WO2004018667A1 - 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

 Description Peptides and pharmaceutical technology fields containing them

 The present invention relates to a novel peptide effective as a cancer vaccine, a medicament for treating and preventing a tumor containing the peptide, and a novel diagnostic agent for hepatocellular carcinoma. Background art

 Primary hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Due to the worldwide outbreak of hepatitis B and C, the incidence of HCC in Asia and European countries is increasing rapidly (Schafer, DF and Sorrell, MF, Lancet 353, 1253-1257 (1999)). This trend is expected to continue over the next 50 years, given the long latency period from HCC infection to disease onset. The prognosis of advanced HCC is poor and new treatment strategies are urgently needed.

 On the other hand, recent advances in molecular biology and tumor immunology have identified a number of genes encoding tumor antigens and antigenic peptides recognized by tumor-responsive cytotoxic T lymphocytes (CTLs). And the potential of antigen-specific tumor immunotherapy is increasing (Boon, T. and van der Bruggen, P., J. Exp. Med. 183, 725-729 (1996); Rosenberg, SA , J. Natl. Cancer Inst. 88, 1635-1644 (1996)). It has been reported that α-photoprotein (AFP) is expressed only in the fetal period in normal tissues, but its expression is reactivated in many HCCs (Fujiyama, S. et al., Oncology 62, 57-63). (2002)). The mouse and human T cell repertoires

Recognize AFP-derived peptide epitopes presented by MHC class I molecules (Butterfield, L. H. et al., Cancer Res. 59, 3134-3142; Jr Vollmer, CM. Et al.,

Cancer Res. 59, 3064-3067 (1999); Butterfield, L. H. et al., J. Immunol. 166,

5300-5308 (2001)). Despite high plasma levels of exposure to this oncofetal protein during fetal development, mature T cells do not achieve full tolerance to AFP, Specific T cells are peripheral blood Detected during. Thus, oncofetal proteins may be targets for immunotherapy. AFP and PIVKA-II (Fujiyama, S. et al., Oncology 62, 57-63 (2002)) are well-known tumor markers for HCC.

 In addition, cDNA microarray technology is rapidly evolving to enable researchers to obtain comprehensive data on gene expression profiles. Several studies have demonstrated that this technique is useful for identifying novel cancer-related genes and for classifying human cancers at the molecular level (Golub, TR et al., Science 286, 531-537 (1999)). Alizadeh, AA et al., Ature 403, 503-511 (2000); 0no, K. et al., Cancer Res. 60, 5007-5011 (2000); Kitahara, 0. et al., Cancer Res. 61, 3544-3549 (2001). Kihara, et al., Cancer Res. 61, 6474-6479 (2001)). The inventors have previously reported the identification of genes whose expression changes during the development of HCC by using a genome-wide cDNA microarray containing 23,040 genes. And 20 types of primary

The expression profiles of these genes in HCC were examined (0kabe, H. et al., Cancer Res. 61, 2129-2137 (2001)).

In 1996, Pilia et al. Reported that glypican-3 (GPC3), a gene encoding one member of the glypican family, had a genetic power of S, and that the Simpson-Golabi-Behmel syndrome (SGBS) syndrome Mutations were reported (Pilia, G. et al., Nat-Genet. 12, 241-247 (1996)). SGBS is an X-linked hereditary disorder characterized by prenatal overgrowth and a wide range of clinical symptoms ranging from very mild phenotypes in female carriers to fatal symptoms in male infants (Neri, G. et al. Am. J. Med. Genet. 79, 279-283 (1998)). The clinical features of SGBS include distinct facial appearance, cleft palate, syndactyly, polydactyly, accessory breast, cystic and dysplastic kidneys, and congenital heart defects (Behmel, A. Hum. Genet. 67, 409-413 (1984); Garganta, CL and Bodurtha, JN, Am. J. Med. Genet. 44, 129-135 (1992); Golabi, M. and ぴ Rosen, L. , Am. J. Med. Genet. 17, 345-358 (1984); Gurrieri, F. et al., Am. J. Med. Genet. 44, 136-137 (1992)). Most of the GPC3 mutations have been reported to be point mutations or deletions of small genes containing some 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, 57-58 (1999)). In addition, because of the lack of correlation between the patient's phenotype and the location of the mutation, SGBS is characterized by functional GPC3 protein deficiency and other genetic factors that produce phenotypic differences within and between families. (Hughes-Benzie, RM, et al., Am. J. Med. Genet. 66, 227-234 (1996)), and studies of GPC3-deficient mice support this hypothesis. (Cano-Gauci, DF et al., J. Cell Biol. 146, 255-264 (1999)). These mice have some of the abnormalities found in patients with SGBS, including overgrowth, cystic and dysplastic kidneys.

 Antigens that are specifically overexpressed in cancer and have negligible expression levels in various normal tissues may be ideal targets for immunotherapy. In these cases, C

TL is expected to show cytotoxicity only against cancers that overexpress the antigen, and not show any side effects on normal tissues. From studies using Northern plot analysis, GP C

3 mRNA has been reported to be overexpressed in HCC, placenta, fetal liver, fetal lung, and fetal kidney (Zhu, ZW. Et al., Gut 48, 558-564 (2001); Hsu. H.C. et al., Cancer

Res. 57, 5179-5184 (1997); Pellegrini, M. et al., Dev Dyn. 213, 431-439 (1998)). In addition, GPC3 has an inhibitory effect on cell growth and can induce apoptosis in certain types of tumor cells (Duenas Gonzales, A. et al., J. Cell Biol. 141,

1407-1414 (1998); Cano-Gauci, DF et al., J. Cell Biol. 146, 255-264 (1999)),

There are reports that GPC3 expression is suppressed in tumors of various origins. Lin et al. Have shown that GPC3 is expressed in normal ovaries but is not detectable in certain ovarian cancer cell lines (Lin, H. et al., Cancer Res. 59, 807-810).

(1999)). In all cases where there was no GPC3 expression, there was no mutation in the coding region, the GPC3 promoter was hypermethylated, and GPC3 expression was restored by treatment with a demethylating agent. Furthermore, ectopic expression of GPC3 has been reported to inhibit colony forming activity in several ovarian cancer cell lines. Other data linking GPC3 to cancer include those obtained from differential mRNA display studies between normal rat mesothelial cells and mesothelioma cell lines (Murthy, SS et al., Oncogene 1992). , 410-416 (2000)). In this study, GPC3 was found to be constantly down-regulated in tumor cell lines. Was done. Furthermore, similar suppression of expression has been observed in primary rat mesothelioma and cell lines derived from human mesothelioma. As in ovarian cancer, no mutations have been found in the GPC3 coding sequence, but most cell lines show abnormal methylation of the GPC3 promoter region. As reported (Duenas Gonzales, A. et al., J. Cell. Biol. 141, 1407-1414 (1998)), ectopic kissing of GPC3 in mesothelioma cell lines inhibits colony forming activity It was shown to be. More recently, Xiang et al. Reported that GPC3 was not expressed in human breast cancer (Xiang, YY et al., Oncogene 20, 7408-7412 (2001)). These data suggest that GPC 3 may act as a negative regulator of cell proliferation in these cancers. That is, GPC3 expression is reduced during tumor progression in cancers arising from adult GPC3-positive tissues, and this reduction appears to play a role in the development of the malignant phenotype.

