TWI333958B - - Google Patents

Description

1333958 玫,发明说明: [Technical Field of the Invention] The present invention relates to a novel peptide which is effective as a cancer vaccine, a medicine for treating and preventing tumors, and a new type of diagnosis of hepatocellular carcinoma including the peptide Agent. [Prior Art] Primary hepatocellular carcinoma (HCC) is one of the most common malignant diseases in the world. Due to the worldwide prevalence of hepatitis B and C, the incidence of HCC has risen sharply in Asia and Europe (Schafer, DF· and Sorrell, MF, Lancet 353, 1253-1257 (1999)), if considering HCC infection to The long-term potential period of onset can predict that this trend will continue in the next 50 years. The worsening the HCC of the disease, the new treatment strategy is urgently needed. On the other hand, in recent years, with the advancement of molecular biology and tumor immunology, a number of genes encoding tumor antigens and antigenic peptides recognizable by tumor-responsive cytotoxic T lymphocytes (Ctl) have been identified. Increase the likelihood of antigen-specific tumor immunotherapy (Boon, T. and van der Bruggen, P., J.

Exp. Med. 183, 725-729 (1996); Rosenberg, SA, J. Natl. Cancer Inst. 88, 1635-1644 (1996)) « 〇c-fetal protein (AFP), although only in normal tissues In the fetal life performance 'but the report will be reactivated in multiple HCC (Fujiyama, S. et al. Oncology 62, 57-63 (2002)). In addition, mouse and human T cell repertorys can recognize peptide epitopes from AFPs presented by MHC class I molecules (Butterfield, LH et al., Cancer Res. 59, 3134-3142, Jr Vollmer) , CM et al., Cancer Res. 59, 3064- 87544 『1333958 The heterogeneous performance of GPC3 in plants showed inhibition of community formation activity. Furthermore, Xiang et al. recently reported that GPC3 was 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, from GPC3-positive tissues in adults. In cancer, the expression of GPC3 is reduced during tumor progression, which is thought to play a role in the occurrence of malignant phenotypes. Conversely, in the case of HCC, tumors are generated from liver tissue that only expresses GPC3 in the fetal period. GPC3 has a tendency to be re-expressed when it is converted to malignant. In recent years, it has been known that Heparan sulfate proteoglycan (HSPG) is a fibroblast growth factor (FGF) and Wnt. B The heparin-binding growth factor is required for optimal activity (Yayon, A. et al., Cell 64, 841-848 (1991);

Schlessinger, J. et al., Cell 83, 357-360 (1995)). Heparin glycoside (GPC) is a GPI-anchored cell surface HSPG family. In terms of the relationship between tumor formation and GPC3 expression, the tissue-specific differences were presumed to be due to GPC3's different methods of regulating growth and survival factors in each tissue. GPC3 is thought to operate at least in such organs as cancerous fetal proteins. In general, cancerous fetal proteins do not play an important role in tumor progression, but they can be used as targets for tumor markers or 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 the cancerous behavior of GPC3 can be used for clinical use 87544 -10- 『1333958 and the re-expression of this heparin (glypican) in HCC deterioration is still important to study. Although there are various treatments for HCC, it has a poor prognosis compared with other cancers and is one of refractory cancers. HCC basically has cirrhosis, which is due to the poor liver function of the original patient, and even if it treats one cancer, it produces cancer from another. It requires an urgent and innovative treatment strategy. If immunotherapy with specific high-performance antigens in HCC can be developed, it will be possible to obtain a treatment that does not harm normal organs and only effectively excludes cancer. Further, it is possible to obtain a treatment method which can be used for patients who are unable to perform other treatments regardless of the end stage patient or liver function. Furthermore, there are currently more than 2 million people infected with hepatitis C in the HCC population. Such immunotherapy can be used for the prevention of HCC in such infected persons. Although AFP and PIVKA-II are known to be used as tumor markers for HCC, there are cases in which patients have not been detected, and cases in which patients with liver cirrhosis and chronic hepatitis of benign liver disease become false positives, especially early in HCC. Diagnosis is quite difficult. Therefore, other useful tumor markers for early diagnosis of HCC are needed. Disclosure of the Invention The present inventors have identified a novel cancer-preferred protein, glypican 3 (GPC3), which is specifically expressed in human hepatocellular carcinoma based on the data of cDNA microarray interpretation. ), and discover novel peptides that can be potential candidates for immunotherapy. Next, the inventors and the like detected soluble GPC3 protein in the serum of HCC patients, and understood

