阅读说明：本技术 Hla-a0201限定性kif15特异性抗肿瘤ctl优势表位肽及应用 (HLA-A0201 limiting KIF15 specific anti-tumor CTL (cytotoxic T lymphocyte) dominant epitope peptide and application thereof ) 是由 魏敏杰 于 2019-08-19 设计创作，主要内容包括：一种HLA-A0201限定性KIF15特异性抗肿瘤CTL优势表位肽,为HLA-A2限制性抗肿瘤CTL表位肽,能与人HLA-A2分子的结合位点进行结合,为九肽,其序列为：Leu-Leu-Asp-Ser-Ala-Ser-Ala-Gly-Leu。本发明的能特异性诱导CTL的优势表位肽是使用配体组学、计算机模拟软件及药效学实验相结合的方法筛选得到,该短肽体外容易合成,能够诱导产生显著抗肿瘤免疫反应,具有非常大的开发应用价值,应用于肿瘤免疫治疗技术领域中。(An HLA-A0201 restrictive KIF15 specific anti-tumor CTL dominant epitope peptide is an HLA-A2 restrictive anti-tumor CTL epitope peptide, can be combined with a binding site of a human HLA-A2 molecule, is a nonapeptide, and has the sequence: Leu-Leu-Asp-Ser-Ala-Ser-Ala-Gly-Leu. The dominant epitope peptide capable of specifically inducing CTL is obtained by screening by using a method combining ligand omics, computer simulation software and pharmacodynamics experiments, is easy to synthesize in vitro, can induce and generate remarkable anti-tumor immune response, has very large development and application values, and is applied to the technical field of tumor immunotherapy.)

1. An anti-tumor dominant CTL epitope peptide derived from a tumor-associated antigen KIF15, which is characterized in that: the anti-tumor dominant epitope peptide has the specific binding with human HLA-A2 molecules, activates specific cytotoxic T lymphocytes, and has the sequence of Leu-Leu-Asp-Ser-Ala-Ser-Ala-Gly-Leu.

2. The HLA-a 2-restricted KIF 15-specific CTL epitope peptide of claim 1, wherein: the HLA-A2-restricted KIF 15-specific CTL epitope peptide is a mutant peptide consisting of an amino acid sequence derived from the amino acid sequence shown in claim 1 by adding, deleting or substituting one or more amino acids.

3. HLA-a 2-restricted ECM 1-specific CTL epitope peptides according to claim 1 and claim 2, characterized in that: the HLA-A2-restricted KIF 15-specific CTL epitope peptide is a free polypeptide, a fusion polypeptide and a chimeric polypeptide with the amino acid sequence of claim 1 and claim 2; and polymers in which one of the above-mentioned polypeptides is a monomer in various forms.

4. The method for obtaining an anti-tumor CTL dominant epitope peptide according to claim 1, claim 2, and claim 3, wherein: the anti-tumor CTL dominant epitope peptide can be artificially synthesized by adopting a solid phase synthesis method or obtained by expression and purification of prokaryotic cells or eukaryotic cells.

5. Use of the anti-tumor CTL epitope peptide according to any one of claims 1 to 4 for preparing a medicament for treating tumors.

Technical Field

The invention belongs to the technical field of polypeptides in the technical field of tumor immunotherapy, and particularly relates to an MHC-I (major histocompatibility complex) -class restricted anti-tumor CTL (cytotoxic T lymphocyte) dominant epitope peptide and application thereof in preparation of anti-tumor drugs.

Background

In recent years, with the intensive study of the molecular mechanism of immune response, it has been intensively recognized that immune cells of the body are not integral molecules against various pathogens or natural antigens, but are directed against epitopes of various antigenic molecules. At present, the role of Cytotoxic T Lymphocytes (CTL) in tumor suppression is greatly regarded, antigens recognized by CD8+ T cells need to be treated by antigen-presenting cells, and then presented on the surface of the antigen-presenting cells or target cells in the form of "antigen peptide-MHC class I molecule" complexes, and the corresponding antigen peptides bound to MHC class I molecules are CTL epitope peptides. The epitope peptide can activate and induce specific CTL cells and has a certain killing effect on tumors successfully matched with human leukocyte antigen HLA. Therefore, research and development of an anti-tumor CTL epitope peptide are new issues to be solved.

