CN114085281A

CN114085281A - Tumor antigen epitope peptide, polymer and application thereof

Info

Publication number: CN114085281A
Application number: CN202111202861.4A
Authority: CN
Inventors: 张强; 宋瑾; 任树成; 沈宁; 张恒辉
Original assignee: Beijing Zhenzhi Medical Technology Co ltd
Current assignee: Beijing Zhenzhi Medical Technology Co ltd
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2022-02-25

Abstract

The present invention is in the fields of molecular biology and immunology. The amino acid sequence of the tumor epitope peptide is shown in any one of SEQ ID NO.1, 3-10, 12, 13, 16-18 and 20-27; or the amino acid sequence of the tumor antigen epitope peptide has more than 90 percent of similarity with the sequence shown in any one of SEQ ID NO.1, 3-10, 12, 13, 16-18 and 20-27 through amino acid substitution, deletion or addition; or the amino acid sequence of the tumor antigen epitope peptide comprises a sequence shown in any one of SEQ ID NO.1, 3-10, 12, 13, 16-18 and 20-27 or a sequence which has more than 90% similarity with the sequence shown in any one of SEQ ID NO.1, 3-10, 12, 13, 16-18 and 20-27 through amino acid substitution, deletion or addition. The antigen peptide of the polymer is the tumor antigen epitope peptide. On one hand, the invention provides a thought for clinical adoptive immunotherapy of cancer, and can better serve the research and development of drugs and the patients with cancer.

Description

Tumor antigen epitope peptide, polymer and application thereof

Technical Field

The invention belongs to the field of molecular biology and immunology, and particularly relates to a tumor epitope peptide, a polymer thereof and application thereof.

Background

The identification and analysis of immune cells currently available in the art mainly includes the following major techniques:

enzyme-linked immunosorbent spot (Elispot) assay

As shown in FIG. 1, the Elispot plates have been coated with a cytokine-specific primary antibody and the exogenously added antigen is presented by the presenting cells in the Elispot plates to the T cells to be tested. After incubation, specific T cells produce effector cytokines by recognizing the corresponding specific antigen, and cytokines are captured by the coated antibody and can ultimately be detected in the form of spots. The Elispot assay can provide information on the frequency of immune responses to a specific antigen, cytokine profile, and specificity of the antigen.

Elispot can only reflect the amount of killer immune cells by the amount of secreted cytokines and cannot determine which cell subset secretes the cytokines, i.e. cannot distinguish T cells from other cells in the sample, such as NK. Since Elispot detects cytokine-secreting cells and makes it difficult to perform phenotypic analysis simultaneously, T cells with specificity cannot be identified and the number of each cytokine-secreting T cell is difficult to quantify. Elispot can only identify specific immune cells generated by antigen stimulation, and cannot sort specific immune killer T cells, so that specific T cells with high purity cannot be obtained.

Tetramer analysis technique

As shown in fig. 2, the antigen is ubiquitinated and degraded in the APC, pMHC is assembled and secreted, and finally expressed on the cell membrane of the APC in the form of pMHC complex, the TCR expressed by the T cell can recognize and bind to pMHC on the surface of the APC, and then transmits an activation signal into the T cell, and the activated CTL kills the target cell by recognizing MHC class I molecules having antigen peptides bound to the surface of the target cell. At the in vitro level, the antigen peptide can be recognized by TCR on the surface of T cells after being combined with MHC-I to form a monomer complex, but the activity of the monomer complex recognized by the T cells is limited, and the pMHC monomer complex is prepared into a polymer which can effectively enhance the capability of being recognized by the T cells. The Tetramer of pMHC can be prepared by labeling the alpha 3 terminus of MHC-I heavy chain with biotin in a mode that 4 moles of biotin can be bound to 1 mole of streptavidin, and recruiting and capturing the pMHC-biotin monomer complex using streptavidin coupled with fluorescein.

