WO2007049394A1

WO2007049394A1 - Peptides originating in hepatitis c virus

Info

Publication number: WO2007049394A1
Application number: PCT/JP2006/316979
Authority: WO
Inventors: Kyogo Itoh; Michio Sata; Shigeru Yutani; Akira Yamada
Original assignee: Kurume University
Priority date: 2005-10-25
Filing date: 2006-08-29
Publication date: 2007-05-03
Also published as: JP2009018990A

Abstract

A peptide which is recognized by an antibody against hepatitis C virus and has one of the following amino acid sequences: IVGGVYLLPR (SEQ ID NO:1), YLLPRRGPR (SEQ ID NO:2), YLLPRRGPRL (SEQ ID NO:3) and LIRACMLVR (SEQ ID NO:4). It is also intended to provide a medicinal composition for treating an HCV-related disease which contains the above peptide as the active ingredient and a composition for diagnosing an HCV-related disease or presuming the progress of an HCV-related disease. The above-described peptide and compositions are useful in treating an HCV-related disease patient having the HLA-A3 supertype, diagnosing the disease and presuming the progress of the disease.

Description

Hepatitis c virus-derived peptide

Technical field

[0001] The present invention relates to treatment of hepatitis C virus-related diseases.

Background art

[0002] Hepatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flaviviridae family, with approximately 1.7-200 million people infected with HCV worldwide and approximately 1.5-2 million people in Japan. It is said that. Many of them develop chronic hepatitis, and as the disease progresses, it shifts to cirrhosis and hepatocellular carcinoma. Hepatocellular carcinoma develops from 1 to 2% annually from chronic hepatitis and from 6 to 8% annually from cirrhosis. About 70% of patients with hepatocellular carcinoma in Japan are infected with HCVlb. Nearly 40,000 deaths per year due to cirrhosis and liver cancer caused by HCVlb infection.

[0003] Currently, interferon (IFN) therapy is widely used for patients with chronic infection with hepatitis C virus, and a total effect of about 30% has been obtained as a whole (Kasahara et al., Hepatology). 27, 1394-1402, 1998: Non-patent document 1). However, the type of virus (HCVlb type), which is widely used in Japan, is resistant to these current therapies, and the response rate is about 50-60%. In particular, the response rate for chronic hepatitis patients with hyperviremia (blood RNA levels of 500 KYUZml or more) is 30% or less. There are also many cases that are not indicated for IFN treatment and cannot be completed due to adverse events of IFN. The combined use of interferon and ribavirin has been effective to some extent even in cases with a high viral load, but the response rate is about 20% (Non-patent Document 2).

[0004] The present inventors have previously identified several types of human leukocyte antigen (HLA), HLA-A2 and HLA-A24-restricted cytotoxic T lymphocytes (CTLs). IgG reactive with any peptide is detected in the majority of HCV-infected patients, and the peripheral blood mononuclear cell (PBMC) strength of HCV patients with HLA types to which these peptides correspond is also peptide-specific CTL It was found that can be induced. Based on these results, HCVlb-derived peptides were administered to patients with HLA-A24 or HLA-A2 who were ineffective with standard treatment (IFN therapy or ribavirin combination therapy) However, immunostimulatory action by peptides was observed in the majority of cases, and a decrease in viral load was observed in more than half of cases (WO2005 / 028503: Patent Document 1). However, antigenic peptides targeting HLA-A2 and A24 positive individuals cannot cover 70% of Japanese patients.

[0005] HLA-A11, HLA-A31 and HLA-A33, and HLA-A0301 and HLA-A6801 share similar antigen-binding motifs and are therefore classified as HLA-A3 supertypes (Takedatsu H, et al., Clin Cancer Res 200 4; 10: 1112-1120: Non-patent document 2). This supertype is relatively dominant in the human population, and the frequency of the phenotype of the HLA-A3 supertype, except for HLA-A6801, is white, North American African black, Japanese, Chinese , About 30%, about 35%, about 44%, about 52%, and about 35%, respectively, among Hispanics (Sette, A., and Sidney, J., Immunogenetics, 50: 201-212). 1999: Non-patent document 3). To date, there are no reports on HLA-A3 supertype-restricted antigen peptides derived from HCV.