 Conversely, in the case of HCC, tumors arise from liver tissue that expresses GPC3 only in the fetus, and GPC3 expression tends to reappear in transformation to malignancy. G

It is not clear whether re-expression of PC3 is important in the progression of these tumors. Over the past few years, paran sulfate proteodalican (HS PG) has been required on the cell surface for the optimal expression of heparin-binding growth factors such as fibroblast growth factor (FGF) and Wnt. (Yayon, A. et al., Cell 64, 841-848 (1991);

Schlessinger, J. et al., Cell 83, 357-360 (1995)). Glypican is a family of GPI-anchored cell-surface HSPGs, and this tissue-specific difference in the relationship between tumorigenesis and GPC3 expression levels indicates that GPC3 regulates growth and survival factors in different ways in each tissue It is presumed that it is because. GPC 3 appears to act as an oncofetal protein at least in these organs. In general, oncofetal proteins have not been found to play a significant role in tumor progression, but have been used as tumor markers or immunotherapeutic targets (Coggin,

J. H. 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)). The Potential of the Oncofetal Behavior of GPC 3 to be Used for Clinical Use Whether or not this dalippican re-expression is important in the progression of HCC requires more research. You.

 Despite various treatments for HCC, it has a poorer prognosis than other cancers and is one of the refractory cancers. This is due to the cirrhosis at the base of HCC, the poor liver function of the original patient, and the fact that treating one cancer causes it to come from another location. There is an urgent need for new treatment strategies. If immunotherapy targeting antigens that are specifically expressed in HCC could be developed, it could be a therapeutic method that effectively eliminates only cancer without damaging its own normal organs. It can also be a treatment that can be used for any terminally ill cancer patient whose liver function is too poor for other treatments. In Japan, it is said that there are more than 2 million hepatitis C infected people in the HCC reserve group. Such immunotherapy could be used to prevent HC C in these infected individuals. AFP and PIVKA-II are known as tumor markers for HCC, but may not be detected in some patients or may be false-positive in patients with benign liver disease cirrhosis or chronic hepatitis. Diagnosis of HCC is considered difficult. Other useful tumor markers are needed for early diagnosis of HCC. Disclosure of the invention

 The present inventors have identified Daribican 3 (GPC 3) as a novel oncofetal protein specifically overexpressed in human hepatocellular carcinoma based on cDNA microarray interpretation data, Novel peptides have been discovered that could be potential candidates for target antigens. We then detected soluble GPC3 protein in the serum of HCC patients and clarified that GPC3 could be a new tumor marker for HCC.

 That is, the present invention provides the following (1) to (18).

 (1) A peptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 5 to 16.

(2) A peptide having one or two amino acids substituted or added in the amino acid sequence shown in any of SEQ ID NOs: 5 to 16, and having an ability to induce cytotoxic T cells.

(3) The second amino acid from the N-terminus is phenylalanine, tyrosine, The peptide according to the above (2), which is nin or tributophan.

 (4) The peptide according to the above (2) or (3), wherein the amino acid at the C-terminus is phenylalanine, leucine, isoleucine, tryptophan or methionine.

 (5) A medicament for treating and / or preventing or treating a tumor, comprising at least one peptide according to any one of the above (1) to (4).

 (6) An exosome presenting on its surface a complex containing the peptide according to any of (1) to (4) above and an HLA molecule.

 (7) The exosome according to (6), wherein the HLA molecule is HLA-A24.

 (8) The exosome according to (7), wherein the HLA molecule is HLA-A * 2402.

 (9) A method for inducing antigen-presenting cells having high cytotoxic T cell inducing ability using the peptide according to any one of the above (1) to (4).

 (10) Introduction of a gene encoding a glypican-3 (glypican-3; GPC3) protein or a partial peptide thereof including the peptide according to any of (1) to (4) above into antigen-presenting cells. A method for inducing antigen presenting cells having high cytotoxic T cell inducing ability.

 (11) A method for inducing cytotoxic T cells using the peptide according to any of (1) to (4).

 (12) An isolated cytotoxic T cell induced by using the peptide according to any one of the above (1) to (4).

 (13) An antigen presenting cell that presents a complex of an HLA molecule and the peptide according to any of (1) to (4).

 (14) The antigen-presenting cell according to (13), which is induced by the method according to (9) or (10).

 (15) A diagnostic agent for hepatocellular carcinoma (HCC), comprising an antibody against GPC3.

(16) A method for diagnosing HCC, comprising contacting a sample with an antibody against GPC3. (17) The method according to (16), further comprising quantifying GPC3 in the sample.

 (18) A kit for diagnosis of HC C, comprising an antibody against GP C3. This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 2002-245831, which is a priority document of the present application. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows HCC-specific expression of GPC3 mRNA in adult tissues.

The relative ratio (RR) of human GPC3 mRNA expression in 20 cases of HCC and various normal tissues is shown. The RR in HCC indicates the tumor: non-tumor strength ratio in each case. RR in normal tissue indicates the intensity ratio of each normal tissue: normal liver. FIG. 2 shows the expression of mRNA of GPC 3 and β-actin (control) detected by RT-PCR in a human HCC cell line.

Lane 1: HepG2, Lane 2: Hep3B, Lane 3: PLC / PRF / 5,

Lane 4: SK—Hep-1; Lane 5: HuH-7

FIG. 3 shows that PBMCs stimulated with GPC 3 peptide (peptides (pep) 1 to 12: SEQ ID NOs: 5 to 16) and further expanded were subjected to CTL using a 6 hr ⁵¹ Cr release assay. A part of the results obtained by examining the activity is shown. The value on the vertical axis indicates the specific cell lysis rate (%) calculated based on the average value of three assays. HCC cell lines as ^{HLA- A 24 + GPC 3 + +} + in HepG2, using HLA- A 24+ GPC 3- Roh SK- Hep- 1, HLA one A 24- GPC 3 ⁺⁺⁺ of Hep3B and HuH- 7 , Patient 1, peptide 1,

CTL induced with 3, 7, or 12; CTL induced with peptide 5, 6, 10, or 11 for patient 2, CTL induced with peptide 2 or 12 for patient 3, patient CTL induced with peptide 1, 2, 3, 4, 7, or 10 for 4; CTL induced with peptide 1, 3, 4, 11, or 12 for patient 5; for patient 6 CTL induced with peptides 6 or 9 vs. peptide 7 versus patient 7

Shown are the results of CTLs induced with 4, 5, 6, 8, 9, 11, or 12 and CTLs induced with peptide 5, 7, or 10 for patient 8. The horizontal axis is Effector / Target ratio (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 using Western blot.

Lane 1, 3, 5, and 7: 6, 1 2, 24 , HepG2 cells 1 X 1 after culture及Pi 48 hours 0 ^five lysate

Lanes 2, 4, 6, and 8: 20 μl of HepG2 culture supernatant after culturing for 6, 12, 24, and 48 hours

FIG. 5 shows the results of quantification by ELISA of GPC3 protein secreted into the culture supernatant of three types of HCC cell lines (HepG2, Hep 3B and SK-Hep-1). The concentration of GPC 3 protein in the culture supernatant 1 m 1 after HepG2 cells 1 X 1 0 ⁵ or 24 hours of culture was defined as lUZm l.

 FIG. 6 shows detection of soluble GPC3 protein in serum of HC C patients by Western blot.

 FIG. 7 shows the results of ELISA quantification of GPC3 protein in the sera of 28 HCC patients and 54 healthy donors (HD). 1.71 is the upper normal limit of soluble GPC3 protein in serum, defined as the mean of GPC3 protein in serum from 54 HDs + 3SD. BEST MODE FOR CARRYING OUT THE INVENTION

 The present inventors used the method described in Okabe, H. et al. (Cancer Res. 61, 2129-2137 (2001)) to specifically overexpress human hepatocellular carcinoma based on cDNA microarrays. Glypican 3 (GPC3) was identified as a novel oncofetal protein expressed. According to the results of detecting GPC3 in serum, GPC3 was negative in cancers other than hepatocellular carcinoma, such as stomach, esophagus, lung, breast, pancreas, bile duct, and colon, and liver cirrhosis and chronic The results were confirmed to be negative for benign liver diseases such as hepatitis. Furthermore, the present inventors have also confirmed that postoperative serum GPC 3 becomes negative in patients after surgical treatment for hepatocellular carcinoma.

