JP2009018990A - Peptides originating in hepatitis c virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for treating an HCV-related disease, which can be applied to a patient having an HLA-A3 supertype. <P>SOLUTION: This invention discloses 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 also a composition for diagnosing an HCV-related disease or predicting the progress of an HCV-related disease. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to the treatment of hepatitis C virus related diseases.

  Hepatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flaviviridae family. It is said that there are about 1.7-200 million people infected with HCV in the world, and about 1.5-2 million people in Japan. Yes. Many of them develop chronic hepatitis, and when the disease progresses, it progresses to cirrhosis and hepatocellular carcinoma. Hepatocellular carcinoma develops from 1 to 2% annually from chronic hepatitis and 6-8% annually from cirrhosis. About 70% of patients with hepatocellular carcinoma in Japan are infected with HCV1b. The annual number of deaths from cirrhosis and liver cancer caused by HCV1b infection is nearly 40,000.

  Currently, interferon (IFN) therapy is widely used for patients with chronic infection with hepatitis C virus, and a total effect of about 30% is obtained as a whole (Kasahara et al., Hepatology 27, 1394 -1402, 1998: Non-patent document 1). However, the type of virus (HCV type 1b), which is often found in Japan, is resistant to these current therapies and has a response rate of about 50-60%. In particular, the response rate for chronic hepatitis cases in a hyperviremia state (blood RNA level of 500 KIU / ml or more) is 30% or less. In addition, there are 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 is effective to some extent even in cases with a large amount of virus, but the response rate is about 20% (Non-patent Document 2).

  We have previously described several peptides recognized by HLA-A2 and HLA-A24-restricted cytotoxic T lymphocytes (CTL), which are human leukocyte antigen (HLA) types. Reactive IgG can be detected in the majority of HCV-infected patients and can also induce peptide-specific CTL from peripheral blood mononuclear cells (PBMC) of HCV patients with the corresponding HLA type of these peptides I found out. Based on these results, HCV1b-derived peptide was administered to patients with HLA-A24 or HLA-A2 who were ineffective with standard treatment (IFN therapy or ribavirin combination therapy). In many cases, a decrease in viral load was observed in more than half of cases (WO2005 / 028503: Patent Document 1). However, antigen peptides targeting HLA-A2 and A24 positive persons can cover only 70% of Japanese patients.

  HLA-A11, HLA-A31 and HLA-A33, and HLA-A0301 and HLA-A6801 share a similar antigen-binding motif and are therefore classified as HLA-A3 supertypes (Takedatsu H, et al ., Clin Cancer Res 2004; 10: 1112-1120: Non-Patent Document 2). This supertype is relatively prevalent 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% among Hispanics (Sette, A., and Sidney, J., Immunogenetics, 50: 201-212, 1999: Non-patent document 3). So far, there has been no report on HCV-derived HLA-A3 supertype-restricted antigen peptides.

WO2005 / 028503 Kasahara et al., Hepatology 27, 1394-1402, 1998 Takedatsu H, et al., Clin Cancer Res 2004; 10: 1112-1120 Sette, A., and Sidney, J., Immunogenetics, 50: 201-212, 1999

  An object of the present invention is to provide a method for treating an HCV-related disease that can be applied to patients of the HLA-A3 supertype.

The present invention provides a peptide recognized by an antibody against hepatitis C virus, which has any of the following amino acid sequences:
Core 30-39: IVGGVYLLPR (SEQ ID NO: 1)
Core 35-43: YLLPRRGPR (SEQ ID NO: 2)
Core 35-44: YLLPRRGPRL (SEQ ID NO: 3)
NS2 918-926: LIRACMLVR (SEQ ID NO: 4).

  Preferably, the peptides of the present invention are capable of inducing HLA-A11, HLA-A31 or HLA-A33 restricted cytotoxic T cells.

