acid sequence. Peptide synthesis can be performed according to methods used in normal peptide chemistry. Commonly used synthesis methods include, for example, Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Experiment, Maruzen Co., Ltd., 1985; Drug Development Continued Volume 14: Peptide Synthesis, Hirokawa Shoten, 1991, and other publications, and International Publication WO99 / 67288.

  The peptide of the present invention can also be synthesized by a known genetic engineering technique. Examples of the genetic engineering synthesis method include the following methods. That is, a DNA encoding a target peptide is introduced into an appropriate host cell to produce a transformed cell. By recovering the peptide produced from this transformed cell, the peptide of the present invention can be obtained.

  The peptide of the present invention can also be obtained by once producing it as a fusion protein and decomposing it with an appropriate protease. A method for producing a fusion protein is as follows: a polynucleotide encoding the peptide of the present invention and a polynucleotide encoding another peptide are linked so that the frames coincide with each other, introduced into an expression vector, and expressed in a host. Well, techniques known to those skilled in the art can be used. Other peptides to be subjected to fusion with the peptide of the present invention include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), and 6 His (histidine) residues. 6 × His, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck Known peptides such as tag, α-tubulin fragment, B-tag, protein C fragment and the like can be used. The peptide of the present invention can also be a fusion protein with GST (glutathione-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, MBP (maltose binding protein), and the like. . The peptide of the present invention can be obtained by treating the fusion protein thus prepared with an appropriate protease and recovering the target peptide. The peptide can be recovered by a method known to those skilled in the art, such as affinity chromatography.

  The binding to the HLA antigen can be measured using a technique in which a cell having an HLA antigen on the cell surface, such as a dendritic cell, is isolated and the peptide is bound to the cell.

  Alternatively, binding of various peptides to HLA antigens using software available on the Internet, such as those described in Parker KC, J. Immunol. 152, 163-75 (1994) Affinity can also be calculated in silico. The binding affinity with HLA antigen is, for example, Parker, KC, J. Immunol., 152, 163-75 (1994); Nukaya, I., Int. J. Cancer, 80, 92-7 (1999), etc. Can be measured.

  As the HLA antigen, for example, A-02 type, which is said to be expressed abundantly in Japanese, is preferable for obtaining effective results, and more preferably a subtype such as A-0201. In clinical practice, by examining the type of HLA antigen in patients in need of treatment in advance, appropriately select a peptide having high binding affinity with this or cytotoxic T cell (CTL) inducing activity by antigen presentation Can do.

  The peptide of the present invention can be used as a cancer vaccine capable of inducing CTL in vivo as one or a combination of two or more. Administration of the peptide of the present invention induces a CTL that specifically presents the peptide to the HLA antigen of the antigen-presenting cell and reacts specifically to the complex of the presented peptide and the HLA antigen, and the target cell The attack power against the vascular endothelial cells in the tumor cells to be increased. Alternatively, by taking out dendritic cells from the patient and stimulating with the peptide of the present invention, antigen-presenting cells with the peptide of the present invention bound on the cell surface can be obtained, and this is administered to the patient again, so that CTLs are expressed in the patient It can induce and increase the attack power against the target cell.

  That is, the present invention provides a pharmaceutical composition for treating and / or preventing a tumor or suppressing tumor growth and / or metastasis, comprising at least one of the peptides of the present invention.

  In the present invention, “tumor growth” refers to a state in which tumor cells increase in an unlimited or irreversible manner and are not in harmony with surrounding cells / tissues. Tumor cells cause “metastasis” by attaching to the vascular endothelium and invading surrounding tissue. The metastasized tumor cells remain there, creating a new independent tumor at a remote site. Such tumor metastasis re-starts tumor cell growth, destroying normal tissue and organ function. In addition, other metastases may arise from metastasized tumors. In the present invention, “treatment” of a tumor refers to extinguishing or reducing the tumor cells thus generated, or suppressing tumor metastasis. Furthermore, “prevention” of a tumor refers to inhibiting tumor cell growth and metastasis in advance. The tumor in the present invention is not particularly limited. As a preferable example, for example, gastric cancer or colorectal cancer is included in the tumor of the present invention.

