阅读说明：本技术 Hla-a1101限制性ecm1特异性ctl表位肽及其制备方法与用途 (HLA-A1101 restriction ECM1 specific CTL epitope peptide, preparation method and application thereof ) 是由 魏丽杰 于 2019-08-20 设计创作，主要内容包括：本发明为一种HLA-A1101限制性CTL表位肽,用于激活DC细胞产生或扩增特异性T细胞群。两条表位肽分别具有下列氨基酸序列为：QY-9,Gln Val Asn Cys Phe Asn Ile Asn Tyr；NK-9,Asn Ile Ser Ser Thr Ser Glu Pro Lys。该发明通过使用计算机预测,能与MHC-I类分子HLA-A1101特异性结合的,ECM1特异性CTL表位肽,并诱导其识别特异性肿瘤相关抗原的T细胞,增强了T细胞群治疗效应,而产生显著的抗肿瘤免疫反应,可有效应用于肿瘤免疫治疗技术领域中。(The invention relates to an HLA-A1101 restrictive CTL epitope peptide, which is used for activating DC cells to generate or amplify specific T cell groups. The two epitope peptides respectively have the following amino acid sequences: QY-9, Gln Val Asn Cys Phe Asn Ile Asn Tyr; NK-9, Asn Ile Ser Ser Thr Ser Glu Pro Lys. The invention predicts the specific combination of ECM1 specific CTL epitope peptide and MHC-I molecule HLA-A1101 by using a computer, induces the T cell of specific tumor-related antigen to be recognized, enhances the treatment effect of a T cell group, generates obvious anti-tumor immune response and can be effectively applied to the technical field of tumor immunotherapy.)

HLA-a 1101-restricted ECM 1-specific CTL epitope peptide, characterized by: can bind with the MHC-I molecule binding site of HLA-A1101+ patient to activate specific cytotoxic T lymphocyte. The two epitope peptides have the following amino acid sequences: QY-9, Gln Val Asn Cys Phe Asn Ile Asn Tyr; NK-9, Asn Ile Ser Ser Thr Ser Glu Pro Lys.

2. The method for obtaining the MHC class I restricted anti-tumor CTL epitope peptide according to claim 1, wherein the HLA-A1101 restricted ECM1 specific CTL epitope peptide can be obtained by artificial synthesis, expression and purification in prokaryotic cells or eukaryotic cells.

3. HLA-a 1101-restricted ECM 1-specific CTL epitope skin according to claim 1 and claim 2, characterized in that: the HLA-A1101 restriction ECM1 specific CTL epitope skin is free polypeptide, fusion polypeptide and chimeric polypeptide with the amino acid sequence in 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 an HLA-a 1101-restricted ECM 1-specific CTL epitope peptide according to any one of claims 1 to 2 for the manufacture of a medicament for the treatment of a tumor.

Technical Field

The invention belongs to the technical field of polypeptide in the technical field of tumor immunotherapy, and relates to an MHC-I-class restricted anti-tumor Cytotoxic T Lymphocyte (CTL) dominant epitope peptide and application thereof in preparation of anti-tumor drugs.

Background

Malignant tumors are the most serious public health problem in the world, with asia accounting for nearly 60% of 960 ten thousand cancer patients who die, and with an increasing annual incidence and mortality. Immunotherapy has been attracting much attention, and the establishment of therapeutic DC immune cell-mediated tumor immunotherapy studies based on antigen loading of Dendritic Cells (DCs) has been a hot spot in tumor immunotherapy. ECM1 is a secreted protein molecule closely related to the development of various malignant tumors, and HLA-A1101 (human leukocyte antigen) restrictive ECM 1-specific CTL epitope peptide can bind to the binding site of human MHC-class I (major histocompatibility complex) molecule and activate specific CTL.