The amino acid sequence of the human GPC3 protein is known, and is registered in, for example, GenBank's protein database as Accession No. NP 004475. Can be easily obtained. The present inventors next considered that various proteins are presented in vivo on antigen-presenting cells after being decomposed into a peptide (nonapeptide) consisting of 9 amino acids. We synthesized partial amino acids consisting of 9 amino acids or 10 amino acids derived from GPC3 having a binding motif for HLA-A24, which accounts for 60% of the total human body.

 Selection of a peptide having a binding motif for HLA-A24 can be performed, for example, based on the method described in (J. Immunol., 152, 3913, 1994; J. Immunol. 155: 4307, 1994). . Similarly, peptides can be selected for HLA types other than HLA-A24. Alternatively, various peptides and HLA molecules (also called HLA antigens) may be used using software recently available on the Internet, such as those described in Parker KC, J. Immunol. 152, 1994. Can be calculated in insilico. The binding affinity with the HLA molecule can be determined by, for example, using the above-mentioned software. BIMAS: HLA Binding Prediction: http://bimas.dcrt.nih.gov/molbio/hla_bind/ , 152, 1994), or Nukaya, I., Int. J. Cancer, 80, 1999 and the like.

 9-mer and 10-mer peptides can be obtained by synthesizing a peptide from any position based on the entire amino acid sequence of the obtained GPC3 protein. The peptide can be synthesized according to the method used in ordinary peptide chemistry. Synthetic methods commonly used include, for example, Peptide Synthesis, Interscience, New

York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide synthesis, Maruzen Co., Ltd., 1975; Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd., 1985; Vol. 14 * It is described in literatures such as Peptide Synthesis, Hirokawa Shoten, 1991, and in gazettes such as International Publication W099 / 67288. The actual binding between the HLA molecule and the peptide is determined by incubating the peptide and the transfectant expressing the HLA gene in a TAP-deficient T2 or RMA-S cell line and expressing it on the cell surface.

LA class I molecules can be quantified and measured using a flow cytometer (for example, Immunol. Lett. 2002 Aug 1, 83 (1): 21-30; Immunogenetics, 44: 233-241,

1996; Nature 346: 321-325, 1990). As the HLA molecule, it is preferable to use the A24 type possessed by many Japanese (60%) in order to obtain effective results, and more preferably a subtype such as A * 2402. It is. However, in the clinic, the type of HLA molecule in patients requiring treatment is determined in advance, and a peptide with high binding affinity to the HLA molecule or the ability to induce cytotoxic T cells (CTL) by antigen presentation can be appropriately selected. You can choose. Further, in order to obtain a peptide having a high binding affinity and a high CTL inducing ability, one or two amino acids may be substituted or added based on the amino acid sequence of a naturally occurring GPC tri-partial peptide. it can. In addition to peptides presented in nature, the sequence regularity of peptides presented to HLA molecules is already known (J. Immunol., 152, 3913, 1994; Imraunogenetics. 41). : 178, 1995; J. Immunol. 155: 4307, 1994), and the obtained peptide may be modified based on these regularities. For example, those with high HLA-A24 binding affinity have the amino acid at the N-terminus of the peptide replaced with phenylalanine, tyrosine, methionine or tributophane, or the C-terminal amino acid with phenylalanine. Peptides substituted with leucine, isoleucine, tryptophan or methionine can also be suitably used.

 However, when the sequence of the peptide is identical to a part of the amino acid sequence of an endogenous or exogenous protein having another function, side effects such as an autoimmune disease may occur, or a specific substance Because of the possibility of causing allergic symptoms, it is preferable to perform a homology search using an available database to avoid a match with the amino acid sequence of another protein. Furthermore, if it is clear from the homology search that no peptide differs by one or two amino acids, the above amino acid sequence for enhancing the binding affinity to the HLA molecule and the ability to induce Z or CTL Does not cause such a problem.

As described above, a peptide having a high binding affinity to an HLA molecule is expected to be highly likely to be effective as a cancer vaccine, but a candidate selected as an index because it has a high binding affinity It is necessary to examine whether the peptide actually has the ability to induce CTL. Confirmation of CTL inducibility can be performed, for example, using antigen-presenting cells (eg, B-lymphocytes, macrophages, dendritic cells) containing HLA molecules Induces dendritic cells derived from human peripheral blood mononuclear cells, stimulated with peptides, and stimulated CD

Mix with 8 positive cells and measure cytotoxic activity against target cells. Alternatively, peptide-specific CTL can be derived from PBMC based on the method described in Nakatsura, T. et al. (Eur. J. Immunol. 32, 826-836 (2002)). Furthermore, as a reaction system, transgenic animals prepared to express human HLA molecules (for example, Hum. Immunol. 2000 Aug .; 61 (8): 764-79 Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A * 0201 / 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.) Can also be used. The cytotoxic activity can be calculated from the radioactivity released from the target cell, for example, by radiolabeling the target cell with ^{5 ×} Cr or the like. Alternatively, CTLs are produced in the presence of peptide-loaded antigen-presenting cells. Observation can be made by measuring spots visualized on the medium by the released IFN-γ and anti-IFN-γ monoclonal antibodies. .

 As a result of examining the CTL inducing ability of the peptide as described above, non-peptides or decaptides selected from peptides having the amino acid sequences shown in SEQ ID NOs: 5 to 16 have particularly high CTL-inducing ability It became clear.

 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

 Trp-Tyr-Cys-Ser-Tyr-Cys-Gln-Gly-Leu (SEQ ID NO: 10)

 Lys-Tyr-Trp-Ar g-Glu-Tyr-11 e-Leu-Ser-Leu (SEQ ID NO: 11)

 Glu- Tyr-lie- 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-lie (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 (system (I number 16) The present invention further provides a peptide in which one or two amino acids are substituted or added in the amino acid sequence shown in any of SEQ ID NOs: 5 to 16, and which has an ability to induce cytotoxic T cells. Substitution or addition of one or two amino acids may be capable of inducing CTL as long as there is no match with the amino acid sequence of another protein. In particular, amino acid substitutions such as substitution of the second amino acid from the N-terminus with phenylalanine, tyrosine, methionine or tryptophan, and substitution of a C-terminal amino acid with phenylalanine, leucine, isoleucine, tryptophan or methionine are not allowed. This is a preferred example.

 The above-mentioned peptide of the present invention can be used as one or a combination of two or more thereof as a cancer vaccine capable of inducing CTL in vivo. By administration of the peptide of the present invention, the peptide is presented at a high density on HLA molecules of antigen-presenting cells, and CTLs that specifically react with the complex of the presented peptide and the HLA molecule are induced. As a result, the offensive power against hepatocellular carcinoma cells to be targeted cells increases. Alternatively, dendritic cells are removed from a subject and stimulated with the peptide of the present invention to obtain antigen-presenting cells loaded with the peptide of the present invention on the cell surface. It can induce CTLs and increase the attack power on target cells.

That is, the present invention provides a medicament for treating a tumor or preventing the growth and metastasis of a tumor, which comprises one or more peptides of the present invention. Incidentally, in i _n vivo及Pi in vitro, peptide stimulation of antigen-presenting cells according to the present invention, by the presence of high concentrations of peptide in to cells, the exchange of the peptide which is previously loaded into the cell Occurs and is easily performed. For this reason, the binding affinity with the HLA molecule needs to be higher than a certain level.

 The medicament of the present invention may be directly administered with the peptide of the present invention alone, or may be administered as a pharmaceutical composition formulated by a commonly used pharmaceutical method. In that case, in addition to the peptide of the present invention, carriers, excipients and the like usually used for pharmaceuticals can be appropriately contained, and there is no particular limitation.