87544 -11 - 1333958 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 by any one of SEQ ID NOs: 5 to 16. (2) A peptide having cytotoxic T cell inducing ability, wherein one or two amino acids in the amino acid sequence shown in any one of the sequence numbers 5 to 16 are substituted or added. (3) The peptide of (2) above, wherein the second amino acid from the N-terminus is phenylalanine, valine, methionine or tryptophan. (4) The peptide of (2) or (3) above, wherein the amino acid at the C-terminus is amphetamine, leucine, isoleucine, tryptophan or guanidine. (5) A medicine for the treatment and/or prevention of a tumor, which comprises the peptide of any one of the above (1) to (4). (6) An exosomes which exhibit a complex comprising a peptide of any one of (1) to (4) and an HLA molecule on a surface. (7) The exosome of (6) above, wherein the HLA molecule is HLA-A24. (8) Ex vivo according to (7), wherein the HLA molecule is HLA-A*2402. (9) A method for inducing an antigen-presenting cell having a high cytotoxic T cell-inducing ability, which comprises using the peptide of any one of the above (1) to (4) for induction of the antigen-presenting cell. (10) A method for inducing an antigen-presenting cell having a high cytotoxic T cell inducing ability, comprising introducing a gene encoding a heparin proteoglycan-3 (GPC3) protein or a part of the peptide thereof into an antigen-presenting cell, wherein The partial peptide comprises the peptide of any one of the above (1) to (4). 87544 -12- 1333958 (π) A method for inducing cytotoxic tau cells, wherein the induction is carried out using the peptide of any one of (1) to (4). (12) A cytotoxic tau cell which is induced and isolated by the peptide of any one of (1) to (4). (13) An antigen-presenting cell which exhibits a complex of HLA and a peptide of any one of (1) to (4). (14) The antigen-presenting cell of the above (13), which is induced by the method of the above (9) or (10). (15) A diagnostic agent for hepatocellular carcinoma (HCC) comprising an antibody against gPC32. (16) A diagnostic method for HCC, which comprises contacting a sample with an antibody against ope]. (17) The method according to (16) above, which further comprises gpC3 in the quantitative sample. (18) A kit for diagnosing HCC comprising an antibody against GPC3. The present specification contains the contents of the specification and/or drawings of the patent application No. 2002-245 831, which is based on the priority of the present application. [Embodiment] The best mode for carrying out the invention The present invention uses the method described in Okabe, Η. et al. (Cancer Res. 61, 2129-2137 (2001)) to identify human hepatocytes according to the cDna microarray. A novel cancer-preferred protein called heparin proteoglycan-3 (GPC3) that is overexpressed in cancer. In the results of detecting GPC3 in serum, it was confirmed that GPC3 was negative in cancers other than hepatocellular carcinoma such as stomach, esophagus, lung, breast, % visceral, bile duct, and colon, in cirrhosis and chronic liver 87544 -13- 1333958 Also negative in benign liver diseases such as inflammation. Furthermore, the inventors confirmed that GPC3 in the serum of the postoperative patients was negative in hepatocellular carcinoma patients after surgical treatment. The amino acid sequence of the human GPC3 protein is well known, for example, it is registered in GenBank's protein database with a naturalization number of NP 004475, which will be readily available to those skilled in the art. The inventors then take into consideration the fact that various proteins are present on the antigen-presenting cells in the living body, and are present after being decomposed into peptides composed of nine amino acids (nonapeptides), and thus the synthesis has an effect against HLA-A24 ( A binding motif of this type, which accounts for 60% of the total population of Japan, and a partial peptide consisting of 9 amino acids or 10 amino acids of GPC3. The selection of a peptide having a binding motif for HLA-A24 can be carried out according to, for example, the method described in J. Immunol., 152, 3913, 1994 and J. Immunol. 155: 4307, 1994. For HLA types other than HLA-A24, the peptide can be selected in the same manner. Or use the software available on the Internet recently, for example, as described in Parker KC, J. Immunol. 152, 1994, in silico can be used to calculate the binding affinity of various peptides to HLA molecules (sometimes referred to as HLA antigens). Sex. Binding affinity to HLA molecules, for example, the above-mentioned software 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 peptide can be synthesized from any position depending on the total amino acid sequence of the obtained GPC3 protein. The synthesis of the peptide can be carried out according to the method used in the usual peptide chemistry. Synthetic method commonly used 87574 -14- 1333958 ^'J Peptide Synthesis, Interscience, New York, 1996; The Proteins, Vol 2, Academic Press Inc., New York, 1976; peptide synthesis, Maruzen Co., Ltd., 1975; The basis and experiment of peptide synthesis, Maruzen Co., Ltd., 1985; Development of pharmaceutical products, continued in Volume 14, Winning Peptide Synthesis, Hirokawa Shoten, 1991, etc.; and International Publication WO 99/67288. The actual binding of the HLA molecule to the peptide can be incubated with the peptide by a transformant that can express the HLA gene in the TAP-deficient T2 or RMA-S cell line, and quantified and assayed in the cell by flow cytometry. Surface type I HLA molecules (see, for example, Immunol. Lett. 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 an effective result, it is preferable to use the A24 type which is mostly owned by the Japanese (60%), and it is preferable to use the isotype. However, clinically, by investigating the type of HLA molecule of a patient to be treated in advance, it is possible to appropriately select a peptide having high binding affinity with the antigen or exhibiting a high ability to induce noxious T cells (CTL) by the antigen. Further, in order to obtain a peptide having high binding affinity and high conductivity of CTL, one or two amino acid substitutions or additions may be carried out based on the amino acid sequence of the naturally occurring GPC 3 partial peptide. In addition to the peptides presented in nature, the sequence of the peptides known to bind to and present in the existing HLA molecules is known (J. Immunol., 152, 3913, 1994; Immunogenetics. 41:178, 1995; J. Immunol. 155:4307, 1994), and therefore for the peptide obtained, it is preferred to vary according to the regularity. For example, HLA-A24 binds to a higher affinity to replace the second amino acid 87544 -15 - 1333958 from the N-terminus of the peptide to amphetamine, tyrosine, methionine or color Amino acids, and the c-terminal amino acid is substituted with a peptide of phenylalanine, leucine, isoleucine, tryptophan or methionine. However, in the case where the sequence of the peptide is identical to one of the other amino acid sequences of the functional intrinsic or foreign protein, since it may cause side effects such as autoimmune diseases or cause allergic symptoms to specific substances, the use may be It is preferred to use a database for homology searches and to avoid alignment with amino acid sequences of other proteins. Furthermore, in the similarity search, if it is apparent that one or two amino acid different peptides are not present, the above amine group is used in order to improve the binding affinity to the HLA molecule and/or the ability to induce CTL. Changes in the acid sequence will not cause the above problems. As the above-mentioned peptide with high binding affinity to HL A molecule, although it is predicted that it is highly likely to be an effective cancer vaccine, it is necessary to review the actual candidate peptide selected with high binding affinity as an index. Whether it has CTL induction ability. Confirmation of CTL conductivity, for example, by antigen-presenting cells (β-lymphocytes, giant sputum cells, and dendritic cells) having HLA molecules, specifically, dendritic cells derived from peripheral blood mononuclear cells, And after being stimulated with the peptide, it was mixed with CD8-positive cells, and then the cytotoxic activity against the target cells was determined. Alternatively, peptide-specific CTLs can be induced from PBMCs according to the method described by Nakatsura, Τ. et al. (Eur. J. Immunol. 32, 826-836 (2002)). Further, as a reaction, a transgenic animal which can be expressed as a human HLA molecule can be used (for example, Hum. Immunol. 2000 Aug.; 61(8): 764-79 Related Articles, Books, Linkout 87544 -16- 1333958

Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response., BenMohamed L., Krishnan R., Longmate J., Auge C·, Low L ., Primus J., Diamond DJ, etc.). The cytotoxic activity can be calculated, for example, by radiolabeling the target cells with 5iCr or the like and then liberating the radioactivity from the target cells. Alternatively, it is observed that the antigen of the load peptide exhibits the production of CTL in the presence of cells. It can be observed by measuring the visual point of the IFN-γ and anti-IFN-γ monoclonal antibodies released on the medium. As a result of the above-mentioned review of the CTL inducing ability of the peptide, the nonapeptide or decapeptide selected from the peptide containing the amino acid sequence shown in the following SEQ ID NO: 5 to 16 has high CTL inducing ability.