The CTL epitope peptide-DC immune cell therapy developed in the project is to modify DC by using originally discovered tumor-associated antigen CTL epitope peptide, so that the DC has HLA-A1101 molecule to restrict and induce CD8⁺The T cell becomes CTL with memory and target killing of tumor cells, and can target killing of tumor cells with high expression of epitope peptide source protein. The project has good application prospect, has important theoretical significance and clinical practical value for improving the cure rate, life cycle and life quality of tumor cancer patients, and is expected to provide a new safe and effective clinical immunotherapy means for breast cancer and lung cancer patients.

Disclosure of Invention

The first purpose of the invention is to provide two HLA-A1101 restriction ECM1 specific CTL epitope peptides QY-9 and NK-9 which can effectively stimulate CTL-mediated specific cellular immune response and thus exert anti-tumor effect, and the amino acid sequences are respectively shown as SEQ No.1 and SEQ No. 2.

The second object of the present invention is to provide a method for producing an HLA-A1101-restricted ECM 1-specific CTL epitope peptide QY-9/NK-9, comprising the steps of:

(1) preparation of crude epitope peptide

Extending a peptide chain from a carboxyl end to an amino end according to an epitope peptide sequence, synthesizing, cutting by using TFA/DCM, drying epitope peptide collection liquid under reduced pressure at normal temperature, precipitating by using precooled ether, and performing suction filtration to obtain the polypeptide;

(2) purification of crude epitope peptide product

Dissolving the crude epitope peptide by using DMS0, diluting with water, filtering by using a 0.22um fiber membrane, purifying by using HPLC (high performance liquid chromatography), and performing purity analysis and relative molecular mass determination to obtain the surface peptide with the purity of more than 95%;

(3) preservation of surface peptides

The prepared surface peptide is subjected to freeze-drying treatment and then stored at-70 ℃ for later use.

In the HPLC purification, the mobile phase was selected from 0.1% TFA in water and 0.1% TFA in acetonitrile, and the surface peptide was purified using a C18 preparative column (Waters, U.S.A., 7.0um, 100A, 7.8 mm. times.150 mm).

For the purity analysis, C18 analytical column (Waters, USA, 5.0um, 100A, 3.9 mm. times.150 mm) was used.

The relative molecular mass determination is carried out conventionally on a mass spectrometer of the API type 2000 (Waters, USA).

The third purpose of the invention is to provide the application of the surface peptide QY-9 and NK-9 in preparing anti-tumor drugs.

The dosage form of the anti-tumor medicament is preferably injection.

The fourth purpose of the invention is to provide the application of the surface peptides QY-9 and NK-9 in the preparation of vaccines for detecting and preventing neoplastic diseases.

The HLA-A1101 restriction ECM1 specific CTL epitope peptides QY-9 and NK-9 can be obtained by artificial synthesis and purification through expression of prokaryotic cells or eukaryotic cells.

The invention has the beneficial effects that: (1) the present invention provides HLA-a 1101-restricted ECM 1-specific CTL epitope peptides capable of binding to MHC class I molecules with high affinity, stable complexes thereof inducing peptide-specific CTL immune responses, and stimulating peptide-specific CTLs to secrete high levels of IFN- γ, thereby producing specific killing effects on tumor cells.

(2) The invention discloses HLA-A1101 limiting CTL (cytotoxic T lymphocyte) dominant epitope peptide of a tumor-associated antigen, wherein CTL can stimulate peptide-specific CTL to secrete high-level IFN-gamma and induce specific CTL immune response, and the peptide has a good application prospect in the field of tumor immunotherapy.

(3) The HLA-A1101 restrictive ECM1 specific CTL epitope peptide provided by the invention is identified as an effective epitope peptide according to the interaction mechanism of the epitope peptide and MHC-I molecules and the characteristic of activating specific T lymphocytes, and meanwhile, the two epitope peptides are respectively 9 amino acid sequences in length, so that the epitope peptide is easy to synthesize in vitro and convenient for clinical application.

Drawings

FIG. 1 is a diagram of a spatial structure simulation of QY-9.

FIG. 2 is a simulation diagram of the spatial structure of NK-9.