A medicament for treating and / or preventing hepatocellular carcinoma containing the peptide of the present invention as an active ingredient can be administered with an adjuvant so that cellular immunity can be effectively established, It can be administered together with other active ingredients such as anticancer agents, or can be administered in the form of particles. As the adjuvant, those described in the literature (Clin. Microbiol. Rev., 7: 277-289, 1994) can be applied. Also, a ribosome preparation, a particulate preparation bonded to beads having a diameter of several μπι, a preparation bonded to a lipid, and the like can be considered. As an administration method, oral administration, intradermal administration, subcutaneous administration, intravenous injection and the like can be used, and systemic administration or local administration near a target tumor can be used. The dose of the peptide of the present invention can be appropriately adjusted depending on the disease to be treated, the age and weight of the patient, the administration method, etc., but is usually 0.001 mg to; L000 mg, preferably 0.001 to 1000 mg, More preferably, the dose is 0.1 mg to: 10 mg, and it is preferable to administer it once every few days to several months. Those skilled in the art can appropriately select an appropriate dose.

 Alternatively, the present invention provides an intracellular vesicle called exosome, which displays a complex of the peptide of the present invention and an HLA molecule on the surface. Exosomes can be prepared, for example, using the methods described in detail in JP-A No. 11-151,073 and JP-A No. 2000-512,161. However, it is preferably prepared using antigen-presenting cells obtained from a subject to be treated and / or prevented. The exosome of the present invention can be inoculated as a cancer vaccine in the same manner as the above-mentioned peptide of the present invention.

 The HLA 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, it is often preferable to set HLA-A24, especially HLA-A * 2402.

 The present invention also provides a method for inducing antigen-presenting cells using the peptide of the present invention. After dendritic cells are derived from peripheral blood monocytes, they can be contacted (stimulated) with the peptide of the present invention in vitro or in vivo to induce antigen-presenting cells. When the peptide of the present invention is administered to a subject, antigen-presenting cells loaded with the peptide of the present invention are induced in the subject. Alternatively, the peptide of the present invention can be administered as a vaccine to a subject after in vitro loading the peptide of the present invention on the antigen-presenting cells.

The present invention also provides a cytotoxic T cell-inducing ability, which comprises introducing a gene encoding a partial peptide thereof containing the GPC3 protein or the peptide of the present invention into an antigen-presenting cell in vitro. Methods for inducing high antigen presenting cells are provided. Guidance The gene to be input may be in the form of DNA or RNA. The method of introduction may be any of various methods usually used in this field, such as ribofusion, electroporation, and calcium phosphate method, and is not particularly limited. Specifically, for example, it is described in Cancer Res., 56: 5672, 1996; J. Immunol., 161: 5607, 1998; J. Exp. Med., 184: 465, 1996; It can be done in this way. By introducing a gene into an antigen-presenting cell, the gene is subjected to transcription, translation, and other processing in the cell, and the resulting protein undergoes HLA class I or class II processing and presentation, and a partial peptide is displayed. Is done.

 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 targeting hepatocellular carcinoma cells is enhanced. Alternatively, exogenous antigen-presenting cells and CD8-positive cells or peripheral blood mononuclear cells from a subject are contacted (stimulated) with the peptide of the present invention in vitro to induce CTL and then return to the subject. It can also be used for treatment.

 The present invention further provides an isolated cytotoxic T cell induced using the peptide of the present invention. The cytotoxic T cells induced based on stimulation by the antigen-presenting cells presenting the peptide of the present invention are preferably derived from a subject to be treated and / or prevented, alone or in the present invention. It can be administered for the purpose of antitumor effect together with other drugs including the peptide of the present invention, exosomes and the like.

 The present invention further provides an antigen-presenting cell that presents a complex of an HLA molecule and a peptide of the present invention. The antigen-presenting cells obtained by contacting the peptide of the present invention, or the GPC3 protein containing the peptide of the present invention, or a gene encoding a partial peptide thereof, are preferably targets for treatment and prevention or prevention. And can be administered alone or as a vaccine together with other drugs including the peptide of the present invention, exosomes, cytotoxic T cells and the like.

The present invention further provides a diagnostic agent for hepatocellular carcinoma, comprising an antibody against GPC3. Antibodies to GPC3 may be either polyclonal or monoclonal antibodies, and can be prepared by methods known to those skilled in the art (for example, see “Shinsei Kagaku Jikken Kozai 1, Protein 1, 389-406, Tokyo Kagaku Dojin”). It is possible. GPC 3 protein The amino acid sequence of the protein is known as described above, and can be produced using ordinary protein expression techniques based on the amino acid sequence, or a commercially available product (Santa Cruz, CA) can be used. When a commercially available GPC 3 is used, it is preferable to use SDS-Out ™ (Sodium Dodecyl Sulfate Precipitation Reagent; purchased from PIERCE, Rockford, IL) and remove SDS as necessary. The partial peptide of GPC3 can be produced by selecting an appropriate partial sequence from the amino acid sequence of GPC3 and using a general peptide synthesis technique.

 In preparing a polyclonal antibody, first, for example, a GPC3 protein or a partial peptide thereof is administered as a sensitizing antigen to animals such as rabbits, guinea pigs, mice, rats, hamsters, -birds, and monkeys. Administration may be with an adjuvant (FIA or FCA) that promotes antibody production. Administration is usually performed every few weeks. Multiple immunizations can increase antibody titers. After the final immunization, antiserum can be obtained by collecting blood from the immunized animal. A polyclonal antibody can be prepared by subjecting the anticoagulant to, for example, ammonium sulfate precipitation, fractionation by anion chromatography, and affinity purification using protein A or immobilized antigen.

 For preparation of a monoclonal 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, a spleen or lymph node is collected from the immunized animal. Antibody-producing cells contained in the spleen or lymph nodes and myeloma cells are fused using polyethylene glycol or the like to prepare a hybridoma. Cell fusion is basically performed by known methods, for example, the method of Kohler and Milstein et al. (Kohler. G. and Milstein, Methods Enzymol. (1981)

73, 3-46) etc. The obtained hybridomas are selected by culturing them in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Culture in the above HAT culture solution is continued for a time sufficient for killing cells other than the target hybridoma (non-fused cells). Next, the desired hybridoma is screened, cultured, and a monoclonal antibody can be prepared from the culture supernatant.

Further, the antibody of the present invention can be expressed by an expression vector containing an antibody gene by a genetic engineering technique. May be produced in a host transformed with-. For example, a recombinant antibody produced by cloning an antibody gene from a hybridoma into a suitable vector, introducing this into a host, and producing the same using a gene recombination technique can be used as a monoclonal antibody. (See, for example, Vanda Thigh e, AM et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).

 For the production of the antibodies used in the present invention, any expression system can be used, for example a eukaryotic or prokaryotic cell system. Eukaryotic cells include, for example, established mammalian cell lines, insect cell lines, animal cells such as filamentous fungal cells and yeast cells, and prokaryotic cells include, for example, bacterial cells such as E. coli cells. Is mentioned. Next, the transformed host cells are cultured in vitro or in vivo to produce the desired antibody. Culture of the host cell is performed according to a known method.

 The monoclonal antibody can be purified, for example, by precipitation with ammonium sulfate or fractionation by anion chromatography, or by affinity chromatography using protein A or immobilized antigen. In addition, the separation and purification methods used for ordinary proteins may be used.For example, a chromatography column other than the above-mentioned affinity column, a filter, ultrafiltration, salting-out, dialysis, etc. may be appropriately selected. By combining them, antibodies can be separated and purified. · The antibody used in the present invention is not limited to the whole antibody molecule, and may be an antibody fragment or a modified product thereof as long as it binds to GPC3, and includes both bivalent antibodies and monovalent antibodies. For example, as the antibody fragment, Fab, F (ab ') 2, Fv, Fab / c having one Fab and complete Fc, or Fv of H chain or L chain were linked by an appropriate linker. Single-chain Fv (scFv). For the purpose of the present invention, the antibody may recognize any epitope of GPC3.