Disclosure of Invention

The invention aims to find the CTL dominant epitope peptide which effectively stimulates the specific cellular immune response mediated by CTL and plays the role of anti-tumor effect. The invention utilizes the polypeptide obtained by the ligand omics method to predict, screen and identify, and precisely screens the epitope peptide which can be combined with the binding site of human HLA-A2 molecule and can activate specific Cytotoxic T Lymphocyte (CTL), thereby effectively killing tumor cells and achieving the purpose of treating tumors, and the application of the epitope peptide in preparing the vaccine for clinical treatment and detecting neoplastic diseases.

The implementation method of the invention comprises the following steps: the invention provides an MHC-I restricted CTL epitope derived from tumor associated antigen, which can be combined with MHC-I molecules with high affinity, and the formed complex is stable, can induce peptide specific CTL immune response, and shows that the stimulatory peptide specific CTL secretes high-level IFN-gamma and generates specific killing effect on tumor cells.

The tumor-associated antigen dominant epitope peptide is nonapeptide, and the amino acid sequence of the nonapeptide is as follows: Leu-Leu-Asp-Ser-Ala-Ser-Ala-Gly-Leu.

The MHC-I restricted targeting anti-tumor CTL dominant epitope peptide can be obtained by artificial solid phase synthesis or purification through expression of prokaryotic cells or eukaryotic cells.

The anti-tumor CTL dominant epitope peptide is identified as an 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 HLA-A2 molecules and can activate specific T lymphocytes, and the epitope peptide is only 9 amino acid sequences in length, is easy to synthesize in vitro and is convenient for clinical application.

The key points of the invention are as follows: provides epitope peptides capable of binding to the binding site of human HLA-A2-class molecule and activating specific Cytotoxic T Lymphocytes (CTL). The basic principle is as follows: the method comprises the following steps of (1) accurately screening ligand omics to obtain a difference peptide segment, verifying the theoretical binding capacity of epitope peptide and human HLA-A2 molecules by the bioinformatics principle, and further verifying the binding force of the epitope peptide and HLA-A2 molecules by computer simulation in combination with a T2 affinity experiment; then epitope peptide is synthesized by Fmoc solid phase synthesis method and purified by HPLC, and the purity and molecular weight of the epitope peptide are analyzed by HPLC-MS; finally, the DC loaded epitope peptide is used for inducing CTL cells, and the limitation of HLA-A2 molecules of the target cells for the immunological killing of the CTL cells is observed by mixed culture with tumor target cells with MHC-I molecular phenotype.

The invention has the beneficial effects that the invention discloses the natural CTL dominant epitope peptide derived from tumor-associated antigen, the CTL dominant epitope peptide can induce the specific CTL immune response of the peptide, the expression is that the peptide-specific CTL can be stimulated to secrete high-level IFN-gamma and generate specific killing effect on the positive tumor cells of the epitope peptide source protein, and the invention has good application prospect in the field of tumor immunotherapy.

Drawings

The invention is described in detail below with reference to the figures and examples.

FIG. 1 is a schematic diagram showing the spatial structure of the epitope peptide.

FIG. 2 is a schematic diagram showing the spatial structure of the epitope peptide bound to human HLA-A2 molecule.

FIG. 3 is a space electron cloud density chart of the binding of the epitope peptide and human HLA-A2 molecule.

FIG. 4 is a diagram of mass spectrometry analysis of the epitope peptide.

FIG. 5 is a high performance liquid chromatography analysis chart of the epitope peptide.

FIG. 6 shows the killing effect of the epitope peptide-induced effector cells on breast cancer MDA-MB-231 cells and breast epithelial cells.

Detailed Description

The following examples will help to understand the present invention, but they are only for illustrative purposes and the present invention is not limited to these contents.

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

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

The anti-tumor CTL dominant epitope peptide is identified as an 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 HLA-A2 molecules and can activate specific T lymphocytes, and the epitope peptide is only 9 amino acid sequences in length, is easy to synthesize in vitro and is convenient for clinical application.

The following examples will help to understand the present invention, but they are only for illustrative purposes and the present invention is not limited to these contents.