The pMHC tetramer technology can mark specific T cells as target detection cells on a single cell level, analyze the cells through a flow cytometry technology, can quickly detect the antigen specific T cells, can simultaneously carry out qualitative and quantitative analysis on the antigen specific T cells, and can separate the antigen specific T cells through flow screening, thereby obtaining the specific T cells with higher purity.

Antigen recognition patterns and characteristics of T cells:

the method comprises the following steps: naive T cells do not recognize antigen directly and require antigen presenting cell processing. Due to the characteristics of double recognition of T cells, the research on the specificity of the T cells needs to consider the properties of the antigen peptide and the MHC molecular type of the presented antigen peptide. That is, the initial α β T cells need to complete recognition of the presented antigen by recognizing and binding to both the polypeptide and MHC in the pMHC complex, and subsequently activate the T cell's immune killing activity.

The method is characterized in that: the α β T fine recognition antigen is MHC restricted.

Because of the low affinity of TCR, and the rapid dissociation from the pMHC complex, monomeric pMHC complexes are difficult to use for detecting antigen-specific T cells. Tetramer technology was developed for the detection of antigen-specific T cells. The tetramer enhances the affinity of the TCR, allowing accurate detection of antigen-specific T cells in vitro using flow cytometry. The Tetramer of pMHC can be prepared by labeling the alpha 3 terminus of MHC-I heavy chain with biotin in a mode that 4 moles of biotin can be bound to 1 mole of streptavidin, and recruiting and capturing the pMHC-biotin monomer complex using streptavidin coupled with fluorescein. 4 pMHC monomers in the tetramer can be combined with specific T cell receptors, so that the affinity activity is effectively enhanced.

The application value of the identification of the antigen-specific T cells is the potential application in the tumor immunotherapy (screening and preparation of DC-CTL and TCR-T)

The tumor evading immune killing is usually caused by the fact that T cells cannot effectively recognize tumor-associated antigens or tumor-specific antigens, so that the effect of killing the tumor cells again by the T cells can be achieved by stimulating or modifying the T cells to recognize the tumor antigens.

DC-CTL therapy is an adoptive immunotherapy method of presenting antigen peptides to T cells and activating killer T Cells (CTLs) by Dendritic Cells (DCs) having the strongest antigen presenting ability, and then the CTLs kill target cells. The specificity of the antigen peptide presented by DC determines the specific killing effect of CTL on tumor. The antigenic peptide herein is a neoantigen encoded by a mutant gene of a tumor cell, a novel abnormal protein different from a protein expressed by a normal cell, mainly produced by a point mutation of a gene, etc. Polypeptide fragments formed by enzymolysis of the proteins can be presented to T cells as antigen peptides, and can promote the T cells to become mature activated T cells which specifically recognize tumor neoantigens.

TCR-T therapy is another adoptive immune cell therapy, and is characterized in that the TCR of the T cell is modified to specifically recognize and process presented antigens, and the TCR-T has a broader spectrum in target selection. The T lymphocyte can be improved in the capacity of specifically recognizing tumor-associated antigens by modifying the TCR, so that the T lymphocyte can efficiently recognize specific target cells. The tetramers prepared based on the novel antigens herein can be used to efficiently identify engineered TCR-ts, thereby preliminarily assessing the effectiveness of TCR-ts.

The second is the application in tumor immunotherapy with diagnosis (combined with other phenotypic and functional markers).

Adoptive immunotherapy approaches enhance the immune killing activity by ex vivo engineering of T cells and reinfusion back into the patient, with sustained killing activity dependent on the high survival of the T cells after reinfusion. Further confirmation of whether specific killer T cells remain in the patient after adoptive immunotherapy and whether the T cells in the fraction have infiltrated tumor tissue is necessary. Tetramers prepared based on tumor neoantigens can be used to track CTLs or TCR-ts that specifically recognize these antigens, to reflect whether sustained immune killing is still present in the patient and to determine the distribution of this fraction of T cells. Therefore, the Tetramer technique can serve well for the concomitant diagnosis after adoptive immunotherapy of tumors, more closely reflecting the activity of specific killing in vivo.

