KR20090125629A - Immunogenic peptide and composition for preventing or treating hpv-related diseases - Google Patents

Abstract

PURPOSE: An E7 protein-derived E7 immunogenic peptide is provided to induce generation of cytotoxic T lymphocyte and prevent or treat HPV-relating disease. CONSTITUTION: An immunogenic peptide contains LEDLLMGTLGIVCPI of sequence number 13 or GTLGIVCPICSQKP of sequence number 14. A composition for preventing or treating HPV-relating disease comprises the immunogenic peptide and pharmaceutically acceptable carrier. A microparticle, liposome or immunostimulating complex(ISCOM) contains the immunogenic peptide or a nucleic acid encoding the same as an active ingredient.

Description

Immunogenic Peptide and Composition for Preventing or Treating HPV-related diseases

The present invention relates to an immunological peptide and a composition for preventing or treating HPV-related diseases comprising the peptide, and more particularly, to a composition for preventing or treating HPV-related diseases comprising an E7 immunopeptide derived from an E7 protein of HPV type 16 and the peptide. It is about.

Cervical cancer is the second most common malignant tumor in women, with more than 350,000 people dying from cervical cancer each year, and the number continues to increase (zur Hausen H .. Nat Rev). Cancer 2002. 2 (5): 342-50). The main cause of cervical cancer is known as human papilomavirus (HPV), and it has been found that 40 out of 100 HPV infections cause female genital infections (de Villiers EM, Fauquet C, Broker TR, Bernard HU and zur). Hausen H. Classification of papillomaviruses. Virology. 2004. 324: 17-27). Among the various types of HPV, HPV type 16 and HPV type 18 are most commonly found in cervical cancer patients and are known to be important in tumor development. Among them, HPV type 16 is detected in most cervical cancer patients. (Munoz N, et al. N. Engl. J. Med. 2003. 348: 518-527).

When HPV is infected with normal cells, viral oncogenes are inserted into genes of host cells to express various viral proteins, of which E6 and E7 are potent oncoproteins that inhibit the actions of tumor suppressors p53 and RB. It has been shown to promote the progression and continued growth of cancer cells (Werness BA, et al. Science. 1990. 248: 76-79; Dyson N, et al. Science. 1989. 243: 934-937). E7 inhibits the function of RB, a tumor suppressor (Kiyono T, et al. Nature. 1998. 396: 84-88), promotes the activities of the S-phase genes cyclin A and cyclin E (Zerfass K, et al. J.). Virol. 1995. 69: 6389-6399), and also inhibit the function of the cyclin-dependent kinase inhibitors CIP1 (p21) and KIP1 (p27) (Jones DL, et al. Genes Dev. 1997. 11: 2101-2111). (Funk JO, et al. Genes Dev. 1997. 11: 2090-2100; Zerfass-Thome K, et al. Oncogene. 1996. 13: 2323-2330) inhibit the apoptosis of infected cells and induce conversion into tumors. It is known. These viral oncoproteins are not found in normal cells, making them very promising targets in immunotherapy for various types of cancer, and also for producing cytotoxic T lymphocytes (CTLs) for the treatment of cervical cancer. It is the main subject of research to identify HPV epitopes.

Therefore, in the present invention, after synthesis of 14 candidate peptides using the E7 amino acid sequence to find HLA-A * 0201 specific HPV type 16 E7 epitope showing a high immune response, flow cytometry and cytotoxicity Experiments were carried out to identify novel HPV type 16 E7 epitopes that could induce cytotoxic T lymphocyte production for the treatment of cervical cancer patients with HLA-A * 0201 type, resulting in E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7. _{85 -98} (GTLGIVCPICSQKP) epitope revealed.

A new HPV type 16 E7 epitope is proposed to induce the production of cytotoxic T lymphocytes that can be treated in cervical cancer patients with HLA-A * 0201 type.

The present invention has been made by the above necessity, an object of the present invention to provide a peptide for the prevention or treatment of HPV-related diseases.

The present invention to achieve the above object

Peptides containing the LEDLLMGTLGIVCPI of SEQ ID NO: 13 or the GTLGIVCPICSQKP amino acid sequence of SEQ ID NO: 14 are provided.

The present invention also provides a nucleic acid encoding the peptide of SEQ ID NO: 13 or the peptide of SEQ ID NO: 14 of the present invention.