[0006] References cited in the present specification are as follows. All the contents described in these documents are cited herein as part of this specification. Any of these documents is not admitted to be prior art to this specification.

Patent Document 1: WO2005 / 028503

Non-patent literature l: Kasahara et al., Hepatology 27, 1394-1402, 1998

Non-Patent Document 2: Takedatsu H, et al, Clin Cancer Res 2004; 10: 1112-1120 Non-Patent Document 3: Sette, A., and Sidney, J., Immunogenetics, 50: 201-212, 1999 Disclosure of the Invention

Problems to be solved by the invention

[0007] An object of the present invention is to provide a method for treating HCV-related diseases, which can be applied to HLA-A3 supertype patients.

Means for solving the problem

[0008] The present invention relates to a peptide recognized by an antibody against hepatitis C virus, which has the following sequence:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1) HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

A peptide having an amino acid sequence represented by any of the above is provided:

[0009] In another aspect, the present invention provides a pharmaceutical composition for treating an HCV-related disease, comprising any of the peptides described above as an active ingredient, and a method for treating a patient having an HCV-related disease. What is provided is a method comprising administering to a patient any peptide as described above.

[0010] In another aspect, the present invention relates to a composition for predicting HCV-related disease progression or progression of HCV-related disease, and HCV in a subject, comprising any of the peptides described above. A method for predicting the power of diagnosing a related disease or the progression of an HCV-related disease, comprising measuring an antibody titer against hepatitis C virus in the blood of the subject using any of the aforementioned peptides provide.

[0011] In another aspect, the present invention provides the following sequence:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

A pharmaceutical composition for treating an HCV-related disease in a patient having HLA-A11, HLA-A31, and HLA-A33, comprising a peptide having an amino acid sequence represented by any of the above as an active ingredient I will provide a. In another aspect, the invention provides a method of treating a patient having an HCV-related disease in a patient having HLA-All, HLA-A31, and HLA-A33, comprising the following sequence:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

A method comprising administering to a patient a peptide having an amino acid sequence represented by any of the above: [0012] In yet another aspect, the present invention provides the following sequences:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

In patients with HLA-A11, HLA-A31, and HLA-A33, containing HLA-A11, HLA-A31, or HLA-A33 or HLA-A33 restricted A composition for inducing sex cytotoxic T cells is provided. In another embodiment, a method of inducing HLA-All, HLA-A31 or HLA-A33-restricted cytotoxic T cells in a patient with HLA-A11, HLA-A31, and HLA-A3 3, comprising: Array of:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

A method comprising administering to a patient a peptide having an amino acid sequence represented by any of the above:

[0013] In yet another aspect, the present invention provides the following sequences:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

HLA—All, HLA—A31, or HLA—A33-restricted cytotoxic T cells in patients with HLA—All, HLA—A31, or HLA—A33, comprising a peptide having an amino acid sequence represented by A kit is provided for testing for induction.

[0014] As used herein, "HCV-related disease" means a disease caused by infection with hepatitis C virus, such as acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. included. The progression of HCV-related disease is typically from chronic hepatitis to cirrhosis Progression to metastasis, cirrhosis, and progression to liver cancer.

Brief Description of Drawings

[0015] FIG. 1 shows the detection of peptide-specific IgG in HCV-infected patients.

[Figure 2] Figure 2 shows the induction of peptide-specific CTL from PBMC in HCVlb patients.

[Fig. 3] Fig. 3 shows the cytotoxic activity of peptide-stimulated PBMC against cells pre-loaded with peptides.

[FIG. 4] FIG. 4 shows the cytotoxic activity of peptide-stimulated PBMC against cells pre-loaded with peptides.

