KR100209295B1 - Peptide controlling human immune response against human papiloma virus type16e6 - Google Patents

Abstract

The present invention relates to a novel peptide capable of inducing cytotoxic T lymphocytes (CTL) against human papillomavirus type 16 E6 protein antigen or modulating the immune system of the human body against the virus.

Description

Peptides with human immunity control function against human papillomavirus type 16 E6 protein

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 a cytotoxic T lymphocyte induced by each synthetic peptide kills a T2 cell that binds the synthetic peptide to the surface.

[Object of the invention]

The present invention relates to a novel peptide having a function of regulating the immune response of the human body to the E6 protein of the human papilloma virus type 16. More particularly, the present invention relates to a method for inducing cytotoxic T lymphocytes (CTL) against human papillomavirus type 16 E6 protein antigens, And relates to novel peptides.

[TECHNICAL FIELD OF THE INVENTION AND RELATED ART OF THE SAME]

Several species belonging to human papillomaviruses (eg, 16 and 18) are known to cause female uterine cancer by the action of the carcinogenic proteins E6 and E7. Therefore, when the human body is infected with the virus, the body produces an antibody to remove the toxic substance, and on the other hand, an immune reaction to destroy the virus-infected cells occurs and ultimately can be recovered from the virus infection. In particular, the destruction of these infected cells is accomplished by the induction of cell activation by CTL, which has cytotoxic ability. CTL must be activated to recognize some of the infected virus proteins in order to have cytotoxicity. In general, virus proteins synthesized in infected cells are converted into peptides composed of 8 to 15 amino acids through intracellular degradation, and some of them are found in the first major histocompatibility complex molecule (MHC) (class I), it appears on the surface of the cell, and CTL recognizes it and activates it and destroys the infected cells.

However, the 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 this specific amino acid sequence is identified, a peptide corresponding to the amino acid sequence can be synthesized by a chemical method. Therefore, the immune response can be controlled by artificially inducing CTL that specifically recognizes a virus in the human body, It becomes possible to prevent and cure the diseases caused.

[Technical Problem]

Therefore, the present inventors intensively studied to identify specific peptide sequences that can induce cytotoxicity by recognizing the E6 protein sequence of human papilloma virus type 16 by CTL as described above. As a result, the three peptides shown in Table 2 Can achieve the desired purpose in accordance with this purpose, and the present invention has been completed.

[Structure and operation of the invention]

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

The present invention relates to a novel peptide sequence capable of inducing CTL or inducing immunomodulation against human papillomavirus type 16 E6 protein antigen.

Since the entire amino acid sequence of human papillomavirus type 16 E6 protein is known in the literature (Virol. 145: 181 and J. Virol. 61: 962), the present inventors firstly identified 10 peptides were synthesized.

The 10 peptides used in this study were selected to contain a common motif of the sequence part known to be capable of binding to HLA-A2 in other types of proteins already reported. That is, a peptide containing 10 or 11 amino acids in the entire amino acid sequence of the known human papillomavirus type 16 E6 protein, wherein the second amino acid is leucine or methionine and the 9th to 11th 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 10 peptides thus selected actually has a specific property meeting the object of the present invention, the inventors of the present invention prepared MHC class I < / RTI > molecules, while testing whether T2 cells with synthetic peptides can be killed by cytotoxic T lymphocytes.

First, T2 cells were used to investigate whether synthetic peptides bind to MHC class I molecules. The T2 cell line was a cell line that mutated to T1 cell, a primary cell line expressing HLA-A2 and type 1 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, in this case, when a peptide capable of binding MHC class I molecules is present in the cell culture medium, the peptide binds to MHC class I molecules on the cell surface, resulting in stable molecular structure, resulting in increased expression of MHC class I molecules The ability to bind specific peptides to MHC class I molecules can be measured.

In this experiment, the degree of expression of MHC class I molecules on the surface of T2 cells was measured using this principle and the degree of binding of specific peptides was measured (see Example 1). The results are shown in FIG. In FIG. 1, T1 and T2 represent the fluorescence intensity values of the corresponding cell lines, that is, the fluorescence intensity values in the absence of the specific peptide. By measuring the activity of each synthetic peptide based on this, the synthetic peptides P3, P5 and P7 were found to exhibit high binding affinity for MHC class I molecules.