In the present invention, the nucleic acid sequence is SEQ ID NO: 18 for the peptide of SEQ ID NO: 13 (ttg gaa gac ctg tta atg ggc aca cta gga att gtg tgc ccc atc) and SEQ ID NO: 19 for the peptide of SEQ ID NO: 14 ggc aca cta gga att gtg tgc ccc atc tgt tct cag aaa cca) It is not limited to this.

The present invention also provides a composition for preventing or treating a disease associated with HPV infection, comprising (a) the peptide of the present invention and (b) a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the HPV infection-related disease is bowenoid papulosis, anal dysplasia, respiratory or conjunctival papilloma, cervical dysplasia, cervical cancer, vulval cancer, Or prostate cancer, but is not limited thereto.

The present invention also provides a microparticle, liposome, or immune stimulating complex (ISCOM) comprising the peptide of the present invention as an active ingredient.

The present invention also provides a microparticle, liposome, or immune stimulating complex (ISCOM) comprising the nucleic acid of the present invention as an active ingredient.

In one embodiment of the present invention, the composition of the present invention is preferably HLA-A * 3303 type patient specific, but is not limited thereto.

The peptides or nucleic acids of the invention may also optionally be incorporated into microparticles, liposomes, or immune stimulating complexes (ISCOM) (which may include only saponins as active ingredients) or other carriers suitable for supplying the peptides of the invention to a subject. It may be formulated. When using microparticles it preferably has a polymer matrix which is a copolymer such as poly-lactic-co-glycolic acid (PLGA).

In mammals, an immune response (e.g., a cellular immune response comprising an MHC class I-mediated or class II-mediated immune response) is a mammal having an MHC molecule to which an immunogenic peptide binds, ie humans, apes, dogs, cats, and animals. It can be triggered by administering immunogenic peptides to cows, mice, and rats. The peptide of the present invention may be administered as a microparticle, liposome, or ISCOM, or as part of a solution.

Another method of administering a peptide of the invention is to use an expression vector comprising a nucleic acid sequence encoding the peptide of the invention. The nucleic acid sequence may optionally encode a signal sequence linked to the above peptide of the present invention. When the nucleic acid encodes a signal sequence, preferably it is a signal sequence Met Ala Ile Ser Gly Val from HLA-DR.alpha. Pro Val Leu Gly Phe Phe Ile Ala Val Leu Met Ser Ala Gln Glu Ser Trp Ala (SEQ ID NO: 20). In that case, the sequence of the present invention may have the sequence of SEQ ID NO: 3 or 9, for example. Preferably the nucleic acid does not contain the viral gene causing the nucleic acid infection and does not encode the intact E7 protein.

Nucleic acids of the invention can be included in the plasmid and optionally provided in microparticles comprising a polymer matrix. In a preferred embodiment the polymer matrix consists essentially of a copolymer of PLGA. Preferably the microparticles have a diameter of, for example, 0.02 to 20 microns or less than about 11 microns. Preferably the plurality of microparticles has a diameter of 0.02 to 20 microns or less than about 11 microns.

Cells containing the plasmid of the present invention are also within the scope of the present invention. The cell can be, for example, a B cell or other antigen presenting cell (APC). The cell is cultured or maintained under conditions that allow peptide expression from the plasmid encoding it.

Nucleic acids and plasmids of the present invention are useful in methods of inducing an immune response in mammals, eg, humans, by administering the aforementioned plasmids to the mammal, eg, as "naked DNA." The mammal may have a risk or disease of HPV infection, cervical dysplasia, and / or cervical cancer. Nucleic acids and plasmids of the invention can also be inserted into microparticles, liposomes, ISCOMS, or other suitable means of transport as described above.

Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Peptides disclosed herein and nucleic acids encoding the peptides can be used to elicit an immune response against HPV E7 protein.

The peptide of the present invention may be linked to a transporting sequence that carries the peptide to intracellular organs. The transport sequence is an amino acid sequence that functions to regulate intracellular transport (directing movement from organelles to organelles or to the cell surface) of the peptide to which it is attached. Such transport sequences can transport the polypeptides to ER, lysosomes or endosomes and signal peptides (N-terminal sequences that direct the protein to the ER during translation), for example ER retention sequences such as KDEL, and KFERQ, Or a lysosomal target sequence such as QREFK.

Short amino acid sequences can act as signals to target proteins to specific intracellular organs. For example, a hydrophobic signal peptide is found at the amino terminus of the protein towards ER.