  In another aspect, the present invention provides a pharmaceutical composition for treating a patient having an HCV-related disease, comprising the above-described peptide of the present invention as an active ingredient. Preferably, patients have HLA-A11, HLA-A31, and HLA-A33. The present invention also provides a composition for diagnosing an HCV-related disease or predicting the progression of an HCV-related disease, comprising the above-described peptide of the present invention.

  As used herein, “HCV-related disease” means a disease caused by infection with hepatitis C virus, and includes, for example, acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The progression of HCV-related diseases is typically progression from chronic hepatitis to cirrhosis, and further from cirrhosis to liver cancer.

  The present invention provides an immunogenic HCV1b-derived peptide that can be used to treat patients with HLA-A11, HLA-A31, and HLA-A33 infected with HCV. As shown in the following examples, in the present invention, as a result of examining an antigen peptide (CTL epitope) recognized by CTL in a patient having HLA-A11, HLA-A31 or HLA-A33 having an HCV1b-related disease, Four novel candidate peptides were identified from 48 peptides that are widely conserved in HCV-1b and have motifs that bind to HLA-A11, A31, or A33. The core protein-derived peptide located at positions 30-39 (IVGGVYLLPR (SEQ ID NO: 1)) induced peptide-specific CTL from HLA-A11, A31, and A33 positive patient peripheral blood mononuclear cells (PBMC). On the other hand, core protein-derived 35-43 peptide (YLLPRRGPR (SEQ ID NO: 2)) and 35-44 peptide (YLLPRRGPRL (SEQ ID NO: 3)) were derived from non-structural protein NS-2 from HLA-A31 and A33-positive patient PBMC. The 918-926 peptide (LIRACMLVR (SEQ ID NO: 4)) induced peptide-specific CTL from HLA-A11 and A33 positive patient PBMC. IgG antibodies reactive to these peptides were detected in the plasma of HCV-1b infected patients.

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

  Preferably, the hepatitis C virus-derived peptide according to the present invention is capable of inducing CTLs, and the CTLs thus induced target HCV-infected cells and attack these cells.

  The peptide of this invention should just have said amino acid sequence to such an extent that there exists a desired effect, and the additional sequence | arrangement may be further contained. A person skilled in the art can naturally understand how much sequence is allowed to be added to the N-terminal side and the C-terminal side in order to achieve a desired effect as an antigen peptide. Therefore, for example, an amino acid sequence useful for promoting immunization, an amino acid sequence for facilitating formulation, or a peptide as a fusion protein on the N-terminal side and / or C-terminal side of the peptide according to the present invention. An amino acid sequence convenient for expression or an amino acid sequence convenient for peptide production and purification may be added. In addition, 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.

  The HCV peptide according to the present invention is produced by an ordinary chemical synthesis method, an enzymatic degradation method of a protein molecule, a gene recombination technique using a host transformed to express a base sequence encoding a target amino acid sequence, and the like. can do.

  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, solid phase synthesis using a peptide synthesizer. Can be synthesized by the 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, affinity chromatography, etc. Can be purified by.

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

  As a method for transforming host cells using expression vectors in this peptide production technique, known methods such as calcium chloride method, calcium phosphate coprecipitation method, DEAE dextran method, lipofectin method, electroporation method and the like are used. It is possible to select appropriately based on the host cell to be used. The obtained peptide can be purified from the cell extract or culture supernatant recovered from the cultured medium by the purification method described above.

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

  The peptide vaccine can be administered, for example, orally or by a transdermal route such as 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 generally 0.1-10 mg is preferred as the peptide amount per day for an adult, It is preferable to administer once every few days to several months. Moreover, you may use together a peptide vaccine and an interferon.

  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 presence of both peptide reactivity and CTL of the anti-HCV antibody. Here, “peptide reactivity of anti-HCV antibody” means that any peptide 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 peptide suitable for each person, so-called tailor-made administration. Experience in clinical trials for cancer peptide vaccines has demonstrated that the tailor-made administration induces secondary immunity promptly, resulting in a safer and better clinical effect (Mine et al., Clin. Can Res. 929-937, 2004).

