TW200413406A - Peptides and drugs containing the same - Google Patents

Abstract

It is intended to provide a novel and useful immunotherapy for HCC and a clinically useful diagnostic for hepatocellular carcinoma. More specifically speaking, a peptide comprising an amino acid sequence represented by any of SEQ ID NOS:5 to 16 from which an HLA-A24-restricted and HCC-reactive CTL can be prepared. It is also intended to provide a diagnostic for hepatocellular carcinoma which contains an antibody against GPC3.

Description

200413406 (1) Description of the invention: [Technical field to which the invention belongs] The present invention relates to a novel peptide effective as a cancer vaccine, a medicine for treating and preventing tumors containing the peptide, and a novel diagnostic agent for hepatocellular carcinoma. [Prior art] Primary hepatocellular carcinoma (HCC) is one of the most common malignant diseases in the world. Due to the worldwide epidemic of hepatitis B and C, the incidence of HCC has increased sharply in Asian and European countries (Schafer, DF · and Sorrell, MF ·, Lancet 353, 1253-1257 (1999)). If HCC infection is considered To the long potential period of onset, this trend can be predicted to continue in the next 50 years. The worsening condition of HCC is worse, and new treatment strategies are urgently needed. On the other hand, with the development of molecular biology and tumor immunology in recent years, a number of genes encoding tumor antigens and antigenic peptides that can be recognized by tumor-responsive cell nociceptive T-lymphocytes (CTLs) have been identified, thus increasing The possibility of antigen-specific tumor immunotherapy (Boon, T., and van der Brugegen, P., J. Exp. Med. 183, 725-729 (1996); Rosenberg, SA, J. Natl. Cancer Inst. 88, 1635-1644 (1996)). Alpha-fetal protein (AFP), although expressed only in the fetal stage in normal tissues, has been reported to be reactivated in multiple HCCs (Fujiyama, S. et al. Oncology 62, 57-63 (2002) ). In addition, mouse and human T cell repertory (repertory) can recognize the peptide epitope from AFP presented by type I MHC molecules (Butterfield, LH et al., Cancer Res. 59, 3 134-3 142; Jr Vollmer, CM et al., Cancer Res. 59, 3064-87544 200413406 3067 (1999); Butterfield, LH et al., J. Immunol. 166, 5 300-5308 (2001)). Although exposed to high plasma concentrations of cancerous fetal proteins during fetal development, mature T cells cannot obtain complete immune tolerance to AFP, and AFP-specific T cells can be detected in peripheral blood. That is, cancer fetal proteins can be the target of immunotherapy. AFP and PIVKA-II (Fujiyama, S. et al., Oncology 62, 57-63 (2002)) are well-known tumor markers of HCC. Furthermore, researchers are rapidly developing cDNA microarray technology that can provide general information about gene expression patterns. Several studies have proven useful for the identification of novel cancer-related genes and the molecular-level classification of human cancers (Golub, TR et al., Science 286, 531-537 (1999); Alizadeh, AA et al., Nature 403, 503-511 (2000); Ono, K. et al.,

Cancer Res. 60, 5007-5011 (2000); Kitahara, O. et al., Cancer Res. 61, 3544-3549 (2001); Kihara, C. Cancer Res. 61, 6474-6479 (2001)). The present inventors first identified genes that behaved differently when HCC occurred by using a cDNA microarray containing 23,040 genes. The expression patterns of these genes in 20 primary HCCs were then reviewed (Okabe, H. et al, Cancer Res. 61, 2129-2137 (2001)). In 1996, Pilia et al. Reported that the heparin proteoglycan-3 (GPC3) gene encoding a member of the heparin proteoglycan (glypican) family was mutated in Simpson-Golabi-Behmel syndrome (SGBS) patients (Pilia, G et al., Nat. Genet. 12, 241-247 (1996)). SGBS is an X-linked genetic disease, which is characterized by excessive growth before and after birth and a wide range of clinical symptoms (from very mild phenotypes of female carriers to lethal symptoms of male infants) 87544 200413406, etc. (Neri, G. et al., Am. J. Med. Genet. 79, 279-283 (1998)). The 6¾ bed of SGBS is characterized by distinctive facial appearance, cracked lids, joint fingers, multi-fingers, paramilitary, cystic and heterogeneous kidneys, and congenital heart defects (Behmel, A. et al., Hum. Genet. 67, 409-413 (1984); Garganta, CL and Bodurtha, J. N., Am. J. Med. Genet. 44, 129-135 (1992); Golabi, M. and Rosen, L., Am J. Med. Genet. 17, 345-358 (1984); Currieri, F. et al., Am. J. Med. Genet. 44, 136-137 (1992)). It has been reported that most of the GPC3 mutations are point mutations or deletions of small genes containing several exons (Hughes-Benzie, RM et al., Am. J. Med. Genet. 66, 227-234 (1996); Lindsay, S. et al., J. Med. Genet. 34, 480-483 (1997); Veugelers. M. et al., Hum. Mol. Genet. 9, 1321-1328 (2000); Xuan, JY et al. J. Med. Genet. 36, 57-5 8 (1999)). In addition, the patient's phenotype has no correlation with the location of the mutation, so SGBS may be caused by a defect in the GPC3 protein and other genetic factors that cause phenotypic differences within and between families (Hughes_Benzie, R. M. et al., Am J. Med. Genet. 66, 227-234 (1996)), which can support this hypothesis from the study of GPC3-deficient mice (Can0-Cauci, DF etal.,

J. Cell Biol 46, 25 5-264 (1999)). These mice have several abnormalities seen in patients with SGBS, including overgrowth, cysticity, and heteronephrosis. Specific overexpressive manifestations in I cancer, but antigens that can be ignored in various normal tissues can be used as ideal targets in immunotherapy. It can be expected that CTL will only show cell damage to cancers with excessive expression of antigen 'and will not show side effects on normal tissues. Excessive expression of GPC3 mRNA in HCC, placenta, fetal liver, fetal lung, and fetal kidney was reported from a study using the analysis of the southern point 87544 200413406 (Zhu, Z.W. et al., Gut 48, 558-564 (2001); Hsu. HC et al., Cancer Res. 57, 5179-5184 (1997); Pellegrini, M. et al., Dev Dyn. 213, 431-439 (1998)). In addition, GPC3 can inhibit apoptosis in certain tumor cells due to its inhibitory effect on cell proliferation (Duenas Gonzales, A. et al., J. Cell Biol. 141, 1407-1414 (1998); Cano-Gauci , DF et al., J. Cell Biol. 146, 255-264 (1999)), so GPC3 expression was suppressed in tumors of various origins. Lin et al. Pointed out that although GPC3 is expressed in normal ovarian tissue, it cannot be detected in certain ovarian cancer cell lines (Lin, H. et al., Cancer Res. 59, 807-810 (1999)). In all cases where GPC3 performance was not seen, there was no variation in the coding domain, but the GPC3 promoter was overmethylated, and GPC3 performance could be restored when treated with a demethylating agent. Furthermore, GPC3 heterogeneity is shown to inhibit colony-forming activity in several ovarian cancer cell lines. Other data on the correlation between GPC3 and cancer can be shown by studying the differences between normal rat mesothelial cells and mesothelial tumor cell lines (Murthy, S.S. et al., Oncogene 19, 410-416 (2000 )). It was found in this study that the expression of GPC3 in tumor cells has been inhibited. The same kind of expression inhibition was also found in cell lines derived from primary rat mesothelial tumors and antitumor cells in humans. Similar to ovarian cancer, although mutations were observed in the GpC3 coding sequence, the GpC3 promoter domain was abnormally methylated in most cell lines. As reported (Duenas Gonzales, A. et. Amelh Biol. 141, 1407_1414 (1998)), heterotopic manifestations of GPC3 in mesothelial tumor cell 87544-9-200413406 strains have been shown to inhibit colony-forming activity. Furthermore, Xiang et al. Recently reported that GPC3 is not expressed in human breast cancer (Xiang, Y. Y. et al., Oncogene 20, 7408-7412 (2001)). These data suggest that GPC3 can be used as a negative regulator of cell proliferation in these cancers. That is, in cancers generated from adult GPC3-positive tissues, the expression of GPC3 decreases during tumor progression, and this decrease is considered to play a role in the occurrence of malignant phenotypes. In contrast, in the case of HCC, tumors are generated from liver tissues that only express GPC3 during the fetal period, and when they become malignant, GPC3 tends to be re-expressed. In recent years, it has been understood that Heparan sulfate proteoglycan (HSPG) on the cell surface is necessary for the optimal activity of Heparin-binding growth factors such as fibroblast proliferation factor (FGF) and Wnt (Yayon, A. et al., Cell 64, 841-848 (1991);

