CN113105528B - Tumor-associated antigen CTL epitope peptide and application thereof - Google Patents

Abstract

An antitumor dominant CTL epitope peptide derived from a tumor-associated antigen, which is HLA-A2 restricted antitumor CTL epitope peptide, can be directly combined with an MHC molecule without processing of APC, has the same effect as a natural endogenous peptide in activating an immune system, and is a fourteen peptide, and has the sequence as follows: AALVLTYLAVASA; the antitumor dominant CTL epitope peptide is free polypeptide, fusion polypeptide and chimeric polypeptide with the amino acid sequence; and various forms of the polymers described above as monomers. The present invention provides epitope peptides that bind to the binding site of a human HLA-A2 molecule and activate tumor-specific CTL cells. The HLA-A2 restrictive CTL epitope peptide provided by the invention can be used for developing therapeutic peptide vaccines for targeting tumors, and provides a theoretical basis for accurate immunotherapy of epithelioid malignant tumors.

Description

Tumor-associated antigen CTL epitope peptide and application thereof

Technical Field

The invention relates to the field of tumor immunotherapy, in particular to a CTL epitope peptide specific to HLA-A2 restricted ECM1 and application thereof, and in particular relates to a tumor specific T lymphocyte of an organism through tumor vaccine specific induction and stimulation activation, excitation and enhancement of an anti-tumor immune function of the organism and an application scheme thereof in specific anti-tumor immunotherapy.

Background

In recent years, with intensive research into the mechanism of immune response molecules, it has been increasingly recognized that immune cells of the body are not whole molecules corresponding to various pathogens or natural antigens, but rather epitopes against various antigen molecules, i.e., protein antigens, exhibit their immunological specificity through their epitopes. The current research shows that the target antigen identified by CD8+ T cells needs to be treated by antigen presenting cells, and then is presented on the surface of the antigen presenting cells or the target cells in the form of an antigen peptide-MHC-I molecule complex, and the corresponding antigen peptide combined with the MHC-I molecule is CTL epitope. The epitope peptide can activate and induce specific cytotoxic T lymphocytes (CTL cells), and has a certain killing effect on Human Leukocyte Antigen (HLA) matched tumors. The most common subtype of MHC class I in the Chinese population is HLA-A2. Thus, screening for HLA-A2 is limiting and recognizes CTL epitopes of ECM1, enabling targeted immunotherapy against HLa-matched malignant epithelial tumors.

Disclosure of Invention

Synthetic peptide vaccines are a new vaccine research direction that has emerged in recent years, and synthetic peptides can be directly combined with MHC molecules without processing of APCs, which have the same effect as natural endogenous peptides in activating the immune system, so synthetic peptide vaccines are widely used in antiviral immunotherapy. A variety of synthetic peptide vaccines based on polypeptide epitopes are currently in the clinical research stage or marketed. The CTL epitope peptide vaccine based on CTL epitope identification is safe, stable, has few toxic and side effects and greatly shortens the development period because the artificially synthesized polypeptide does not contain irrelevant components of pathogenic organisms and only induces specific CTL response. More importantly, multivalent CTL epitope peptide vaccines against one or more pathogens can also be designed based on a combination of multiple CTL epitopes developed by the CTL epitope. Therefore, the CTL epitope synthetic peptide vaccine has become a new strategy for the research of treating tumors, and the CTL epitope peptide specific to HLA-A2 restricted ECM1 has become the key of the research;

since binding of the CTL epitope to the Major Histocompatibility Complex (MHC) -class I molecule is a key factor for inducing CTL immune response, and an important basis for binding of the CTL epitope to the MHC-class I molecule is an anchor residue, the purpose of enhancing immunogenicity without affecting specificity can be achieved by changing the anchor residue or amino acid residues at adjacent positions thereto. The present invention provides a tumor-associated antigen-derived MHC-I restricted CTL epitope capable of inducing a peptide-specific CTL immune response, which is manifested by stimulating a high level of IFN-gamma secretion by the peptide-specific CTL and producing a specific killing effect on tumor cells;

the tumor-associated antigen dominant epitope peptide is trideceth, and the amino acid sequence is as follows: AALVLTYLAVASA