BEST MODE FOR CARRYING OUT THE INVENTION

[0016] The present invention provides an immunogenic HCVlb-derived peptide that can be used for the treatment of patients having HLA-All, HLA-A31, and HLA-A33 infected with HCV. As shown in the following examples, in the present invention, an antigenic peptide (CTL epitope) recognizing CTL in a patient having HLA-All, HLA A31 or HL A-A33 having an HCVlb-related disease is examined. As a result, four new candidate peptides were identified from 48 peptides that are widely conserved in HCV-lb and have motifs that bind to HLA-A11, A31, or A33. A peptide derived from the core protein located at positions 30-39 (IVGGVYLLPR (SEQ ID NO: 1)) induced peptide-specific CTL from peripheral blood mononuclear cells (PBMC) of HLA-All, A31, and A33 positive patients. On the other hand, 35-43 peptides derived from the core protein (sushi 0¾1¾ ^ 1? (SEQ ID NO: 2)) and 35-44 peptides (sushi 0 ³ RRGPRL (SEQ ID NO: 3)) are HLA-A31 and 918-926 peptide (LIRACMLVR (SEQ ID NO: 4)) derived from unstructured protein NS-2 induced peptide-specific CTL from HLA-A11 and A33-positive patient PBMC from ABMC positive patient PBMC. IgG antibodies reactive to these peptides were detected in the plasma of HCV-lb infected patients.

[0017] The hepatitis C virus-derived peptide (HCV peptide) of the present invention contains a partial amino acid sequence of a protein derived from hepatitis C virus, and an HLA binding motif compatible with the patient's HLA in the sequence. . This peptide can be recognized by blood antibodies from patients infected with hepatitis C virus.

[0018] Preferably, the hepatitis C virus-derived peptide according to the present invention induces CTL. The CTL thus induced can target HCV-infected cells and attack them.

[0019] The peptide of the present invention only needs to have the amino acid sequence described above to the extent that a desired effect can be obtained, and may further include an additional sequence. A person skilled in the art naturally understands how much sequence addition is allowed on both the N-terminal side and the C-terminal side in order to achieve the desired action and effect as an antigen peptide. Can do. Therefore, for example, an amino acid sequence useful for promoting immunization, an amino acid sequence that facilitates formulation, or a fusion protein on the N-terminal side and Z or C-terminal side of the peptide according to the present invention. As an amino acid sequence convenient for expressing a peptide, an amino acid sequence convenient for peptide production and purification, etc. may be added. Moreover, the peptide according to the present invention may be chemically modified, or a polymer or sugar chain may be added, as long as the specificity of binding to the antibody is not lost.

[0020] The HCV peptide according to the present invention is a gene recombination technique using a normal chemical synthesis method, an enzymatic decomposition method of a protein molecule, or a host transformed to express a base sequence encoding the target amino acid sequence. Etc. can be manufactured.

[0021] When the target peptide is produced by a chemical synthesis method, it can be produced by a conventional method known per se in ordinary peptide chemistry, for example, a peptide synthesizer. Can be synthesized by a solid phase synthesis method. The crude peptide thus obtained can be purified by purification methods commonly used in protein chemistry, such as salting out, ultrafiltration, reverse phase chromatography, ion exchange chromatography, It can be purified by tea chromatography.

[0022] On the other hand, when a desired peptide is produced by gene recombination technology, for example, a synthetic or cloned DNA fragment encoding the target amino acid sequence is incorporated into an appropriate expression vector, and this expression vector is used. The desired peptide can be obtained by transforming microorganisms or animal cells using, and culturing the resulting transformant. As expression vectors that can be used, plasmids, virus vectors, and the like known in the art can be used.

[0023] A method for transforming a host cell using an expression vector in this peptide production technique; For example, a method known per se, such as calcium chloride method, calcium phosphate coprecipitation method, DEAE dextran method, lipofectin method, electopore position method, etc. can be used, and it should be selected as appropriate based on the host cell to be used. Good. The obtained peptide can be purified from the cell extract or culture supernatant collected from the cultured medium by the purification method described above.

[0024] The peptide of the present invention is useful as a pharmaceutical composition for treating a patient having an HCV-related disease, that is, as a peptide vaccine. A vaccine consisting of a hepatitis C virus-derived peptide is prepared by mixing the peptide produced as described above with a pharmaceutically acceptable adjuvant and Z or a carrier as appropriate. Adjuvants that can enhance the immune response, such as Freund's incomplete adjuvant, aluminum hydroxide gel, can be used. As the carrier, for example, a diluent such as phosphate buffered saline (PBS) or distilled water, physiological saline, or the like can be used.