For accuracy as a diagnostic agent, the antibody is preferably a human antibody or a human antibody. For example, in the case of a mouse-human chimeric antibody, a human antibody is isolated from a mouse cell that produces an antibody against the GPC3 protein, and the H chain constant region is recombined into a human IgE H chain constant region gene. It can be prepared by introduction into mouse myeloma cells. In addition, the human antibody binds the immunoglobulin gene to human. It can be prepared by immunizing the replaced mouse with GPC3 protein.

 In the diagnostic agent of the present invention, the antibody can be used at, for example, but not limited to, a concentration of 1 ^ g / ml. In addition to the antibody against GPC3 described above, a diagnostically acceptable carrier or the like can be appropriately contained in the diagnostic agent, if necessary.

 The present invention further provides a method for diagnosing hepatocellular carcinoma, which comprises contacting a sample with an antibody against GPC3. The diagnostic method may further include quantifying GPC3 in the sample. In the present invention, the sample includes serum, saliva, urine, and the like obtained from a subject who may be suffering from HCC, and is particularly preferably serum. The contact between the sample and the above antibody may be performed by a method generally used in the art, and is not particularly limited. Diagnosis is, for example, after contact between the sample and the above antibody, the specific binding between the antibody and GPC3, which may be present in the sample, is determined using a fluorescent or luminescent substance, or a secondary antibody labeled with an enzyme This can be done by quantitative detection. In addition, the reaction for diagnosis may be performed in a liquid phase such as a well, or may be performed on a solid support on which an antibody against GPC3 is immobilized. In this case, the measured value is HCC-positive by comparing with a standard value prepared in advance using a normal sample without HCC or a sample known to be HCC. Can be determined. At the time of diagnosis, it is preferable to set the cut-off value by measuring the amount of GPC3 in the serum of a large number of HCC patients and healthy persons.

 The diagnostic method of the present invention can be used for diagnosing whether or not the subject has HCC, and can be performed over time to confirm the effect of treatment for HCC. Furthermore, the present invention provides a kit for diagnosing HCC, comprising an antibody against GPC3. The kit can include an antibody against GPC3, a secondary antibody, a standard sample for quantification, a buffer, and the like. Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.

[Example 1] Identification of GPC3 gene specifically overexpressed in HCC Profiling of gene expression by cDNA microarray was performed as previously reported (Okabe, H. et al., Cancer Res. 61, 2129-2137 (2001)). Primary HCC and corresponding non-cancerous liver tissue were obtained from 20 patients who underwent hepatectomy. Of these, 10 were positive for hepatitis B surface antigen, 10 were positive for hepatitis C virus (HCV), and none were infected with both HBV and HCV. HBV-positive and ぴ HCV-positive, with significant differences in age, gender, degree of differentiation, vascular invasion, and tumor stage. A “genome-wide” cDNA microarray containing 23,040 cDNAs selected from the UniGene database of the National Center for Biotechnology Information was generated. Comparison of expression profiles between HCC and corresponding non-cancerous liver tissues to search for genes that are specifically over-expressed in HCC, and as a result, are candidates for immunotherapy for HCC patients, possibly GP C3, which could be an ideal target, was identified. In 16 of the 20 HCCs, the expression of GPC 3 niRNA in cancerous tissues was more than 5-fold higher than in non-cancerous tissues (FIG. 1). That is, GPC3 was overexpressed in most HC C and was not associated with hepatitis B virus (HBV) or HCV infection. On the other hand, GPC3 mRNA was highly expressed in the placenta, fetal liver, fetal lung, and fetal kidney, and was low in most adult normal tissues (Figure 1). These data for GPC3 were consistent with those published based on Northern plotting studies (Zhu, ZW 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)). These results revealed that GPC3, like α-phytoprotein (AFP), is a novel carcinoembryonic antigen in HCC.

 [Example 2] Expression of GPC3 mRNA and HLII-A24 in human HC C cell line

 Reverse transcription-PCR to select target HC C cell lines for CT L-assay

GPC 3 mRNA expression using (RT-PCR), anti-HLA class I ( _W 6/32,

Immunostaining and flow cytometry using IgG2a) or anti-HLA-A24 (IgG2) monoclonal antibody (VERITAS, Tokyo) and FITC conjugation anti-mouse IgG (ICN / CAPPEL, Aurora, 0H) Departure of HLA—Class I and A24 The current was considered. The HCC cell lines Hep G2, Hep 3B, PLC / PRF / 5, and HuH-7 were obtained from the Medical Cell Resources Center, Institute of Aging and Medicine, Tohoku University. SK-Hep-1 was provided by Dr. K. Itoh of Kurume University.

 RT-PCR was performed according to a known method (for example, Nakatsura, T. et al., Biochem. Biophys. Res. Comraun. 281, 936-944 (2001)). A GPC3 gene-specific PCR primer was designed to amplify a 939 bp fragment and was used with RT-P consisting of 30 amplification cycles at 94 ° C, 5 min initial transformation, and 58 ° C annealing temperature. A CR reaction was performed. The GP C 3 P CR primer sequence used was

Sense: 5'-GTTACTGCAATGTGGTCATGC-3 '(SEQ ID NO: 1)

Antisense: 5'-CTGGTGCCCAGCACATGT-3, (SEQ ID NO: 2)

The β-actin PCR primer sequence for control experiments

Sense: 5'-CCTCGCCTTTGCCGATCC-3 '(SEQ ID NO: 3)

Antisense: 5, -GGATCTTCATGAGGTAGTCAGTC-3 '(SEQ ID NO: 4).

 After normalization with the control β-actin mRNA, the expression of GPC3 mRNA in HCC cell lines was compared.

 The results showed strong expression of GPC 3 mRNA in HepG2, Hep3B, and HuH-7 HCC cell lines, and moderate expression in PLC / PRF / 5 cell lines, but SK-Hep-1 No such expression was seen (Figure 2). Note that HepG2 and SK-Hep-1 expressed 3-24, but Hep3B and HuH-7 did not.

Example 3 Induction of Tumor-Responsive CTL by Stimulating Peripheral Blood Mononuclear Cells (PBMC) Based on the prior art (Kubo, RT et al., J. Immunol. 152, 3913-3924 (1994)), HLA-A24 We searched for GPC3-derived peptides that are expected to bind to the molecule and synthesized and used 12 different peptides (Table 1). These peptides were synthesized using the Fffloc / PyBOP method (see Reference Examples 1 to 12 below) and purchased from biologica (Tokyo). The purity of the peptide was confirmed to exceed 95% in all cases by HP LC. Table 1 Induction rate of HLA-A24-restricted hepatocellular carcinoma-reactive CTL from PBMC in HLA-A24-positive HCC patients stimulated with GPC3 peptide Induction of CTL from PBMC by ぺ: ^

 GPC3 peptide, HLA-A24 positive HCC patient

No rank Nishiki row Binding score ^a 1 2 3 4 5 6 7 8 GTL induction rate

1 Q PP ^ AO- 4-Q FF0RL0PGL 24 ― + + 3/8

2 GPC3 41-9 FFQRLQPG 36 2/8

3 GPC3 129-137 MFKNNYPSL 20 +-+ + 3/8

4 GPC3 148-157 FTDV 8S 〇LYIL 24 ― + + + 3/8

5 GPC3 247-256 KFSKDCGRML 40 + + + 3/8

6 GPC3 260-268 WYCSYCQGL 240 + + + 3/8 t 7 GPC3 294-303 KYWREY! LSL 400 + + 3/8

 8 GPC3 298-306 EYILSLEEL 330 + 1/8

9 GPC3 313-321 IYDMENVLL 200 + + 2/8

10 GPC3 360-368 AYYPEDLFI 60 + ― + 3/8

11 GPC3 401-409 FYSALPGYI 60 + + + 3/8

12 GPC3 521-530 RFLAELAYDL 72 + + 4/8

 Conductivity 4/12 4/12 2/12 6/12 5/12 2/12 7/12 3/12

\ Evaluation of the half-life of dissociation of molecules with each sequence

(BIMAS: H-A Binding Prediction: From the study of Dr. Kenneth Parker: http://bimas.dcrt.nih.gov/molbio/hla_bind/; b, + are peptide-specific and respond to HLA-A24 + tumor cells This indicates that the induction of sex CTL was successful.