  In the pharmaceutical composition of the present invention, the peptide of the present invention can be used alone as a pharmaceutical composition. Alternatively, it may be formulated by a commonly used pharmaceutical method. In that case, in addition to the peptide of the present invention, carriers, excipients and the like that are usually used in medicine can be appropriately included, and there is no particular limitation. Examples of the use of the pharmaceutical composition of the present invention include treatment and / or prevention of various tumors such as gastric cancer, colorectal cancer, duodenal cancer, colon cancer, lung cancer, breast cancer, prostate cancer, brain tumor and the like. Includes treatment and / or prevention of gastric cancer or colorectal cancer.

  A pharmaceutical composition for treating and / or preventing a tumor comprising the peptide of the present invention as an active ingredient is administered together with an adjuvant so that cellular immunity is effectively established, or administered together with an active ingredient such as another anticancer agent. Or can be administered in a particulate dosage form. As the adjuvant, those described in the literature (Johnson AG., Clin. Microbiol. Rev., 7: 277-289, 1994) can be applied. In addition, a liposome preparation, a particulate preparation bound to beads having a diameter of several μm, a preparation bound to lipid, and the like are also conceivable.

  As the administration method, oral administration, intradermal administration, subcutaneous administration, intravenous injection, and the like can be used, and systemic administration or local administration in the vicinity of the target tumor may be used. The dose of the peptide of the present invention can be appropriately adjusted depending on the disease to be treated, the age, weight, administration method, etc. of the patient, but is usually 0.001 mg to 1000 mg, preferably 0.01 mg to 1OO mg, more preferably O. It is preferably 1 mg to 1 O mg, and is preferably administered once every several days to several months. A person skilled in the art can appropriately select an appropriate dose.

  Alternatively, the present invention provides intracellular vesicles called exosomes, which display a complex of the peptide of the present invention and HLA antigen on the surface. The preparation of exosomes can be carried out using the methods described in detail in, for example, JP-T-11-510507 and JP-T2000-512161, but preferably obtained from a patient to be treated or prevented. Prepared using the antigen-presenting cells. The exosome of the present invention can be inoculated as a cancer vaccine in the same manner as the peptide of the present invention.

  The HLA antigen needs to be of the same type as the HLA antigen of the patient in need of treatment and / or prevention. For example, as described above, HLA-A02, particularly HLA-A0201, is often suitable for Japanese.

  The present invention also provides a method for producing an antigen-presenting cell having cytotoxic T cell-inducing activity, which comprises the step of contacting the peptide of the present invention with a cell having an antigen-presenting ability. In the present invention, the “cell having an antigen-presenting ability” refers to a cell that is capable of presenting an antigen to a T cell or a B cell by being activated. For example, dendritic cells prior to antigen sensitization, skin Langerhans cells and the like are included in “cells having antigen-presenting ability”. A cell having an antigen-presenting ability derived from peripheral blood monocytes becomes an antigen-presenting cell by contacting with the peptide of the present invention in vitro or in vivo. When the peptide of the present invention is administered to a patient, antigen-presenting cells with the peptide of the present invention bound are produced.

  Furthermore, the present invention provides a method for producing antigen-presenting cells having cytotoxic T cell-inducing activity, comprising the step of contacting at least one polynucleotide encoding the peptide of the present invention with cells having antigen-presenting ability. To do. The polynucleotide to be introduced may be in the form of DNA or RNA, and can be inserted into an appropriate vector and used depending on the purpose. For example, it can be appropriately selected from virus vectors such as retrovirus vectors, adenovirus vectors, and vaccinia virus vectors, and non-viral vectors such as cationic liposomes and ligand DNA complexes. Moreover, it is also conceivable to introduce a naked plasmid DNA (naked pDNA) together with a large volume of aqueous solution without using a carrier.