Example one

The CTL dominant epitope peptide is obtained by utilizing ligand omics screening.

The epitope peptide which is compatible with HLA-A2 is obtained by affinity chromatography, the structure of the epitope peptide is confirmed by LC/MS, the source protein of the epitope peptide is confirmed by NCBI Blast, and the dominant CTL epitope peptide derived from KIF15 is obtained by screening.

And (3) performing affinity verification on the epitope peptide and human MHC-I molecules.

The binding property of the designed polypeptide sequence and MHC-I molecules is verified by a hyper-motif method.

1. The super-motif method is based on peptide motifs in which antigenic peptides taken up by HLA allotypes in the same Human Leukocyte Antigen (HLA) family, or even in different HLA families, have identical or similar anchor residues, the anchor residues for the main function of the super-motif 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 residues of P2 and P9 are A, I, L, M, V, T, and the second position is aromatic amino acid and the ninth position is hydrophobic amino acid, 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 of the polypeptide molecule to which the MHC-I molecule binds, and determine the activity profile of the polypeptide in binding to the MHC-I molecule.

Although the method for predicting CTL epitopes by the hyper-motif method overcomes the false negative result easily generated by the conventional simple motif method, the prediction method has the same weakness as the motif method, namely the influence of residues at two and nine positions is only considered in the prediction, and the residue combination condition of other positions is neglected, so that the false positive result is easily generated, and the accuracy of CTL epitope prediction is improved by combining the quantitative motif method (Table 1). Table 1 shows that the epitope peptide HLA-A2 molecule has good binding capacity by the super-base sequence method; table 2 is an abbreviated list of 20 amino acids.

TABLE 1 super motif method scores for binding of polypeptide amino acid sequences to HLA-A2.

Name (R)	123456789	Super motif method
			CTL dominant epitope peptide	LLDSASAGL	25

2. HLA-A2 restricted analysis of KIF15 dominant epitope peptide was performed using the analysis software provided by the internationally recognized epitope peptide prediction website http:// tools. iedb. org/mhci, with a percent rank <2 and IC50<50nM, with high affinity (Table 2).

IEDB is a published experimental data for providing the public of the scientific community with relevance to immune epitope recognition, which records experimental data for antibodies and T cell epitopes studied in human, non-human primate and other animal species in infectious disease, allergy, autoimmune and transplantation settings. IEDB also provides tools to aid in the prediction and analysis of epitopes. IEDB as a public and reliable experimental database can be used to test the recognition of immune epitopes by adaptive immune receptors.

Analysis of the amino acid sequence of a polypeptide can be accomplished by using MHC class I molecules as provided in IEBD in combination with predictive analysis software (http:// tools. iedb. org/mhci /).

IEDB prediction shows that the epitope peptide HLA-A0201 molecule has good binding capacity; table 3 is an abbreviated list of 20 amino acids.

TABLE 2 IEDB Scoring binding of polypeptide amino acid sequences to HLA-A1101

Name (R)	123456789	Percentage ranking	Ann_IC50	Smm_IC50
					KIF15	LLDSASAGL	1.6	48.34	94.43

TABLE 3 amino acid abbreviations Table

Binding of epitope peptides to human MHC class I molecules was in silico.