Such a transport sequence may be the HLA-DR.alpha leader sequence Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Ile Ala Val Leu Met Ser Ala Gln Glu Ser Trp Ala (SEQ ID NO: 20). If that portion is sufficient to transport the polypeptide of the invention to the ER, it may comprise only a portion of the above specified 25 residue sequences (eg at least 10 amino acid residues) of the signal peptide.

In some cases, it is desirable to modify the portion linking the transport coding sequence with the sequence encoding the HPV E7 antigenic peptide of the present invention to enable processing (ie cleavage) by signal peptides. Recognition sequences for signal peptides are described in Von Heijne, NAR 14: 4683, 1986.

Standard techniques can be used to construct DNA encoding the peptides of the invention (see eg the technique described in WO 94/04171). The construct can be located on additional sequences that increase expression in human cells, such as appropriate promoters, RNA stable 5 'and 3' sequences of coding sequences, introns (5 'or 3' in the sequence being encoded). ), And poly (A) addition sites, as well as their constructs, may include replication origin and selection markers that allow for selection and replication for prokaryotic and / or eukaryotic hosts.

The plasmid of the present invention may have a kanamycin resistance gene (519-1313 site), an SV40 early promoter (131-484 site), and a thymidine kinase (TK) polyadenylation site (1314-1758 site). Kanamycin resistance genes and related regulatory sequences are for selection purposes only and can be removed from the plasmid if selection is not required or desired.

The peptides and nucleic acids of the invention can be used as prophylactic or therapeutic vaccines in subjects known to be infected by HPV, suspected of being infected by HPV, or susceptible to infection by HPV. Other suitable subjects include those who exhibit or are likely to develop symptoms of HPV-related diseases. Peptides and vaccines of the invention may be used to treat diseases associated with infection of HPV strain 16, such as bowenoid papulosis, anal dysplasia, respiratory or conjunctival papillomas, cervical dysplasia, cervical cancer, and vulvar cancer. vulval cancer, or as a vaccine to treat or prevent prostate cancer.

Peptides or nucleic acids of the invention can be administered alone or in combination with other therapies known in the art, such as chemotherapeutic agents, radiation and surgery to treat HPV infection or diseases associated with HPV infection. Peptides and nucleic acids of the invention can also be administered in combination with other therapies designed to enhance an immune response, for example adjuvant or cytokines (or nucleic acids encoding cytokines), as is well known in the art. .

Peptides or nucleic acids of the invention can be used in the manufacture of a medicament for the prevention or treatment of HPV infection or a disease associated with HPV infection.

The delivery systems of the present invention can be used to deliver a peptide or DNA construct that expresses the peptide to appropriate cells that are intended to promote an immune response against HPV.

Peptides of the invention or nucleic acids encoding the peptides are standard methods, for example Donnelly et al., J. Imm. Methods 176: 145, 1994, and Vitiello et al., J. Clin. Invest. 95: 341, may be administered using a method such as that described in 1995.

Peptides or nucleic acids of the invention can be injected in a form known in the art to a subject, such as intramuscular injection, intravenous injection, arterial injection, intradermal injection, intraperitoneal injection, intranasal, intravaginal, enema, subcutaneous or they For example, it may be administered to the gastrointestinal tract, mucosa or respiratory tract by inhalation of a powder or solution containing microparticles. Administration can be local (eg cervical or other infectious site) or systemic.

The peptides of the present invention or nucleic acids encoding the peptides can be carried in pharmaceutically acceptable carriers such as colloidal suspensions, powders, saline, lipids, liposomes, microspheres, or nanospheres. They may be complexed with, associated with, or naked with a vehicle and may be lipids, liposomes, microparticles, gold, nanoparticles, polymers, catalyzers, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. The delivery can be carried out using a delivery system known in the art.

Preferably, a dose of about 0.1 to 100 micromoles of peptide or about 1 to 200 micrograms of DNA is administered per kg of body weight once. Of course, as is well known in the art, a dose for a given patient may be based on a number of factors including the patient's height, body surface area, age, the specific compound administered, sex, time and route of administration, general health and other agents administered simultaneously. Depends on Determination of the appropriate dosage is skillful within the capabilities of pharmacists of ordinary skill.

Other standard delivery methods may be used, such as biolistic delivery or ex vivo treatment. In vitro treatment, for example, antigen presenting cells (APCs), dendritic cells, peripheral blood mononuclear cells, or bone marrow cells can be obtained from a patient or a suitable donor and activated in vitro with the present immune composition and then administered to the patient. have.