FIG. 3 is a schematic diagram showing the spatial structure of the binding of QY-9 and MHC-I molecules.

FIG. 4 is a schematic diagram showing the spatial structure of NK-9 and MHC-I molecule binding.

FIG. 5 is a diagram of mass spectrometry of QY-9.

FIG. 6 is a diagram of mass spectrometry of NK-9.

FIG. 7 is a high performance liquid chromatography analysis chart of QY-9.

FIG. 8 is a high performance liquid chromatography analysis chart of NK-9.

FIG. 9 shows INF-gamma secretion changes of T lymphocytes before and after induction of QY-9 and NK-9 loaded DCs.

FIG. 10 shows INF- γ secretion by CTL induced by QY-9 and NK-9 load DC.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that: in the present invention, all embodiments and preferred methods mentioned herein may be combined with each other to form a new technical solution, if not specifically stated; in the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated; in the present invention, unless otherwise specified, the individual reactions or operation steps may be performed sequentially or may be performed in sequence; preferably, the reaction processes herein are carried out sequentially. Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art; in addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

According to one aspect of the present invention, there is provided an HLA-a 1101-restricted ECM 1-specific CTL epitope peptide represented by:

(1) a surface peptide consisting of the amino acid sequence shown in SEQ ID No. 1;

(2) a surface peptide consisting of the amino acid sequence shown in SEQ ID NO. 2.

Wherein SEQ ID NO.1 is Gln Val Asn Cys Phe Asn Ile Asn Tyr;

SEQ ID NO.2 is Asn Ile Ser Ser Thr Ser Glu Pro Lys.

The HLA-A1101 restrictive ECM1 specific CTL epitope peptide is designed and synthesized by a bioinformatics means, can be combined with a binding site of a human MHC-I molecule, can specifically activate cytotoxic T lymphocytes, and has a strong killing effect on tumor cells.

The HLA-A1101 restriction ECM1 specific CTL epitope peptide can be obtained by artificial synthesis, prokaryotic cell expression or eukaryotic cell expression and purification and the like.

In addition, the invention also provides application of the HLA-A1101 restriction ECM1 specific CTL epitope peptide in preparation of products for preventing and/or treating tumors.

The product is a vaccine or a medicament.

In some embodiments, the vaccine further comprises an adjuvant; the medicament also comprises a pharmaceutically acceptable carrier, and the preferred dosage form of the medicament is injection.

The HLA-A1101-restricted ECM1 specific CTL epitope peptide provided by the invention can be combined with MHC-I molecules with high affinity, a stable compound of the HLA-A1101-restricted ECM1 specific CTL epitope peptide can induce peptide-specific CTL immune response, and stimulate peptide-specific CTL to secrete high-level IFN-gamma, so that a specific killing effect is generated on tumor cells.

The basic principle of the invention is as follows: HLA-A1101 restrictive CTL epitope peptide sequences which may be present in the ECM1 protein sequence are predicted by using an IEDB database, and candidate sequences HK-9, SK-9, QY-9, KK-9 and NK-9 with IC50<500mM are selected. HLA-A1101 healthy volunteers were first sensitized with recombinant ECM1 protein in Peripheral Blood Mononuclear Cells (PBMC) and stimulated with candidate epitope peptides, and intracellular flow cytometry examined the secretion level of IFN-. gamma.with the most significant effect of QY-9, NK-9 in 5 candidate sequences.

The following examples may help to better understand the present invention, but are only for illustrative purposes and the present invention is not limited to the following examples.

Example 1 epitope peptides have been predicted for affinity to human MHC class I molecules.

The binding of The designed polypeptide sequences of The present invention to MHC-class I molecules was predicted by using IEDB (Immune Epitope Database, The Immune Epitope Database).

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 predict 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 provided in IEDB in combination with predictive analysis software (http:// tools. IEDB. org/mhci /).

IEDB prediction shows that the epitope peptide derived from ECM1 has good binding capacity with HLA-A1101 molecules; table 2 is an abbreviated list of 20 amino acids.