Ser-Phe-Phe-Gln-Arg-Leu-Gln-Pro-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 (SEQ ID NO: 7) Phe-Thr-Asp-Val-Ser-Leu-Tyr-Ile-Leu (SEQ ID NO: 8) Lys-Phe-Ser-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 ( SEQ ID NO: 13) Ala-Tyr-Tyr-Pro-Glu-Asp-Leu-Phe-Ile (SEQ ID NO: 14) Phe-Tyr-Ser-Ala-Leu-Pro-Gly-Tyr-Ile (SEQ ID NO: 15) Arg- Phe-Leu-Ala-Glu-Leu-Ala-Tyr-Asp-Leu (SEQ ID NO: 16) -17- 87544 1333958 The present invention further provides one of the amino acid sequences shown in any one of SEQ ID NOs: 5 to 16. Or 2 amino acids are substituted or added, and have a peptide that induces cytotoxic tau cells. The substitution or addition of one or two amino acids is not consistent with the amino acid sequence of other proteins and has CTL inducing ability. Particularly with regard to the substitution of an amino acid, the second amino acid is substituted with phenylalanine, tyrosine, methionine or tryptophan from the N-terminus, and the amino acid at the c-terminus is substituted with phenylalanine. A preferred example is leucine, isoleucine, tryptophan or guanidine thioglycolic acid. The above-described peptides of the present invention may be used alone or in combination of two or more, and may be used as a cancer vaccine for inducing CTL in the body. By administering the peptide of the present invention, the peptide is present at a high density on the HLa molecule of the antigen-presenting cell, embedding a CTL which is specifically reactive with the complex of the presented peptide and the HL A molecule, It has a high attack power for liver cancer cells that are the target cells. Alternatively, by extracting the dendritic cells from the subject and stimulating with the peptide of the present invention, an antigen-presenting cell which loads the peptide of the present invention on the cell surface can be obtained, and by re-administering it to the subject, the subject can be Inducing CTL in vivo and mentioning the attack power of the target cells. That is, the present invention provides a medicine for treating a tumor or preventing proliferation and metastasis of a tumor, and the like, which comprises the above-mentioned ones of the present invention. Further, in the living body and in the test f, the sputum-producing cells are stimulated by the peptide of the present invention, and the peptide is present in a high concentration relative to the cells to compete with the peptide originally loaded on the cell. Exchange will be easy to carry out. Therefore, the binding affinity to molecules must be above a certain level. In the case of the medicament of the present invention, I隹·g is directly administered to the peptide of the present invention, 87544, 〇1333958, but is formulated into a pharmaceutical composition formulated by a commonly used formulation method. And as well. In this case, in addition to the peptide of the present invention, a carrier and an excipient which are usually used in medicine may be appropriately contained, and are not particularly limited.

The medicine for treating and/or preventing hepatocellular carcinoma using the peptide of the present invention as an active ingredient can be administered together with an adjuvant in a manner effective for cellular immunity, and can also be administered together with other active ingredients such as an anticancer agent. And, or into a granular dosage form. As an adjuvant, those described in the literature (Clin. Microbiol. Rev., 7:277-289, 1994) can be used. Further, a liposome preparation, a particulate preparation which is bound to beads having a diameter of several μη, a preparation which is bound to a lipid, and the like can be considered. The administration method can be carried out by oral administration, intradermal administration, subcutaneous administration, intravenous injection, etc., or can be administered systemically or locally to the target tumor. The dosage of the peptide of the present invention may be appropriately adjusted depending on the disease to be treated, the age, body weight, and administration method of the patient, but usually it is 0.001 mg to 1000 mg, preferably 0.001 mg to 1000 mg. It is preferably 0.1 mg to 10 mg, preferably 1 day to several months. If the skilled person is to be able to properly select the appropriate amount of investment. Still alternatively, the present invention provides an intracellular small cell which is a so-called "exosome" which presents a complex of the peptide of the present invention and an HL A molecule to the surface. For the preparation of the foreign body, the method described in detail in JP-A No. 5, 10,507 and JP-A No. 2000-512161 can be used, but it is preferred to use an antigen-presenting cell obtained from a subject for treatment and/or prevention. To modulate. The exosome of the present invention can be made into a cancer vaccination in the same manner as the above-described peptide of the present invention. Regarding the HLA molecule, it must be the same type as the HLA molecule of the subject in need of treatment and/or prevention. For example, in the case of the Japanese, it is appropriate to make HLA-A24, 87544 -19- 1333958, especially HLA-A*2402. The present invention further provides a method of inducing antigen-presenting cells using the peptide of the present invention. After the dendritic cells are induced from the peripheral mononuclear cells, they are contacted (stimulated) with the peptide of the present invention in a test tube or in a living body, and the antigen-presenting cells can be induced. In the case where the peptide of the present invention is administered to a subject, in the subject, the antigen-presenting cells loaded with the skin of the present invention are induced. Alternatively, the peptide of the present invention may be loaded into an antigen-presenting cell in a test tube, and then a vaccine is administered to the subject. Further, the present invention provides a method for inducing antigen-presenting cells having high cytotoxic T cell inducing ability, which comprises introducing a gene encoding a GPC3 protein or a partial peptide thereof (including the above-described peptide of the present invention) into an antigen-presenting cell. The introduced gene may be in the form of DNA or in the form of RNA. The method of introduction can be carried out by various methods such as a lipofective method (lip 〇 〇 。), an electroporation method, and a calcium phosphate method which are usually carried out, and is 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 Special Table 2000-509281 . By introducing a gene into an antigen-presenting cell, the gene is subjected to transcription, translation, and the like in the cell, and the obtained protein is subjected to processing and presentation of the class I or class II HLA to present the peptide. The present invention further provides a method for inducing CTL using the above-described peptide of the present invention. When the peptide of the present invention is administered to a subject, CTL is induced in the subject, and immunity against hepatocyte cancer cells is enhanced. Or you can use the so-called "presentation of antigens from the subject and cells."

87544 1333958 CD8-positive cells or peripheral blood mononuclear spheres are contacted with a peptide of the present invention in a test tube (stimulation), and an in vitro treatment method is administered to a subject after induction of CTL. The present invention further provides cytotoxic T cells induced by the peptide of the present invention and isolated. The cytotoxic T cells induced by the stimulation of the antigen presenting the peptide of the present invention, preferably from a subject who is a subject for treatment and/or prevention, may be used alone or in combination for the purpose of antitumor effect. The other peptides such as the peptide or the exosome of the present invention are administered together.