  The method for introducing a polynucleotide is not particularly limited, and a method commonly used in this field can be used. Examples thereof include various methods such as lipofection, electroporation, and calcium phosphate method. Specifically, for example, Reeves ME, et al., Cancer Res., 56: 5672, (1996); J. Immunol., 161: 5607, (1998); J. Exp. Med., 184: 465, ( 1996); can be carried out as described in JP-T 2000-509281. By introducing the polynucleotide into a cell having the antigen-presenting cell ability, the polynucleotide undergoes treatment such as transcription and translation in the cell. The produced protein is presented on the cell surface as a partial peptide via MHC class I or class II processing and presentation pathways.

  The present invention also provides an agent for inducing antigen-presenting cells having cytotoxic T cell-inducing activity, comprising the peptide of the present invention. By bringing the inducer of the present invention into contact with cells having antigen-presenting ability in vitro or in vivo, antigen-presenting cells having cytotoxic T cell-inducing activity can be obtained. In addition, administration of the inducer of the present invention to a patient can induce antigen-presenting cells having cytotoxic T cell-inducing activity in the patient's body. As described above, the inducer of the present invention can be formulated by a commonly used pharmaceutical method and administered as, for example, a vaccine.

  The present invention further provides a method for producing activated cytotoxic T cells, which comprises the step of contacting the peptide of the present invention with a cell having an antigen-presenting ability. The definition of “cell having antigen-presenting ability” is as described above. When the peptide of the present invention is brought into contact with a cell having an antigen-presenting ability, an antigen-presenting cell is obtained. The antigen-presenting cell activates T cell progenitor cells to cytotoxic T cells. It further increases the activity of cytotoxic T cells.

  The present invention also provides an inducer of activated cytotoxic T cells comprising the peptide of the present invention. By administering the inducer of the present invention to a patient, CTL is induced in the patient's body, and the immunity targeting the endothelial cells in the tumor tissue is enhanced. Alternatively, the inducer of the present invention and an antigen-presenting cell can be contacted in vitro to induce CTL, and then used for ex vivo treatment of returning to the patient.

  The present invention further provides isolated cytotoxic T cells (CTL) induced by the peptides of the present invention. In the present invention, the target CTL can be obtained by contacting an antigen-presenting cell presenting the peptide of the present invention with a T cell progenitor cell derived from peripheral blood. Purification of T cell progenitor cells from peripheral blood can be performed by using magnetic beads or fluorescence activated cell sorter (FACS) from peripheral blood mononuclear cells (PBMC). In addition, cytotoxic T cells activated by contact with antigen-presenting cells can be isolated by culturing with IL-2 and irradiation feeder cells.

  It is preferable that the cytotoxic T cell induced based on the stimulation by the antigen-presenting cell presenting the peptide of the present invention is derived from a patient to be treated and / or prevented. CTL can be administered to a patient for the purpose of antitumor effect alone or together with other pharmaceutical compositions containing the peptides, exosomes and the like of the present invention. As the administration method of CTL, oral administration, intradermal administration, subcutaneous administration, intravenous injection and the like can be used, and systemic administration or local administration in the vicinity of the target tumor may be used. The dose can be appropriately adjusted in consideration of the disease to be treated, the patient's age, weight, administration method, and the like.

  The resulting cytotoxic T cells act specifically on target cells presenting the peptides of the present invention, preferably the same peptides used for induction. The target cell may be a cell that endogenously expresses the CXADRL1 or GCUD1 gene, or a cell that forcibly expresses the CXADRL1 or GCUD1 gene. Even cells that present the peptide can be attacked.