Microparticles, including those described in US Pat. No. 5,783,567, can be used as vehicles for transporting macromolecules such as DNA or peptides into cells. They contain macromolecules enclosed within the shell of the polymer or entered into the polymer matrix. Microparticles act to maintain the properties of macromolecules, for example to keep the DNA intact. Microparticles can also be used for pulsed delivery of macromolecules and for delivery to specific locations or to specific cells or target cell populations.

The polymer matrix may be a biodegradable copolymer such as poly-lactic-co-glycolic acid, starch, gelatin or chitin. Microparticles can be used in particular to maximize the transport of DNA molecules into the phagocytes of the subject. Or the microparticles can be injected or implanted into tissue.

Peptides of the invention can be administered to a subject via lipids, dendrimers or liposomes well known in the art. For example, liposomes carrying an immunopeptide or nucleic acids encoding the peptide are known to cause a CTL response in vivo (Reddy et al., J. Immunol. 148: 1585, 1992; Collins et al., J. Immunol. 148: 3336-3341, 1992; Fries et al., Proc. Natl. Acad. Sci. USA 89: 358, 1992; Nabel et al., Proc. Nat. Acad. Sci. (USA) 89: 5157, 1992).

Peptides and nucleic acids of the invention can be administered using Immune Stimulating Complexes (ISCOMS), a 30-40 nm sized (-) occupied cage-like structure produced by mixing saponin alone or cholesterol and Quil A (saponin) simultaneously. have. Peptides and nucleic acids of the invention can be administered simultaneously or separately from ISCOMS.

Protected immunity has been generated in several experimental models of infections including toxoplasmosis and Epstein-Barr virus induced tumors using ISCOMS as a vehicle for antigen (Mowat et al., Immunology Today 12: 383-385, 1991). ). Low antigen doses of 1 ug encapsulated in ISCOMS have been found to produce class I mediated CTL responses (Takahashi et al., Nature 344: 873-875, 1990).

The ability of the peptides and nucleic acids of the invention to elicit an immune response can be analyzed using methods for measuring immune responses well known in the art. For example, the generation of cytotoxic T cells can be expressed using MHC tetramers, measuring intracellular cytokine expression or in a standard ⁵¹ Cr release assay. Standard assays such as ELISA or ELISPOT can also be used to determine cytokine profiles that contribute to T cell activation. T cell proliferation can also be measured using other assays known in the art and assays such as the ³ H-thymidine uptake. B cell responses can be measured using an assay such as ELISA.

Other methods such as digital imaging, cytoscopy, and histological examination can be used to determine the effect of the peptides and nucleic acids of the invention on papilloma virus related lesions or papilloma virus levels.

Hereinafter, the present invention will be described.

Cervical cancer is the second most common malignant tumor in women and its risk is well known. Human papillomavirus (HPV) is known to be the major cause of cervical cancer, among which HPV type 16 has been found to be the most detected in cervical cancer patients. HPV types 16 E6 and E7 are viral oncoproteins that have been shown to cause HPV-infected cervical cells to progress into cancer cells and promote the continued growth of cancer cells. HPV type 16 E7 inhibits the function of Rb, a tumor suppressor, inhibits apoptosis of infected cells and induces the transition to tumors. HPV type 16 E7 is a potent target antigen in the development of immune cell therapy using cytotoxic T lymphocytes for the treatment of cervical cancer. It is being studied as a target antigen.

E7 candidate peptides as a result of flow cytometry using HPV type 16 E7 candidate peptides synthesized to develop specific HPV type 16 E7 epitopes that induce the production of HLA-A * 0201 type cytotoxic T lymphocytes Among them, E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) epitopes induced high levels of differentiation and activity of cytotoxic T cells, and cytotoxicity experiments showed that E7 _{79 -93} ( _{LEDLLMGTLGIVCPI} ) it was found that LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) epitopeuyi well identified E7 _{82 -90} (LLMGTLGIV) and E7 _{86 -93} (TLGIVCPI) peptide induced the same or superior cervical cancer cell killing effects in previous studies. Thus, we identified that E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) epitopes are novel HLA-A * 0201 epitopes that can be effectively applied to the treatment and vaccine development of cervical cancer patients.