Further, the present invention provides an antigen-presenting cell which exhibits a complex of an HLA molecule and a peptide of the present invention. The antigen-presenting cell obtained by contacting the gene encoding the peptide of the present invention, the GPC3 protein comprising the peptide of the present invention or a partial peptide thereof, preferably from a subject for treatment and/or prevention, It can be administered as a vaccine either alone or in combination with other pharmaceuticals comprising the peptide, 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 may be any of a plurality of strains or a single antibody, and can be prepared by a method known to those skilled in the art (for example, refer to "New Chemistry Experiment Lecture 1, Protein 1, 389-406, Tokyo Chemical Co., Ltd."). The GPC3 protein, whose amino acid sequence is known as described above, can be produced by a usual protein expression technique depending on the amino acid sequence, or can be used by a commercially available person (Santa Cruz, CA). In the case of commercially available GPC3, it is preferred to use SDS-OutTM (sodium dodecyl sulfate precipitation reagent; from PIERCE, if necessary)

Rockford, IL purchased) used after removing SDS. Further, a partial peptide of GPC3 can be selected from an amino acid sequence of GPC3 and prepared by a conventional peptide synthesis technique. 87544 -21 - 1333958 Gene' was introduced into small air myeloma cells and modulated. Further, human antibodies can be prepared by replacing the mouse immunoglobulin gene with a human gene and then immunizing the mouse with 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. In the diagnostic agent, in addition to the above-mentioned antibody against GPC3, a pharmaceutically acceptable carrier or the like may be contained as needed.

The invention further provides a method of diagnosing hepatocellular carcinoma comprising contacting a sample with an antibody against GPC3. The diagnostic method may additionally comprise GPC3 in a quantitative sample. In the present invention, the sample is serum, saliva, urine, and the like obtained from a subject suffering from HCC, and serum is particularly preferable. The contact of the sample with the above antibody can be carried out according to the methods generally used in the field. Further, the reaction for diagnosis may be carried out in a liquid phase such as a cavity, or the antibody against GPC3 may be immobilized on a solid support. In this case, it is possible to determine whether or not the measured value is HCC positive by comparison with a standard value pre-produced with a normal specimen that does not have HCC or a specimen that is judged to be HCC. Further, at the time of diagnosis, it is preferred to measure the amount of GPC3 in the serum of a plurality of HCC patients and healthy persons and set a cut-off index. The diagnostic method of the present invention, in addition to being useful for diagnosing whether or not suffering from HCC, can be performed at various stages of the course of treatment to confirm the therapeutic effect on HCC. Furthermore, the present invention provides a kit for diagnosing HCC comprising an antibody against GPC3. The kit may contain, in addition to the antibody against GPC3, a secondary antibody, a standard sample for quantification, a buffer, and the like. The invention will now be described in more detail by way of examples, but the invention is not limited by the examples. [Example 1] Identification of GPC3 gene with specific excess expression in HCC As previously reported, the pattern of gene expression was determined by cDNA microarray (Okabe, H. et al., Cancer Res. 61, 2129-2137 (2001) )). Primary HCC and corresponding non-cancerous liver tissue were obtained from 20 patients undergoing hepatectomy. Among them, 10 cases were positive for hepatitis B surface antigen, 10 cases were positive for hepatitis C virus (HCV), and there were no cases of HBV and HCV infection. 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 "radionomic diversity" of 23,040 cE)NAs selected from a database of the National Center for Biotechnology Information was produced. Comparing the performance patterns between HCC and corresponding non-cancerous liver tissues and exploring genes that are specifically overexpressed in HCC, the results identified GPC3, which is a candidate for immunotherapy for HCC patients and perhaps the ideal target. In 16 of 20 HCC cases, GPC3 mRNA expression was 5 times higher in cancer tissues than in non-cancerous tissues (Fig. 1). That is, GPC3 is excessively expressed in most HCCs and is not associated with 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 low in most tissues of adults (Fig. 1). These GPC3-related data are consistent with those published by the Southern Point Institute (21111, 2·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 this result, it is understood that GPC3 is a novel cancer fetal antigen in HCC, similarly to a-fetal protein (AFP). 87544 -25 - 1333958 [Example 2] Expression of GPC3 mRNA and HLA-A24 in human HCC cell line

In order to select the target HCC cell line for CTL assay, anti-class I HLA (w6/32, IgG2a) or anti-HLA-A24 (IgG2) was used by using GPC3 mRNA expression by reverse transcription-PCR (RT-PCR). Individual antibody (VERITAS,

The immunostaining of goat anti-mouse IgG (ICN/C APPEL, Aurora, OH) combined with FITC, 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 Ageing Medicine, Tohoku University. Also, SK-Hep-Ι was provided by Dr. K. Jtoh of the University of Kurume. RT-PCR is according to well-known methods (eg Nakatsura, T. et al.,

Biochem. Biophys. Res. Commun. 281, 936-944 (2001)). A GPC3 gene-specific PCR primer for the 939 bp fragment was designed. This primer was used for the RT-PCR reaction, which contained an initial denaturation at 94 ° C / 5 minutes and 30 amplification cycles at a bonding temperature of 58 ° C. GPC3 PCR used

The primer sequence is: Meaning primer: 5'-GTTACTGCAATGTGGTCATGC-3' (SEQ ID NO: 1) Antisense primer: 5,-CTGGTGCCCAGCACATGT-3' (Sequence 歹J No. 2) /3 - Actin PCR primer for control experiments Sequence Meaning Primer: S'-CCTCGCCTTTGCCGATCC-S1 (SEQ ID NO: 3) Antisense primer: 5, -GGATCTTCATGAGGTAGTCAGTC-3, (SEQ ID NO: 4) 〇

The performance of GPC3 mRNA in the HCC 87544 -26- 1333958 cell line was compared by normalization to the control /3 -actin mRNA. As a result, GPC3 mRNA was strongly expressed in HepG2, Hep3B, and HuH-7 HCC cell lines, and moderately expressed in the PLC/PRF/5 cell line, and no such expression was observed in SK-Hep-Ι (Fig. 2). Further, although HepG2 and SK-Hep-1 exhibited HLA-A24, Hep3B and HuH-7 did not exhibit HLA-A24.

