KR980009278A - Peptides with human immunity control function against human papillomavirus type 18 E6 protein - Google Patents

Abstract

The present invention relates to a novel peptide capable of inducing cytotoxic T lymphocyte (CTL) against human papillomavirus type 18 E6 protein antigen or controlling immunity of the human body against virus.

Representative figure: The second figure

Description

Peptides with human immunity control function against human papillomavirus type 18 E6 protein

The present invention relates to a novel peptide having a function of regulating the immune response of the human body to the human papilloma virus type 18 E6 protein. More specifically, the present invention relates to a method for inducing cytotoxic T lymphocytes (CTL) against human papillomavirus type 18 E6 protein antigen, Lt; / RTI > peptide.

Several species belonging to the human papilloma virus (eg, 16 and 18) are known to induce uterine cancer in women by the action of carcinogenic proteins such as E6 and E7.

Accordingly, when a human body is infected with a virus, the body produces an antibody against the body to remove toxic substances, while an immune response is generated to destroy the virus-infected cells, and ultimately, the virus is recovered from the infection. In particular, the destruction of infected cells is caused by the induction of the activity of CTLs, which have cell killing ability. In order for CTL to activate and kill cells, it is necessary to recognize some of the proteins of infected viruses. The recognition of viral proteins is generally accomplished through the intracellular degradation of the viral proteins synthesized in infected cells into a peptide composed of 8 to 15 amino acids and a major histocompatibility complex molecule, MHC) group 1 (cuss I), it appears to be activated by the CTL to be activated and destroys the infected cells.

However, the specific site (peptide) of the protein recognized by the CTL must have a specific amino acid sequence among the peptides generated as a result of the degradation of the viral protein. When the specific amino acid sequence is identified, the peptide corresponding to the amino acid sequence can be synthesized by a chemical method. Therefore, the immune response such as artificially inducing CTL under the specific virus in the human body can be controlled, It is possible to prevent and treat the diseases caused by the diseases.

Therefore, the inventors of the present invention conducted intensive studies to identify specific peptide sequences that can induce cytotoxicity by recognizing the E6 protein sequence of human papillomavirus type 18 by CTL as described above. As a result, the three peptides shown in Table 2 Tight can achieve the desired purpose in accordance with this purpose, and the present invention is completed.

FIG. 1 shows that the amount of MHC class I molecules expressed on the surface of T2 cells varies depending on the type of synthetic peptide present in the culture medium.

FIG. 2 shows the manner in which each peptide-induced cytotoxic T lymphocyte kills T2 cells bound to the surface of the synthetic peptide.

Hereinafter, the configuration of the present invention will be described in detail

The present invention relates to a novel peptide sequence capable of inducing CTLs or inducing immunomodulation against human papillomavirus type 18 E6 protein antigen.

Since the entire amino acid sequence of the human papillomavirus type 18 E6 protein is known in the literature (Journal of Molecular Biology 193, 599), the present inventors have identified 18 peptides as shown in the following Table 1 on the basis of this known sequence Were synthesized first.

[Table 1] Synthetic peptide sequence

The 18 peptides used in this study were selected to contain a motif common to the sequence part known to be capable of binding to HLA-A2 in other types of proteins that have already been reported. That is, a peptide containing 9 or 10 amino acids in the entire amino acid sequence of the known human papillomavirus type 18 E6 protein, wherein the second amino acid is leucine or methionine and the 9th or 10th amino acid is leucine, valine or isoleucine (Nature 351: 2900, Science 255: 1261, Cell 74: 929 and J. Immunol 152-3904).

In order to select which of the 18 peptides thus selected actually has a specific property meeting the purpose of the present invention, the present inventors examined each of the synthesized peptides and examined MHC class I T2 cells were used to measure the amount of MHC Class I molecules in the HLA-A2.1 type of T2 cells, while T2 cells with synthetic peptides were killed by cytotoxic T lymphocytes Or not.

In order to investigate whether synthetic peptides bind to MHC class I molecules, T2 cell lines were used to mutate T1 cells, the primary cell line expressing HLA-A2.1 type MHC class I molecules. The enzyme involved in peptide migration in cells inhibits normal binding of MHC class I molecules to peptides, resulting in an unstable molecular structure and consequently exhibits low MHC class I expression under normal growth conditions. However, when peptides capable of binding MHC class I molecules exist in the cell culture medium, the peptides bind to MHC class I molecules on the cell surface, resulting in stable molecular structure, resulting in an increase in the expression of MHC class I molecules This allows us to measure the probability of binding specific pptides to MHC class I molecules.

In this experiment, the degree of binding of specific peptides was measured by measuring the expression level of MHC class I molecules on the surface of T2 cells using this principle (see Example 1), and the results are shown in FIG. In FIG. 1, T1 and T2 represent the fluorescence intensities of the respective cell lines, ie, the fluorescence intensities in the absence of specific peptides, and A8, B4 and C1 Were found to have high binding affinity for MHC class I molecules.

In order to recognize a specific peptide by CLT, it must be recognized by T cell receptor of T lymphocyte in combination with MHC class I. Therefore, the peptide having relatively high binding power shown in FIG. It was recognized by the CLT recognizing the Roman virus, and thus it was examined whether T2 cells (target cells) sensitized with the synthetic peptide were killed by CLT (efficacy cells) (see Example 2).