Schlessinger, J. et al., Cell 83, 357-360 (1995)). Glypican (GPC) is a GPI anchored cell surface HSPG family. In terms of the relationship between tumor formation and GPC3 expression, the tissue-specific differences are presumed to be due to the fact that GPC3 regulates growth and survival factors in different tissues in different ways. GPC3 is thought to function as a cancer-fetal protein in at least these organs. In general, cancer fetal proteins do not play an important role in tumor progression, but they can be used as tumor markers or targets for immunotherapy (Coggin, JH Jr., CRC Critical Reviews in Oncology / Hematology 5, 37- 55 (1992); Matsuura, H. and Hakomori S.-I., Proc. Natl. Acad. Sci. USA. 82, 6517-6521 (1985))). Whether GPC3 cancerous fetal sex can be used for clinical purposes 87544 -10- 200413406 and whether the re-expression of the heparin proteoglycan (glypican) is important in the deterioration of HCC remains to be studied. Although there are various treatment methods for HCC, its prognosis is poor compared with other cancers, and it is one of the refractory cancers. HCC basically has cirrhosis, which is due to poor liver function of the original patient, and even if one cancer is treated, the cancer is generated from another. It requires urgent and novel treatment strategies. If we can develop immunotherapies that target antigens that are highly specific in HCC, we will be able to obtain treatments that do no harm to normal organs and only exclude cancer. In addition, it is possible to obtain a treatment method which can be used for patients with terminal stage or patients with poor liver function who cannot perform other treatments. In addition, at present, there are more than 2 million people with hepatitis C infection in the HCC susceptible group. For the prevention of HCC in these infected persons, such immunotherapy can be used. Although AFP and PIVKA-II are known as tumor markers for HCC, there are cases where patients are not detected, and there are cases where patients with benign liver disease, cirrhosis and chronic hepatitis become false positive, especially in the early stages of HCC. The diagnosis is quite difficult. Therefore, other useful tumor markers for early diagnosis of HCC are needed. [Summary of the Invention] According to the disclosure of the invention, the inventors identified a novel cancer fetal protein, specifically heparin proteoglycan 3 (GPC3), which is specifically expressed in human hepatocellular carcinoma based on the data of cDNA microarray interpretation ), And novel peptides found to be potential candidates for immunotherapy. Secondly, the present inventors have detected soluble GPC3 protein in the serum of HCC patients, and it is understood that 87544 200413406 GPC3 can be a novel tumor marker for HCC. That is, the present invention provides the following (1) to (18) (1) a peptide comprising the amino acid sequence shown in any one of sequence numbers 5 to 16. (2) A peptide having a cytotoxic τ cell defensive ability, which is one in which one or two amino acids are substituted or added in the amino acid sequence shown in any one of sequence numbers 5 to 16. . (3) The peptide as described in (2) above, wherein the second amino acid from the N-terminus is phenylalanine, tyrosine, methionine or tryptophan. (4) The peptide according to (2) or (3) above, wherein the amino acid at the C-terminus is phenylalanine, leucine, isoleucine, tryptophan, or methionine. (5) A medicine for treating and / or preventing tumors, which comprises one or more of the peptides according to any one of (1) to (4). (6) A kind of exosomes (exosomes), which presents on the surface a complex comprising a peptide of any of ⑴ to ⑷ and an HLA molecule. (For example, the exosome (6) above, where the Hla molecule is HLA_A24. For example, the exosome, where the hla molecule is hla_a * 24〇2. (9) Presentation of an antigen with high cell-deficient T cell slander ability Cell defamation method 'uses the above-mentioned peptides of any one of the above-mentioned items to induce the original king's present cells. (10)-A method for inducing antigen-presenting cells with high cell-injurious T-cell induction ability 'It comprises a gene-leading antigen-presenting cell encoding a heparin proteoglycan-3 (Gpc3) protein or a partial peptide thereof, wherein the partial peptide comprises any one of (1) to (4) above Peptide. 87544 -12- 200413406 (11) A method for inducing cellular nociceptive T cells, wherein the inducing is performed using the epidermis of any one of (1) to (4). (12) — a kind of cellular nociceptive T Cells derived from the peptide of any one of (1) to (4) and isolated. (13) An antigen-presenting cell that exhibits HLA and any of (1) to (4) The complex of one item of peptide. (14) The antigen-presenting cell as described in (13) above, which is induced by the method of ⑼ or (10) above. (15) — A diagnostic agent for J? H cell carcinoma (HCC), which includes an antibody against gpc3. (16) — A diagnostic method for HCC, which comprises contacting a specimen with an antibody against gpc3. (17) As described above (16) The method further comprises quantifying Gpc3 in the specimen. (18) —A kit for diagnosing HCC, which includes antibodies against Gpc3. The winter specification includes the present patent application, which is the basis of priority of the present application. 2002-245831 No. of description and / or drawings. [Embodiment] The best mode for carrying out the invention The present inventor used Okabe, H. et al. (Cancer Res. 61, 2129-2137 (2001)) According to the documented method, a novel cancer fetal protein that is specifically overexpressed in human hepatocellular carcinoma is identified based on the cDNA microarray * 'that is, heparin proteoglycan-3 (GPC3). The result of detecting GpC3 in serum' was confirmed GPC3 is negative in cancers other than hepatocellular carcinoma, such as stomach, esophagus, lung, breast, kidney, bile duct, and colon. It is also negative in benign liver diseases such as cirrhosis and chronic liver 87544-13-200413406 inflammation. Furthermore, after the inventors confirmed the surgical treatment, In patients with hepatocellular carcinoma, the postoperative serum GPC3 is negative. The amino acid sequence of the human GPC3 protein is well known. For example, those registered in the protein database of GenBank with the citizenship number NP 004475 will be easily Obtained. The inventors then considered that various proteins are presented on antigen-presenting cells in the living body, which are presented after being decomposed into peptides consisting of nine amino acids (nine peptides), so the synthesis has HLA- The binding motif of A24 (having this type accounts for 60% of the whole person) and a part of the peptide composed of 9 amino acids or 10 amino acids from GPC3. The selection of a peptide having a binding motif for HLA-A24 can be performed according to the methods described in J. Immunol., 152, 3913, 1994 and J. Immunol. 155: 4307, 1994, for example. For HLA types other than HLA-A24, peptides can be selected in the same manner. Or use software available on the Internet recently, such as those described in Parker K. C., J. Immunol. 152, 1994, which can use in silico to calculate various peptides and HLA molecules (sometimes referred to as HLA antigens) Binding affinity. For binding affinity to HLA molecules, for example, the above-mentioned BIMAS: HLA Binding Prediction: http://bimas.dcrt.nih.gov/molbio/hla_bind/ (Parker, KC et. Al., J. Immunol. , 152, 1994) or the method described in Nukaya, I., Int. J. Cancer, 80, 1999. The 9-mer and 10-mer peptides can be synthesized from arbitrary positions based on the entire amino acid sequence of the GPC3 protein obtained. The synthesis of the peptide can be carried out according to a method used in ordinary peptide chemistry. The commonly used synthesis methods 87544 -14- 200413406 are, for example, Peptide Synthesis, Interscience, New York, 1996; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Corporation, 1975; Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1985; Development of Pharmaceutical Products, Continued Volume 14, Peptide Synthesis, Hirokawa Shoten, 1991, etc .; and those described in International Publication No. WO 99/67288. The actual binding of HLA molecules and peptides can be achieved by culturing the transformants that can express the HLA gene in TAP-deficient T2 or RMA-S cell lines with peptides, and quantify and measure in cells by flow cytometry. Class I HLA molecules represented on the surface (for example, see Immunol·Let. 2002 Aug 1, 83 (1): 21-30; Immunogenetics, 44: 233-241, 1996 and Nature 346: 321-325, 1990) ° About HLA For the numerator, in order to obtain effective results, it is better to use the A24 type held by the majority of the Japanese (60%), and it is better to use the subtype such as A $ 2402. However, clinically, by investigating in advance the type of HLA molecules in patients in need of treatment, it is possible to appropriately select peptides with high binding affinity to them, or antigens capable of demonstrating noxious T cells (CTL). Furthermore, in order to obtain a peptide with high binding affinity and CTL defamatory ability, one or two amino acids may be substituted or added based on the amino acid sequence of the naturally occurring GPC3 partial peptide. In addition to the peptides that are present in nature, the sequence of the peptides is known to bind to existing HLA molecules and is presented in the 4th chapter (J. Immunol., 152, 3913, 1994; Immunogenetics 41: 178, 1995; J. Immunol. 155: 4307, 1994), so for the obtained peptide, it is preferable to change according to this regularity. For example, HLA-A24 binds to those with high affinity to use the second amino acid from the N-terminus of the peptide. 87544 -15-200413406 is replaced with phenylalanine, tyrosine, methionine or chromosome Amino acid, and the c-terminal amino acid is substituted with a peptide of phenylalanine, leucine, isoleucine, tryptophan or methionine. However, when the peptide sequence is part of the amino acid sequence of other functional intrinsic or foreign proteins, it may cause side effects such as autoimmune diseases or cause allergic symptoms for specific substances. It is better to use the database for homology search and avoid the amino acid sequence agreement with other proteins. Furthermore, in the similarity search, if it is clear that there are also no one or two peptides with different amino acids, in order to improve the binding affinity with the HLA molecule and / or the ability to defame CTL, Changes made to the acid sequence will also not cause the above problems. As described above, peptides with high binding affinity to HLA molecules, although predicted to be highly effective as cancer vaccines, must be reviewed for candidate peptides selected with high binding affinity as an indicator. Whether it has CTL slander ability. Confirmation of CTL defamatory ability, for example, by inducing antigen-presenting cells (β-lymphocytes, macrophages, and dendritic cells) with HLA molecules, specifically, dendritic cells from peripheral blood mononuclear cells, etc., and After being stimulated with peptide, it was mixed with CD8 positive cells, and then the cytotoxic activity against the target cells was measured. Alternatively, peptide-specific CTLs can be induced from PBMCs according to the method described by Nakatsura, T. et al. (Eur. J. Immunol. 32, 826-836 (2002)). Moreover, as a responder, transgenic animals made of human HLA molecules can be used (for example, Hum. Immunol. 2000 Aug .; 61 (8): 764-79 Related Articles, Books, Linkout 87544 -16- 200413406 丄 nauction οί CJLL · response bv a mi ”. I · · y Minimal epitope vaccine in HLA A * 0201 / DR1 transgenic mice: dependence 〇n HLA ciass II restricted T (H) response., BenMohamed L„ Krishnan R., (Longmate J., Auge C., Low L., Primus ;, Diamond DJ, etc.). The cytotoxic activity can be calculated, for example, by radiolabeling the target cells with MCr or the like and then releasing the radioactivity from the target cells. Alternatively, CTL production was observed in the presence of peptide-loaded antigen-presenting cells. It can be observed by measuring visualized points caused by IFN_Y and anti-IFN-Y monoclonal antibodies released on the culture medium. As described above, the results of the CTL inducing ability of the serapyrin showed that the nonapeptide or decapeptide selected from the peptides containing the amino acid sequences shown in the following sequence numbers 5 to 16 has high CTL defensive ability.