The T cell epitope is a peptide segment with special functions on a T cell antigen, and can be specifically recognized by T cells, so that the immune response of the T cells is stimulated. The epitope peptide therefore comprises a peptide fragment of all epitopes on an antigen, i.e. the epitope is part of the antigen peptide. Based on the above principle, T cells, when bound to an antigen, do not recognize the entire antigen, but rather primarily recognize linear epitopes on the antigenic molecule. However, there are many epitopes on an antigen, so it is crucial to determine which epitope on the antigen the T cell can perform the best antigen recognition and produce the strongest killing effect.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a tumor epitope peptide, a polymer thereof and application thereof. The invention screens out the antigen peptide with tumor specificity; adoptive immunotherapy DC-CTL or TCR-T can be developed based on these antigen peptides; multimers (such as but not limited to tetramers) are prepared based on these antigenic peptides to detect the antigen specificity of DC-CTL or TCR-T, while providing concomitant diagnosis for clinical treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

the tumor epitope peptide comprises a tumor neogenesis epitope peptide or a tumor related epitope peptide; wherein the content of the first and second substances,

the amino acid sequence of the tumor neogenesis antigen epitope peptide is shown in any one of SEQ ID NO.1, 3-10 and 12; or the amino acid sequence of the tumor neoantigen epitope peptide has more than 90 percent of similarity with the sequence shown in any one of SEQ ID NO.1, 3-10 and 12 through amino acid substitution, deletion or addition; or the amino acid sequence of the tumor neoantigen peptide comprises a sequence shown in any one of SEQ ID NO.1, 3-10 and 12 or a sequence which has more than 90% similarity with the sequence shown in any one of SEQ ID NO.1, 3-10 and 12 through amino acid substitution, deletion or addition.

The amino acid sequence of the tumor-associated epitope peptide is shown in any one of SEQ ID NO.13, 16-18 and 20-27; or the amino acid sequence of the tumor neogenesis and related antigen epitope peptide has more than 90 percent of similarity with the sequence shown in any one of SEQ ID NO.13, 16-18 and 20-27 through amino acid substitution, deletion or addition; or the amino acid sequence of the tumor-associated antigenic peptide comprises a sequence shown in any one of SEQ ID NO.13, 16-18 and 20-27 or a sequence which has more than 90% similarity with the sequence shown in any one of SEQ ID NO.13, 16-18 and 20-27 through amino acid substitution, deletion or addition.

The tumor neoantigen epitope peptide is a peptide fragment containing the mutation sites defined on the basis of the gene mutation in the tumor cells to cause the tumor cells to be different from normal cells, and the tumor-associated antigen is a peptide fragment related to some genes defined on the basis of the abnormal expression of the genes in the tumor cells relative to the normal cells. The tumor neogenesis antigen epitope peptide and the related antigen epitope peptide can identify tumor specific genes, and are tumor antigen epitope peptides.

The screening method of the tumor epitope peptide comprises the following steps:

1) adopting an antigen epitope prediction algorithm to predict new antigen epitopes of mutation sites which occur at high frequency in tumors to obtain candidate antigen epitope polypeptide sequences, and screening the predicted affinity scores of the epitope peptides and HLA-A1101 MHC compounds to obtain a first batch of candidate high-affinity epitope peptides;

2) evaluating the compounding of the candidate epitope peptides and HLA-A1101 MHC by adopting a competitive enzyme-linked immunosorbent assay method for the first batch of candidate high-affinity epitope peptides obtained in the step 1), and further screening a second batch of candidate high-affinity epitope peptides with stronger affinity;

3) detecting whether the second batch of candidate high-affinity epitope peptides screened in the step 2) have immunogenicity by using a PBMC sample of a tumor patient of HLA-A1101 type by adopting an enzyme-linked immunosorbent assay, stimulating PBMC to secrete immune effector factor IFN gamma, and screening out neogenetic epitope peptides or related epitope peptides with immunogenicity to obtain the tumor epitope peptides.