TABLE 1 IEDB Scoring binding of ECM1 dominant polypeptide amino acid sequence to HLA-A1101

Name (R)	123456789	Initiation of	Terminate	Percentage ranking	Ann method	Smm method
							QY-9	QVNCFNINY	495	503	1.4	141.64	266.87
NK-9	NISSTSEPK	530	538	1.6	195.72	267.49

TABLE 2 amino acid abbreviations Table

Name of Chinese	English name	Symbols and abbreviations	Name of Chinese	English name	Symbols and abbreviations
						Alanine	Alanine	A or Ala	Leucine	Leucine	L or Leu
Arginine	Arginine	R or Arg	Lysine	Lysine	K or Lys
						Asparagine	Asparagine	N or Asn	Methionine	Methionine	M or Met
Aspartic acid	Aspartic acid	D or Asp	Phenylalanine	Phenylalanine	F or Phe
						Cysteine	Cysteine	C or Cys	Proline	Proline	P or Pro
Glutamine	Glutamine	Q or Gln	Serine	Serine	S or Ser
						Glutamic acid	Glutamic acid	E or Glu	Threonine	Threonine	T or Thr
Glycine	Glycine	G or Gly	Tryptophan	Tryptophan	W or Trp
						Histidine	Histidine	H or His	Tyrosine	Tyrosine	Y or Tyr
Isoleucine	Isoleucine	I or Ile	Valine	Valine	V or Val

Binding of epitope peptides to human MHC class 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 structural state most suitable for molecular docking with a compound of HLA-A1101, and then exporting the optimized epitope peptide into a MOL2 format for three-dimensional docking with a binding groove of HLA-A1101. (2) Complexes of HLA-a1101 were prepared prior to molecular dynamics simulation. The initial coordinates of the HLA-A1101 are from a protein crystal structure-specific website http:// www.rcsb.org/pdb/, the obtained HLA-A1101 structure data are 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 pre-Docking structure modification (side chain repair and dehydration hydrogenation) on the HLA-A1101 structure, and after the structure modification is finished, three-dimensional information of a binding groove of the HLA-A1101 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-A1101 obtained in the columns of a Docking Model and a filing, selecting Mole2 File in the column of a Ligand Source, selecting optimized epitope peptide data in MOL2 format and with the lowest energy level, respectively Docking, and finally obtaining a Docking simulation score, and predicting the binding force of epitope peptide molecules and HLA-A1101 through the Docking simulation score. The results of computer modeling showed that QY-9, NK-9 and HLA-A1101 all bound well (see FIGS. 3 and 4).

Example 2 synthesis, purification and molecular weight determination of epitope peptides.

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 5 and figure 6, and an HPLC analysis chart is shown in figure 7 and figure 8, which shows that the molecular weight theoretical values of the CTL dominant epitope peptide are all similar to the measured values, the CTL dominant epitope peptide is within an allowable error range, the purity 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 3 the epitope peptide provided in example 2 of the present invention was loaded with IFN-. gamma.secretion of CTL induced by DC cells.

The method specifically comprises the following steps:

1. separation and preparation of peripheral blood mononuclear cells: adding the human lymphocyte separation solution into a centrifuge tube, and mixing the human lymphocyte separation solution according to the proportion of 1:2, adding the concentrated white blood cells of the HLA-A2 positive healthy volunteers at the upper part of the human lymphocyte separation liquid along the tube wall of the centrifuge tube. 2300r, centrifuging for 15min, sucking white membrane layer cells with 1ml microinjector into 10ml centrifuge tube, diluting with PBS, centrifuging for 5min at 1400r, collecting precipitate, re-suspending the cells with complete culture medium, and culturing in incubator at 37 deg.C and 5% CO2 saturation humidity. After culturing for 2 hours, the culture medium supernatant and the suspension cells are sucked, the non-adherent cells are washed by PBS, the non-adherent cells are collected and marked as lymphocytes, and the adherent cells are marked as monocytes.