  Confirmation of the therapeutic effect of HCV-related disease is achieved by monitoring the blood antibody titer of a subject suffering from HCV-related disease, that is, the blood concentration of an antibody having reactivity with a peptide by a conventional method such as ELISA. It can be carried out. In addition, the amount of virus can be monitored by measuring the amount of HCV RNA by a conventional method such as RT-PCR.

  The peptides of the present invention are also useful as compositions for diagnosing patients with HCV-related diseases or predicting disease progression. By using the antigen-antibody reaction well known in the art, the antibody titer in the blood of a subject can be measured using the peptide of the present invention. For example, when a typical ELISA method is used, measurement can be performed as follows. The peptide as an antigen is bound to a conventional ELISA plate such as 96 well, and the plate is appropriately blocked to prevent non-specific adsorption. Next, serum prepared from the blood of the subject is appropriately diluted and added to each well of the plate, and reacted for a predetermined time. After the plate is washed to remove unbound components, an antibody that can bind to a human antibody (for example, a rabbit anti-human antibody) is added. If it is desired to detect IgG, gamma chain specific anti-human IgG can be used. After reacting for a predetermined time, the plate is washed, and a detectably labeled antibody (for example, 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, biotin, radiation compounds and the like. After reacting the plate for a predetermined time, the label is detected by adding an appropriate substrate and measuring the decrease of the substrate or the increase of the product, or by measuring the fluorescence, luminescence or radioactivity. In this way, the amount of antibody against a specific peptide in the serum of the subject can be measured. Furthermore, the progress of HCV-related diseases can be predicted by monitoring the amount of antibody.

  Among such methods for detecting antibodies by antigen-antibody reaction, there is xMAP technology as one of highly sensitive methods. This is a flowmetry measurement method using a fluorescent microbead array system developed by Luminex, in which peptide-bound microbeads are brought into contact with serum, and then a fluorescently labeled secondary antibody is bound, followed by flowmetry. The fluorescence intensity is measured by

  In addition, immunochromatography can be used when measuring antibody titer in a hospital laboratory. In this method, a portion in which antigen (or antibody) is linearly distributed on a test paper is prepared, and when a complex of an antibody in a specimen and an antigen labeled with colored particles moves on the test paper, In this method, qualitative analysis is performed based on the presence or absence of a colored line that appears by being intensively captured by an antigen (or antibody). If this method is used, a result can be obtained in a short time (within 20 to 30 minutes) with simple equipment.

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

Subjects HCV1b infected patients with HLA-A3 supertype and healthy donors were subjects. These subjects had any HLA type of HLA-A11, HLA-A31, and HLA-A33, and none was infected with HIV. In order to obtain peripheral blood mononuclear cells (PBMC), 20 ml of peripheral blood was collected and PBMC was prepared by Ficoll-Conlay density gradient centrifugation. All samples were stored cold until used for experiments. Expression of HLA-A11, HLA-A31, and HLA-A33 molecules in PBMC of HCV1b patients was measured by flow cytometry using the following antibodies: anti-HLA-A11 monoclonal antibody (mAb) (Cat # 0284HA; One Lambda Inc., Canada, CA), anti-HLA-A31 mAb (Cat # 0273HA; One Lambda), and anti-HLA-A33 mAb (Cat # 0612HA; One Lambda).

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

Based on the peptide HLA-A11, HLA-A31, and HLA-A33 molecule binding motifs (Parker KC et al., J Immunol 1994; 152: 163-75), 47 types of peptides derived from HCV-1b protein were prepared. . All peptides are from Biologica Co. (Nagoya, Japan) and was 90% or more pure. Control peptides that bind to the HLA-A3 supertype include influenza (Flu) virus-derived peptide (NVKNLYEKVK (SEQ ID NO: 5)), Epstein Barr virus (EBV) -derived peptide (AVFDRKSDAK (SEQ ID NO: 6)), tyrosinase-related protein 2 ( A peptide derived from TRP2) (LLGPGRPYR (SEQ ID NO: 7)) and a peptide derived from HIV (RLRDDLLIVTR (SEQ ID NO: 8)) were used. All peptides were dissolved in dimethyl sulfoxide and diluted to 10 μg / ml before use.