After obtaining informed consent, a blood sample of 30 ml from HLA-A24 + HCC patients under treatment was obtained at the second course of Kumamoto University School of Medicine, and as previously reported (Nakatsura, T. et al. Eur. J. Immunol. 32, 826-836 (2002)),

PBMC was isolated by Ficoll-Conray density gradient centrifugation. Eight HLA-A2 + The ability to induce HLA-A24-restricted and tumor-responsive CTL from PBMC obtained from HCC patients (Pt 1 to 8) was examined (Figure 3, Table 1). The method for inducing peptide-specific CTL from P BMC is based on the method reported previously (Nakatsura, T. et al., Eur. J. Immunol. 32,

826-836 (2002)). The surface phenotype of CTL was examined by direct immunofluorescence staining using a FITC-conjugated anti-CD3, CD4, or CD8 monoclonal antibody (Nichirei, Tokyo). To determine effector cells and HLA restriction, 20 μg Zm1 of each anti-HLA—Class I

(W6 / 32, IgG2a), anti-HLA-A24 (0041HA, IgG2a), anti-CD8 (Nu-Ts,

IgG2a), anti-HLA-DR (H-DR-1, IgG2a), and anti-CD4 (Nu-Th / I, IgGl) monoclonal antibodies were added. Anti-CD 1 as isotype matched control

3 (MCS-2, IgG2a) and anti-CD14 (JML-H14, IgGl) monoclonal antibodies were used. After 3-4 weeks, the number of these CTL strains increased about 10-fold compared to the number before culture of PBMC. It was then examined by ⁵¹ C r release Atsusi of 6 hours cytotoxic activity against HC C cell lines of these CTL lines. The results are shown in FIG. 3 and Table 1. 9 CTL lines six 3 three (34.4%) is, HLA- A 2 4- GPC 3 ⁺ of the He _P 3B及Pi HuH- 7, and HLA- A 2 4 + GPC 3- Bruno SK- Hep- HL than against one

AA24 ⁺ GPC3 + showed strong cytotoxicity against HepG2. This cytotoxicity indicates peptide specificity, anti-HLA-class I, anti-CD8 or anti-H

Inhibition by the LA-A24 monoclonal antibody indicates that these T cell responses are mediated by HLA-A24-restricted CD8 + CTL. Table 1 shows the induction rate of CTL in each peptide or each patient. From these results, all of the 12 peptides derived from GPC3 have CTL-inducing ability, and HL

A— A 24 restricted GPC 3 derived peptide-specific CD 8 + ^ 5 with 8 shares It is shown that harm was induced in all of the persons.

 [Example 4] Expression of GPC3 protein in HC C

 Western blotting analysis and immunohistochemical analysis of GPC3 in HCC and non-cancerous regions around liver tissues excised from four HCC patients were performed.

Western blotting was performed as follows. Dissolve the sample in an appropriate volume of lysis buffer (1 50 mM NaC1, 50 mM Tris, pH 7.4, 1% Nonidet P_40, 1 mM sodium orthovanadate (Wako Pure Chemical Industries, Ltd.), 1 mM EDTA and protease inhibitor tablets (Amersham, Arlington Heights, Ill.). Lysate supernatants were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). After blocking in Tris-buffered saline containing 5% skim milk, 0.2% Tween20, the membrane was ligated against an anti-GPC3 antibody prepared against a recombinant protein corresponding to the amino acid GPC3303-464.ン Incubate with a heron polyclonal antibody (Santa Cruz, California) and wash well with PBS. Peroxidase conjugation anti-Egret Ig, horseradish peroxidase-bound F (ab ') ₂ fragment (from donkey ) Chemiluminescence detection was performed using (Amersham) and ECL kit (Amercham).

 Immunohistochemical analysis was performed according to methods known in the art (Nakatsura, T. et al., Biochem. Biophys. Res. Coramun. 281, 936-944 (2001)). Anti-GPC was prepared by diluting a 4 μm-thick tissue sample section 1: 200 diluted in OCT embedding compound.

Stained with 3 antibodies. GPC 3 303-464 (Santa Cruz, CA) produced from E. coli as a 45 kDa tagged fusion protein for positive control and FluoReporter Mini-Biotin for biotinting of anti-glypican-3ansagi polyclonal antibody -XX

Protein Labeling Kit (F-6347) (Molecular Probes, Inc., Eugene) was used.

96-well ELISA plate (Nunc, Denmark) at 4 ° C-PBS, pH

Coated with anti-human GP C 3 303-464 (Santa Cruz) at 0. The plates were then blocked for 1 hour at room temperature with 4% Pserum albumin / PBS. The positive control standard sample and culture supernatant were biotinylated anti-GPC

The antibody was added together with 3 antibodies, and incubated at room temperature for 2 hours. After washing three times with PBS, add HRP—Conjugate Streptavidin (END0GEN, Woburn) was added. After a 30 minute incubation, the plates were washed three times with PBS and TMB substrate solution (END0GEN) was added. An ELI SA — der (model 550, Bio-Rad) was used at 405 nm for analysis.

 After obtaining informed consent, tissue samples were obtained from HCC patients being treated at Kumamoto University School of Medicine. Similar results were obtained in all four tumors in which the expression of GPC3 protein was as low in HCC cells as in non-cancerous hepatocytes. Thus, there is a contradiction between GPC3 niRNA expression and GPC3 protein expression in HCC cells. GPC3 is a GPI-anchored membrane protein and has been reported to be a secreted protein (Filmus J., Glycobiology 11, 19R-23R (2001)). Therefore, we next attempted to detect the secreted GPC3 protein.

 [Example 5] Detection of soluble GPC 3 protein in culture supernatant of HC C cell line and serum of HCC patient

To determine whether soluble GPC3 protein was present in the culture supernatant of HepG2, Western blotting was performed on the HepG2 cell lysate and the culture supernatant collected after a predetermined culture period. 6 in serum free medium, 1 2, 24, and 1 X 1 0 cell lysates prepared from ^five HepG2 cells after 48 hours culture, showed the presence of GPC 3 protein similar amounts of 60 kDa (Fig. Lanes 4, 3, 5, and 7 of 4). On the other hand, in the HepG2 culture supernatant (20 μl of 1 ml / well) after culturing for 6, 12, 24, and 48 hours, the 60 kDa GPC3 protein was gradually increased. Was secreted into the culture supernatant from HepG2.