Establishing two-dimensional and three-dimensional structures of the epitope peptide by using ChemDraw Ultra and ChemDraw 3D Ultra in a ChemOffice software package; modifying the energy and structure of the epitope peptide and HA-A2.1 by MOE software, simulating the three-dimensional structure of the combination of the epitope peptide and HA-A2.1, and performing molecular dynamics combination simulation and estimation of physiological activity and application value, wherein the method mainly comprises the following steps of (1) constructing an epitope peptide molecular model. Constructing a two-dimensional structure of an epitope peptide Molecule by using ChemDraw Ultra in a ChemOffice software package, introducing the two-dimensional structure of the epitope peptide into ChemDraw 3D Ultra to obtain a three-dimensional model, storing the three-dimensional model in a Mol2 format, introducing the three-dimensional structure into an MOE software in a docking simulation link, sequentially selecting a pull-down menu computer-Minimize-Molecule, and generating a Minimize dialog box to optimize the energy level of the polypeptide Molecule to enable the energy level to reach the minimum energy and structure state most suitable for molecular docking with a compound of HLA-A2.1, and then exporting the optimized epitope peptide into a MOL2 format for three-dimensional docking with a binding groove of HLA-A2.1. (2) Complexes of HLA-A2.1 were prepared prior to molecular dynamics simulation. The initial coordinate of HLA-A2.1 is from a protein crystal structure exclusive website http:// www.rcsb.org/pdb/, the obtained HLA-A2.1 structure data is imported into MOE software, pull-down menu Applications-Docking Suite-Docking Ligands are sequentially selected, a Docking dialog box appears, Define is selected on the right side of a Filename column to carry out structural modification (side chain repair, dehydration and hydrogenation) on the HLA-A2.1 structure before Docking, and after the structural modification is completed, three-dimensional information of a binding groove of HLA-A2.1 is obtained through analysis and stored for Docking with epitope peptide. (3) And (5) constructing a docking model. Sequentially opening Applications-Docking Suite-Docking Ligands in MOE software, generating a Docking dialog box, selecting three-dimensional information of a binding groove of HLA-A2.1 obtained in columns of a Docking Model and a filing Model, selecting Mole2 File in a column of a Ligand Source, selecting optimized epitope peptide data in a MOL2 format and with the lowest energy level, respectively Docking and finally obtaining a Docking simulation score, estimating the binding force of epitope peptide molecules and HLA-A2.1 through the Docking simulation score, and because the binding force between the epitope peptide molecules and HLA-A2 molecules is mainly recognized through weak interactions such as hydrophobic interaction, H bond, salt bond and the like, the stronger the secondary bond interactions are, the tighter the binding is. The results of computer simulation show that the CTL epitope peptide can be well combined with HLA-A2.1 (shown in figure 2).

Example two

Synthesizing and purifying epitope peptide and measuring molecular weight.

The synthesis of the polypeptide was carried out using a standard Fmoc protocol using an ABI43IA model polypeptide synthesizer, manufactured by PE, USA, as follows: extending a peptide chain from a carboxyl terminal to an amino terminal according to a polypeptide sequence, after synthesis, selecting TFA/DCM for cutting, drying epitope peptide collection liquid to 1-2mL under reduced pressure at normal temperature, then precipitating with at least 50mL of precooled ether, and then carrying out suction filtration to obtain a crude polypeptide product. The crude epitope peptide obtained was dissolved in a small amount of DMS0, diluted to the desired volume with water at a concentration of 10mg/mL, filtered through a 0.22um fiber membrane, purified by HPLC on model Delta600 from Waters, USA, and analyzed for purity. The mobile phase was selected from 0.1% TFA in water and 0.1% TFA in acetonitrile. Purification of each peptide was performed by using a C18 preparative column (Waters, U.S.A., 7.0um, 100A, 7.8 mm. times.150 mm) and a C18 analytical column (Waters, U.S., 5.0um, 100A, 3.9 mm. times.150 mm) for purity analysis of each peptide. The relative molecular mass of each purified polypeptide is determined on an API 2000 (Waters corporation) mass spectrometer according to a conventional method, a mass spectrum analysis chart is shown in figure 4, and an HPLC analysis chart is shown in figure 5, which shows that the molecular weight theoretical value of the CTL dominant epitope peptide is similar to the measured value, the CTL dominant epitope peptide is within an allowable error range, the purity of the CTL dominant epitope peptide is more than 95%, and the CTL dominant epitope peptide is good in synthesis effect and can be used for the next experiment. The polypeptide is lyophilized and stored at-70 deg.C for use.

EXAMPLE III

The T2 affinity assay detects the binding of epitope peptides to MHC class I molecules.