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

Detection of peptide-specific IgG First, using a 1: 100 diluted sample of sera from 12 patients infected with HCV-1b, the level of peptide-specific IgG reactive to each of the 48 peptides was determined. It was measured. 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 μL of plasma (1: 100 dilution) together with 25 μL of peptide-conjugated color-coded beads (Luminex Corp. (Austin, TX)) in a 96 well filter plate (MABVN1250; Millipore Corp., Bedford, MA) Incubated for 2 hours at room temperature on a plate shaker. After 2 hours, the plates were washed with 0.05% Tween 20-PBS and incubated with 100 μL of biotinylated goat anti-human IgG for 1 hour on a plate shaker at room temperature. The plates were then washed and 100 μL streptavidin-PE (5 μg / ml) was added to the wells and incubated for 30 minutes at room temperature on a plate shaker. The fluorescence intensity (FI) was measured by 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 a value higher than the average of healthy donor FI values + 2SD.

  As a result, IgG reactive to HCV cores 30-39, 35-43, 35-44, and HCV NS 2918-926 peptide was detected in 7, 7, 7, and 9 plasma in 12 patients, respectively. It was done. 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 HCV NS 2916-926 peptides in patients were statistically significant compared to healthy donors, respectively. It was high (Figure 1).

HCV1b-infected patients four above recognized frequently by IgG induction HCV1b-infected patient blood peptide-specific CTL from PBMC of the peptide has an HLA-A11, HLA-A31 or HLA-A33, HCV- It was investigated whether peptide-specific CTL could be induced from PBMC of 1b patients. 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 index.

The assay for detecting peptide-specific CTLs was modified according to previously reported methods (Hida N et al., Cancer Immunol Immunother 2002; 51: 219-28). Briefly, PBMC (1 × 10 ⁵ cells / well) were incubated with 10 μl / ml of each peptide in a 200 μl culture in a U-bottom 96-well microculture plate (Nunc, Roskilde, Denmark). The experiment was performed in quadruplicate. The cultures consisted of 45% RPMI 1640, 45% AIM-V medium (Gibco-BRL, Gaithersburg, MD), 10% FCS, 100 U / ml interleukin-2 (IL-2), and 0.1 mM MEM non-essential amino acids It consisted of a 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-A11, C1R-A31, or C1R-A33 cells pulsed with the corresponding peptide, and the other half of the cells were C1R-A11 pulsed with HIV peptide, Cultured with C1R-A31 or C1R-A33 cells. After 18 hours of incubation, the supernatant was collected and the level of IFN-γ was measured by ELISA.

  In response to C1R-A11, C1R-A31, or C1R-A33 cells pulsed with the corresponding peptide, P compared to C1R-A11, C1R-A31, or C1R-A33 cells pulsed with the control group HIV peptide. When the value was lower than 0.05 and when more than 50 pg / ml of IFN-γ was produced, it was determined that the induction of peptide-specific CTL was successful. As a result, the HCV peptides (core proteins 30-39, 35-43 and nonstructural protein 2 918-926) were 3/7, 0/7, and 2/7, respectively, of HLA-A11 positive HCV1b patients. , HLA-A31 positive HCV1b patients 1/4, 0/4 and 0/4, HLA-A33 positive HCV-1b patients 3/5, 3/5, and 3/5 PBMCs to corresponding peptides Reactive CTL was induced (FIG. 2).

  It is already known that HCV core 35-44 peptide can induce HLA-A2 restricted CTL (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 HCV1b patients.