 Subsequently, detection was performed by an enzyme-linked immunosorbent assay (ELISA) using an anti-GPC3303-464 antibody and a biotinylated anti-GPC3 antibody. Using a commercially available recombinant protein corresponding to GPC 3 303-464, the accuracy of quantification of GPC 3 in the ELISA system was confirmed. Using serial dilution of HepG2 culture supernatant, based on OD data

A standard curve for quantifying GPC3 protein was evaluated. The 1 X 1 0 ⁵ or concentrations of GP C 3 protein in the culture supernatant 1 m 1 after 24 hours of culture the HepG2 cells was defined as 1 m 1. The amount of GPC3 protein in the HepG2 culture supernatant was much higher than in Hep3B, whereas it was not detectable in SK-Hep-1 (Figure 5). Next, we detected soluble GPC3 protein in the serum of HCC patients (Fig. 6). Blood samples were collected from eighteen HCC patients, and patient profiles were collected from medical records to determine the clinical stage based on the UICC T-Wake Classification. A band of 60 kDa GPC3 protein was detected in 20 μl of serum from patient 7 (Pt7, lane 3 in Figure 6), but serum from two other HCC patients and four healthy donors Was not detected. The serum levels of GPC3 protein in 28 HCC patients and 54 healthy donors (HD) were then assessed by ELISA (Figure 7). The mean of GPC3 protein in the HD serum of 54 patients was 0.75 U / ml and the standard deviation (SD) was 0.32. There was no difference in expression between males and females, and therefore weak expression of GPC3 in the ovaries was considered negligible for this system. The mean for 28 HCC patients was 1,98 UZ ml. To determine the upper normal limit of GPC3 protein in serum, 1.71 was defined as the average of GPC3 protein + SD of 54 HD sera. 35.7% (10/28) of HCC patients and 0% (0/54) of HDs were GPC3 protein positive (> 1.71). The concentration of GPC3 protein in the serum of HCC patients was significantly higher than in HD (P <0.0001). The concentrations of GPC3 evaluated by ELISA for patients 4, 5 and 7 were 0.94, 1.73 and 69.4 U / m, respectively, as shown in FIG. 6 (Table 2). That is, Western blotting could detect 69.4 UZ ml of GPC3 protein, but could not detect 1.73 UZm1 of GPC3.

As is evident from the results in Table 2, two of the ten GPC3-positive patients (patients 6 and 25) had negative AFP and PIVKA-II, and one of The patient (patient 6) was classified as a relatively early UICC stage II. That is, GPC3 may be positive in patients with negative AFP and PIVKA-II in both cases, indicating that GPC3 is useful as a novel tumor marker for HCC. Table 2 Measurement results of AFP, PIVKA-Π and GPC3 in serum of 28 HCC patients

Pt lD age (y), gender virus ^a UICC Stage AFP (ng / ml) ^u (<20) ^c PIVKA-ll (mAU / ml) ^Q (<40) GPC3 (U / ml) ^a (<1.71)

3 56 HBV IVA 54 ^e 957 1.73

5 71M HCV IIIB 8900 31577 1.73

11 69 Non-B, non-C IVA 9400 319 3.23

1 64M HCV IIIA 50 15 9.40

2 53M HCV II 45 38

 8 69M HCV II! 21 21 2.22

9 73M HCV IIIA 25 242

 12 61M HCV IVA 349 169

 17 0M HBV IVA 56 133

 18 71F HBV 1! 930 994 0.38

19 77M HCV II 163 96 0.42

 291 0.28

 OF, 0.87

 HCV IIIA 178 28 0.94

15 HCV II 100 32 0.83

22 72 HCV IIIA 25 12 1.67

7 62M HCV IVA 10 239 69.39

16 72M HCV II <1 840 1.94

14 62M HCV II 3 69 0.90

20 75F Non-B, non-C IIIA <1 63 0.35

27 71M HCV IVA 10 19 288 0.83

6 72F HCV II 9 20 3.09

25 63 HCV IVB <1 10 62.45

10 58M non-B, non-C IIIA 3 18 0.66o o

13 69 HCV IVB 6 10 1.53

21 71M HBV, HCV IVA 2 26 0.52

23 59 HBV IIIA 3 18 1.70

28 69M HCV IIIA <1 28 0.80 ^a HCV was detected using RT-PCR. HBsAg was examined using Radioimnoassy (R).

b, AFP quantified using RIA fc

c, The value in parentheses indicates the cutoff index.

d, PIVKA-II and GPG3 were quantified using Enzymimnoassay (E1A).

e, Positive measurements are underlined.

[Reference Example 1] Synthesis of Ser-Phe-Phe-Gin-Arg_Leu-Gin-Pro-Gly-Leu (SEQ ID NO: 5) Schedule using Fmoc-Leu-Wang resin (l00-200mesh) as the starting resin Start the synthesis according to A, proceed to step 5, and then return to step 2 to repeat the deprotection, washing, coupling, and washing of the α-amino group to obtain Fmoc-Gly-0H, Fmoc-Pro_0H

Fmoc-Gln (Trt) -0H, FmocHLeu-0H Fmoc- Arg (Pbf)-0H Fmoc Gin (Trt)-0H

Fmoc-Phe-0H Fmoc-Phe-0H Fmoc-Ser (tBu) -OH was subjected to random coupling to obtain a peptide-bound resin. Is the peptide a resin by reacting with the reagent shown in Step 6? It was cut off, filtered into cold methyl tart butyl ether (MTBE) and precipitated. The precipitated peptide was washed twice with cold MTBE and lyophilized under nitrogen. Table 3 Schedule A

 Process time Processing

 (Minutes)

1. (Wash) DMF 2 2. (Deprotection) 20% piperidine / DMF 10ml 1

 20% piperidine / DMF 10ml 30 1

3. (washing) DMF 4

 DCM 1

4. (Coupling) Each α-amino group protected amino acid lmraole 30

 / 0.45M HBTU / H0BT 2.lral (lmmole),

 DIEA 348 1 (2mmole)

 5-(wash) DMF

 DCM

(Clearage) 5% H ₂ 0 120

 5% fenore

 3% thioanisol

 3% ethanedithiol

 3% triisopropyl silene

 81% TFA

 7. (Precipitation) MTBE

 8. (Washing) MTBE

9. (Lyophilization) The obtained crude peptide was subjected to reverse phase HPLC using BioCad 60 (Perkin-Elmer, Foster City CA) using a C18 column (Phenomenex, Torrance, CA) with a particle size of 10 microns. Purified by the method. Flow rate 20ml / min, 10% B to 80 ° / in 45 minutes. B (solvent A: 0. 05% TFA / H 2 0, solvents 0. 05% TFA / Asetonitoriru) eluted with a linear gradient of was monitored eluate A220nm. Mass spectrometry of the main peak occupying 90% or more in area ratio was performed using Lasermat 2000 (Finnigan Mat, San Jose, Calif.) Using MALD! [-TOF method, and the theoretical value was [MH +] 1109.3. On the other hand, the measured value was 1109.9.

 [Reference Example 2] Synthesis of Phe-Phe-Gin-Arg-Leu-Gin-Pro-Gly-Leu (SEQ ID NO: 6) Fmoc-Leu-Wang resin was used as a starting resin as in [Reference Example 1].

Fmoc-Gly-0H, Fmoc-Pro-0H, Fmoc-Gin (Trt)-0H, FmocHLeu-0H, Fraoc-Arg (Pbf)-0H, Fmoc-Gin (Trt)-0H, Fmoc-Phe-0H, Fmoc -Phe-OH was coupled sequentially.

 As a result of mass spectrometry, the measured value was 1130.4, whereas the theoretical value was [MH +], 1131.3.

 [Reference Example 3] Synthesis of Met-Phe-Lys-Asn-Asn-Tyr-Pro-Ser-Leu (SEQ ID NO: 7) • As in [Reference Example 1], Fmoc-Leu-Wang resin was used as a starting resin.

Fmoc-Ser (tBu) -OH, Fmoc-Pro-0H, Fmoc-Tyr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH , Fmoc-Phe-0H and Fmoc-Met-OH were sequentially bonded.

 As a result of mass spectrometry, the measured value 1032.3 was obtained against the theoretical value [MH +] 1031.2.

 [Reference Example 4] Synthesis of Phe-Thr-Asp-Val-Ser-Leu-Tyr-Ile-Leu (SEQ ID NO: 8)

Fmoc-Leu-Wang resin was used as the starting resin as in [Reference Example 1].

 Fmoc-lie-0H ヽ Fmoc-Tyr (tBu) -OH, Fmoc-Leu-OH ヽ Fmoc-Ser (tBu)-OH ヽ Fmoc- Vato 0H,

Fmoc-Asp (Otbu) -OH, Fmoc-Thr (tBu) -0H, and Fmoc-Phe-OH were sequentially bonded.

 As a result of mass spectrometry, the measured value 1122.1 was obtained against 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)

Fmoc-Leu-Wang resin was used as the starting resin as in [Reference Example 1].