The targeted anti-tumor CTL dominant epitope peptide capable of specifically activating CTL in combination with MHC-I molecules can be obtained by artificial synthesis or expression and purification of prokaryotic cells or eukaryotic cells;

the anti-tumor CTL dominant epitope peptide is identified as effective epitope peptide according to the interaction mechanism of the epitope peptide and human HLA-A2 molecules and the characteristic that the epitope peptide can be effectively combined with the HLA-A2 molecules and can activate specific T lymphocytes, the length of the epitope peptide is 13 amino acid sequences, and the epitope peptide is easy to synthesize in vitro and convenient for clinical application;

the gist of the present invention is to provide epitope peptides which bind to the binding site of human HLA-A2 molecules and activate specific Cytotoxic T Lymphocytes (CTLs). The basic principle is as follows: analyzing the expression of E protein in various tumor cells by using bioinformatics, screening E protein epitope peptide by using the whole protein expression sequence of the E protein, verifying the theoretical binding capacity of the epitope peptide and human HLA-A2 molecules, and further verifying the binding force of the epitope peptide and the HLA-A2 molecules by using a T2 affinity experiment; then synthesizing by Fmoc solid-phase synthesis, purifying the epitope peptide by HPLC, and analyzing the purity and molecular weight by HPLC-MS; finally, using DC load epitope peptide to induce CTL cells, and observing the limitation of HLA-A2 molecules of the CTL cell immune killing target cells by mixed culture with tumor target cells of MHC-I molecular phenotype;

the invention has the beneficial effects that: the present invention provides epitope peptides capable of binding to the binding site of human MHC class I molecules and activating antigen-specific CTL cells. The accuracy of predicting epitope peptide is improved through a strategy combining bioinformatics means, a computer software simulation technology and a molecular biology experimental method; the epitope peptide is easy to synthesize in vitro, can induce and generate remarkable anti-tumor immune response, has the advantages of great commercial industrialization value and the like, and can be widely applied to the technical field of tumor immunotherapy.

Drawings

The invention will now be described in detail with reference to the accompanying drawings and examples.

FIG. 1 shows the results of the verification of the affinity of the epitope peptide for T2 cells.

FIG. 2 is a mass spectrometry diagram of the epitope peptide.

FIG. 3 is a high performance liquid chromatography analysis chart of the epitope peptide.

FIG. 4 shows the killing result of the epitope peptide on HLA-A.times.0201 positive breast cancer cell line MDB-MA-231.

Fig. 5 is the killing effect of the epitope peptide on HLA-A x 0201 breast cancer cell line BT 549.

Detailed Description

The tumor-associated antigen dominant CTL dominant epitope peptide can be combined with a binding site of a human HLA-A2 molecule, can activate specific cytotoxic T lymphocytes, and has the following amino acid sequence:

AALVLTYLAVASA

the human HLA-A2 restriction targeting anti-tumor CTL dominant epitope peptide can be obtained by artificial synthesis or expression and purification of prokaryotic cells or eukaryotic cells.

The anti-tumor CTL dominant epitope peptide is identified as effective epitope peptide according to the interaction mechanism of the epitope peptide and human HLA-A2 molecule and the characteristic that the epitope peptide can be effectively combined with the HLA-A2 molecule and can activate specific T lymphocytes, the length of the epitope peptide is 13 amino acid sequences, and the epitope peptide is easy to synthesize in vitro and convenient for clinical application.

The following examples will aid in the understanding of the present invention, but are merely illustrative of the invention and the invention is not limited thereto.

Example 1

Verification of epitope peptide affinity with human MHC-class I molecules:

1. through a supermotif method, the binding characteristics of the polypeptide sequence designed by the invention and the MHC-I molecule are verified.

Supermotif methods are based on peptide motifs consisting of identical or similar anchor residues in antigenic peptides taken up by HLA allotypes of the same or even different families of the Human Leukocyte Antigen (HLA) family, the anchor residues that the supermotif mainly acts on being the amino acid residue at position 2 (P2) and the amino acid residue at position 9 (P9) of the carboxyl terminus of the peptide chain. When the residues of P2 and P9 are A, I, L, M, V, T, the second position is aromatic amino acid, the ninth position is hydrophobic amino acid, and the amino acid has higher affinity with HLA-A2 molecules. The specific motifs of these amino acids are the active sites or key amino acids at which the polypeptide molecules bind to MHC-I molecules, and determine the activity profile of binding polypeptides to MHC-I molecules.