[0025] The peptide vaccine can be administered by a transdermal route such as oral or intravenous administration or subcutaneous administration, depending on the form of use. Examples of the dosage form include tablets, granules, soft capsules, hard capsules, liquids, oils, and emulsifiers. The dosage of such a pharmaceutical composition may vary depending on the symptoms of the patient to whom it is administered, but in general, 0.1 to 10 mg of peptide per day is preferable for adults. The interval is preferably administered once every few days to several months. You can also use peptide vaccine and interferon together.

[0026] In a preferred embodiment, the hepatitis C virus-derived peptide to be administered is selected based on the peptide reactivity of anti-HCV antibodies present in the patient's blood or the presence of CTL. More preferably, the selection is based on the peptide reactivity of the anti-HCV antibody and the presence of CTL. Here, “peptide reactivity of anti-HCV antibody” means that any of the peptides to be administered is recognized by the anti-HCV antibody present in the blood of the patient. Higher therapeutic effects can be expected by examining the peptide reactivity of anti-HCV antibodies in each patient and administering only the appropriate peptides for each patient, so-called tailor-made administration. Experience in cancer peptide vaccine clinical trials promptly induces secondary immunity activation by tailor-made administration, so it is safer and better. It has been demonstrated that the floor effect appears (Mine et al., Clin. Can. Res. 929-937, 2004).

[0027] The therapeutic effect of HCV-related diseases can be confirmed by monitoring the blood antibody titers of subjects suffering from HCV-related diseases, that is, the blood concentration of antibodies reactive to peptides, by conventional methods such as ELISA. Can be done. In addition, the amount of virus can be monitored by measuring the amount of HCV RNA by conventional methods such as RT-PCR.

[0028] The peptide of the present invention is also useful as a composition for diagnosing a patient having an HCV-related disease or predicting the progression of the disease. Using the peptide of the present invention, the antibody titer in the blood of a subject can be measured using an antigen-antibody reaction well known in the art. For example, when using a typical ELISA method, the measurement can be performed as follows. The antigen peptide is bound to a conventional ELISA plate such as 96well, and the plate is appropriately blocked to prevent nonspecific adsorption. Next, dilute the prepared blood serum of the subject appropriately and add it to each well of the plate, and react for a predetermined time. After washing the plate to remove unbound components, add antibodies that can bind to human antibodies (eg, rabbit anti-human antibodies). If it is desired to detect IgG, γ chain specific anti-HgG can be used. After reacting for a specified time, the plate is washed and detectably labeled antibody (eg anti-rabbit IgG) is added. Labeling can be performed by methods well known to those skilled in the art using enzymes, fluorescent dyes, chemiluminescent substances, piotin, radiation compounds and the like. After reacting the plate for a specified time, the label is detected by adding an appropriate substrate and measuring the decrease in substrate or increase in product, or by measuring fluorescence, luminescence, or radioactivity. In this way, the amount of antibody against a specific peptide in the serum of the subject can be measured. In addition, the progression of HCV-related disease can be predicted by monitoring the amount of antibody.

[0029] Among such methods for detecting antibodies by antigen-antibody reaction, xMAP technology is one of highly sensitive methods. This is a flowmetry measurement method developed by Luminex using a fluorescent microbead array system. Microbeads to which peptides are bound are brought into contact with serum, and then a fluorescently labeled secondary antibody is bound to the flow. Fluorescence intensity by measurement Measure.

[0030] In addition, when the antibody titer is measured in a hospital laboratory or the like, an immunochromatography method can be used. In this method, a portion in which the antigen (or antibody) is linearly distributed is made on the test paper, and the complex of the antibody in the sample and the antigen labeled with the colored particles moves on the test paper. Qualitative analysis is based on the presence or absence of colored lines that appear by intensive capture by antigens (or antibodies). Using this method, results can be obtained in a short time (within 20 to 30 minutes) with simple equipment.

[0031] The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, all the contents described in the specification and drawings of Japanese Patent Application No. 2005-310203, which is the application on which the priority of the present application is based, are cited herein as part of this specification.

Example

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are given for illustrative purposes only, and the present invention is not limited to the examples.