 Fmoc-Met-0H, Fmoc-Arg (Pbf)-0H, Fmoc-Gly-0H, Fmoc-Cys (Trt) -0H,

 Fmoc-Asp (Otbu)-0H, Fmoc-Lys (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-0H,

 Fmoc-Lys (Boc) -OH was linked sequentially.

 As a result of mass spectrometry, the measured value was 1111.4 compared 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)

Using Fmoc-Leu- Wang resin as the starting resin as in [Reference Example 1], Fraoc-Gly-0H, Fmoc-Gin (Trt)-0H, Fmoc-Cys (Trt) -OH, Fraoc-Tyr (tBu)-0H,

Fmoc-Ser (tBu) -0H, Fmoc-Cys (Trt) -0H, Fmoc-Tyr (tBu) -0H, and Fmoc-Trp (Boc) -OH were sequentially bonded.

 As a result of mass spectrometry, an actual measured value of 115.7 was obtained with respect to a theoretical value of [thigh +] of 114.3.

 [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, Fmoc-Ser (tBu) -0H, Fraoc-Leu-0H, Fmoc-lie-0H, Fmoc-Tyr (tBu) -0H,

Fmoc-Glu (Otbu) -OH, Fmoc-Arg (Pbf)-0H, Fmoc-Trp (Boc) -OH, Fmoc-Tyr (tBu)-0H, and Fmoc-Lys (Boc)-0H were sequentially bonded.

 As a result of mass spectrometry, an actual measured value of 1105.3 was obtained against a theoretical value of [MH-] of 1104.3.

 [Reference Example 8] Synthesis of Glu-Tyr-Ile-Leu-Ser-Leu-Glu-Glu-Leu (SEQ ID NO: 12)

Fmoc-Leu-Wang resin was used as the starting resin as in [Reference Example 1].

Fmoc-Glu (Otbu) -OH, Fmoc-Glu (Otbu) -OH, Fmoc-Leu-0H, Fmoc-Ser (tBu) -OH, Fmoc-Leu_0H, Fmoc-Ile-0H, Fmoc- Tyr (tBu)- 0H and Fmoc-Glu (Otbu) -OH were sequentially bonded.

 As a result of mass spectrometry, the measured value was 130.7.

 [Reference Example 9] Synthesis of Ile-Tyr-Asp-Met-Glu-Asn-Val-Leu-Leu (SEQ ID NO: 13)

Using Fmoc-Leu-Wang resin as the starting resin as in [Reference Example 1],

Fmoc-Leu-0H, Fmoc-Vato 0H, Fmoc- Asn (Trt)-0H, Fmoc-Glu (Otbu)-0H, Fmoc-Met-0H,

Fmoc-Asp (Otbu) -0H, Fmoc-Tyr (tBu) -0H, and Fmoc-lie-OH were sequentially bonded.

 As a result of mass spectrometry, the theoretical value [MH +] 1211.

 [Reference Example 10] Synthesis of Ala-Tyr-Tyr-Pro-Glu-Asp-Leu-Phe-lie (SEQ ID NO: 14)

Using Fmoc-lie-Wang resin as an open resin, Fmoc-Phe-0H, Fmoc-Leu-0H,

Fmoc-Asp (Otbu)-0H, Fmoc-Glu (Otbu)-0H, Fmoc-Pro-0H, Fmoc-Tyr (tBu)-0H,

Fraoc-Tyr (tBu) -OH and Fmoc-Ala-OH were sequentially bonded.

 As a result of mass spectrometry, the measured value was 1217.4 in comparison to the theoretical value [MH-] 1216.4.

 [Reference Example 11] Synthesis of Phe-Tyr-Ser-Ala-Leu-Pro-Gly-Tyr-lie. (SEQ ID NO: 15)

Reference Example 1 0] Like the Fmoc - lie- initiate Wang resin (100- ² 00 mesh) I½oc-Tyr (tBu) -0H, Fraoc-Gly-0H, Fmoc-Pro-0H, Fmoc-Leu-0H Fmoc-Ala-0H, Fmoc- Ser (tBu) -0H, Fmoc-Tyr (tBu ) -0H and Fmoc-Phe-OH were sequentially bonded.

 As a result of mass spectrometry, the measured value was 1196.8 compared to the theoretical value of [view +] 1193.

 [Reference Example 1 2] Synthesis of Arg-Phe-Leu-Ala-Glu-Leu-Ala-Tyr-Asp-Leu (SEQ ID NO: 16)

 Use Fmoc—Leu Wang resin as in [Reference 1]! / ヽ, Fmoc — Asp (Otbu) — 0H, Fmoc-Tyr (tBu)-0H, Fmoc-Ala-0H, FmocHLeu-0H, Fffloc-Glu (Otbu)-0H, Fmoc-Ala-0H, Fmoc-Leu- 0H, Fmoc-Phe-0H, and Fmoc-Arg (Pbf) -OH were sequentially bonded.

 As a result of mass spectrometry, the theoretical value [MH-] was 1183.5, but the measured value was 1186.7. Industrial potential

The present inventors have identified 12 peptides from GPC3, an oncofetal protein, as target candidates for immunotherapy of HLA-A24 + HCC patients. Regardless of GPC3 overexpression in HCC patients, GPC3 expression is significantly lower in normal adult organs, except for the placenta, making GPC3 an ideal target for HCC immunotherapy It has been found. In addition, the method of the present invention has been shown to be a very useful method for diagnosing whether or not the subject has HCC. The present inventors have further identified a GPC3-derived peptide capable of preparing HLA-A24-restricted and HCC-reactive CTL. The HLA-A24 allele is owned by 60% of all Japanese and 95% of its genotype is A * 2402. It is 20% in Caucasians and 12% in 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 Ed.) 1065-1220 (Oxford University Press, Oxford, 1992)). These results demonstrate that GPC3 is very useful for specific immunotherapy or use in cancer diagnosis and prevention for many HCC patients worldwide. All publications, patents and patent applications cited herein are referred to as they are. This specification ^j '

Claims

The scope of the claims

1. A peptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 5 to 16.

2. A peptide having one or two amino acids substituted or added in any of the amino acid sequences shown in SEQ ID NOs: 5 to 16, and having an ability to induce cytotoxic T cells.

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

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

 5. A medicament for treating and / or preventing a tumor, which comprises one or more peptides according to any one of claims 1 to 4.

 6. An exosome displaying on its surface a complex comprising the peptide according to any one of claims 1 to 4 and an HLA molecule.

 7. The exosome according to claim 6, wherein the HLA molecule is HLA-A24.

8. The exosome according to claim 7, wherein the HLA molecule is HLA-A * 2402.

 9. A method for inducing antigen-presenting cells having a high ability to induce cytotoxic T cells using the peptide according to any one of claims 1 to 4.

 10. Introduction of a gene encoding a glypican-3 (glypican-3; GPC3) protein or a partial peptide thereof including the peptide according to any one of claims 1 to 4 into an antigen-presenting cell. A method for inducing antigen-presenting cells having high cytotoxic T cell-inducing ability, comprising:

 1 1. A method for inducing cytotoxic T cells using the peptide according to any one of claims 1 to 4.

 1 2. An isolated cytotoxic T cell induced using the peptide of any one of claims 1-4.

1 3. An antigen-presenting cell that presents a complex of an HLA molecule and the peptide according to any one of claims 1 to 4.

14. The antigen presenting cell according to claim 13, which is induced by the method according to claim 9 or 10.

 1 5. A diagnostic agent for hepatocellular carcinoma (HCC), including an antibody against GPC3.

 1 6. A method for diagnosing HCC, comprising contacting a sample with an antibody against GPC3.

 17. The method of claim 16, further comprising quantifying GPC 3 in the sample.

 1 8. A kit for diagnosis of HC C, comprising an antibody against GP C3.