The hypermotif analysis of the polypeptide amino acid sequence was performed using http:// tools.

Table 1 shows that the supermotif method shows that the epitope peptide HLA-A2 molecule has good binding capacity;

table 2 is an abbreviation table for 20 amino acids.

TABLE 1 supermotif method for scoring binding of polypeptide amino acid sequence to HLA-A2

TABLE 2 amino acid abbreviations

T2 affinity assay to detect binding of epitope peptides to MHC-class I molecules

This example uses the characteristics of the T2 cell line to detect the affinity of the epitope peptide to human MHC class I molecules. T2 cells express MHC class I molecules, but cannot transport endogenous antigens due to defects in the antigen polypeptide Transporter (TAP) necessary for their endogenous antigen presentation pathway, and only empty HLA-A2 molecules can be presented on the cell surface, whereas empty HLA-A2 molecules are unstable on the cell surface, degrade quickly or return to the inside of the cell. However, the exogenously administered antigenic polypeptide, when combined with the HLA-A2 molecule into a complex, enhances the stability of the cell surface HLA-A2 molecule while remaining on the cell surface. The stronger the binding force of the antigenic peptide to the HLA-A2, the higher the expression level of the HLA-A2 molecule on the surface of the T2 cell. Therefore, the increase of the HLA-A2 molecular expression on the surface of the T2 cell treated by the antigen polypeptide can be detected, the binding force between the foreign antigen polypeptide and the HLA-A2 molecule and the binding stability can be intuitively reflected, and the effectiveness of the polypeptide can be further proved.

In the examples, T2 cells were washed 2 times with PBS and resuspended in serum-free IMDM medium and plated 1X 10 in 6-well plates ⁶ The density planting of the holes, after dissolving the antigen polypeptide by dimethyl sulfoxide (DMSO), adding the antigen polypeptide into the culture solution of T2 cells at the concentration of 50ug/ml, setting a blank control group and a positive control group, and adding 30ug/ml influenza virus matrix protein polypeptide into the positive control group. 3ug/ml human beta 2 microglobulin was added to each well. T2 cells 37 ℃,5% CO ₂ Culturing for 24 hours under saturated humidity. After the incubation, the T2 cells were centrifuged at 1000rpm for 5min, the pelleted cells were washed 3 times with ice PBS, then resuspended with 100u1 ice PBS, 5ul of murine anti-human HLA-A2.1-FITC mab was added, incubated at 4℃for 15min, the supernatant was centrifuged off and resuspended with 300u1 ice PBS,the Mean Fluorescence Intensity (MFI) was measured on a flow cytometer. Table 3 shows the results of the affinity experiments for epitope peptides and MHC-molecules.

The result takes the fluorescence coefficient (FI) as a measurement index, and the calculation formula is as follows: fluorescence coefficient (FI) = (polypeptide average fluorescence intensity- β2 microglobulin average fluorescence intensity)/β2 microglobulin average fluorescence intensity, criterion: FI >1.5 is high binding force of polypeptide and HLA-A2.1, 1.0< FI <1.5 is medium binding force, FI >0.5 is low binding force.

TABLE 3 results of affinity experiments of epitope peptides with MHC-I molecules

The experimental results show that: the CTL dominant epitope peptide has high binding force with MHC-I molecules

Example two

Synthesis, purification and molecular weight determination of epitope peptides:

the synthesis of the polypeptides was performed using a standard Fmoc protocol using an ABI43 IA-type polypeptide synthesizer manufactured by PE company, USA, and is briefly described below: and (3) extending a peptide chain from a carboxyl end to an amino end according to a polypeptide sequence, after synthesis, cutting by using TFA/DCM, drying an epitope peptide collection liquid to 1-2mL under reduced pressure at normal temperature, precipitating by using at least 50mL of precooled diethyl ether, and then filtering by suction to obtain a crude polypeptide product. The epitope peptide crude product obtained was dissolved with a small amount of DMS0, diluted with water to the desired volume at a concentration of 10mg/mL, filtered through a 0.22um fiber membrane, purified on Delta600 HPLC, product of Waters company, USA, and analyzed for purity. The mobile phase is selected from aqueous solution containing 0.1% TFA and acetonitrile solution containing 0.1% TFA. The purification of each peptide is carried out by selecting C18 preparation column