The invention also provides a polymer, and the antigen peptide of the polymer is the tumor antigen epitope peptide. Preferably, the polymerization unit of the polymer is pMHC, and the polymer can be, but not limited to, tetramer, pentamer or hexamer or polymer prepared by polymerizing pMHC with other vectors, and the like.

The multimer according to the present invention is preferably a tetramer, and the tetramer further comprises streptavidin, which binds to four groups of biotin attached to the heavy chain of MHC α 3, each MHC being specifically attached to a tumor epitope peptide.

The present invention also preferably provides a method for producing the above tetramer, comprising the steps of:

1) pMHC preparation and peptide replacement:

preparation of biotin-carrying sensitive peptide p by pMHC monomer preparation method_senseMHC, to prepare the resulting sensitive peptide p_senseOn the basis of MHC, incubating the antigen epitope peptide of the tumor as a target peptide after ultraviolet irradiation to prepare a target peptide pMHC monomer compound;

2) co-incubating the pMHC monomer complex prepared in the step 1) with streptavidin, and combining the streptavidin with biotin connected to an MHC alpha 3 heavy chain to prepare a tetramer.

According to the preparation method of the present invention, preferably, the sensitive peptide sequence is KILGFVVFJV.

According to the preparation method of the present invention, preferably, before adding avidin in step 1), the target peptide pMHC is incubated with an ELISA plate coated with β 2M antibody to capture the target peptide pMHC on the ELISA plate, the prepared target peptide pMHC is detected by the ELISA method, and a tetramer is further prepared after the success of the preparation of the target peptide pMHC is confirmed.

According to the preparation method of the present invention, preferably, the streptavidin in step 2) is streptavidin with fluorescence.

The invention also provides application of the tumor epitope peptide in detecting the antigen specificity of DC-CTL or TCR-T. For example, in the preparation of a medicament or detection agent for use in adoptive immunotherapy of DC-CTL or TCR-T.

The invention further provides the use of the multimer in detecting the antigen specificity of DC-CTL or TCR-T. For example, in the preparation of a medicament or detection agent for use in adoptive immunotherapy of DC-CTL or TCR-T.

The tumor antigens herein are generated based on various mutations in the KRAS and like gene loci and CEACAM5 and like related genes, and multimers (such as but not limited to tetramers) are prepared based on such tumor neogenesis and related antigens. In DC-CTL, presenting antigen peptide through DC stimulates T cells together to generate specific CTL, and the polymer can detect the effect of antigen peptide stimulation to generate specific CTL; and in TCR-T therapy, screening specific TCR gene sequences based on the neoantigen and preparing TCR-T, and then testing the activity of the TCR-T on the specific antigen through the multimer.

According to the invention, a batch of cancer specific antigen peptides are screened by an independently developed antigen epitope prediction algorithm and evaluation of molecular level and cell level, and more specific adoptive immunotherapy including DC-CTL, TCR-T and the like can be provided for cancers, especially liver cancers based on the antigen peptides, so that possibility is provided for cancer cure. On the basis, a polymer preparation technology based on the antigen peptide is established, and the prepared polymer can effectively identify the antigen specificity of the DC-CTL and other T cells prepared by stimulating the antigen peptide, so that a detection means is provided for non-clinical efficacy evaluation and pharmacological explanation. Meanwhile, the clinical diagnosis of the medicine can be provided, namely, the polymer (such as Tetramer) prepared based on the antigen peptide is used for evaluating the survival ability and distribution of the medicine in the body of a patient, and the activity of the medicine can be evaluated more intuitively. Therefore, on one hand, the invention provides a thought for clinical adoptive immunotherapy of cancer, particularly liver cancer, and simultaneously provides a detection means for drug research and development, so that the invention can better serve the drug research and development, and can enable the effective therapeutic drug to serve cancer patients as quickly as possible.