In other words, in the present invention, in order to find a peptide having a higher anticancer effect than the known E7 epitope, a PBMC was supplied from a normal HLA-A * 0201 type blood donor and treated with a synthesized E7 peptide, followed by incubation for 1 week or 2 weeks, and then IFN- γ expression was measured to select candidate peptides to induce differentiation into CD8 + T cells, and the actual anti-cancer effect was confirmed using ⁵¹ Cr release assay, a cytotoxicity test. As it was shown in Figure 1, effective immunity by showing the production of IFN-γ of higher than the cost of the synthetic peptides E7 14 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) peptide other E7 peptide ( immunogenic).

⁵¹ Cr release assay technique to determine whether E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) epitopes screened using fluid cytometry actually have anticancer effects on HLA-A * 0201 type cervical cancer cells As a result of measuring the cell death effect, the experimental group treated with E7 _{79 -93} (LEDLLMGTLGIVCPI) peptide showed significantly higher cell killing effect than the positive control group treated with CMVpp65 _{495 -503} (A02, NLVPMVATV) peptide. (Figure 2). On the other hand, the expression level of IFN-γ in the fluid cytometry E7 _{85 -98 79} (GTLGIVCPICSQKP) E7 appears similar to the eptiope _-93 (LEDLLMGTLGIVCPI) eptiope is rather low cell killing effect as compared to E7 _{85 -98} (GTLGIVCPICSQKP) eptitope Although similar to the previously reported positive controls, cancer cell death was shown (Figures 2a and 2b). This result is expected to be due to the different degree of immunity to actual cervical cancer cells due to the difference in amino acid sequence of the two E7 epitopes. In addition, E7 _{82 -90} (LLMGTLGIV) and E7 _{86 -93} (TLGIVCPI) peptides, which have been shown to be immune in the preceding studies, and E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) peptides, which have been shown to be immunosuppressive in this study. Comparison of the apoptosis effects of E7 _{85 -98} (GTLGIVCPICSQKP) peptide showed the highest anti-cancer effect (Fig. 3).

In conclusion, HPV type 16 E7 E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) peptides were used to develop vaccines and adoptive immune cell therapy for HLA-A * 0201 type cervical cancer. It was found that it was an effective epitope for the production of the necessary cytotoxic T lymphocytes.

As can be seen in the present invention HPV type 16 E7 E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) peptide is HLA-A * 0201-type vaccine development of cervical cancer and cytotoxic T required for adoptive immune therapy cell The epitope was found to be effective for lymphocyte production.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example :One Peptide ( peptide ) synthesis

The entire amino acid sequence of HPV type 16 E7 protein was synthesized by using computer algorithms to overlap each of 15mer in size, and then purified using a high performance liquid chromatography (HPLC) instrument. A total of 14 peptides were synthesized by finally selecting more than 95% of peptides (Table 1). Peptide synthesized in this way was dissolved in 1% DMSO PBS and stored frozen at -20 ℃ and thawed at room temperature immediately before use.

Peptide Number (SEQ ID NO) Amino acid position Amino acid sequence One 1-15 MHGDTPTLHEYMLDL 2 7-21 TLHEYMLDLQPETTD 3 13-27 LDLQPETTDLYCYEQ 4 19-33 TTDLYCYEQLNDSSE 5 25-39 YEQLNDSSEEEDEID 6 31-45 SSEEEDEIDGPAGQA 7 37-51 EIDGPAGQAEPDRAH 8 43-57 GQAEPDRAHYNIVTF 9 49-63 RAHYNIVTFCCKCDS 10 61-75 CDSTLRLCVQSTHVD 11 67-81 LCVQSTHVDIRTLED 12 73-87 HVDIRTLEDLLMGTL 13 79-93 LEDLLMGTLGIVCPI 14 85-98 GTLGIVCPICSQKP

Table 1 shows the synthesized HPV type 16 E7 peptide.

Example  2: Preparation of Cells

Peripheral blood mononuclear cells (PBMCs) are isolated by centrifugation of the blood of HLA-A * 0201 type blood donors (Table 2) who signed the study agreement. Briefly, after forming a density gradient using Ficoll-Hypaque 1.077, the blood of the donor was carefully flowed thereon, followed by centrifugation at room temperature for 15 minutes at a rate of 2000 rpm, followed by a leukocyte layer ( Only buffy coat) was collected and washed twice with PBS and used for the experiment.