[Example 3] Tumor-responsive CTL induced by stimulation of peripheral blood mononuclear sphere (PBMC) Based on prior art (Kubo, RT et al., J. Immunol. 152, 3913-3924 (1994)), exploration was predicted The peptide derived from GPC3 was bound to the HLA-A24 molecule, and 12 different peptides were synthesized and used (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 peptide was confirmed by HPLC to be more than 95%. 87544 27- a, and the age of the slope as -"今屮"-醤卡_helmet~牮翕b-v"^薛泽恭驷薛il庠琢111^^241^551& oblique 溆谇~011^蹀I am rushing. 87544 ^^%^CTUf^— 4/12 4/12 2/12 6α2 5/12 2/12 7/12 3/12 1 OPC3 έ- 49 2 GPC3 41-49 3 GPC3 129-137 Private GPC3 1400-157 5 GPC3 247-256 6 GPC3 260-268 7 G^C3 294-303 8 GPC3 298-306 9 GPC3 313-321 10 GPC3 360-3600 UoPG4ori 12 GPC3 521-530 SFFSSPQL 24 FFORLOPGL 36 MFKNNYPSL 20 FrDVSLYP 24 KFSKDCGRML 40 WYOSYOoGr240 ICYWREYPSr 400 EYILSLEEL 330 IYDMENVLL 200 AYYPEDLFI 60 FYSALPGYI 60 HFLAELAYDL 72 + + + ?· s GPC3 Xue Axe + + + + + + + + 3 10 + + + + + + + + + + + + + + + + + + 3 /S ,2/8 3/S 3/8 3/S 3/8 3/S 1/8 2/8 3/S 3/8 4/8 -28- 5 6 HLA-A24It^Hcpt Bee CTUf·^ ^ ^au_S^PBMCf^fcTL~#^ 3⁄4GPC3 Xuexu坌绛衿HLA->24l%^HCpe Bee~PBMC 庠芩HLA-A24^i?>}f 溆冲;ti^CTLif朵-# 1333958 After informed consent, 30 ml blood samples were obtained from HLA-A24+ HCC patients being treated in the second lecture of the Department of Surgery at Kumamoto University, as previously reported (Nakatsura, T. et al., Eur. J) Immunol. 32, 826-836 (2002)), PBMC were isolated by Ficoll-Conray density gradient centrifugation. For 12 peptides derived from heparin-3 (glypican-3) with HLA-A24 binding motif (corresponding to Table 1, No. 1~12: SEQ ID NO: 5-16), review from 8 HLA- The ability of PBMCs from A24+HCC patients (patients 1-8) to induce HLA-A24 binding and tumor-responsive CTLs (Fig. 3, Table 1). A method for inducing a peptide-specific CTL by PBMC using the previously reported method (Nakatsura, T. et al., Eur. J. Immunol. 3 2, 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 cell and HLA binding, 20 pg/ml of each anti-HLA-class I (W6/32, IgG2a), anti-HLA-A24 (0041HA, IgG2a) was added at the beginning of the culture. , anti-CD8 (Nu-Ts/c, IgG2a) 'anti-HLA-DR (H-DR-1, IgG2a) and anti-CD4 (Nu-Th/I, IgGl) monoclonal antibodies. Anti-CD13 (MCS-2, IgG2a) and anti-CD14 (JML-H14, IgGl) monoclonal antibodies were used as isotype control groups. After 3 to 4 weeks, the number of such CTL strains increased to about 10 times compared with the number of PBMCs before culture. The cytotoxic activity of these CTL strains against HCC cell lines was then examined by a 6 hour 51Cr release assay. The results are shown in Figure 3 and Table 1. 33 of the 96 CTL strains (34.4%) showed cytotoxic activity against HLA-A24+ GPC3+ HepG2, which was for Hep3B and HuH-7 of HLA-A24· GPC3 + and 87544 -29- 1333958 of HLA-A24+ GPC3· SK-Hep-l is more active. These cytotoxicity, showing peptide specificity, and due to inhibition by anti-HLA-class I, anti-CD8 or anti-HLA-A24 monoclonal antibodies, indicates that these T cell responses are constrained by HLA-A24 The sex CD8+CTL served. The peptides and CTL induction rates in each patient are shown in Table 1. These results show that all of the 12 peptides from GPC3 have CTL-inducing ability, are HLA-A24 binding and are specific for CD8+ CTL strains specific for GPC3 peptides in all 8 HCC patients. Out. [Example 4] Expression of GPC3 protein in HCC

Western blot analysis and immunohistochemical analysis were performed on HCC around the liver tissue excised from 4 HCC patients and GPC3 in the non-cancerous field.

The western point collapses as follows. Dissolve the sample in an appropriate amount of dissolution buffer (150 mM NaCl' 50 mM Tris' pH 7.4» 1% Nonidet P-40' 1 mM sodium orthovanadate (Wako Pure Chemical Co., Ltd.), 1 mM EDTA and protease Inhibitor Lozenge (Amersham, Arlington Heights, IL)). The supernatant of the lysate was electrophoresed on an SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). After blocking in 5% skim milk and 0.2% Tween 20 in Tris buffered physiological saline, the membrane was incubated with an anti-GPC3 rabbit polyclonal antibody against a recombinant protein corresponding to amino acid of GPC3 303-464 ( Santa Cruz, California) - incubated, washed thoroughly with PBS, using anti-rabbit Ig in combination with peroxidase and F(ab')2 (from scorpion) (Amersham) combined with mustard peroxidation, and using ECL The kit (Amersham) was tested for chemiluminescence. Immunohistochemical analysis was performed according to methods well known in the art 87544 -30- 1333958 (Nakatsura, T: et al., Biochem. Biophys. Res. Commun. 281, 936-944 (2001)). Tissue sections embedded in the 〇CT-embedded compound and having a thickness of 4 μm were stained with an anti-GPC3 antibody diluted to 1:200. In order to serve as a positive control, GPC3 303-464 (Santa Cruz, CA) produced from Escherichia coli was made into a fusion protein with a 45 kDa tag, and anti-heparin proteoglycan-3 rabbit polyclonal antibody was used with FluoReporter mini-bio The prime-XX protein marker # group (F-6347) (Molecular Probes, Inc., Eugene) was biotinylated. A 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 pg per well. The plate was then blocked with 4% bovine serum albumin/PBS for 1 hour at room temperature. The positive control standard sample and the culture supernatant were added together with the biotinylated anti-GPC3 antibody, and cultured together at room temperature for 2 hours. After washing three times with PBS, streptavidin (ENDOGEN, Woburn) which binds to HRP was added to each well. After 30 minutes of incubation, the plate was washed 3 times with PBS, and then TMB substrate solution (ENDOGEN) was added. For analysis, it was measured at 405 nm using an ELISA reader (Model 550, Bio-Rad). After receiving the informed consent, the tissue samples were obtained from HCC patients being treated in the second lecture of the Department of Surgery, Kumamoto University. In all four tumors, the result that the "GPC3 protein is expressed as low as that of non-cancerous hepatocytes in HCC cells" is obtained. Therefore, there is a contradiction between GPC3 mRNA expression and GPC3 protein expression in HCC cells. GPC3 has been reported to be a GPI-anchored membrane protein as well as a secreted protein (Fnmus J., Glycobiology 11, 19R-23R (2001)). Therefore, the next attempt to detect the secreted GPC3 protein was 87544 -31 - 1333958. [Example 5] Detection of soluble GPC3 protein in culture supernatant of HCC cell line and serum of HCC patient In order to examine whether or not soluble GPC3 protein was present in the culture supernatant of HepG2, HepG2 was recovered after the predetermined culture period. Cell lysates and culture supernatants were subjected to Western point collapse analysis. Modulated from 1 X 105 HepG2 cells in serum-free medium after 6, 12, 24 and 48 hours of culture.