Drawings

FIG. 1 is a flow chart of an Elispot experiment;

FIG. 2 is a tetramer technical roadmap;

FIG. 3 is a process of screening high affinity, high immunogenic epitope peptides of the present invention;

FIG. 4 shows the technical principle of the cELISA for verifying epitope peptide-MHC affinity according to the present invention;

FIG. 5 shows the results of the cELISA method of the present invention for determining the binding force between HLA-A1101 and high affinity positive epitope peptides (Pos, EBV-derived antigen peptide group; Neg, negative peptide group; UV only, no new peptide group added by UV irradiation; Pos _ monomer, p without UV irradiation)_senseMHC; blank, Blank control group);

FIG. 6 shows the results of the cELISA method of the present invention for detecting the binding force between the epitope peptide with high affinity and HLA-A1101 (Pos, EBV-derived antigen peptide group; Neg, negative peptide group; UV only, no new peptide group added by ultraviolet irradiation; Pep1-27, tumor antigen peptide group);

FIG. 7 shows that the highly immunogenic epitope peptide of the present invention stimulates HLA-A1101 type PBMC to secrete IFN γ;

FIG. 8 shows the flow cytometry analysis and staining results of the self-made EBV Tetramer of the present invention and the existing commercial EBV Tetramer;

FIG. 9 shows the result of flow cytometry analysis and staining of the tumor epitope peptide Tetramer of the present invention.

Detailed Description

The experimental method, the detection method and the preparation method disclosed by the invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture and recombinant DNA technology in the technical field and the conventional technology in the related field. The method can be specifically carried out according to a specific method listed in a molecular cloning experimental manual (fourth edition, J. SammBruk et al), or according to a kit and a product instruction; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Abbreviations and Key term definitions (referred to in this application)

TCR, T cell receptor.

APC, antigen-presenting cell, antigen presenting cell.

CTL, cytoxic T lymphocyte, killer T lymphocyte.

MHC, major histocompatibility complex, histocompatibility complex.

Monomer: is a monomeric complex, an MHC-peptide (pMHC) building block formed by MHC and a peptide.

Multimers: the pMHC building blocks are polymerized into complexes by means of a streptavidin-biotin (1:4) system.

KRAS gene: kras is a murine sarcoma virus oncogene, located on chromosome 12, and K-ras gene encodes a 21kD ras protein, also known as the p21 gene. The Kras gene is also a common oncogene in human tumors, and somatic mutation of the gene is common in various malignant tumors, with the mutation rate of 15% -30% in lung cancer patients and 20-50% in colorectal cancer patients. KRAS plays an important role in tumor signaling as the most important effector downstream of the EGFR signaling pathway. The mutation is the most malignant and difficult carcinogenic mutation in history, so that the KRAS gene mutation is detected, and a more exact basis can be provided for the individualized treatment of tumor patients.

TP53 gene: also known as P53, is the first cancer suppressor gene identified, and the protein encoded by this gene is a transcription factor that regulates cell growth, proliferation, and repair of damage. The gene in tumor cells is often mutated, resulting in uncontrolled proliferation and damage repair of tumor cells, and malignant evolution of tumors.

CTNNB1 gene: also named as beta-catenin, the coded protein regulates gene transcription and intercellular adhesion, and has a regulating effect on the form maintenance of cells. The gene mutation can promote the malignant evolution of the tumor by influencing the epithelial mesenchymal transition of the tumor cells.

CEACAM5 gene: also known as CD66e, belongs to the carcinoembryonic antigen gene family, and mediates cell-to-cell adhesion during cancer cell invasion and metastasis.

MSLN gene: mesothelin, a cell surface glycoprotein with a molecular weight of 40kDa, mediates intercellular adhesion. The gene is highly expressed in various tumor cells, and the encoded protein can promote the diffusion of the tumor cells by interacting with CA125 to influence the adhesion process.