2. Preparation of DC cells: the above monocytes were cultured in VIVO-X medium and supplemented with 2% fetal bovine serum, IL-4, GM-CSF. After 3 days, half the volume was changed and IL-4, GM-CSF was supplemented. And adding TNF-alpha while supplementing IL-4 and GM-CSF on the 7 th day of culture, and obtaining the induced mature human DC cells after 48 hours.

3. Preparation of lymphocytes: lymphocytes were cultured in T cell expansion medium supplemented with anti-CD 3 and anti-CD 28 mabs, IL-2 and IL-7.

4. Epitope peptide-loaded DC cells induced effector T lymphocytes: mature human DCs were washed with PBS and resuspended in serum-free IMDM media and 50ug/ml epitope peptide was added. After incubation at 37 ℃ overnight, mitomycin C was added at a concentration of 30ug/ml and incubated at 37 ℃ for 30 min.

DCs were washed with ice PBS, counted, co-cultured with lymphocytes at a 1:20 ratio, and IL-2 was added to stimulate T cells in PBMCs. T cells in PBMC were induced to become CTL by 1 stimulation 1 time for 1 week and 2 times in total.

5. The change process of the cell morphology of the T lymphocytes before and after induction was obtained by microscopic observation and stored by photographing, and the scale bar of the microscope was 200 ×, and the result is shown in fig. 9.

6. Intracellular flow cytometry is used for detecting IFN-gamma secretion of effector T cells after induction of epitope peptide loaded DC. First, a cell suspension is prepared, 5X 10⁵Per well, epitope peptide-loaded DC restimulated T lymphocytes. The activator activates cells at 37 ℃ with 5% CO₂Culturing for 4-6 h under the condition, staining cell surface antigens CD3 and CD8, fixing cells by 4% paraformaldehyde, breaking membranes, adding IFN-gamma-APC, and incubating for 30min at room temperature in a dark place. Flow cytometry detection (fig. 10).

As shown in FIG. 9, no aggregation of T lymphocytes was observed before induction, as observed by an inverted microscope; after the DC cells loaded with QY-9 and NK-9 induce T lymphocytes, the cells are obviously gathered around the DC cells, and the cell proliferation is obvious.

As shown in FIG. 10, CD8 was first selected by flow cytometry⁺ T lymphocytes. Next, CD8 in the control group, QY-9 group, NK-9 group was compared⁺ IFN-gamma secretion by T lymphocytes. The results showed that IFN-. gamma.was secreted at 46.7% in the control group, 70.54% in the QY-9 group, and 72.74% in the NK-9 group. The statistical results of the repeated experiments show that the IFN-gamma secretion of the QY-9 group is 1.509 times that of the control group, and the IFN-gamma secretion of the NK-9 group is 1.518 times that of the control group.

The experimental result shows that HLA-A1101 restriction CTL epitope peptide QY-9 and NK-9 load DC cells to induce T lymphocytes to generate CTL, IFN-gamma secretion is increased, and immune effect is enhanced.

According to the invention, by predicting, screening and identifying the advantageous polypeptide of ECM1, epitope peptides QY-9 and NK-9 which can be combined with HLA-A1101 binding site of human MHC-I molecules and can activate specific Cytotoxic T Lymphocytes (CTL) can be accurately screened, and CTL immune effects can be effectively activated, so that the effects of treating tumors, preparing vaccines for clinical treatment and detection of neoplastic diseases and the like can be achieved.

Sequence listing

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

<120> LA-A1101 restriction ECM1 specific CTL epitope peptide, and preparation method and application thereof

<140> 2019107669743

<141> 2019－08－20

<160> 2

<170> SIP0SequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> human (Homo sapiens)

<400> 1

Gln Val Asn Cys Phe Asn Ile Asn Tyr 9

<210> 2

<211> 9

<212> PRT

<213> human (Homo sapiens)

<400> 2

Asn Ile Ser Ser Thr Ser Glu Pro Lys 9