Sei: -Phe-Phe-Gln-Arg-Leu-Gln-Pr0-Gly-Leu (SEQ ID NO: 5) Phe-Phe-Gln-Arg-Leu-Gln-Pro-Gly-Leu (SEQ ID NO: 6) Met- Phe-Lys-Asn-Asn-Tyr-Pro-Ser-Leu (Serial No. 7) Phe-Thr-Asp-Val-Ser-Leu-Tyr-Ile-Leu (Serial No. 8) Lys-Phe-Se and Lys- Asp-Cys-Gly-Arg-Met-Leu (SEQ ID NO: 9) Trp-Tyr-Cys-Ser-Tyr-Cys-Gln-Gly-Leu (SEQ ID NO: 10) Lys-Tyr-Trp-Arg-Glu-Tyr- Ile-Leu-Ser-Leu (SEQ ID NO: 11) Glu-Tyr-Ile-Leu-Ser-Leu-Glu-Glu-Leu (SEQ ID NO: 12) Ile-Tyr-Asp-Met-Glu-Asn-Val-Leu- Leu (serial number 13) Ala-Tyr-Tyr-Pro-Glu-Asp-Leu-Phe-Ile (serial number 14) Phe-Tyr-Ser-Ala-Leu-Pro-Gly-Tyr-Ile (serial number 15)