  The present invention further provides an antigen-presenting cell obtained by presenting a complex of the HLA antigen and the peptide of the present invention. The antigen-presenting cell obtained by contact with the peptide of the present invention or the nucleotide encoding the peptide of the present invention is preferably derived from a patient to be treated and / or prevented, alone or in accordance with the present invention. Can be administered as a vaccine together with other pharmaceutical compositions, including peptides, exosomes, cytotoxic T cells and the like.

  EXAMPLES Hereinafter, although an Example is given and this invention is further demonstrated, this invention is not limited to these Examples.

[Example 1] Induction of CTL by anchor modified peptide (CXADRL1) The present inventors have previously identified an epitope peptide of CXADRL1-9mer-207 (SEQ ID NO: 1). The binding score for is relatively low. Therefore, in order to obtain an epitope peptide having a larger binding score with MHC class I molecules, a modified peptide was prepared from CXADRL1-9mer-207.

  The HLA-A * 0201 allele-binding peptide is often 9mer. Positions 2 and 9 of this peptide are thought to be major anchor residues that bind only to the HLA class I peptide binding groove and do not contact the TCR. In the HLA-A02 antigen motif previously reported by Smith et al. (Smith et al., Mol Immunol: 35 1033-43 (1998)), leucine (Leu) and isoleucine (Ile) are HLA-A * 0201 It was found to be the optimal anchor residue at position 2, which enhances the binding affinity of the peptide for the molecule. Similarly, valine at position 9 (Val) is also preferred for the 9mer peptide. Based on these findings, CXADRL1-9mer-207 has an amino acid substitution that includes an optimal HLA-A * 0201 allele-binding amino acid as an anchor residue to increase binding affinity to the HLA-A * 0201 molecule. Two anchor modified peptides were designed and synthesized.

  Table 1 shows the modified amino acid sequences and binding scores of the peptides with increased binding affinity for HLA-A * 0201 molecules among the synthesized anchor-modified peptides.

^*これらの改変ペプチドの結合スコアは、BIMASのエピトープ予測アルゴリズムにより算出した。

^* The binding score of these modified peptides was calculated by the epitope prediction algorithm of BIMAS.

  The binding scores for these peptides were calculated by the BIMAS epitope prediction algorithm as previously described, and both modified peptides showed higher HLA-A * 0201 binding scores than the wild type CXADRL1 peptide. These anchor-modified peptides were tested for their ability to induce CTL and whether the induced CTL recognized not only the modified peptide but also the parent CXADRL1-9mer-207 peptide on T2.

When CTL was induced with CXADRL1-9V (SEQ ID NO: 3) and CXADRL1-9L (SEQ ID NO: 4), CTL strain 5 and CTL clone 69 shown in FIGS. 1A and B were obtained by stimulation with CXADRL1-9V peptide. It was. These CTLs cross-react with wild-type CXADRL1-9mer-207 peptide on T2 cells, and endogenously processed CXADRL1-derived peptides as measured by the ⁵¹ Cr release assay shown in FIG. 1C. The tumor cell line SNU475 presented in was killed. Several published studies have shown the utility of such modified peptides. Peptides with high affinity for MHC class I molecules are presented on the cell surface for a longer time than peptides with low affinity. Thus, peptides with increased affinity for MHC class I are believed to have a higher potential to induce T cell-mediated immune responses (Keogh et al., J Immunol 167: 787-96 (2001) ); Tourdot et al., J Immunol 159: 2391-8 (1997); Sette et al., J Immunol 153: 5586-92 (1994)).

  The binding score of CXADRL1-9mer-207 (SEQ ID NO: 1) calculated by Bimas prediction software was 11.4, relatively low compared to viral antigens or other TAAs (Bednarek et al., J Immunol 147: 4047-53 (1991); Sette et al., J Immunol 153: 5586-92 (1994)). To increase the immunogenicity of the natural epitope peptide, a modified peptide was designed and synthesized by replacing the 2nd or 9th anchor residue with an amino acid optimal for HLA-A * 0201 (Vierboom et al., J Immunother 21 : 399-408 (1998); Irvine et al., Cancer Res 59: 2536-40 (1999); Dyall et al., J Exp Med 188: 1553-61 (1998); Muller et al., J Immunol 147: 1392-97 (1991)).