  Next, it was examined whether the HCV core 35-44 peptide could induce HLA-A3 supertype-restricted CTL. PLA of HLA-A2 and HLA-A3 supertype positive HCV1b patients were stimulated with HCV core 35-43 and 35-44 peptides or control peptides in vitro and pulsed with the corresponding peptide T2 (having HLA-A2 ), IFN-γ production in response to C1R-A11, C1R-A31, and C1R-A33 cells was examined. As a result, peptides of core protein 35-44 were found to be HLA-A11 positive HCV1b patients (0/7), HLA-A31 positive HCV1b patients (1/2), and HLA-A33 positive HCV-1b patients (1/2). Peptide-reactive CTLs were induced from PBMCs (FIG. 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 HLA-A2 positive HCV-1b patients, respectively (data not shown) .

  These findings indicate that HCV core 30-39, 35-43, core 35-44, and peptide-specific CTLs in PBMC of HCV-1b patients whose HCV NS2918-926 peptide has HLA-A11, HLA-A31 or HLA-A33. It can be induced.

PBMC stimulated with cytotoxic peptides of peptide-stimulated PBMCs, C1R-A11, C1R-A31, and C1R pre-pulsed with either the corresponding peptide or HIV peptide by standard 6 hour ⁵¹ Cr-release assay. -The cytotoxic activity against A33 cells was examined. 2000 ⁵¹ Cr-labeled cells per well were cultured together with effector cells in a 96 round bottom well plate at a predetermined effector / 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 the supernatant of target cells incubated with 1% Triton-X (Wako Pure Chemical Industries, Osaka, Japan). In some experiments, at the beginning of the culture, 10 μg / ml of anti-HLA-class I (W6 / 32: mouse IgG2a), anti-HLA-DR (L243: mouse IgG2a), anti-CD4 (NU-TH / I: Mouse IgG1), anti-CD8 (NU-TS / C: mouse IgG2a), or anti-CD14 (H14: mouse IgG2a) monoclonal antibody was added to the wells.

  Although HLA-A3 supertype positive PBMC stimulated and cultured with HCV peptides were able to show cytotoxic activity against C1R-A11, C1R-A31, or C1R-A33 cells pre-loaded with the corresponding peptides. , HIV peptide was not shown (FIG. 3).

In addition, cytotoxic activity against C1R-A11, C1R-A31, or C1R-A33 cells pulsed with each peptide was inhibited by HLA-class I (W6 / 32) or CD8 monoclonal antibody (mAb), but was tested. It was not inhibited by HLA-class II (DR-1) or CD4 or CD14 mAb. This indicates that the expressed peptide-specific cytotoxic activity by mainly CD8 ⁺ T cells HLA- class I restricted (Figure 4).

  From the above results, these four peptides were shown to be useful for peptide immunotherapy for HLA-A11, HLA-A31, or HLA-A33-positive HCV-1b-related disease patients.

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

FIG. 1 shows the detection of peptide specific IgG in HCV infected patients. FIG. 2 shows the induction of peptide specific CTL from PBMC of HCV1b patients. FIG. 3 shows the cytotoxic activity of peptide-stimulated PBMC against cells preloaded with peptide. FIG. 4 shows the cytotoxic activity of peptide-stimulated PBMC against cells preloaded with peptide.

Claims (6)

A peptide that is recognized by an antibody against hepatitis C virus and has an amino acid sequence represented by any one of SEQ ID NOs: 1-4. The peptide according to claim 1, which can induce HLA-A11, HLA-A31 or HLA-A33-restricted cytotoxic T cells. A pharmaceutical composition for treating an HCV-related disease, comprising the peptide according to claim 1 or 2 as an active ingredient. A pharmaceutical composition for treating an HCV-related disease in a patient having HLA-A11, HLA-A31, and HLA-A33, comprising the peptide according to claim 1 or 2 as an active ingredient. A composition for diagnosing an HCV-related disease or predicting the progression of an HCV-related disease, comprising the peptide according to claim 1 or 2. A composition for diagnosing HCV-related disease or predicting progression of HCV-related disease in a patient having HLA-A11, HLA-A31, and HLA-A33, comprising the peptide according to claim 1 or 2.

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