Cell lysate, showing the same amount of 60 kDa of GPC3 protein (lines 1, 3, 5 and 7 of Figure 4). On the other hand, in the Hep'G2 culture supernatant (1 ml/well 20 μΐ) after 6, 12, 24 and 48 hours of culture, the 60 kDa GPC3 protein was gradually increased, indicating that the GPC3 protein was secreted from HepG2 to Culture the supernatant.

Next, detection was carried out by an enzyme-binding immunosorbent assay (ELIS A) using an anti-GPC3 303-464 antibody and a biotinylated anti-GPC3 antibody. The commercially available recombinant protein corresponding to GPC3 303-464 was used and the quantitative accuracy of GPC3 in the ELIS A system was confirmed. A serial dilution of the supernatant of HepG2 culture was used to evaluate the standard curve of quantitative GPC3 protein based on OD data. The concentration of GPC3 protein in 1 ml of the culture supernatant after incubation of 1 X 105 HepG2 cells for 24 hours was defined as 1 U/ml. The amount of GPC3 protein in the HepG2 culture supernatant was higher than in Hep3B, and on the other hand, it was not detected in SK-Hep-1 (Fig. 5). Next, detection of soluble GPC3 protein in serum of HCC patients was performed (Fig. 6). Blood samples were taken from 28 HCC patients, patient records collected from medical records and clinical stages determined according to the UICC TNM classification. In the patient's 7 87544 -32- 1333958 (pt7, line 3 of Figure 6) serum 60 μΐ detected a 60 kDa GPC3 protein band, but not from the other 2 HCC patients and 4 healthy donors serum Detected in. Next, the amount of GPC3 protein in the serum of 28 HCC patients and 54 healthy donors (HD) was evaluated by ELIS A (Fig. 7). The average amount of GPC3 protein in 54 HD serum was 0.75 U/ml with a standard deviation (SD) of 0.32. There is no difference in performance between men and women, so the weak performance of GPC3 in the ovary is considered to be negligible in relation to the department. The mean value of the 28 HCC patients was 1.98 U/m. In order to determine the normal upper limit of the GPC3 protein in the serum, the mean value of the GPC3 protein of the 54 HD serum was +3 SD, i.e., 1.71, which was the normal upper limit. 35.7% (10/28) of HCC patients and 0% (0/54) of HD were positive for GPC3 protein (> 1.71). The concentration of GPC3 protein in the serum of HCC patients was significantly higher than that in HD (p < 0.0001). The GPC3 concentrations evaluated by ELIS A in patients 4, 5 and 7 were 0.94, 1.73 and 69.4 U/ml as shown in Fig. 6, respectively (Table 2). Even though the GPC3 protein of 69_4 U/ml was detected by Western blotting, the GPC3 protein of 1.73 U/ml could not be detected. As can be seen from the results in Table 2, 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 the initial UICC. Phase II. That is, GPC3 is positive in patients with negative AFP and PIVKA-II, so GPC3 is clearly a novel tumor marker for HCC. 87544 -33 - 1333958 AFP of 28 patients with HCC, PIVKA-Π and GPC3 determination results 2^3mm?m(10)5MMJMMM1FMMMMM M MMM5FM M MM MM M <^56716964536973617071775063696772627262757172635869715969 5X1128 91217181924-26415227161420276251013212328

Virus 3 · HBV HCV 卯B, non-C HCV HCV HCV HCV HCV HBV HBV HCV HCV H8V HCV HCV HCV HCV HCV HCVmt no HCV HCV HCV Non-B, non-C HCV HBV, MCV HBV HCV segment c lc ul IVAllleIVAIIIAulil AAA A ru o ^ { I - IVV i I 丨lv IA-IA ]n ( VA= =]iava=b illIV, ΠIV111^1^111.111. AFP(ng/ml)D(<20)c PIVKA-IICmAU/min< 40) GPC3(U/ml)u(<1.71) 54" 25Z 8900 .^1577 1.73 MP.Q 313. 3,23 504512125119301^29232^1510-7 37109<1-23<1 5ι81 2 a ciil34i 6 118 2 2 91 οι 9* 318 1322416113. 幽94JJ423123184166928 ^812j S 9 282 87 4 3 7 9 A 9 2 14 5 3 4 2 8 9 8 6 3 a2121 1 1 ο ο ο ο ο ο ο 1 9

ο ο 8 0 6 8 8 21 11212 < V 3 0 5 3 2r. ·9·3·8cj·4 °·0·0·3^62· 6 3 2 0 0 ·6·5·5·7· 8 0-1·0·1·0· a, HCV was detected by RT-PCR. HBsAg was investigated by radioimmunoassay (RIA). b, AFP is quantified by RIA. c, the value in parentheses indicates the critical index (cut-〇ffindex). d, PIVKA-Π and GPC3 were quantified by enzyme immunoassay. e, underline the positive measurement. 34- 87544 1333958 [Reference Example 1] Synthesis of S.er-Phe-Phe-Gln-Arg-Leu-Gln-Pro-Gly-Leu (SEQ ID NO: 5) Using Fmoc-Leu-Wang resin (over 100-200) The sieve is used as the starting resin, and the synthesis is started according to the following procedure A. After proceeding to step 5, the process returns to step 2 to repeatedly perform deprotection, washing, coupling and washing of the α-amino group. Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH 'Fmoc-Arg(Pbf)-OH 'Fmoc-Gln(Trt)-OH ' corpse 111〇(: -?116-01'1, ?111〇.-?116-011 and ?111〇(:-861'(1811)-01'1 are sequentially coupled to obtain a peptide-binding resin. The reagent reaction shown in Figure 6 was excised from the resin, filtered in cold methyl tert-butyl ether (MTBE), and precipitated. The precipitated peptide was washed twice with cold MTBE and frozen under nitrogen. Dry. 87544 35- 1333958