As a result, three peptides of A8, B4 and C1 as shown in Table 3 were found to be highly active in CLT recognizing human papillomavirus antigens present in the human body, Type of peptide.

Hereinafter, the present invention will be described more specifically based on the following examples. However, these examples are provided only for the understanding of the present invention, and the scope of the present invention is not limited to these examples in any sense.

However, the reagents used in the following examples were products of Sigma, and the antibodies were obtained from Bioreource. Cell line BB7.2 and T1 and T2 cells secreting antibody were purchased from ATCC.

[Example 1]

The binding potency of the shake peptide according to the present invention to MHC class I molecules

Eighteen peptides including the peptides listed in Table 1 were synthesized by chemical methods from the amino acid sequence of human papillomavirus type 18 E6 protein and dissolved in phosphate buffered saline (PBS, pH 7.6). The amino acid sequences of the 18 kinds of peptides used are as shown in Table 1.

In this experiment, the degree of expression of MHC class I molecules on the surface of T2 cells was measured and the binding degree of a specific peptide was measured as follows.

The synthesized peptides were added to 0.5 ml of the T2 cell culture at a concentration of 10 μg / ml, reacted at 37 ° C for 3 hours, and unreacted peptides were removed with PBS. The cells were reacted with 100 μl of the culture medium of BB7.2, an anti-MHC class I antibody, at 4 ° C for 40 minutes, and then washed with anti-mouse immunoglobulin antibody (FITC) with fluorescein isothiocyanate immunoglobulin antibody diluted 50: 1, reacted at 4 ° C for 30 minutes, washed, and the fluorescence intensity of the cells was measured by a flow cytometer.

Fig. 1 shows the fluorescence intensities measured in terms of the average of fluorescence indices obtained from the following formula (1).

Fluorescence index = (fluorescence intensity - basic fluorescence intensity) / fluorescence intensity of T2 cell line itself x100 (1)

Here, the basic fluorescence intensity refers to the fluorescence intensity which appears by nonspecific antibody reaction.

As a result of the experiment, it was confirmed that the eight peptides shown in Table 2 below have higher binding capacity than the other peptides. Therefore, the present inventors conducted the following experiment to the eight synthetic peptides in the following Example 2 Respectively.

[Table 2] Location of the peptides recognized by CTL in the human papilloma virus type 18 E6 protein and its amino acid sequence

[Example 2]

The cytotoxicity of peptide-specific T cells on sensory scaffold cells of synthetic peptides according to the present invention

Leukocytes were separated from the blood of general blood donors by centrifugation, and the above peptides were added at a concentration of 10 쨉 l / ml and cultured in a constant temperature and humidity incubator containing 5% of carbon dioxide. After 1 week, the cells were incubated with the same amount of the same peptide added at 3 to 5 times the number of cells cultured with leukocyte cells of the same tissue-compatible antigen inactivated with gamma rays as stimulating cells. At this time, irritating cells induce CLT to induce carbohydrate proliferation. If gamma rays do not inactivate proliferation of stimulated cells, they will proliferate with original T cells and can not be distinguished. Thus, irritated cells inactivated with gamma rays are used Respectively. One week after the incubation, 10 units of interleukin-2 was added thereto.

The extent to which a peptide-specific hemocyte recognizes and destroys a T2 cell line adhering a specific peptide antigen to the surface was measured according to the following method. The measurement results are shown in Table 3 and FIG.

Each peptide was added to T2 cells at a concentration of 10 μl / ml. The cells were reacted at 37 ° C for 3 hours, washed, and treated with octadecanoylaminofluorescine (OAF) at a concentration of 100 mg / Followed by washing at 37 ° C. After the reaction, the T2 cell line was mixed with hemocytes at the ratio shown in Table 3, reacted at 37 ° C, treated with propidium iodide (PI) at a concentration of 3 μl / ml for 30 minutes, And the amount of destroyed T2 cells was measured. At this time, the T2 cell line used as the target cell is fluorescence due to OAF, and the dead cell is fluorescence due to PI, and thus, the T2 cell line showing both fluorescence by OAF and PI is destroyed.

As shown in the following Table 3, among the eight synthetic peptides used, the three peptides A8, B4 and C1 not only activate the CTL recognizing the human papillomavirus antigen present in the human body, It has been confirmed that this is a new class of peptides that can be recognized.

[Table 3] The cytotoxicity of peptide-specific T cells on target cells sensitized with synthetic peptides

As confirmed specifically in Examples 1 and 2 above, the synthetic peptides A8, B4 and C1 according to the present invention possess a high binding ability to MHC class I molecules, while activating CTLs that recognize human papilloma virus antigens And the activated CTL serves to kill the virus-infected cells again. Therefore, it is anticipated that it will be able to regenerate from the peptide virus infection of the present invention, and ultimately, to regulate the immune mechanism in the human body

Claims (2)

A peptide or a variant thereof selected from the following SEQ ID NOS: 1 to 3, which has a function of regulating the immune response of the human body to the human papilloma virus type 18 E6 protein antigen. Sequence 1: Ser-Leu-Gln-Asp-Ile-Glu-Ile-Thr-Cys-Val Sequence 2: Glu-Leu-Thr-Glu-Val-Phe-Glu-Phe-Ala Sequence 3: Lys-Leu-Thr-Asn-Thr-Gly-Leu-Tyr-Asn-Leu 2. The peptide of claim 1, wherein the peptide is SEQ ID NO: 1. ※ Note: It is disclosed by the contents of the first application.