On the other hand, in order to be recognized by CTL, a specific peptide must be recognized by T cell receptor of T lymphocyte in a form associated with MHC class I. The relatively high binding force peptides shown in FIG. 1 are actually recognized by CTLs that recognize human papillomaviruses in the human body, and thus T2 cells (target cells) sensitized with synthetic peptides are killed by CTLs (See Example 2).

As a result, as shown in Table 3, the three peptides P3, P5 and P7 highly activate the CTL recognizing the human papillomavirus antigen present in the human body, as well as a new kind that can be recognized again by the CTL ≪ / RTI >

Hereinafter, the present invention will be described more specifically based on the following examples. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

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

[Example 1]

[Binding ability of synthetic peptides according to the present invention to MHC class I molecules]

From the amino acid sequence of human papillomavirus type 16 E6 protein, 10 kinds of peptides including the peptides listed in Table 1 were synthesized by chemical methods and dissolved in phosphate buffered saline (PBS). The amino acid sequences of the 10 kinds of peptides used are as shown in Table 2.

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.

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

FIG. 1 shows the fluorescence intensity value converted into an average value of the fluorescence indices obtained by the following formula (1), respectively. Here, the basic fluorescence intensity means a fluorescence intensity value exhibited by a nonspecific antibody reaction.

As a result of the experiment, it was confirmed that among the 10 peptides synthesized, the three peptides shown in the following Table 2 show higher binding ability than the other peptides. The present inventors conducted the experiments of the following Example 2 Respectively.

[Example 2]

[Cytotoxic activity of peptide-specific T cells on target cells sensitized with synthetic peptides according to the present invention]

Leukocytes were separated from the blood of a general blood donor by centrifugation, and the above peptide was added at a concentration of 10 / / ml and cultured in a constant-temperature and constant-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 and proliferate CTLs. If irreversible stimulation cells are not inactivated by gamma rays, irreproducible cells can not be distinguished from original T cells. Therefore, stimulation cells inactivated by gamma rays were used. Ten weeks after the incubation, 10 units of interleukin-2 were added and cultured.

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

Each peptide was added to the T2 cells at a concentration of 10 μg / ml and reacted at 37 ° C. for 3 hours, washed, and treated with octadecanoylaminofluorescine (OAF) at a concentration of 100 ng / ml for 30 minutes at 37 ° C. Lt; / RTI > The reacted 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 μg / ml for 30 minutes, The fluorescence intensity of the mixed solution was measured to determine the amount of destroyed T2 cells. At this time, the T2 cell line used as the target cell is fluorescence due to OAF, and the dead cell shows fluorescence due to PI, and thus the T2 cell line that has two fluorescent fluorescence due to OAF and PI is destroyed.

As shown in the following Table 3, not only the three peptides P3, P5 and P7 among the ten peptides used, but also the CTL that recognizes 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.

[Effects of the Invention]

As shown in the above Examples 1 and 2, the synthetic peptides P3, P5 and P7 according to the present invention possess high binding ability to MHC class I molecules and activate CTLs recognizing human papillomavirus antigen And the activated CTL serves to kill the virus-infected cells again. Therefore, the peptide of the present invention is expected to play a role of regulating the immune mechanism in the human body as well as the recovery from virus infection.

Claims (2)

Any one of the peptides or variants thereof selected from the following SEQ ID NOS: 1 to 3 having the function of regulating the immune response of the human body to the human papilloma virus type 16 E6 protein antigen. Sequence 1: Glu-Leu-Gln-Thr-Thr-Ile-His-Asp-Ile-Ile Sequence 2: Leu-Leu-Arg-Arg-Glu-Val-Tyr-Asp-Phe-Ala Sequence 3: Thr-Leu-Glu-Gln-Tyr-Asn-Lys-Pro-Leu 2. The peptide of claim 1, wherein the peptide is SEQ ID NO: 1.