Blood donor gender HLA-A HLA-B HLA-C One south A * 0201/2402 13, 40 03, 03 2 south A * 0201/3303 44, 44 03, 14 3 south A * 0201/3303 44, 44 03, 14 4 south A * 0201/2402 40, 40 03, 04 5 south A * 0201/3001 13, 35 03, 06

Table 2 shows HLA-A * 0201 type blood donors.

Example  3: PBMC From autologous dendritic cells ( autologous dendritic cell ) Generation

PBMC obtained by centrifugation was placed in a T75 flask containing RPMI culture (10% FBS, 5% antibiotics) and incubated at 37 ° C. for 2 hours. Thereafter, the suspended cells were removed and Interleukin-4 (IL-4, 1000 U / ml) and granulocyte-macrophage stimulating factor (GM-CSF, 800 U / ml) were added to the monocytes attached to the flask. IL-4 (1000U / ml) and GM-CSF (1600U / ml) were added at 2 and 4 days after incubation. RPMI culture medium was changed from time to time as the cells grow. At 5 days after the start of the culture, tumor necrosis factor-α (TNF-α, 1000u / ml) was added.

Example  4: On peptides  Specific polyclonal  Generation of Cytotoxic T Cells

PBMCs of cryofreezing HLA-A * 0201 type blood donors were thawed and placed in the appropriate number of cells in a 24 well culture vessel filled with 2 ml RPMI culture medium per well. At 1 day after the start of the culture, the peptide (10 μg / ml) and Interleukin-2 (IL-2, 100 U / ml) to be tested were added to each well. IL-2 (1000U / ml) was added at intervals of two days during the incubation period, and the culture medium was replaced. At 7 days after the start of the culture, the dendritic cells (at least 10/10 the number of cytotoxic T cells) and peptides (20 μg / ml) generated by the above-mentioned method were added.

Example 5 Intracellular Interferon-γ (IFN-γ) Production Measurement Using Flow Cytometry

Seven days after the start of the culture, dendritic cells and cytotoxic T cells to which the peptide was added were incubated at 37 ° C. for 1 hour. At this time, phytohemagglutinin (PHA, 0.25 μg / ml) was added to one of the experimental groups. After incubation, 10 μg of brefeldin A (BFA) was added thereto, and then incubated at 37 ° C. for 5 hours. Then, cells were collected from each well and washed with PBS (phosphate-buffered saline). After washing, put PBS containing 1 mM concentration of EDTA and incubated for 10 minutes at 37 ℃. After the incubation, the cells were washed twice with PBS containing 5% FBS. CD8 + antibodies were put in a ratio of 1: 100, respectively, and then incubated in a dark state at 4 ° C. for 15 minutes. After incubation, the lysing solution and permeabilization solution were treated, and fluorescein isothiocyanate (FITC) -conjugated mouse anti-human IFN-γ antibody was added and incubated in dark at 4 ° C for 30 minutes. Thereafter, the cells were washed twice with PBS containing 5% FBS, fixed with 1% formaldehyde, and analyzed by FACS (fluorescence activated cell sorting).

Example  6: cytotoxicity test ( cytotoxicity assay )

Cytotoxicity experiments were performed using a ⁵¹ Cr release assay using cervical cancer cell lines as target cells. Briefly, 0.1 mCi Na ₂ ⁵¹ CrO ₄ was added to HLA-A * 3303 type cervical cancer cell line cultured in RPMI culture and incubated at 37 ° C. for 45 minutes. After washing twice with PBS, an appropriate number of target cells were put in various ratios to cytotoxic T cells and incubated at 37 ° C for 5 hours. After incubation, centrifuge at 1500 rpm for 5 minutes and transfer 100μl to the prepared γ-counter tube. Measure and analyze using γ-ray counter. The extent of cytotoxic T cell specific target cell death is calculated using the formula [(experimental cpm-spontaneous cpm) / (maximum cpm-spontaneous cpm)] × 100.

The result of the above Example is as follows.