Arg-Phe-Leu-Ala-Glu-Leu-Ala-Tyr-Asp-Leu (SEQ ID NO: 16) 87544 -17- 200413406 The present invention further provides an amino group sequence shown in any one of SEQ ID Nos. 5-16 One or two amino acids that are substituted or attached and have a cytotoxic τ cell-deficient ability. The substitution or addition of one or two amino acids is not required as long as they do not coincide with the amino acid sequences of other proteins and have the ability to induce CTL. Especially regarding amino acid substitution, the second amino acid from the N-terminus was replaced with phenylalanine, tyrosine, methionine, or tryptophan, and the c-terminal amino acid was replaced with phenylalanine , Leucine, isoleucine, tryptophan, or methionamine fe are preferred examples. The above-mentioned peptides of the present invention can be used in combination of one kind or two or more kinds, and can be used as a cancer vaccine for demonstrating CTL in the body. By administering the peptide of the present invention, the peptide is presented on the A molecule of the antigen-presenting cell at a high density, and the CTL having specific reactivity to the complex of the presented peptide and the HL A molecule is derived. Attack on the target cells of liver cancer cells. By taking out the dendritic cells from the subject and stimulating them with the peptide of the present invention, the controller can serve to load the antigen-presenting cells of the peptide of the present invention on the cell surface. The examiner induces CTL in vivo and increases the attack power on the target cells. Also, Mao Maomingdi provides a method for treating tumors or preventing tumor proliferation and transgenic silicon π, tilework, which contains more than one peptide of the present invention. In addition, in the living body and > 4 self-drying organs, the cells were stimulated by the peptides of the present invention, and the cells existed at a high concentration through the cells, so as to be compared with the cells previously loaded on the cells. Exchanges will be made easily. Because of the & molecular affinity (the binding affinity must be higher than a certain degree. Although the peptide of the present invention can be directly administered directly to the peptide of the present invention, 87544 -18- 200413406, but by the commonly used formulation The method is preferably to prepare and prepare a pharmaceutical composition for administration. In this case, in addition to the peptide of the present invention, carriers and excipients commonly used in medicine may be appropriately included without particular limitation. Peptides as medicines for the treatment and / or prevention of hepatocellular carcinoma can be administered with adjuvants in an immunologically effective manner, or with other anticancer agents and other active ingredients, or It is administered in a granular form. As an adjuvant, one described in the literature (Clin. Microbiol. Rev., 277-289, 1994) can be used. In addition, it is possible to consider the use of a microlipid preparation and the number of diameters. μιη bead-like granular preparations and lipid-bound preparations, etc. For administration methods, oral administration, intradermal administration, subcutaneous administration, and intravenous injection can be used, as well as systemic or local administration. Swell Nearly. Although the administration amount of the peptide of the present invention can be appropriately adjusted depending on the disease to be treated, the age, weight of the patient, and the administration method, etc., it is usually 0.001 mg to 1000 mg, and 0.001 mg to 1000. mg is more preferred, 0.1 mg to 10 mg is more preferred, and it is preferably administered once a few days to several months. If a person skilled in the art will be able to appropriately select an appropriate administration amount, or the present invention provides a Intracellular vesicles of so-called "exosomes" present on the surface of the complex of the peptide of the invention and the HL A molecule. Although the preparation of exosomes can be described in detail in Tables 11-5 and 10507 and Table 2000-5 The method described in No. 12161 is preferably prepared using antigen-presenting cells obtained from a subject who is a subject for treatment and / or prevention. The exosomes of the present invention can be made into a cancer vaccine in the same manner as the peptides of the present invention described above. Vaccination. The HL A molecule must be of the same type as the HLA molecule of the subject in need of treatment and / or prevention. For example, in the case of Japanese, HLA-A24, 87544 -19- 200413406, especially HLA-A * Most of 2402 is appropriate. The present invention also provides a method of using the present invention. A method for defensive peptides to induce antigen-presenting cells. After inducing dendritic cells from peripheral monocytes, they can be contacted (stimulated) with the peptides of the present invention in a test tube or in vivo to induce antigen-presenting cells. In the case where the peptide of the invention is administered to the subject, the antigen-presenting cells carrying the peptide of the present invention are defamated in the subject. Alternatively, the peptide of the present invention may be loaded on the antigen-presenting cells in a test tube. The vaccine is administered to a subject. The present invention also provides a method for defamating an antigen-presenting cell with high cytotoxic T cell defensive ability, which comprises encoding a GPC3 protein or a part of a peptide (including the above-mentioned present invention) (Peptide) is introduced into antigen-presenting cells. The introduced gene can be in the form of DNA or RNA. As the method of introduction, various methods such as a lipofection method, an electroporation method, and a calcium phosphate method, which are generally performed, can be used, and are not particularly limited. Specifically, for example, as disclosed in Cancer Res ·, 56: 5672, 1996; J. Immunol., 161: 5607, 1998; J. Exp. Med., 184: 465, 1996 and Japanese Patent Publication No. 2000-509281 get on. By introducing a gene into an antigen-presenting cell, and after the gene undergoes processing such as transcription and translation in the cell, the resulting protein undergoes the processing and presentation pathways of class I or class II HLA, and this portion of the peptide can be presented. The present invention further provides a method for inducing CTL using the peptide of the present invention. When the peptide of the present invention is administered to a subject, the CTL is defamated in the subject, and the immunity against the liver cell cancer cells is enhanced. Or you can use the so-called "expressing antigen-presenting cells and -20- 87544 200413406 CD 8 positive cells or peripheral blood mononuclear spheres from the subject, contacting (stimulating) the peptide of the present invention in a test tube, defamating CTL and giving Subject "in vitro treatment. The present invention further provides cytotoxic T cells that are deflated using the peptides of the invention and isolated. The cytotoxic T cells induced based on the stimulation of the antigen-presenting cells presenting the peptides of the present invention are preferably from subjects who are subjects for treatment and / or prevention. When the purpose of antitumor effect is achieved, they may be used alone or in combination with The peptides, exosomes, and other medicines of the present invention are administered together. Furthermore, the present invention provides an antigen-presenting cell that displays a complex of an HLA molecule and a peptide of the present invention. The antigen-presenting cells obtained by contacting with a gene encoding a peptide of the present invention, a GPC3 protein comprising the peptide of the present invention, or a part of the peptide thereof, preferably come from a subject who is a subject for treatment and / or prevention, It can be administered as a vaccine alone or in combination with other medicines containing peptides, exosomes or cytotoxic T cells of the invention. The present invention further provides a hepatocellular carcinoma diagnostic agent comprising an antibody against GPC3. The antibody against GPC3 can be any of multiple antibodies or single antibodies, and can be prepared by a method known to those skilled in the art (for example, refer to "Seminary Chemistry Experiment Lecture 1, Protein 1,389-406, Tokyo Chemical Associates"). The amino acid sequence of the GPC3 protein is known as described above, and it can be manufactured by ordinary protein expression techniques based on the amino acid sequence, or it can be used by a commercial vendor (SantaCruz, CA). When using commercially available GPC3, it is better to use SDS-Out ™ (sodium dodecyl sulfate precipitation reagent; purchased from PIERCE, Rockford, IL) before removing SDS, if necessary. Moreover, a partial peptide of GPC3 can be produced by selecting an appropriate partial sequence from the amino acid sequence of GPC3 and using conventional peptide synthesis techniques. 87544 -21-200413406 For the preparation of multiple strains of antibodies, first GPC3 protein or a part of its peptides are administered to animals such as rabbits, marmots, mice, rats, guinea pigs, chickens and simians. In the case of sensitization antigen. It can be administered with adjuvants (FIA and FCA) that promote antibody production. Administration is usually performed every few weeks. By performing multiple immunizations, the antibody price can be increased. After the final immunization, antiserum was obtained by collecting blood from the immunized animals. The antiserum can be divided into fractions by ammonium sulfate precipitation and anion chromatography, and affinity-refined using protein A and immobilized antigen to prepare multiple antibodies. On the other hand, for the preparation of a single antibody, for example, an animal is immunized with the GPC3 protein or a partial peptide thereof in the same manner as described above, and after the final immunization, the spleen or lymph node is taken from the immunized animal. The antibody-producing cells contained in the spleen or lymph node are fused with myeloma cells using polyethylene glycol or the like to prepare a fusion tumor. Cell fusion can be performed basically according to a known method, such as the method of Kohler and Milstein (Kohler G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). The obtained fusion tumor can be selected by culturing in a conventional selection medium, such as a HAT medium (including hypoxanthine, aminopterin, and thoracic acid). The culture in the above-mentioned HAT medium is continued for a sufficient period of time in order to eliminate cells (non-fused cells) other than the intended fusion tumor. Next, a target fusion tumor is selected, the fusion tumor is cultured, and a monoclonal antibody is prepared from the culture supernatant. The antibody of the present invention can also be produced from a host transformed by a genetic engineering method using an expression vector containing the antibody gene. For example, monoclonal antibodies can be recombinant antibodies produced by genetically recombining antibodies (see Vandamme, for example, by cloning the antibody gene from a fusion tumor, inserting the appropriate 87544-22-200413406), introducing the vector into a host, and using genetic recombination technology (see Vandamme, AM et al., Eur J. Biochem. (1990) 192, 767-775, 1990). In order to produce the antibody used in the present invention, an arbitrary expression line such as a eukaryotic cell or a prokaryotic cell line can be used. Eukaryotic cells are animal cells such as mammalian cell lines, insect cell lines, fungal cells, and yeast cells, and prokaryotic cells are bacterial cells such as coliform cells. Next, the transformed host cells are cultured in a test tube or in a living body to produce an antibody of interest. The host cell is cultured according to a known method. The purification of the monoclonal antibody is performed, for example, by ammonium sulfate precipitation and anion chromatography, and then fractionated by affinity chromatography using protein A and an immobilized antigen. In addition, separation and purification methods commonly used for proteins can be used, for example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns, filters, ultrafiltration, salting out, and dialysis other than the above-mentioned affinity chromatography. The antibody used in the present invention is not limited to the entire molecule of the antibody, as long as it can bind to GPC3, it may be a fragment of the antibody or a modification thereof, and also includes a bivalent antibody and a monovalent antibody. For example, the antibody fragment may be Fab, F (alV) 2, Fv, Fab / c with 1 Fab and complete Fc, or single-chain Fv formed by linking the Fv of the Η chain or L chain with an appropriate linker (scFv). Furthermore, for the purpose of the present invention, the antibody should preferably be capable of recognizing any epitope of GPC3. For the accuracy of the diagnostic agent, the antibody is preferably a human antibody or a human antibody. For example, if a human antibody is a mouse-human chimeric antibody, the antibody gene can be isolated from mouse cells that produce antibodies against GPC3 protein, and then the constant portion of the Η chain can be recombined into human IgE Η The chain constant portion 87544 -23- 200413406 gene is introduced into mouse myeloma cells and modulated. Furthermore, human antibodies can be prepared by replacing the mouse immunoglobulin gene with a human gene and then immunizing the mouse with a GPC3 protein. In the diagnostic agent of the present invention, the concentration of the antibody is not limited, and for example, it can be used at a concentration of 1 pg / ml. The diagnostic agent may contain a pharmaceutically acceptable carrier or the like in addition to the above-mentioned antibody against GPC3, if necessary. The present invention also provides a method for diagnosing hepatocellular carcinoma, which comprises contacting a specimen with an antibody against GPC3. The diagnostic method may further include quantifying GPC3 in the specimen. In the present invention, the specimen is serum, saliva, urine, etc. obtained from a subject who is at risk of suffering from HCC, and serum is particularly preferred. Contact of the specimen with the above-mentioned antibodies can be performed according to a method generally used in the field. The reaction for diagnosis can be performed in a liquid phase such as a cavity, or an antibody against GPC3 can be immobilized on a solid support. In this case, it is possible to determine whether or not the measured value is HCC positive by comparing with a standard value prepared in advance from a normal specimen that does not suffer from HCC or a specimen that is determined to be HCC. At the time of diagnosis, it is preferable to measure the amount of GPC3 in the serum of a plurality of HCC patients and healthy people and set a cut-off index. The diagnostic method of the present invention can be used at various stages of the course of treatment, in addition to being used to diagnose whether or not HCC is present, to confirm the therapeutic effect on HCC. Furthermore, the present invention provides a kit for diagnosing HCC, which comprises an antibody against GPC3. In addition to the antibodies against GPC3, the kit can also include secondary antibodies, standard samples and buffers for quantification. Although the present invention will be described in more detail with reference to the following examples, the present invention is not limited to these examples. [Example 1] Identification of the GPC3 gene with specific excess expression in HCC as previously reported by cDNA microarray to determine the pattern of gene expression (Okabe, H. et al., Cancer Res. 61, 2129-2137 (2001 )). From 20 patients undergoing hepatectomy, primary HCC and corresponding non-cancerous liver tissue were obtained. Ten of them were positive for hepatitis B surface antigen, 10 were positive for hepatitis C virus (HCV), and no cases were infected with both HBV and HCV. Among HBV-positive and HCV-positive individuals, there were no differences in age, gender, degree of differentiation, vascular invasion, and tumor stage. A cDNA microarray containing 23,040 cDNAs selected from the National Center for Biotechnology Information database, such as "genomic breadth", was prepared. Comparing the expression patterns between HCC and corresponding non-cancerous liver tissues and exploring genes that are specifically overexpressed in HCC, it was identified as a candidate agent for immunotherapy of HCC patients and may be the ideal target GPC3. In 16 of the 20 cases of HCC, the expression of GPC3 mRNA was 5 times higher in cancerous tissues than in non-cancerous tissues (Figure 1). That is, GPC3 is overexpressed in most HCCs and is not related to hepatitis B virus (HBV) or HCV infection. On the other hand, GPC3 mRNA is highly expressed in the placenta, fetal liver, fetal lung, and fetal kidney, and is poorly expressed in most tissues of adults (Figure 1). These GPC3-related data are consistent with those published by the Southern Point Research Institute (Zhu, Z.W. et al., Gut 48, 558-564 (2001); Hsu. HC et al., Cancer Res. 57, 5179- 5184 (1997); Pellegrini, M. et aL, Dev Dyn. 213, 431-439 (1998)). From these results, it is understood that GPC3, like a-fetal protein (AFP), is a novel cancer fetal antigen in HCC. 87544 -25- 200413406 [Example 2] Expression of GPC3 mRNA and HLA-A24 in human HCC cell line To select the target HCC cell line for CTL measurement, GPC3 was used by reverse recording-PCR (RT-PCR) mRNA expression using anti-type I HLA (w6 / 32, IgG2a) or anti-HLA-A24 (IgG2) monoclonal antibodies (VERITAS, Tokyo) and FITC-conjugated goat anti-mouse IgG (ICN / C APPEL, Aurora, OH) immunostaining, and flow cytometry, review the performance of HLA-Class I and -A24. Hep G2, Hep 3B, PLC / PRF / 5 and HuH-7, which are HCC cell lines, were obtained from the Medical Cell Resource Center of the Institute of Aging Medicine, Tohoku University. SK-Hep-1 was provided by Dr. K. Jtoh of Kurume University. RT-PCR is performed according to a known method (for example, Nakatsura, T. et al., Biochem. Biophys. Res. Commun. 281, 936-944 (2001)). A GPC3 gene-specific PCR primer was designed to amplify a 939 bp fragment. An RT-PCR reaction was performed using this primer, which included an initial denaturation of 94 ° C / 5 minutes and 30 amplification cycles at a bonding temperature of 58 ° C. The GPC3 PCR primer sequence used is: sense primer: 5f-GTTACTGCAATGTGGTCATGC-3, (sequence number 1) antisense primer: 5'-CTGGTGCCCAGCACATCAT-3-3 (sequence 歹 J number 2) Protein PCR primer sequence significance primer: 5f-CCTCGCCTTTGCCGATCC-3, (sequence number 3) Antisense primer · 5'-GGATCTTCATGAGGTAGTCAGTC-3, (sequence number 4).