MUC1 gene: also known as Epithelial Membrane Antigen (EMA), is a glycoprotein whose extracellular domain has extensive O-linked glycosylation. In the nucleus, MUC1 protein can regulate the host immune function by regulating the activity of transcription factor complex, and the high expression of the protein is related to tumor.

TERT gene: encodes telomerase reverse transcriptase, which is the catalytic subunit of telomerase. Relaxed regulation of telomerase expression in somatic cells is associated with tumorigenesis.

WT1 gene: a transcription factor, the C terminal of which contains 4 zinc finger structures and the N terminal of which is rich in Pro/Gln DNA binding domain. In connection with the normal development of the urogenital system, genetic mutations may lead to the development of wilm's tumours. Interacts with p53 in cells, and the expression quantity of the gene is increased in tumor cells.

Antigen-specific T cells: cells that play an important role in the defense against viral infections, tumorigenesis, and autoimmune diseases. Detecting the activity of cells to identify the response of lymphocytes to specific stimuli is of great importance in the study of the pathogenesis and treatment of these diseases. Adaptive immune responses and immune pathogenesis are based on the ability of T cells to respond to specific antigens.

The specific implementation mode of the invention is divided into three parts of antigenic peptide prediction screening verification, Tetramer preparation and Tetramer activity evaluation according to the flow. The prediction screening verification of the antigen peptide is performed by a prediction algorithm, and then the immunogenicity of the predicted antigen peptide and the killing activity of the antigen-specific T cells to corresponding antigen-expressing tumor cells are verified by a molecular level and a cell level. The Tetramer is prepared by adopting a prokaryotic expression system and an in-vitro assembly mode to obtain the ultraviolet sensitive peptide p_senseMHC (pMHC) monomer complex, under 366nm ultraviolet irradiation, the sensitive peptide on the monomer complex will break off to expose peptide binding site, providing possibility for binding of target peptide or other peptide. Tetramer activity evaluation the Tetramer was prepared from the polypeptides produced by CMV virus and compared with commercial CMV antigen-active products of the same type by staining on the same sample to test the self-made Tetramer activity, after verification the Tetramer of the antigen peptide was prepared and the target activity was detected on DC-CTL produced by stimulation of the antigen peptide.

Example 1 antigenic peptide prediction, screening, validation

The discovery of epitope peptides is key to the preparation of polypeptide-MHC tetramers. Epitope peptides having a higher affinity to MHC and a higher immunity to elicit an immune response are used to prepare preferred polypeptides for recognizing the polypeptide-MHC tetramer of the antigen-specific T cells. The process for screening epitope peptides with high affinity and high immunogenicity and preparing pMHC in the invention is shown in FIG. 3:

an independently developed antigen epitope prediction algorithm (patent publication No. CN112002374A) is adopted to carry out antigen epitope prediction on mutation sites (CTNNB1_ T41A, KRASpG12D, KRASpG12V, KRASpG12R, KRASpG12C and Tp53pR248W) which occur at high frequency in tumors and high-expression tumor-associated genes (CEACAM5, MSLN, MUC1, TERT and WT1) to obtain candidate antigen epitope polypeptide sequences, and affinity scores of the predicted epitope peptides and HLA-A1101 type MHC compounds are screened to obtain 27 candidate high-affinity epitope peptides in total, which is shown in Table 1.

TABLE 1 27 epitope peptide information obtained by epitope prediction analysis

Synthesizing 27 candidate epitope peptides, and evaluating the complexation of the candidate epitope peptides and HLA-A1101 MHC by using a cELISA (chip-ELISA) experimental method. The specific experimental principle is as follows: when the candidate epitope peptide is combined with MHC complex, fluorescence signal can be excited, and the stronger the combination force, the stronger the fluorescence signal (see the technical principle in figure 4).

The experimental screening results in 22 (Pep1, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27) candidate epitopes with strong affinity, as shown in table 2, fig. 5 and fig. 6.