(1) Confirmation of production of IFN-γ from HPV type 16 E7 epitope-specific CD8 + cytotoxic T lymphocytes using flow cytometry

To select candidates for the E7 epitope that induces HLA-A * 0201 type-specific patient-specific cytotoxic T cell generation among 14 synthesized HPV type 16 E7 peptides, each peptide was treated with PBMCs of blood donor for 1 week. after seen by comparing the IL-2 (negative control), CMVpp65 _{495 -503} (control) each handle (NLVPMVATV, HLA-a * 0201 , positive control) culture. As shown in Figure 1, most of the 14 candidate peptides induced the production of IFN-γ higher than the control group treated with IL-2 only, and E7 among the peptides showing higher levels of IFN-γ production than the negative control. to _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) equal or higher level as the positive control (positive control) in the test group treated with the peptide was found to produce a level of IFN-γ appears. Therefore, it was predicted E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) peptides as potent novel HLA-A * 0201-specific E7 epitopes.

(2) Cervical cancer cytotoxicity experiment of HPV type 16 E7 epitope specific CD8 + cytotoxic T lymphocytes

In order to confirm that HLA-A * 0201 type cervical cancer cell line acts on apoptosis, it was analyzed using ⁵¹ Cr release assay. PBMCs of blood donors were treated with IL-2, CMVpp65 _{495 -503} (NLVPMVATV, HLA-A * 0201, positive control), E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) peptides, and cultured one week later. Each peptide was treated with dendritic cells of the same patient once again, followed by incubation for one week, and then mixed with ⁵¹ Cr-labeled HLA-A * 0201 type cervical cancer cell line and incubated for 5 hours using a gamma radiation meter. Analyzed. The results of the analysis showed that the cervical cancer cell killing effect of the experimental group treated with the E7 _{85 -98} (GTLGIVCPICSQKP) peptide was treated with the positive control group CMVpp65 _{495 -503} (NLVPMVATV) as well as the negative control group treated with IL-2. The cell death effect was much higher than that. E7 _79-93 (LEDLLMGTLGIVCPI) experimental groups treated with the peptides eoteuna indicate the close apoptotic effect levels in positive control group treated with _{CMVpp65 495 -503 (NLVPMVATV) E7 85} -98 (GTLGIVCPICSQKP) shows a rather low effect than the effect of the peptide there was. (Figure 2a, b)

(3) Comparative measurement of the killing effect of HLA-A * 0201 type cervical cancer cells of HPV type 16 E7 epitope and new epitopes E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP)

Other HPV type 16 E7 epitopes E7 _{82 -90} (LLMGTLGIV; SEQ ID NO: 15) and E7 _{86 -93} (TLGIVCPI; SEQ ID NO: 16) and the peptides of the present invention E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _85-98 The direct killing effect of (GTLGIVCPICSQKP) on cervical cancer cells was compared using a ⁵¹ Cr release assay. IL-2 (negative control), CMVpp65 _{495 -503} (NLVPMVATV, HLA-A * 0201, positive control; SEQ ID NO: 17), E7 _{82 -90} (LLMGTLGIV, positive control), E7 _{86 -93} (TLGIVCPI, positive control), E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) peptides were treated separately and each peptide was again treated with dendritic cells of the same patient cultured one week later for one week. After incubation, the cells were incubated with ⁵¹ Cr labeled HLA-A * 0201 type cervical cancer cells for 5 hours and analyzed using a gamma radiation meter. Analysis of the measured results showed that the E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) peptides selected in this study were the same as the known E7 _{82 -90} (LLMGTLGIV) or E7 _{86 -93} (TLGIVCPI) epitopes. Or showed high cervical cancer cell killing effect. Among them, E7 _{85 -98} (GTLGIVCPICSQKP) was found to have the highest cancer cell killing effect. (Figure 3)

1 is a measure of the degree of IFN-γ expression in HLA-A * 0201 type specific CD8 + T cells induced by HPV type 16 E7 candidate epitopes using fluid cytometry.

FIG. 1A shows that HLA-A * 0201 type specific CD8 + T cells induced after stimulation of peripheral blood monocytes extracted from HLA-A * 0201/2401 type patients with HPV type 16 E7 candidate peptides and negative and positive control peptides. It is a measure of the degree of IFN-γ expression. IL-2 (negative control), A02; CMVpp65 _495-493 (HLA-A * 0201, NLVPMVATV, positive control), A24; CMVpp65 _328-336 (HLA-A * 2402, QYDPVAALF, positive control), A33; CMVpp65 _91-100 (HLA-A * 3303, SVNVHNPTGR, Negative Control),

FIG. 1B shows HLA-A * 0203 type specific CD8 + T cells induced after stimulation of peripheral blood monocytes extracted from HLA-A * 0201/3303 type patients with HPV type 16 E7 candidate peptides and negative and positive control peptides. It is a measure of the degree of IFN-γ expression. IL-2 (negative control), A02; CMVpp65 _495-493 (HLA-A * 0201, NLVPMVATV, positive control), A24; CMVpp65 _328-336 (HLA-A * 2402, QYDPVAALF, negative control), A33; CMVpp65 _91-100 (HLA-A * 3303, SVNVHNPTGR, positive control).