Table 3 Procedure A Step 1. (washing) 2. (deprotection) 3. (washing) 4. (coupling) 1 5. (washing) 6. (breaking) 7. (precipitation) 8. (Washing) 9. (Freeze drying) Time X treatment (minutes) Number of times DMF 2 20% p bottom biting / DMF 10 ml 5x1 20% Shouting / DMF 10 ml 30 x 1 DMF 4 DCM 1 Each OC amine protecting amino acid 1 Mmmole / 30 x 1 0.45 M HBTU/HOBT 2.1 ml (1 mmole), DIEA 348 μΐ (2 mmole) DMF 4

DCM 5% H20 5% phenol 3% thioanisole 3% ethanedithiol 3% triisopropyl decane

81% TFA MTBE MTBE 120 x 1 87544 -36- 1333958 The measured value is 1032 ‘.3. [Reference Example 4] Synthesis of Phe-Thr-Asp-Val-Ser-Leu-Tyr-Ile-Leu (SEQ ID NO: 8) Similarly to [Reference Example 1], Fmoc-Leu-Wang resin was used as the starting resin. And 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 results of the mass analysis showed that the measured value was 1122.1 with respect to the theoretical value [MH-] 1121.3. [Reference Example 5] Synthesis of !Lys-Phe-Ser-Lys-Asp-Cys-Gly-Arg-Met-Leu (SEQ ID NO: 9) Similarly to [Reference Example 1], Fmoc-Leu-Wang resin was used as the starting point. The original resin, and 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 results of the mass analysis showed an actual value of 1111.4 relative to the theoretical value [MH-] 1108.3. [Reference Example 6] Synthesis of Trp-Tyr-Cys-Ser-Tyr-Cys-Gln-Gly-Leu (SEQ ID NO: 10)

As in [Reference Example 1], Fmoc-Leu-Wang resin was used as the starting resin, 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 87544 -38- 1333958 . - The results of the mass analysis showed a measured value of 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) was used. )-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBU)-OH and Fmoc-Lys(Boc)-OH . The results of the mass analysis showed an actual value of 1105.3 with respect to the theoretical value [MH-] 1104.3. [Reference Example 8] Synthesis of Glu-Tyr-Ile-Leu-Ser-Leu-Glu-GIu-Leu (SEQ ID NO: 12) Similarly to [Reference Example 1], Fmoc-Leu-Wang resin was used as a starting tree. Moon' and Fmoc-Glu(Otbu)-OH, Fmoc-Glu(Otbu)-OH,

Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fm〇c-Leu-OH,

Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Glu(〇tbu)-OH are sequentially combined. The results of the mass analysis showed a measured value of 1370.7 relative to the theoretical value [MH+] 1371.6. [Scheme 9] Synthesis of Ile-Tyr-Asp-Met-Glu-Asn-Val-Leu-Leu (SEQ ID NO: 13) is the same as [Reference Example 1], using Fmoc-Leu-Wang resin as a starting point. 87544 -39- 1333958 Resin, and Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(Otbii)-OH, Fmoc-Met-OH, Fmoc-Asp(Otbu -OH, Fmoc-Tyr(tBu)-OH and Fmoc-Ile-OH are sequentially combined. The results of the mass analysis showed a measured value of 1213.4 relative to the theoretical value [MH + ] 1211.4. [Reference Example 10] Synthesis of Ala-Tyr-Tyr-Pro-Glu-Asp-Leu-Phe-Ile (SEQ ID NO: 14) - Using Fmoc-Ile-Wang resin as a 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 and Fmoc -Ala-OH is combined in sequence. The results of the mass analysis showed an actual value of 1217.4 with respect to the theoretical value [MH-] 1216.4. [Reference Example 11] Synthesis of Phe-Tyr-Ser-Ala-Leu-Pro-Gly-Tyr-Ile (SEQ ID NO: 15)

As in [Reference Example 10], Fmoc-Ile-Wang resin (over 100-200 sieve) was used as the starting resin, and Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Pro were used. -OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Phe-OH are sequentially bonded. The results of the mass analysis showed a measured value of 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 - 1333958 Same as [Reference Example, using Fmoc-Leu-Wang resin 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 results of the mass analysis showed that the measured value was 1186.7 with respect to the theoretical value [MH-] 1183.5 '. The possibility of industrial use. The inventors identified 12 peptides derived from GPC3, which are cancerous fetal proteins, as HLA-A24+. Candidates for immunotherapy of HCC patients. Despite the excess performance of GPC3 in HCC patients, in normal organs of adults, the performance of GPC3 was significantly lower than that of placenta, so GPC3 was identified as an ideal target for immunotherapy of HCC. Further, it has been demonstrated that the method of the present invention is a very useful method for diagnosing whether or not suffering from HCC. The present inventors have further identified a peptide derived from GPC3 which can be prepared as a HLA-A24 binding and HCC reactive CTL. The HLA-A24 allele is 60% of all Japanese, and 95% of its genotype is 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 llth International Histocompatibility Workshop And Conference (edited by Tsui, K. et al.) 1065-1220 (Oxford University Press,