  CTL generated against CXADRL1-9V (SEQ ID NO: 3) with a binding score of 49 responded not only to the CXADRL1-9V peptide but also to the parent peptide CXADRL1-9mer-207 on T2 cells. Furthermore, these CTLs induced by the GCUD1-9V peptide also recognized natural peptides that were naturally processed by tumor cells.

  However, many recent studies have shown that increased affinity for MHC class I does not necessarily correlate with increased peptide immunogenicity or ability to stimulate CTL (Clay et al., J Immunol 162: 1749-55 (1999); Dionne et al., Cancer Immunol Immunother 52: 199-206 (2003); Slansky et al., Immunity, 13: 529-38 (2000); Sloan-Lancaster et al., Annu Rev Immunol 14: 1-27 (1996); Yang et al., J Immunol 169: 531-9 (2002); Trojan et al, Cancer Res 61: 4761-5 (2001); Dionne et al., Cancer Immunol Immunother 53: 307-14 (2004)). Modification of peptide ligands can result in significantly different phenotypes of T cells, and in some cases can result in peptides acting as partial agonists or even antagonists during T cell activation or TCR signaling. It cannot be determined whether CTL stimulated with the modified peptide act as an effector more efficiently than CTL induced by the native peptide in vivo. However, in this experiment, it was revealed that the modified peptide CXADRL1-9V activates a specific partial TCR repertoire that recognizes the naturally processed wild-type epitope peptide presented by tumor cells. More importantly, this modified peptide was able to induce natural peptide-specific CTLs more frequently and more abundantly than the wild-type peptide. Stimulation with this modified peptide produced natural peptide-specific CTL in 3 out of 4 individuals, whereas the wild-type peptide induced CTL only in 1 healthy person. This improvement in induction of CTL is associated with several advantages in clinical use, which provides selection in clinical trials. That is, the possibility of an anchor modified peptide was demonstrated.

[Example 2] Induction of CTL by anchor modified peptide (GCUD1) The present inventors have already identified an epitope peptide of GCUD1-196 (SEQ ID NO: 2), and the binding score between it and an MHC class I molecule is Relatively low. Therefore, in order to obtain an epitope peptide having a larger binding score with MHC class I molecules, a modified peptide was prepared from the GCUD1-196 peptide.

  As mentioned above, the HLA-A * 0201 allele-binding peptide is often 9mer, and the amino acids at positions 2 and 9 bind only to the HLA class I peptide-binding groove and do not contact the TCR. It is considered a group. In the HLA-A02 antigen motif previously reported by Smith et al. (Smith et al., Mol Immunol: 35 1033-43 (1998)), leucine (Leu) and isoleucine (Ile) are HLA-A * 0201 It was found to be the optimal anchor residue at position 2, which enhances the binding affinity of the peptide for the molecule. Similarly, valine at position 9 (Val) was also preferred for the 9mer peptide.

  Thus, two anchor-modified modified peptides of GCUD1-196 with amino acid substitutions containing the optimal HLA-A * 0201 allele-binding amino acid as an anchor residue to increase binding affinity for HLA-A * 0201 molecules Was designed and synthesized.

  Table 2 shows the modified amino acid sequences and binding scores of the peptides with increased binding affinity for HLA-A * 0201 molecule among the synthesized anchor-modified peptides.

^*N.S.：アミノ酸配列が疎水性であるため、合成されなかった。
^*結合スコア：高いスコアは、MHCクラスI分子に対する親和性が高いことを示す。これらの改変ペプチドの結合スコアは、BIMASのエピトープ予測アルゴリズムにより算出した。

^* NS: Not synthesized because the amino acid sequence is hydrophobic.
^* Binding score: A high score indicates a high affinity for MHC class I molecules. The binding score of these modified peptides was calculated by the epitope prediction algorithm of BIMAS.