(Waters, USA, 7.0um,100A,7.8 mm. Times.150 mm), the purity of each peptide was analyzed using a C18 analytical column (Waters, USA, 5.0um,100A,3.9 mm. Times.150 mm). Relative molecular mass measurements of each purified polypeptide were performed on an API 2000 type (Waters company, usa) mass spectrometer in a conventional manner. The mass spectrum analysis chart is shown in fig. 2, the HPLC analysis chart is shown in fig. 3, and the theoretical molecular weight values of the CTL dominant epitope peptide are similar to the actual measurement values, and the result shows that the synthesis effect is good within the allowable range, and the CTL dominant epitope peptide can be used for the next experiment. The polypeptide is freeze dried and stored at-70 deg.c for further use.

Example III

Calcein (Calcein-AM) releases the targeted immune killing effect of the experimental epitope peptide on tumor cells:

1. preparation of effector cells

Mature human DCs were induced, washed with PBS, resuspended in serum-free IMDM medium, added with 50ug/ml epitope peptide, and incubated overnight at 37 ℃. DC were washed with PBS, counted, mitomycin C was added at a concentration of 30ug/ml and incubated at 37℃for 30min.

DC were washed with ice PBS, counted, co-cultured with the proliferating T cells at a ratio of 1:20, and 50U/ml IL-2 was added to stimulate the T cells. T cells were induced to cytotoxic T Cells (CTLs), i.e., effector cells, by 1 week stimulation 1 for 3 times.

2. Preparation of target cells

Resuscitates breast cancer cell MDA-MB-231 and epithelial cell BT-549, and cultures and passages normally. Both were positive for HLA-A 2.1.

3. Calcifenesin release assay

Labeling target cells with Calcein-AM at a concentration of 10mM, washing, and resuspension; co-culturing effector cells and labeled tumor cells for 4 hours at 37 ℃ according to the effective target ratio of 10:1, 20:1 and 40:1; then, the culture solution was collected and centrifuged at 1000rpm for 5 minutes, 100ul of the supernatant was taken to determine the average fluorescence intensity, and the pure target cells were used as the spontaneous release amount and the pure target cells plus detergent as the maximum release amount.

The killing rate of effector cells against target cells is expressed as cell lysis rate = (experimental release amount-spontaneous release amount)/(maximum release amount-spontaneous release amount). FIGS. 4 and 5 show the results of epitope peptide-induced targeted killing of tumor cells by effector cells.

Note that killing effect is shown in tables of killing rate (% omitted)

Experimental results show that the epitope peptide has the effect of specifically killing tumor cells.

Sequence listing

<110> Liaoning Chinese medical science and technology Co., ltd

<120> a tumor-associated antigen CTL epitope peptide and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13

<212> PRT

<213> human (homosapiens)

<400> 1

Ala Ala Leu Val Leu Thr Tyr Leu Ala Val Ala Ser Ala

1 5 10

Claims (3)

1. An anti-tumor CTL epitope peptide derived from a tumor-associated antigen, characterized in that: an antitumor dominant CTL epitope peptide derived from a tumor-associated antigen is HLA-A2 restrictive antitumor CTL epitope peptide, can be directly combined with an MHC molecule without processing of APC, has the same effect as a natural endogenous peptide in activating an immune system, and is tridecetide, and the sequence of the peptide is as follows: AALVLTYLAVASA.

2. The method for obtaining an anti-tumor CTL epitope peptide according to claim 1, wherein: the anti-tumor CTL dominant epitope peptide can be synthesized artificially by adopting a solid phase synthesis method or obtained by expression and purification of prokaryotic cells or eukaryotic cells.

3. Use of an anti-tumor CTL epitope peptide according to any one of claims 1 and 2 in the manufacture of a medicament for the treatment of breast cancer.