Oxford, 1992)). From these results, it can be understood that GPC3 is very useful for the diagnosis and prevention of specific immunotherapy or cancer in most HCC patients in the world. 87544 -41 - 1333958 CTL induced with peptide 5, 7 or 10. Further, the horizontal axis represents the ratio of the execution cell/target ratio (E/T ratio), that is, the ratio of the number of CTLs to the number of cancer cells. Figure 4 shows the presence of GPC3 protein in the culture supernatant of HepG2 when measured by Western blotting. 1, 3, 5 and 7 behaviors, 1 X 105 lysates of HepG2 cells after 6, 12, 24 and 48 hours of culture, and 2, 4, 6 and 8 behaviors of HepG2 after 6, 12, 24 and 48 hours of culture. The supernatant was cultured for 20 μM. Fig. 5 shows the results of quantification of GPC3 protein secreted into the culture supernatant of three HCC cell lines (Hep G2, Hep 3Β and SK-Hep-Ι) by ELISA. The concentration of GPC3 protein in 1 ml of the culture supernatant after culturing HexG2 cells for 1 hour was defined as 1 U/ml. Figure 6 shows the detection of soluble GPC3 protein in the serum of HCC patients by Western blotting. Figure 7 shows the results of quantification of GPC3 protein in serum of 28 HCC patients and 54 healthy blood donors (HD) by ELISA. 1.71, the normal upper limit of the soluble GPC3 protein in serum as defined by the mean + 3 SD (standard deviation) of GPC3 protein in serum from 54 HD. 87544 43- 1333958 SEQUENCE LISTING <110〉日商麒麟麦酒股份有限公司<120> peptide and medicine containing same<130> PH-1870-PCT <140>092122744 <141> 2003-08- 19 9· <150> JP 2002-245831 <151> 2002-08-26 <160> 16 <170> Patentln Ver. 2. 0

<210〉 1 <211> 21 <212> DNA <213> Homo sapiens <400> 1 gttactgcaa tRtggtcatg c 21

<210> 2 <211> 18 <212> DNA <213> Homo sapiens <400> 2 87544 1333958 ctggtgccca gcacatgt <210> 3 <211> 18 <212> DNA <213><400> 3 cctcgocttt gccgatcc <210> 4 <211> 23 ' <212> DNA <213>human <400> 4 ggatettcat gaggtagtca gtc <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 87544 1333958 <2Π> 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 Ty.r Pro Ser Leu 1 5 &lt;210〉 8 &lt;211&gt; 9 <212> PRT &lt;213>human &lt;400&gt; 8

Phe Thr Asp Val Ser Leu Tyr lie Leu 1 5 87544 1333958 &lt;210&gt; 9 &lt;211> 10 &lt;212> PRT &lt;213>human &lt;400&gt;

Lys Phe Ser Lys Asp Cys Gly Arg Met Leu 1 5 10 &lt;210> 10 &lt;211&gt; 9 &lt;212> PRT &lt;213>human &lt;400&gt;

Trp Tyr Cys Ser Tyr Cys Gin Gly Leu 1 5 &lt;21〇&gt; 11 &lt;211> 10 &lt;212&gt; PRT &lt;213>human &lt;4〇〇&gt; 11

Lys Tyr Trp Arg Glu Tyr lie Leu Ser Leu 1 5 10 -4 - 87544 1333958 &lt;210&gt; 12 &lt;211&gt; 9 &lt;212> PRT &lt;213>human &lt;400&gt;

Glu Tyr lie Leu Ser Leu Glu Glu Leu 1 5 #· &lt;210&gt; 13 &lt;211&gt; 9 &lt;212> PRT &lt;213>human &lt;400&gt; 13

He Tyr Asp Met Glu Asti Val Leu Leu 1 5

&lt;210&gt; 14 &lt;211> 9 &lt;212> PRT &lt;213>human &lt;400> 14

Ala Tyr Tyr Pro Glu Asp Leu Phe lie 1 5 87544 1333958 &lt;210> 15 &lt;211&gt; 9 &lt;212> PRT &lt;213>human &lt;400> 15

Phe Tyr Ser Ala Leu Pro Gly Tyr lie 1 5 &lt;210> 16 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213>human &lt;400> 16

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

Claims (1)

1333958 ___ ___ No. 09212274 such as | seeking interest to read the case female: ί Chinese application for special ^ ^ ^ Han for the month) ---------------------- 1 pick up , apply for Fan Park &quot; 丨? 0曰修(more)本本 I ;---------------i 1. An isolated peptide with cytotoxic tau cell-inducing ability, comprising sequence number 5~ The amino acid sequence shown in any of 16. 2. An isolated peptide having cytotoxic sputum cell inducing ability, comprising: in the amino acid sequence shown in any one of SEQ ID NOs: 5 to 16, the second from the Ν end The amino acid is phenylalanine, tyrosine, methionine or tryptophan, and/or the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan or methionine. Amino acid sequence. A pharmaceutical composition for the treatment and/or prevention of a tumor comprising one or more isolated peptides as claimed in claim 1 or 2. 4. An exosome which exhibits a complex comprising an isolated peptide and an HLA molecule as disclosed in claim 1 or 2 of the patent application. 5. The isolated exosome of claim 4, wherein the HLA molecule is HLA-A24. 6. 6. The exogenous exosome of claim 5, wherein the HLA molecule is HLA-A* 2402. 7. An antigen-presenting method for inducing cytotoxic T cell inducing ability in vitro, wherein the antigen-presenting cell is induced using the isolated peptide as claimed in claim 1 or 2. A method for inducing an antigen-presenting cell having high cytotoxic T cell inducing ability in vitro, which comprises encoding human heparin 3 (glypican-3, GPC3) or containing the first or second item as claimed in the patent application. The gene of the partial peptide of the isolated peptide is introduced into the antigen-presenting cell. 87544.990817.doc 1333958 9. A method of inducing cytotoxic T cells in vitro using an isolated individual of the singularity of claim 1 or 2. 10--Isolated cytotoxic tau cells using an isolated peptide inducer as in claim 1 or 2 of the patent application. 11. A = cells presented by isolated antigens, which are a complex of HLA and the isolated skin of the patent application of claim 1 or 2. 12· = The patented antigen presenting cells of claim 11 of the patent scope, which is induced by the method of claim 7 or 8 of the patent application. 87544-990817.doc 1333958 Announcement Book 壹 壹 贯&quot;:- Chinese translation of the patent application No. 092122744 (June 98 ^. _ — • 沢 沢)) 正 H ^ HCC20 cases. Liver · Brain and lung _ Heart · Kidney - Pancreas · Spleen · Thyroid. Thymus * Tian small intestine · colon 11 bones · breast - bone marrow · ovary · uterus _ anterior gland · testis · placenta · fetal liver · fetal brain 'fetal lung ' fetal kidney · 87544 1333958 Patent application No. 092122744 60kDa ι_ι reward 培养 culture medium line 8 (supernatant) line 7 (cell) line 6 (supernatant) line 5 (cell) line 4 &lt;superscript) line 3 (cell) line 2 (Upper) Line 1 (cell) Figure 4 87544 Ϊ333958 ^ t^mi27J4 patent, please [HD 54 6 87544 -11 -