The performance of GPC3 mRNA in HCC 87544 -26- 200413406 cell line was compared by normalizing the control-actin mRNA. As a result, GPC3 mRNA was strongly expressed in HepG2, Hep3B, and HuH-7 HCC cell lines, and moderately expressed in PLC / PRF / 5 cell lines. No such performance was seen in SK-Hep-1 (Figure 2). Although HepG2 and SK-Hep-1 showed HLA-A24, Hep3B and HuH-7 did not show HLA-A24.

[Example 3] Peripheral blood mononuclear spheres (PBMCs) stimulated tumor-responsive CTLs based on the prior art (Anti-1113, 11.1 \ 61 & 1.,]. 11111111111〇1.152, 3913-3924 (1994) ), Exploring peptides from GPC3 predicted to bind to HLA-A24 molecules, and synthesizing and using 12 different peptides (Table 1). These peptides were synthesized using the Fmoc / PyBOP method (see Reference Examples 1 to 12 below), or purchased from Biologica (Tokyo). The purity of the peptides was confirmed to exceed 95% by HPLC. 87544 27- 200413406 a, and oblique ^^ 2 ^ 今 屮-^ 承 襻 -f 棒 helm elfjf (BIMAS: HLA ^^ f, € fr ^^ Dr · Ke eth parker ^^^: http: // bi Head s.dcrtb ^ b \ v > Lin Xuechuan #_i? 一 庠 琴 HLA-A24ffEf ^ r ^^ rI3ii ^^ CTL ^ lf ^ & rv :; 87544 1 GP03 s' $ 2 GP03 41- 仁 9 3 GPC3 129-137 Ren GPC3 148-157 5 Gpn3 247-256 6 GJP03 260-268 7 GP03 294-303 8 GPC3 298-306 9 GPC3 313-321 10 GPC3 3S-368 11 GPC3 401-409 12 GPC3 521 丨 530 SFFQRLQPGL 24 FFQRLQPGL 36 MFKN2YPSL 20, FDVVSLYIL 24 KFSKDCGRML 40 WYnSYnoGL 240 wYWREYILSr 払 00 EYPSLEEL 330 IYDMENVLL 200 AYYPEDLFI 60 FYSALPGYI 60 RFLAELAYDL Ί2 of 0 β

Gp-i 4/12 4/12 2/12 22 5/12 2/12 7/12 3/12 + + + + + + + + + + + + + + 3 + + + + + + + + + + + + + + + + + + + 3/8 2/8 3/8 3/8 3/8 3/8 3/8 1/8 2/8 3/8 3/8 4/8 -28- 5 6 HLA -A24 Tim, H? HCpt bee CTUf ^ — 5 CFC3 itching through fry HLA-A24 ^ 寐 Hcpe% fry PBMCfs: HiLA-A2_ > ^ > lfr ^ Chechong it 寐 CTL ^ lf _ > ais fry PBMC CTL ~ 棼 200413406 After obtaining informed consent, 30 ml blood samples were obtained from HLA-A24 + HCC patients being treated in the second lecture of the Department of Surgery, Kumamoto University Medical Department, as previously reported (Nakatsura, T et al., Eur. J. Immunol. 3 2, 826-836 (2002)), and isolated PBMC by Ficoll-Conray density gradient centrifugation. For the 12 peptides from glycinan-3 (corresponding to Tables 1, 1-12: sequence numbers 5-16) with HLA-A24 binding motifs, review from 8 HLA- A24 + HCC patients (Patients 1 to 8) received the ability of PBMC to defame HLA-A24 binding and tumor-responsive CTL (Figure 3, Table 1). The method for deriving peptide-specific CTL from PBMC 'uses a previously reported method (Nakatsura, T. et al., Eur. J. Immunol. 32, 826-836 (2002)). The surface phenotype of CTL was reviewed by direct immunofluorescence staining using FITC-conjugated anti-CD3, -CD4 or -CD8 monoclonal antibodies (Nitirei, Tokyo). In order to determine the effector cells and HLA restraint, at the beginning of culture, anti-HLA-type I (W6 / 32, IgG2a) and anti-HLA-A24 (0041HA, IgG2a) were added at 20 pg / ml each. , Anti-CD8 (Nu-Ts / c, IgG2a), anti-HLA-DR (H-DR-1, IgG2a) and anti-CD4 (Nu-Th / I, IgG1) monoclonal antibodies. Anti-CD13 (MCS-2, IgG2a) and anti-CD14 (JML-H14, IgG1) monoclonal antibodies were used as isotype control groups. After 3 to 4 weeks, the number of these CTL strains increased by about 10 times compared to the number before PBMC cultivation. The cytotoxic activity of these CTL strains on HCC cell lines was then reviewed by a 6-hour 51Cr release assay. The results are shown in Fig. 3 and Table 1. Thirty-three (34.4%) of the 96 CTL strains showed cellular nociceptive activity against HLA-A24 + GPC3 + and HepG2, compared with Hep3B and HuH_7 for HLA-A24- GPC3 + and -29 for HLA-A24 + GPC3--87544 200413406 SK- Hep-1 is more harmful. These cells are noxious, show peptide specificity, and because they are inhibited by anti-HLA-type I, anti-CD8 or anti-HLA-A24 monoclonal antibodies, it is shown that these T cell responses are restricted by HLA-A24 Sex of CD8 + CTL. Table 1 shows the peptides and the CTL induction rate in each patient. These results show that all of the 12 peptides from GPC3 have CTL-inducing ability, are HLA-A24 binding and are specific for the peptides from GPC3. The CD8 + CTL strain was vilified in all 8 HCC patients Export. [Example 4] Expression of GPC3 protein in HCC About Western HCC analysis and immunohistochemical analysis of HCC and GPC3 in non-cancerous areas around liver tissue excised from 4 HCC patients. The western point is as follows. Dissolve the specimen in an appropriate amount of dissolution buffer (150 mM NaCn, 50 mM Tris, pH 7.4, 1% Nonidet P-40, 1 mM sodium vanadate (Wako Pure Chemical Industries, Ltd.), 1 mM EDTA, and protease Inhibitor Lozenges (Amersham, Arlington Heights, IL)). The lysate supernatant was electrophoresed on an SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). After blocking in Tris-buffered saline with 5% deprivation and 0.2% Tweeη 20, the membrane and the anti-GPC3 rabbit made against the recombinant protein corresponding to the amino acid of GPC3 303-464 Multiple strains of antibodies (Santa Cruz, California) — raised together, washed thoroughly with PBS, using anti-rabbit 1S that binds to peroxidase and F (ab,) 2 (from donkey) that binds to mustard peroxidation (Amersham) , And ECL kit (Amersham) for chemiluminescence detection. Immunohistochemical analysis was performed according to a method known in the art -30- 87544 200413406 (Nakatsura, T. et al. 9 Biochem. Biophys. Res. Commun. 281, 93 6-944 (2001)). Tissue specimens with a thickness of 4 μm embedded in the OC embedding compound were stained with an anti-GPC3 antibody diluted to 1.200. To serve as a positive control, 'GPC 3 3 0 3-4 6 4 (Santa Cruz, CA) produced from colorectal pain was made into a fusion protein with a 45 kDa label and an anti-heparin proteoglycan-3 rabbit FhoReporter Mini-Biotin-XX Protein Identification Kit (F-6347) (Molecular Probes, Inc.,