The binding scores for these peptides were calculated based on the BIMAS epitope prediction algorithm as previously described, but all three modified peptides achieved higher HLA-A * 0201 binding scores than the wild-type GCUD1 peptide. did. These anchor-modified peptides were tested for their ability to induce CTL and whether the induced CTL recognized the parental GCUD1-196 peptide on T2 in addition to the modified peptide. When CTL was induced with GCUD1-9V (SEQ ID NO: 6) and GCUD1-2L (SEQ ID NO: 5), CTL line 3 and CTL clone 16 shown in FIGS. 2A and 2B were obtained by stimulation with GCUD1-9V peptide. It was. These CTLs cross-react with wild type GCUD1-196 peptide on T2 cells and endogenously processed naturally processed GCUD1-196 peptide as measured by the ⁵¹ Cr release assay shown in FIG. 2C. The presented tumor cell line SNU475 was killed.

  In particular, GCUD1-9V peptide (SEQ ID NO: 6) produced GCUD1-196-specific CTL in 3 out of 4 HLA-A * 0201-positive individuals, whereas GCUD1-196 induced CTL. When used in the above, GCUD1-196 wild type peptide-specific response was observed in 1 out of 4 individuals.

  The GCUD1-196 (SEQ ID NO: 2) binding score calculated by Bimas prediction software was 21.6, which was relatively low compared to viral antigens or other TAAs (Bednarek et al., J Immunol 147: 4047-53 (1991); Sette et al., J Immunol 153: 5586-92 (1994)). In order to increase the immunogenicity of the natural epitope peptide, a modified peptide was designed and synthesized with an anchor residue modification that is a substitution to the amino acid optimal for HLA-A * 0201 at position 2 or 9 (Vierboom et al ., J Immunother 21: 399-408 (1998); Irvine et al., Cancer Res 59: 2536-40 (1999); Dyall et al., J Exp Med 188: 1553-61 (1998); Muller et al. , J Immunol 147: 1392-97 (1991)).

  CTL generated against the GCUD1-9V peptide (SEQ ID NO: 6) with a binding score of 70.3 responded not only to the GCUD1-9V peptide but also to the parental wild type peptide GCUD1-196 on T2 cells. Furthermore, these CTLs induced by the GCUD1-9V peptide also recognized natural peptides that were naturally processed by tumor cells.

  However, as noted above, many recent studies have shown that increased affinity for MHC class I does not necessarily correlate with increased peptide immunogenicity or ability to stimulate CTL (Clay et al ., J Immunol 162: 1749-55 (1999); Dionne et al., Cancer Immunol Immunother 52: 199-206 (2003); Slansky et al., Immunity, 13: 529-38 (2000); Sloan-Lancaster and Allen, Annu Rev Immunol 14: 1-27 (1996); Yang et al., J Immunol 169: 531-9 (2002); Trojan et al, Cancer Res, 61: 4761-5 (2001); Dionne et al. , Cancer Immunol Immunother 53: 307-14 (2004)). Peptide ligand modifications can also result in significantly different phenotypes of T cells, and in some cases, peptides that act as partial agonists or even antagonists during T cell activation or TCR signaling. Therefore, it cannot be determined whether CTL stimulated with the modified peptide act as an effector more efficiently than CTL induced by the natural peptide in vivo. However, in this experiment, it was revealed that the modified peptide GCUD1-9V activates a specific partial TCR repertoire that recognizes naturally processed wild-type epitope peptides presented by tumor cells. More importantly, this modified peptide induced natural peptide-specific CTLs more frequently and more abundantly than the wild-type peptide. Stimulation with this modified peptide produced natural peptide-specific CTL in 3 out of 4 individuals, whereas the wild-type peptide induced CTL only in 1 healthy person. This evidence is associated with several advantages in clinical use and the present invention provides a choice in clinical trials. That is, the possibility of an anchor-modified peptide was provided.