This example utilizes the characteristics of the T2 cell line to detect the affinity of epitope peptides for human MHC class I molecules. T2 cells express MHC class I molecules but are unable to transport endogenous antigens due to a deficiency in the antigen polypeptide Transporter (TAP) essential for the endogenous antigen presentation pathway, presenting only the unloaded HLA-A2 molecule on the cell surface, while the unloaded HLA-A2 molecule is unstable on the cell surface and degrades quickly or returns to the interior of the cell. However, the exogenous antigen polypeptide can enhance the stability of HLA-A2 molecules on the surface of cells after being combined with HLA-A2 molecules to form a complex, and the complex is left on the surface of the cells. The stronger the binding force between the antigen peptide and HLA-A2, the higher the expression level of HLA-A2 molecules on the surface of T2 cells. Therefore, the increase of the expression level of the HLA-A2 molecules on the surface of the T2 cell treated by the antigen polypeptide can be detected, the binding force and the binding stability of the foreign antigen polypeptide and the HLA-A2 molecules can be intuitively reflected, and the effectiveness of the polypeptide can be further proved.

In the example, T2 cells were washed 2 times with PBS, resuspended in serum-free IMDM medium, and plated at a density of 1 × 106/well on a 6-well plate, and after the antigenic polypeptide was dissolved in dimethyl sulfoxide (DMSO), the antigenic polypeptide was added to the culture medium of T2 cells at a concentration of 50ug/ml, and a blank control and a positive control were set, and 30ug/ml of the influenza virus matrix protein polypeptide was added to the positive control. 3ug/ml human beta 2 microglobulin was added to each well. T2 cells were cultured at 37 ℃ under 5% CO2 and saturated humidity for 24 hours. After the incubation, the T2 cells were centrifuged at 1000rpm for 5min, the pelleted cells were washed 3 times with ice PBS, then resuspended in 100u1 ice PBS, 5ul mouse anti-human HLA-a2.1-FITC monoclonal antibody was added, incubated at 4 ℃ for 15min, centrifuged to discard the supernatant, resuspended in 300u1 ice PBS, and the Mean Fluorescence Intensity (MFI) was measured on a flow cytometer. Table 3 shows the results of the affinity test between 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- β 2 microglobulin average fluorescence intensity)/β 2 microglobulin average fluorescence intensity, criterion: FI >1.5 is high binding force between polypeptide and HLA-A2.1, FI <1.5 of 1.0 is medium binding force, FI >0.5 is low binding force.

Table 4 results of affinity experiments of epitope peptides with MHC-I molecules.

Antigenic polypeptides	Mean Fluorescence Intensity (MFI)	Fluorescence coefficient (FI)
			Beta 2 microglobulin	5304	——
Positive control	20917	2.94
			CTL dominant epitope peptide	19682	2.71

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

Example four

The calcium flavin (Calcein-AM) releases the target immune killing effect of the experimental epitope peptide on the tumor cells.

1. Preparation of Effector cells

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

After washing with ice PBS, the DCs were counted, co-cultured with T cells in the propagation phase at a ratio of 1:20, and 50U/ml IL-2 was added to stimulate the T cells. T cells were induced into cytotoxic T Cells (CTL), i.e., effector cells, 1 stimulation and 3 stimulations for 1 week.

2. And preparing target cells. .

Recovering normal culture and passage of breast cancer cell MDA-MB-231 and epithelial cell MCF-10A. Both were positive for HLA-A2.1.

3. Calcein release experiments.

Labeling target cells with Calcein-AM with the concentration of 10mM, washing and resuspending; co-culturing the effector cells and the marked tumor cells for 4 hours at 37 ℃ according to the effective target ratio of 10:1, 20:1 and 40: 1; then collecting the culture solution, centrifuging at 1000rpm for 5min, taking 100ul of supernatant to determine the average fluorescence intensity, and taking the pure target cells as the spontaneous release amount and taking the pure target cells and detergent as the maximum release amount.

The killing rate of effector cells against target cells is expressed as a cell lysis rate = (experimental release amount-spontaneous release amount)/(maximum release amount-spontaneous release amount). FIG. 6 is the result of the target killing of tumor cells by the effector cells induced by the epitope peptide.

Note that killing is reported as the kill rate (% omitted).

The experimental result shows that the epitope peptide has the effect of specifically killing tumor cells.

<110> Liaoning Zhongjian medicine science and technology Co., Ltd

<120> HLA-A0201 limiting KIF15 specific anti-tumor CTL dominant epitope peptide and application thereof

<140> 2019107681035

<141> 2019－08－20

<160> 1

<170> SIP0SequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> human (Homo sapiens)

<400> 1

Leu Leu Asp Ser Ala Ser Ala Gly Leu 9