[0033] Subject

Subjects were HCVlb-infected patients with HLA—A3 supertype and healthy donors. These subjects had one of the HLA types HLA-All, HLA-A31, and HLA-A33, and none of them was infected with HIV. Peripheral blood To obtain mononuclear cells (PBMCs), 20 ml of peripheral blood was collected and PBMCs were prepared by Ficoll-Conley density gradient centrifugation. All samples were stored cold until used for experiments. Expression of HLA-All, HLA-A31, and HLA-A33 molecules in PBMC of HCVlb patients was measured by flow cytometry using the following antibodies: anti-HLA-All monoclonal antibody (mAb) (Cat # 0284HA One Lambda Inc., Canoga, CA), anti-HLA—A31 mAb (Cat # 0273HA; One Lambda), and anti-HLA—A33 mAb (Cat # 0612HA; One Lambda).

[0034] Cell lines

The cell lines used, C1R-A11, C1R-A31, and C1R—A33 are each HL C1R lymphoma subline constitutively transfected with the A-A1101, HLA-A3101, and HLA-A3303 genes. Expression of HLA-A11, HLA-A31, and HLA-A33 molecules in these sublines has been previously reported (Takedats u H, et al., Clinical Cancer Research 2004; 10: 1112-20). All cell lines were maintained in RPMI 1640 (Invitrogen) containing 10% FCS.

[0035] Peptides

Based on the binding motifs of HLA-A11, HLA-A31, and HLA-A33 molecules (Parker KC et al., J Immunol 1994; 152: 163-75), 47 types of peptides derived from HCV-lb protein were prepared. . All peptides were purchased from Biologica Co. (Nagoya, Japan) and were more than 90% pure. Control peptides that bind to the HLA-A3 supertype include influenza (Flu) virus-derived peptides (NVKNLYEKVK (SEQ ID NO: 5)), Epstein Barr virus (EBV) -derived peptides (AVFDRKSDAK (SEQ ID NO: 6)), tyrosinase-related peptides Protein 2 (TRP2) -derived peptide (LLGPGRPYR (SEQ ID NO: 7)) and HIV-derived peptide (RLRDLLLIVTR (SEQ ID NO: 8)) were used. All peptides were dissolved in dimethyl sulfoxide and diluted to 10 g / ml.

[0036] Statistics

Statistical analysis was performed using Student's t test. A value of P 0.05 was considered statistically significant.

[0037] Extraction of peptide-specific IgG

Initially, the level of peptide-specific IgG reactive to each of the 48 peptides was measured using a 1: 100 diluted sample of sera from 12 patients infected with HCV-lb. Plasma levels of peptide-specific IgG were measured by the Luminex® method according to the method described previously (Komatsu N. et al., Scand J Clin Lab Invest, 64, 535-546, (2004 )). Briefly, 200 L of plasma (1: 100 dilution) is added to a 96 well filter plate (MABVN1250; Millipore Corp., Bedford, MA) with 25 L of peptide-conjugated color-coded beads (Luminex Corp. (Austin, TX)). ) In a plate shaker at room temperature for 2 hours. After 2 hours, the plate was washed with 0.05% Tween 20—PBS and shaken with 100 L of pyotinylated goat anti-human IgG. Incubated for 1 hour at room temperature on a shaker. The plates were then washed and 100 L of streptavidin-PE (5 μg / ml) was placed in the well and incubated at room temperature for 30 minutes on a plate shaker. The fluorescence intensity (FI) was measured with Luminex (registered trademark) to determine the level of peptide-specific antibody. Plasma from 15 healthy donors was used as a negative control. The cut-off FI value was determined as the mean FI value of healthy donors + 2SD.

[0038] As a result, IgG reactive to HCV cores 30-39, 35-43, 35-44, and HCVNS2918-926 peptides were found in 7, 7, 7, and 9 of 12 patients, respectively. Was detected in plasma. In contrast, each of these peptides was not reactive to plasma from any of the 15 healthy donors. Mean IgG levels reactive to HCV core 30-39, HCV core 35-43, 35-44, and HCVNS2916-926 peptides in patients were statistically significant compared to healthy donors, respectively. High strength (Figure 1).