  The amino acid sequences of GCUD1-196 (SEQ ID NO: 2) and GCUD1-9V (SEQ ID NO: 6) were subjected to BLAST homology analysis. Peptides derived from any other known molecule with high homology were not detected. These results support the fact that the identified peptides are GCUD-1 specific and are unlikely to have cross-reactivity to other known molecules. This also supports the possibility of using these peptides in clinical applications without causing undesirable side effects.

FIG. 1 shows the cytotoxic activity of CTL induced by the anchor modified peptide CXADRL1-9V. CTL line 5 (A) and CTL clone 69 (B) induced with CXADRL1-9V released 4h ⁵¹ Cr for cytotoxic activity against peptide-pulsed T2 cells (HLA-A * 0201-positive cell lines) and tumor cell lines Tested by assay. Both CTL line 5 and CTL clone 69 recognize not only CXADRL1-9V, but also the parent peptide CXADRL1-9mer-207, which is equivalent or stronger in CTL clone 69 with a lower E / T ratio, and HLA-A * SNU475 cells displaying the wild-type peptide CXADRL1-9mer-207 naturally processed on the molecule were killed (C). FIG. 2 shows the cytotoxic activity of CTL induced by the anchor modified peptide GCUD1-9V. GCh1-9V-induced CTL line 3 (A) and CTL clone 16 (B) released 4h ⁵¹ Cr for cytotoxic activity against peptide-pulsed T2 cells (HLA-A * 0201-positive cell lines) and tumor cell lines Tested by assay. Both CTL line 3 and CTL clone 16 recognize not only GCUD1-9V but also the parent peptide GCUD1-196, which is equivalent or stronger in CTL clone 16 with a lower E / T ratio, and also HLA-A * 0201 molecule SNU475 cells presenting the wild-type peptide GCUD1-196 naturally processed above were killed (C).

Claims (18)

A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 . A pharmaceutical composition for treating and / or preventing a tumor comprising the peptide according to claim 1 . The pharmaceutical composition according to claim 2, wherein the tumor is gastric cancer or colorectal cancer. A vaccine for inhibiting tumor growth and / or metastasis, comprising the peptide according to claim 1 . The vaccine according to claim 4, for administration to a patient whose HLA antigen is HLA-A02. The vaccine according to claim 5, wherein the tumor is gastric cancer or colorectal cancer. An exosome presenting on its surface a complex comprising the peptide of claim 1 and an HLA antigen. The exosome according to claim 7, wherein the HLA antigen is HLA-A02. The exosome according to claim 8, wherein the HLA antigen is HLA-A0201. A method for producing an antigen-presenting cell having cytotoxic T cell-inducing activity, comprising a step of contacting the peptide according to claim 1 with a cell having an antigen-presenting ability in vitro . A method for producing an antigen-presenting cell having cytotoxic T cell-inducing activity, comprising a step of introducing a polynucleotide encoding the peptide according to claim 1 into a cell having an antigen-presenting ability in vitro . An agent for inducing an antigen-presenting cell having cytotoxic T cell-inducing activity, comprising the peptide according to claim 1 . A method for producing activated cytotoxic T cells comprising the step of contacting the peptide according to claim 1 with cells having antigen-presenting ability in vitro . An agent for inducing activated cytotoxic T cells, comprising the peptide according to claim 1 . An antigen-presenting cell obtained by presenting a complex of an HLA antigen and the peptide according to claim 1. The antigen-presenting cell according to claim 15, which is induced by the inducer according to claim 12. An isolated cytotoxic T cell induced by the peptide of claim 1. The cytotoxic T cell of Claim 17 induced | guided | derived with the inducer of Claim 14.

Images