Figure 2 shows the killing effect of HLA-A * 0201 type cervical cancer cells by HPV type 16 E7 _{79 -93} (LEDLLMGTLGIVCPI) and E7 _{85 -98} (GTLGIVCPICSQKP) epitope specific CD8 + cytotoxic T lymphocytes using ⁵¹ Cr release assay technique. It is measured.

2a shows the killing effect of HLA-A * 0201 type cervical cancer cells measured after stimulation of peripheral blood monocytes extracted from HLA-A * 0201/3303 type patients with HPV type 16 E7 candidate peptides and negative and positive control peptides. being. IL-2 (negative control), A02; CMVpp65 _495-493 (HLA-A * 0201, NLVPMVATV, positive control),

Figure 2b shows the killing effect of HLA-A * 0201 type cervical cancer cells measured after stimulating peripheral blood monocytes extracted from HLA-A * 0201/2402 type patients with HPV type 16 E7 candidate peptides and negative and positive control peptides. being. IL-2 (negative control), A02; CMVpp65 _495-493 (HLA-A * 0201, NLVPMVATV, positive control).

Figure 3 shows the existing HPV type 16 E7 _{82 -90} (LLMGTLGIV), E7 _{86 -93} (TLGIVCPI) epitope and new E7 _{79 -93} (LEDLLMGTLGIVCPI), E7 _{85 -98} (GTLGIVCPICSQKP) using a ⁵¹ Cr release assay technique. Comparative measurement of the killing effect of HLA-A * 0201 type cervical cancer cells.

<110> BIOCORE CO., LTD. <120> Immunogenic Peptide and Composition for preventing or treating          HPV-related diseases <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 1 Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu   1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 2 Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp   1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 3 Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln   1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 4 Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu   1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 5 Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp   1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 6 Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala   1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 7 Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His   1 5 10 15 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 8 Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe   1 5 10 15 <210> 9 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 9 Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser   1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 10 Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp   1 5 10 15 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 11 Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp   1 5 10 15 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 12 His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu   1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 13 Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile   1 5 10 15 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 14 Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro   1 5 10 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> Positive control <400> 15 Leu Leu Met Gly Thr Leu Gly Ile Val   1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> Positive control <400> 16 Thr Leu Gly Ile Val Cys Pro Ile   1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> Positive control <400> 17 Asn Leu Val Pro Met Val Ala Thr Val   1 5 <210> 18 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid <400> 18 ttggaagacc tgttaatggg cacactagga attgtgtgcc ccatc 45 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid <400> 19 ggcacactag gaattgtgtg ccccatctgt tctcagaaac ca 42 <210> 20 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Signal sequence <400> 20 Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val   1 5 10 15 Leu Met Ser Ala Gln Glu Ser Trp Ala              20 25  

Claims (8)

A peptide containing the LEDLLMGTLGIVCPI of SEQ ID NO: 13 or the GTLGIVCPICSQKP amino acid sequence of SEQ ID NO: 14. A nucleic acid encoding each of the peptide of SEQ ID NO: 13 or the peptide of SEQ ID NO: 14. The nucleic acid according to claim 2, wherein the nucleic acid sequence is SEQ ID NO: 18 for the peptide of SEQ ID NO: 13 and SEQ ID NO: 19 for the peptide of SEQ ID NO: 14. A composition for preventing or treating a disease associated with HPV infection, comprising (a) the peptide of claim 1 and (b) a pharmaceutically acceptable carrier. The method of claim 4, wherein the HPV infection-related disease is bowenoid papulosis, anal dysplasia, respiratory or conjunctival papilloma, cervical dysplasia, cervical cancer, vulval cancer, or prostate cancer. The composition characterized in that the. Microparticles, liposomes, or immune stimulating complex (ISCOM) comprising the peptide of claim 1 as an active ingredient. Microparticles, liposomes, or immune stimulating complexes (ISCOM) comprising the nucleic acid of any one of claims 2 or 3 as an active ingredient. 6. A composition according to claim 4 or 5, wherein said composition is HLA-A * 0201 type patient specific.

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