Further obtaining a PBMC sample of a tumor patient of HLA-A1101 type, detecting whether the high affinity epitope peptide screened in the last step has immunogenicity by adopting an enzyme linked immunosorbent assay, stimulating PBMC to secrete immune effector factor IFN gamma, and further verifying that the 22 antigen epitope peptides have stronger immunogenicity, wherein the result is shown in figure 7.

The 22 high-affinity and high-immunogenicity antigen epitope peptides obtained by the screening steps have the following sequences (table 2):

TABLE 2 high affinity, high immunogenicity epitope peptide information

Example 2Tetramer preparation

pMHC preparation and peptide replacement

The part is prepared by adopting the existing pMHC monomerThe method (patent publication numbers CN109293779A, CN106397549A) prepares a sensitive peptide pMHC carrying biotin, wherein the sequence of the sensitive peptide is as follows: KILGFVVFJV (HLA-A1101, wherein J means 3-amino-3- (2 nitro) phenylpropionic acid). To prepare the resulting sensitive peptide p_senseMHC (pMHC) as a basis, the target peptide pMHC is prepared by incubating with a target peptide (EBV source antigen peptide after 366nm ultraviolet irradiation, the pMHC is captured on an ELISA plate through incubating with the ELISA plate coated with a beta 2M antibody, then avidin-HRP is added to combine avidin with biotin connected with an MHC alpha 3 heavy chain, and finally HRP substrate is added to reflect the content of pMHC through an absorbance value, so that the separation degree of sensitive peptide after ultraviolet irradiation and the pMHC effect of the newly formed target peptide after re-incubation can be reflected.

Reagent, consumable, material and instrument equipment

Reagent: PBS buffer (pH7.4), Peptide sample, and p_senseMHC, streptavidin-conjugated horseradish peroxidase (Avidin HRP), DMSO, Assay Buffer A, Wash Buffer, Substrate Solution F, Stop Solution.

Consumable material: ELISA strips coated with β 2M antibody, 96-well plates (polystyrene, V-well), sealing membrane, 1.5mL EP tubes.

The instrument equipment comprises: ultraviolet lamp (containing 366nm wavelength), water bath, low temperature centrifuge (suitable for 96-well plate and 1.5mL EP tube), pipetting gun (single gun and row gun), shaking table, and enzyme labeling instrument (containing 450 and 570nm two modules).

Experimental procedure

A. Polypeptide antigen replacement

Taking out the reagents required by the experiment, placing the reagents in an ice bath, and keeping the temperature at 0 ℃;

diluting the target peptide to 400 mu M with PBS, and placing in ice bath for later use;

a96-well plate with V-shaped wells was used, and 1.5. mu.L of the diluted target peptide and 1.5. mu.L of p were added to the wells using a pipette gun_senseMHC (200. mu.g/mL), pipette gun to mix (negative control and UV only group plus 1.5. mu.L of the corresponding peptide (negative peptide sequence of HLA-A1101 is NPKASLLSL) and PBS with 1.5. mu.L of p_sense-MHC mixing);

sealing the cover, and centrifuging at 2500g for 2 minutes at the temperature of 4 ℃ to enable the liquid to sink to the bottom of the hole;

removing the cover, and irradiating with ultraviolet (366nm) under ultraviolet lamp for 30min under ice bath condition of 96-well plate;

then, the plate is covered and incubated for 30min at 37 ℃ in a dark environment to ensure that the target peptide is fully combined;

centrifuging and collecting liquid in the holes for later use. (the liquid contained the prepared pMHC monomer complexes at a concentration of 100. mu.g/mL and 300ng pMHC monomer complexes per well).