Eugene) was biotinylated. The 96-well ELISA plate (Nunc, Denmark) was placed at 4 ° C overnight, and coated with anti-human GPC3 303-464 (Santa Cruz) in PBS (pH 7.1) at 0.1 Kg per well. The plate was then blocked with 4% bovine serum albumin / PBS for 1 hour at room temperature. The standard sample and culture supernatant of the positive control were added with biotinylated anti-GPC3 antibody 'and incubated together at room temperature for 2 hours. After washing 3 times with PBS, in each hole

Add streptoavidin (ENDOGEN, Woburn) that binds to HRP. After 30 minutes of incubation, the plate was washed three times with PBS, and then TMB matrix solution (ENDOGEN) was added. For analysis, an ELIS A reader (type 550, Bio-Rad) was used for measurement at 405 nm. After obtaining informed consent, tissue samples were obtained from HCC patients who were being treated in the second lecture of the Department of Surgery of Kumamoto University Medical Surgery. In all four tumors, the so-called "the expression of GPC3 protein in HCC cells was as low as that in non-cancerous liver cells" was obtained. Therefore, there is a contradiction between GPC3 mRNA expression and GPC3 protein expression in HCC cells. GPC3 has been reported as a GPI-anchored membrane protein and as a secreted protein (Fiimus J., Glycobiology 11, 19R-23R (2001)). Therefore, the next attempt was 87544 -31- 200413406 to detect the secreted GPC3 protein. [Example 5] Detection of soluble GPC3 protein in the culture supernatant of HCC cell lines and the serum of HCC patients In order to review the presence of soluble GPC3 protein in the culture supernatant of HepG2, HepG2 was recovered after a predetermined culture period. Cell lysates and culture supernatants were analyzed by Western spotting. Modified from 1 × 105 HepG2 cells in serum-free medium at 6, 12, 24, and 48 hours after culture

Cell lysates showed the same amount of 60 kDa GPC3 protein (lines 1, 3, 5 and 7 of Figure 4). On the other hand, 60 kDa GPC3 protein was gradually increased in HepG2 culture supernatant (1 ml / hole 20 μΐ) after 6, 12, 24, and 48 hours of culture, showing that GPC3 protein was secreted from HepG2 to the culture Serum. Next, use anti-GPC3 303-464 antibody and biotinylated anti-GPC3

Antibodies were detected by enzyme-linked immunosorbent assay (ELISA). A commercially available recombinant protein corresponding to GPC3 303-464 was used, and the accuracy of GPC3 quantification in the ELISA system was confirmed. A serial dilution of HepG2 culture supernatant was used to evaluate the standard curve for quantifying GPC3 protein based on OD data. The concentration of GPC3 protein in 1 mi of the culture supernatant of 1 X 105 HepG2 cells cultured for 24 hours was defined as 1 U / m. The amount of GPC3 protein in HepG2 culture supernatant was more than that in Hep3B. The aspect cannot be detected in SK-Hep-1 (Figure 5). Next, detection of soluble GPC3 protein in the serum of HCC patients was performed (Figure 6). Blood samples were taken from 28 HCC patients, and patient files collected from medical records were used to determine the clinical stage based on the UICC TNM classification. Although a band of 60 kDa GPC3 protein was detected in 20 μΐ of serum from patient 7 87544 -32- 200413406 (pt7, line 3 of Fig. 6), serum from other 2 HCC patients and 4 healthy blood donors was not detected. Detected. Next, the amount of GPC3 protein in the serum of 28 HCC patients and 54 healthy blood donors (HD) was evaluated by ELIS A (Figure 7). The average amount of GPC3 protein in 54 HD sera was 0.75 U / ml, and the standard deviation (SD) was 0.32. There is no difference in performance between men and women, so the weak performance of GPC3 in the ovaries is considered to be negligible in relation to this line. The average of 28 HCC patients was 1.98 U / ml. In order to determine the normal upper limit of GPC3 protein in serum, the average GPC3 protein of 54 HD sera + 3 SD, which is 1.71, is the normal upper limit. 35.7% (10/28) of HCC patients and 0% (0/54) of HD were GPC3 protein positive (> 1.71). The concentration of GPC3 protein in the serum of patients with HCC was significantly higher than that in HD (p < 0.0001). The GPC3 concentrations evaluated by ELISA in patients 4, 5, and 7 were 0.94, 1.73, and 69.4 U / ml as shown in Fig. 6, respectively (Table 2). That is to say, although the GPC3 protein of 69.4 U / ml can be detected by the Western dot crush method, the GPC3 protein of 1.73 U / ml cannot be detected. From the results in Table 2, it can be seen that 2 of the 10 GPC3-positive patients (patients 6 and 25) were negative in both AFP and PIVKA-II, and one of the patients (patient 6) was classified as a relatively early UICC. Phase II. That is, there are cases where GPC3 becomes positive in patients who are both AFP and PIVKA-II negative, so GPC3 can obviously be used as a novel tumor marker for HCC. 87544 -33- 200413406 Table 2 Measurement results of AFP, ΡννκΑ-11 and GPC3 in serum of 28 patients with HCC

Scale | MMM 4 3 9 6 5 6 T1217S192426415227161420276251013212328 mmmfmmm 3 1 o MX 7 o 3 7677756 MM M 9 7 2 6 6 7 MM 2 2 6 7 ls F M 723 7 6 MM MM M 8 9 19 9 5 6 7 5 6

_Virus a

VKA P 20) cl ιι m Stage ICC

r—II | I Gf-I IVAiIVAT ⑴i] [AIVAIVA = sIVAiIIA = l (IAlvAH = gIVA = IVBlllAIVBivAil! Al

JrISS chooses 56! 122 view glsv " si 252 ^ 15332124211 ^ 2832121169.6319288 3 6 2 3 1 ο o ο 6 8 8 2.1112 12 IJ.231 J.9SI.22I.1S.49.52.3S.42.28.S7.94.83. 57.39 inverse. 90.3583J22.4S.6653.S27080 1l3i CF3 212I ιιοΰο 0 · 0 · 0 · 0 * 1 · 5 · ^ α ° -αu2 · 0 · 1 · 0 · 1 · 0 · 6 6 a, HCV RT- PCR detection. HBsAg was investigated with a radioimmunoassay (RIA). b, AFP was quantified by RIA. c, the value in parentheses indicates the cutoff index. d. PIVKA-II and GPC3 were quantified by enzyme immunoassay. e. Underline the positive measurement. 87544 34- 200413406 [Reference Example 1] Synthesis of Ser-Phe-Phe-Gln-Arg-Leu-Gln-Pro-Gly-Leu (Serial No. 5) using Fmoc-Leu-Wang resin (size 100-200) As the starting resin, the synthesis was started in accordance with the following procedure A. By proceeding to step 5 and returning to step 2, repeated deprotection, washing, coupling, and washing of the alpha amine group were performed. Fmoc -Gly-OH, Fmoc-Pro-OH, Fmoc-Gln (Trt) -OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Phe-OH- Fmoc-Phe-OH ^. Fmoc-Ser (tBu) -OH {ic ^ As a result, a peptide-binding resin was obtained. The peptide was separated from the resin by reacting with the reagent shown in step 6, filtered through cold methyl tert-butyl ether (MTBE), and precipitated. The precipitated peptide was washed twice with cold MTBE and freeze-dried under nitrogen. 35- 87544 200413406

Table 3 Procedure A Step 1. (washing) 2 · (deprotection) 3 · (washing) 4. (coupling) 5 · (washing) 6. (breaking) time X treatment (minutes) times