[0039] Induction of peptide-specific CTL from PBMC in HCVlbi¾¾ patients

The above four peptides that are frequently recognized by IgG in blood of HCVlb infected patients have HLA—All, HLA-A31, or HLA—A33. Whether or not can be induced. PBMCs were stimulated and cultured in vitro with HCV-derived peptides or control peptides, and the reactivity to C1R-A11, C1R-A31, or C1R-A33 cells pulsed with the corresponding peptides was examined using IFN-γ production as an indicator. It was.

[0040] For the detection of peptide-specific CTL, we used the method reported previously, with some modifications (Hida N et al, Cancer Immunol Immunother 2002;

51: 219-28). Briefly, PBMC (IX 10 ⁵ cells Z-well) was incubated with 10 μ 1 Zml of each peptide in a 200 μ 1 culture in a U-bottomed 96-well microphone mouth-pressure plate (Nunc, Roskilde, Denmark). did. The experiment was performed in quadruplicate. The cultures are 45% RPMI1640, 45% AIM-V medium (Gibco—BRL, Gaithersburg, MD), 10% FC S, lOUU / ml interleukin-2 (IL-2), and 0. ImM MEM not essential It consisted of an amino acid solution (Gibco-BRL). Every 3 days, half of the culture was removed and replaced with fresh medium containing the corresponding peptide (10 μg / ml). On day 15 of culture, Half of the cultured cells were stimulated with C1R—Al 1, C1R-A31 or C1R—A33 cells pulsed with the corresponding peptide, and the other half were C1R -Al 1, C1R-A31 pulsed with HIV peptide. Or cultured with C1R-A33 cells. After 18 hours of incubation, the supernatant was collected and IFN-γ levels were measured by ELISA.

[0041] In response to C1R—Al 1, C1R-A31, or C1R—A33 cells pulsed with the corresponding peptide, C1R—Al 1, C1R-A31, or C 1R—A33 pulsed with a control group of HIV peptides When the P value was lower than 0.05 compared to the cells, if IF N—y was produced, which was more than 50pgZmU, it was judged that the induction of peptide-specific CTL was successful. As a result, HCV peptides (30–39, 35–43 for core protein and 918–926 for nonstructural protein 2) were 3Z7, 0/7, and 2/7, respectively, in HLA—All positive HCVlb patients. , HLA—A31-positive HCVlb patients 1Z4, 0/4, and 0Z4, HLA—A33 positive HCV—lb patients 3Z5, 3/5, and 3Z5 PBMCs induced CTL reactive to the corresponding peptide (Fig. 2).

[0042] It is already known that the HCV core 35-44 peptide induces and repels the 1¾ ^ \ 8-2 restriction (Takao Y et al., Microbiol. Immunol, 48 (7), 507). -517, 2004). The above results indicate that the HCV core 35-43 peptide can also induce CTL from HLA-A3 supertype positive HCVlb patients.

[0043] Next, we investigated whether the HCV core 35-44 peptide could induce 111 ^-8-3 supertype restraint. HLA—A2 and HLA—A3 supertype positive HCVlb patients PBMCs stimulated with HCV core 35-43 and 35-44 peptides or control peptides in vitro and pulsed with the corresponding peptides (having HLA-A2 ), IFN-γ production in response to C1R-All, C1R-A31, C1R—A33 cells was examined. As a result, the peptide of core protein 35-44 also showed PBMC activity in HLA—All positive HCVlb patients (0-7), HLA—A31 positive HCVlb patients (1Z2), and HLA—A33 positive HCV—lb patients (1Z2). Peptide-reactive CTLs were induced (Figure 2). As a positive control, HCV core 35-43 or core 35-44 peptide induced peptide-specific CTL in 0 out of 3 or 2 out of 3 HL A-A2 positive HCV-1b patients, respectively (data shown) ) [0044] These findings

39, 35—43, core 35—44, and HCVNS291 8-926 peptide can induce peptide-specific CTL in PBMC of HC V— lb patients with HL A -All, HLA A31 or HL A—A33 Indicates.