DMF 2 20% Lukouding / DMF 10 ml 5x1 20% Shout Lake / DMF 10 ml 30 x 1 DMF 4 DCM 1 Each alpha amino protected amino acid 1 mmole / 30 x 1 0.45 M HBTU / HOBT 2.1 ml (1 mmole), DIEA 348 μΐ (2 mmole) DMF 4 DCM 1 5% H20 120 x 1 5% 酉 3% thioanisole 3% ethylene dithiol 3% triisopropylsilane 81% TFA 7 · ( Precipitation) MTBE 8 · (washed) MTBE 9. (freeze-dried) 87544 -36- 200413406 The crude skin was obtained by BioCad 60 (Perkin-Elmer, Foster City CA) 5 C18 column with a particle size of 10 microns (Phenomenex, Torrance, CA) Purified by reverse phase HPLC. Dissolve with a linear concentration gradient of 10% B to 80% B (solvent A: 0.05% TFA / H20; solvent B: 0.05% TFA / acetonitrile) at a flow rate of 20 ml / min over 45 minutes, and dissolve the eluate in A220 nm monitoring. Mass analysis of the main peak with an area ratio of more than 90% using Lasermat 2000 (Finnigan Mat, San Jose, CA) 'by MALDI-TOF method. The result was 1109.9 relative to the theoretical value [MH +] 1109.3. [Reference Example 2] The synthesis of Phe-Phe-Gln-Arg-Leu-Gln-Pro-Gly-Leu (sequence number 6) was the same as [Reference Example 1], using Fmoc-Leu-Wang resin as the starting resin And add Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Gln (Trt) -OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc- Phe-OH and Fmoc-Phe-OH are combined in sequence. The result of the mass analysis showed that relative to the theoretical value [MH +] 1131.3, the measured value was 1130.4. [Reference Example 3] The synthesis of Met-Phe-Lys-Asn-Asn-Tyr-Pro-Ser-Leu (SEQ ID NO: 7) was the same as that in [Reference Example 1], and Fmoc-Leu-Wang resin was used as the starting tree. The purpose of the month, and Fmoc-Ser (tBu) -〇H, Fmoc-Pro-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Asn (Trt) -OH, Fmoc- Lys (Boc) -OH, Fmoc-Phe-OH, and Fmoc-Met-OH were sequentially combined with 0. The results of mass analysis showed that relative to the theoretical value [MH +] 1031.2, -37- 87544 200413406 was found to be 1032.3. [Reference Example 4] The synthesis of Phe-Thr-Asp-Val-Ser-Leu-Tyr-Ile-Leu (Serial Number 8) was the same as [Reference Example 1], using Fmoc-Leu-Wang resin as the starting resin And add Fmoc-Ile-OH, Fmoc_Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (Otbu) -OH, Fmoc-Thr ( tBu) -OH and Fmoc-Phe-OH are sequentially combined. The result of mass analysis showed that the measured value was 1122.1 relative to the theoretical value [MH-] 1121 · 3. [Reference Example 5] The synthesis of Lys-Phe-Ser-Lys-Asp-Cys-Gly-Arg_Met-Leu (SEQ ID NO. 9) was the same as [Reference Example 1], and Fmoc-Leu-Wang resin was used as the starting resin. And add Fmoc-Met-OH, Fmoc_Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (Otbu) -OH,

Fmoc ~ Lys (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, and Fmoc-Lys (Boc) -OH are sequentially combined. The result of mass analysis showed that the measured value was 1111.4 relative to the theoretical value [MH-] 1108.3. [Reference Example 6] Synthesis of Trp-Tyr-Cys-Ser-Ty and Cys-Gln-Gly-Leu (SEQ ID NO: 10)

As in [Reference Example 1], Fmoc-Leu-Wang resin was used as the starting tree, and Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Cys (Trt) -OH ^ Fmoc-Tyr (tBu) -OH ^ Fmoc-Ser (tBu) -OH ^ Fmoc, Cys (Trt) -OH, Fmoc-Tyr (tBu) -OH and Fmoc-Trp (Boc) -〇H -38- 87544 200413406 Combine in turn. ~ The result of mass analysis shows that the measured value is 1115.7 relative to the theoretical value [MH +] 1114.3. [Reference Example 7] Synthesis of Lys-Tyr-Trp-Arg-Glu-Tyr-Ile-Leu-Ser-Leu (SEQ ID NO: 11)

As in [Reference Example 1], Fmoc-Leu-Wang resin was used as the starting resin, and Fmoc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc_Ile-OH, Fmoc-Tyr (tBu)- OH, Fmoc-Glu (Otbu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Tyr (tBU) -OH, and Fmoc-Lys (Boc) -OH are sequentially combined "Mass The analysis results show that the measured value is 1105.3 relative to the theoretical value [MH-] 1104.3. [Reference Example 8] Synthesis and synthesis of Glu-Tyr-Ile-Leu-Ser-Leu-Glu-Glu-Leu (SEQ ID NO: 12) [Reference Example 1] Similarly, Fmoc-Leu-Wang resin was used as the starting tree, and Fmoc-Glu (Otbu) -OH, Fmoc-Glu (Otbu) -OH, Fmoc-Leu-OH, Fmoc -Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH and Fmoc-Glu (Otbu) -OH are sequentially combined. The results of mass analysis show that relative to the theoretical value [ MH +] 1371.6, and the measured value is 1370.7. [Reference Example 9] The synthesis of Ile-Tyr-Asp-Met-Glu-Asn_Val-Leu-Leu (SEQ ID NO: 13) was the same as that in [Reference Example 1], using Fmoc-Leu- Wang resin is the starting point of 87544 -39- 200413406, and Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Glu ( Otbu) -OH, Fmoc-Met-OH, Fmoc-Asp (Otbu) -OH, Fmoc-Tyr (tBu) -OH, and Fmoc-Ile-OH are combined in sequence. The results of mass analysis show that relative to the theoretical value [MH +] 1211.4 The measured value is 1213.4. [Reference Example 10] The synthesis of Ala-Tyr-Tyr-Pro-Glu-Asp-Leu-Phe-Ile (SEQ ID NO: 14) uses Fmoc-Ile-Wang resin as the starting resin. Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Asp (Otbu) -OH, Fmoc-Glu (Otbu) -OH, Fmoc-Pro-OH, Fmoc_Tyr (tBu) -OH, Fmoc-Tyr (tBu) -OH is sequentially combined with Fmoc-Ala-OH. The result of the mass analysis showed that the measured value was 1217.4 relative to the theoretical value [MH-] 1216.4. [Reference Example 11] The synthesis of Phe-Tyr-Ser-Ala-Leu-Pro-Gly-Tyr-Ile (SEQ ID NO: 15) was the same as that in [Reference Example 10], using Fmoc-Ile-Wang resin (over 100-200 (Sieve) as the starting resin, and Fmoc-Tyr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ser (tBu) -OH, Fmoc-Tyr (tBu) -OH and Fmoc-Phe-OH are sequentially combined. The result of mass analysis showed that the measured value was 1196.8 relative to the theoretical value [MH +] 1193.4. [Reference Example 12] Synthesis of Arg-Phe-Leu-Ala-Glu-Leu-Ala-Tyr-Asp-Leu (SEQ ID NO: 16) 87544 -40- 200413406 As in [Reference Example 1], Fmoc-Leu-Wang was used The resin was used as the starting resin, and Fmoc-Asp (Otbu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ala-OH, Fmoc-Leu-OH-Fmoc-Glu (Otbu) -OH Fmoc- Ala-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, and Fmoc-Arg (Pbf) -OH are sequentially combined. The result of mass analysis showed that the measured value was 1186.7 relative to the theoretical value [MH-] 1183.5. Possibility of Industrial Utilization The inventors identified 12 peptides derived from GPC3, which is a cancer-fetal protein, as candidate targets for immunotherapy in patients with ΑΑ-Α24 + HCC. Although GPC3 is overexpressed in HCC patients, GPC3's performance is significantly lower in the normal organs of adults, except for the placenta. Therefore, GPC3 has been identified as an ideal target for HCC immunotherapy. In addition, the method of the present invention has proven to be very useful for diagnosing the presence or absence of HCC. The present inventors have further identified GPC3-derived peptides that can be regulated as HLA-A24 binding and HCC-reactive CTLs. The HLA-A24 allele accounted for 60% of all individuals, and 95% of its genotype was eight * 2402. 20% of Caucasians and 12% of Africans (Tokunaga, K. et al., Immunogenetics 46, 199-205 (1997); Imanishi, I. Et al., Proceedings of the 11th International Histocompatibility Workshop and Conference (Tsuji, K. et al.) 1065-1220 (Oxford University Press, Oxford, 1992)). From these results, it can be understood that GPC3 is very useful in the diagnosis and prevention of specific immunotherapy or cancer for most HCC patients in the world. 87544 -41-200413406 All publications, patents and patent applications cited in this specification > are incorporated herein by reference. [Brief description of the figure] Figure 1 shows the HCC-specific expression of GPC3 mRNA in adult tissues. It shows the relative ratio (RR) of human GPC3 mRNA performance in 20 cases of HCC and various normal tissues. The RR of HCC is the intensity ratio of tumor to non-tumor in each condition. The RR of normal tissue is the intensity ratio of normal tissues to normal liver. Figure 2 shows the performance of mRNAs of GPC3 and β-actinin (control group) detected by RT-PCR in human HCC cell lines. Line 1: HepG2; Line 2: Hep3B; Line 3: PLC / PRF / 5; Line 4: SK-Hep-1; Line 5: HuH-7. Figures 3 (a) ~ (z) and (af) ~ (g ') show PBMCs stimulated and proliferated with GPC3 peptides (peptide sequence numbers 5-16) stimulated with 51Cr release measurement survey for 6 hours. The result is one of the effects of CTL · activity. The values on the vertical axis represent specific cell lysis rates calculated based on the average of three measurements. HCC cell lines used HLA-A24 + GPC3 +++ HepG2, HLA-A24 + GPC3_ of SK-Hep-1, HLA-A24 GPC3 +++ of Hep3B and HuH-7, and peptide 1, 3, 7 for patient 1 Or 12 CTLs, PTL 5, 6, 10, or 11 for patient 2; CTLs induced by Peptide 2 or 12 for patient 3; Peptides 1, 2, 3, and 4 for patient 4. CTL induced by 4, 7, or 10, CTL induced by peptide 1, 3, 4, 11, or 12 for patient 5, CTL induced by peptide 6 or 9 for patient 6, and peptide 4 for patient 7 , 5, 6, 8, 9, 11 or 12 CTLs, for patients 8 87544 -42- 200413406 CTLs induced using peptides 5, 7 or 10. In addition, the horizontal axis represents the execution cell / standard Babi (E / T ratio), that is, the ratio of the number of CTLs to the number of cancer cells. Fig. 4 shows the presence of GPC3 protein in the culture supernatant of HepG2 when measured by the Western dot crush method. Lines 1, 3, 5 and 7 of HepG2 cells after 6, 12, 24, and 48 hours of culture 1 X 105 lysates, Lines 2, 4, 6, and 8 of HepG2 after 6, 12, 24, and 48 hours of culture The culture supernatant was 20 μΐ. Figure 5 shows the results of quantification of GPC3 protein secreted into the culture supernatant by three HCC cell lines (Hep G2, Hep 3B, and SK-Hep-1) by ELIS A. 1. Define the concentration of GPC3 protein in 1 ml of the culture supernatant of HexG2 cells 1 × lO5 24 hours later as 1 U / ml. Figure 6 shows the detection of soluble GPC3 protein in the serum of HCC patients by Western spot ulceration. Figure 7 shows the results of quantification of GPC3 protein in the serum of 28 HCC patients and 54 healthy blood donors (HD) by ELISA. 1.71 is the normal upper limit of soluble GPC3 protein in serum as specified by the average of GPC3 protein in serum from 54 HDs + 3SD (standard deviation). 87544 43- 200413406 Sequence Listing < 110> Nissho Kirin Barley Co., Ltd. < 120 > Peptide and Medicine Containing the Same

< 130 > PH-1870-PCT < 140〉 092122744 < 141 > 2003-08-19 < 150〉 JP 2002-245831 < 151 > 2002-08-26 < 160> 16 < 170 > Patentln Ver. 2. 0

< 210〉 1 < 211〉 21 < 212〉 DMA < 213 > Homo sapiens < 400 > 1 gttactgcaa tgtggtcatg c 21

< 210 > 2 < 211〉 18 < 212 > DNA < 213 > Homo sapiens < 400 > 2 87544 200413406 18 ctggtgccca gcacatgt < 210〉 3 < 211 > 18 < 212 > DNA < 213〉 Human < 400 > 3 cctcgccttt gccgatcc 18 < 210〉 4 < 211〉 23 < 212 > DNA < 213〉 human &400; 4 ggatcttcat gaggtagtca gtc 23 < 210〉 5 < 211> 10 < 212 > PRT < 213〉 Human &400; 5

Ser Phe Phe Gin Arg leu Gin Pro Gly Leu 1 5 10 < 210〉 6 2- 87544 200413406 < 211 > 9 < 212 > PRT < 213> human < 4〇〇 > 6

Phe Phe Gin Arg Leu Gin Pro Gly Leu 1 5 < 210〉 7 < 211〉 9 < 212 > PRT < 213〉 human < 400 > 7

Met Phe Lys Asn Asn Tyr Pro Ser Lou 1 5 < 210〉 8 < 211〉 9 < 212 > PRT < 213〉 human < 40〇 > 8

Phe Thr Asp Val Ser Leu Tyr lie Leu 1 5 87544 200413406 < 210 > 9-< 211〉 10 < 212 > PRT < 213〉 human < 400 > 9

Lys Phe Ser Lys Asp Cys Gly Arg Met Leu 1 5 10 < 210〉 10 < 211〉 9 < 212 > PRT < 213〉 Human < 400〉 10

Trp Tyr Cys Ser Tyr Cys Gin Gly Leu 1 5 < 210〉 11 < 211〉 10 < 212〉 PRT < 213〉 Human < 4〇〇 > 11

Lys Tyr Trp Arg Glu Tyr lie Leu Ser Leu 1 5 10 -4-87544 200413406 < 210 > 12 < 211〉 9 < 212〉 PRT < 213〉 human < 400 > 12

Glu Tyr lie Leu Ser Leu Glu Glu Leu 1 5 < 210 > 13 < 211〉 9 < 212 > PRT < 213〉 human < 400 > 13 lie Tyr Asp Met Glu Asn Val Leu Leu 1 5 < 210 > 14 < 211 > 9 < 212〉 PRT < 213〉 human < 400 > 14

Ala Tyr Tyr Pro Glu Asp Leu Phe lie 1 5 87544 200413406 < 210〉 15 < 211 > 9 < 212〉 PRT < 213〉 human < 400 > 15

Phe Tyr Ser Ala Leu Pro Gly Tyr lie 1 5 < 210〉 16 < 211〉 10 < 212 > PRT < 213〉 human < 400 > 16

Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu 1 5 10 87544

Claims (1)

200413406 Patent application patent park: 1. A peptide 'comprising an amino acid sequence shown in any one of sequence numbers 5 to 16. 2. A peptide having cytotoxic τ cell-deficient ability, which is one in which one or two amino acids are substituted or added in the amino acid sequence shown in any one of sequence numbers 5 to 16. 3. The peptide according to item 2 of the patent application range, wherein the second amino acid from the n-terminus is phenylalanine, tyrosine, methionine or tryptophan. 4. The peptide according to item 2 or 3 of the scope of patent application, wherein the amino acid at the C-terminus is phenylalanine, leucine, isoleucine, tryptophan or methionine. 5. A medicine for the treatment and / or prevention of tumors, which comprises one or more of the peptides of any one of claims 1 to 4. 6_ — an exosome, which presents on the surface a complex comprising the peptide and any of the HLA molecules in any of claims 1 to 4 of the scope of patent application. 7. The exosome according to item 6 of the patent application, wherein the HLA molecule is HLA-A24. 8. As for the exosomes in the 7th scope of the patent application, the HLA molecule is HLA-A * 2402. 9. A method for defamating an antigen-presenting cell having a high cytotoxic T-cell defensive ability, wherein the antigen-presenting cell is induced using a peptide of any one of claims 1 to 4 of the patent application. 1 〇. — A method for deflecting antigen-presenting cells with high cytotoxic T cell defensive ability, which includes encoding heparin proteoglycan 3 (GPC3) or including in the first to fourth items of the patent application scope Part of the peptide of any one is 87544 200413406 The gene 4 of the peptide is incorporated into the antigen-presenting cell. 11 · A method for inducing a nociceptive τ cell, which uses a peptide of any one of claims 1 to 4 in the patent application. 12. A kind of cell-harmful T cell, which is obtained by inducing and isolating with a peptide of any one of claims i to 4 of the scope of patent application. 13. · An antigen-presenting cell, which is a complex that presents HLA and a peptide according to any one of claims 1 to 4. 14. If the antigen-presenting cells in the scope of the application for item 13 are applied, they are defamated by the method in the scope of the application for item 9 or 10. 15. A diagnostic agent for hepatocellular carcinoma (HCC) comprising an antibody against GPC3. 16. A method of diagnosing HCC ', which comprises contacting a specimen with an antibody against GPC3. 17. The method according to item 16 of the scope of patent application, which further comprises quantifying GPC3 in the specimen. ○ 18 · A kit for diagnosing HCC, which includes antibodies against GPC3. 87544