[0045] Peptide-stimulated PBMC cytotoxic activity

Cytotoxicity of peptide-stimulated PBMCs against C1R—All, C1R-A31, and C1R-A33 cells pre-pulsed with either the corresponding peptide or HIV peptide by a standard 6 hour ⁵¹ Cr-release assay The activity was examined. 2000 ⁵¹ Cr-labeled cells per well were cultured together with effector cells in a 96 round bottom well plate at the specified effector Z target cell ratio. Specific ⁵¹ Cr—release was calculated according to the following formula: (test group cpm spontaneous release cpm). Spontaneous release was determined by the supernatant of target cells incubated without effector cells. Total release was determined by supernatants of target cells incubated with 1% Triton — X (Wako Pure Chemical Industries, Osaka, Japaru. In some experiments, at the beginning of the culture, 10 μg / ml of HLA—class I (W6Z32: mouse IgG2a), anti-HLA—DR (L243: mouse IgG2a), anti-CD4 (NU—THZl: mouse IgGl), anti-CD8 (NU—TSZC: mouse Ig G2a), or anti-CD14 ( H14: Mouse IgG2a) monoclonal antibody was added to the well.

[0046] HLA-A3 supertype-positive PBMC stimulated with HCV peptides should exhibit cytotoxic activity against C1R-All, C1R-A31, or C1R-A33 cells pre-loaded with the corresponding peptide However, HIV peptide was not shown (Fig. 3).

[0047] Furthermore, cytotoxic activity against C1R—All, C1R—A31, or C1R—A33 cells pulsed with each peptide is inhibited by HLA class I (W6 / 32) or CD8 monoclonal antibody (mAb) However, the tested HLA—Class II (DR-1) or CD4 or CD14 mAb was uninhibited. This indicates that peptide-specific cytotoxic activity is expressed mainly by CD8 + T cells in an HLA class I-restricted manner (Fig. 4).

[0048] The above results indicate that these four peptides are useful for peptide immunotherapy in patients with HLA-All, HLA-A31, or HLA-A33-positive HCV-lb-related diseases. Industrial applicability

The peptides of the present invention are useful for the treatment of _HCV related diseases.

Claims

The scope of the claims

[1] A peptide recognized by an antibody against hepatitis c virus,

The following sequence:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

The peptide which has an amino acid sequence represented by either.

[2] A pharmaceutical composition for treating an HCV-related disease, comprising the peptide according to claim 1 as an active ingredient.

[3] A method for treating a patient having an HCV-related disease, the method comprising administering the peptide of claim 1 to the patient.

[4] A composition for predicting HCV-related disease progression or HCV-related disease progression, comprising the peptide of claim 1.

[5] A method for diagnosing HCV-related disease in a subject or a method for predicting the progression of HCV-related disease, wherein hepatitis C in the blood of the subject using the peptide according to claim 1 Measuring the antibody titer against the virus.

[6] The following sequence:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

A pharmaceutical composition for treating an HCV-related disease in a patient having HLA-A11, HLA-A31, and HLA-A33, comprising a peptide having an amino acid sequence represented by any of the above as an active ingredient .

[7] A method for treating a patient having an HCV-related disease in a patient having HLA-A11, HLA-A31, and HLA-A33, comprising the following sequence:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2) HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

A method comprising administering to a patient a peptide having an amino acid sequence represented by any of the above.

[8] The following sequences:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

In patients with HLA-A11, HLA-A31, and HLA-A33, which contain a peptide having an amino acid sequence represented by any of the above as an active ingredient, restrained by HLA-All, HLA-A31 or HLA-A33 Composition for inducing sexual cytotoxic T cells

[9] A method for inducing HLA-A11, HLA-A31 or HLA-A33-restricted cytotoxic T cells in patients with HLA-A11, HLA-A31, and HLA-A33, wherein:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

[10] The following sequences:

HCV core 30—39: IVGGVYLLPR (SEQ ID NO: 1)

HCV core 35—43: YLLPRRGPR (SEQ ID NO: 2)

HCV core 35—44: YLLPRRGPRL (SEQ ID NO: 3)

HCV NS2 918-926: LIRACMLVR (SEQ ID NO: 4)

Including a peptide having an amino acid sequence represented by any of the following: HLA— All, HLA— A kit for examining the induction of HLA-All, HLA-A31, or HLA-A33-restricted cytotoxic T cells in patients with A31, or HLA-A33.