B. Polypeptide replacement activity assay

By ddH₂O, diluting 20 multiplied by the Wash Buffer to 1 multiplied by the Wash Buffer; diluting the pMHC compound sample prepared in the previous step by using Assay Buffer A to ensure that the concentration of the monomer compound reaches 50ng/mL, which is a monomer compound sample mother solution;

taking out the required reagents, placing the reagents at room temperature, calculating the number of the required ELISA strips, taking out the required ELISA strips and placing the ELISA strips in an ELISA plate;

samples (2ng/mL) of appropriate concentration were prepared using 96. mu.L of Assay Buffer A in 4. mu.L of pMHC monomer complex stock solution, Assay Buffer A being set as a control;

adding 50 μ L of Assay Buffer A into the wells of the ELISA plate, and then adding 50 μ L of sample solution or control solution into the corresponding wells; (the concentration of samples after this step was 1ng/mL and 3 replicates per sample) (Pos control was added as an untreated p_sense-MHC; and a set of blank controls, wells with equal amounts of Assay Buffer a);

sealing the ELISA plate with a sealing film, and then performing shaking incubation on a shaking table for 30min (220 rpm can be selected) or standing incubation on a plane for 2h at room temperature;

then, the liquid in the hole is discarded, and the plate is washed for 4 times by 200 mu L of 1 multiplied by Wash Buffer/hole, wherein the liquid is controlled to be dry as much as possible when the liquid is discarded each time, and the plate can be reversely buckled on absorbent paper paved on a table to ensure that the liquid is absorbed and dried;

after the liquid is drained in the last step, 100 mu L of avidin-HRP reagent is added into each hole, and after the sealing film is sealed, the mixture is shaken on a shaking table for incubation for 30min (220 rpm can be selected) or kept standing on a plane for incubation for 30min under the room temperature environment;

then, liquid in the holes is discarded, and the washing is carried out for 5 times according to the plate washing steps, wherein after the last Wash Buffer is added, the liquid is soaked in the holes for 30 s-1 min so as to reduce the background interference to the minimum;

after the liquid is drained in the last step, 100 mu L of substrate solution F is added into each hole and is kept stand and incubated for 10min at room temperature in a dark place; (the color of the liquid in the experimental hole is changed into blue after the substrate is added, and the blue color is gradually deepened along with the increase of the concentration of the sample; the step can be selectively sealed and incubated);

after incubation, 100 μ L of stop buffer was added to each well; (the color of the liquid changed from blue to yellow after the stop solution was added);

after the color development is stopped, the absorbance of both OD450 and OD570 bands is detected within 30min by using a microplate reader. (Absorbance of OD 450-Absorbance of OD570 was used for calculation).

Results and analysis of the experiments

As shown in fig. 5, from the results: 1. intact p after UV irradiation relative to non-UV irradiated group_sense-a significant decrease in MHC content and almost total disappearance; the EBV-derived antigen peptide addition group significantly increased the amount of newly produced pMHC relative to the UV only group, and almost leveled with the Pos _ monomer group; the Neg group had little newly generated intact pMHC. It can be concluded that: 1.p_senseMHC under UV irradiation, sensitive peptides can degrade and lead to p_sense-MHC complex disassembly; 2. after the sensitive peptide is detached, the target peptide can be effectively bound to an exposed peptide binding site on MHC; 3. the MHC prepared is selective for the antigenic peptide, i.e. binds only the corresponding peptide of the type with affinity. Thus, p_senseSuccess in MHC production.

Preparation of Tetramer

Reagent, consumable, material and instrument equipment

Reagent: PBS buffer (pH7.4), Peptide sample, and p_sense-MHC、DMSO、50mM D-Biotin、10％(w/v)NaN₃、Fluorophore-conjugated Streptavidin。

Consumable material: 1.5mL EP tube.

The instrument equipment comprises: ultraviolet lamp (containing 366nm wavelength), water bath, low temperature centrifuge (suitable for 96-well plate and 1.5mL EP tube), liquid transfer gun (single gun and row gun), and shaking table.

Experimental procedure

Preparing 36 mu L of pMHC compound according to the experimental conditions in '1. pMHC preparation and peptide replacement' and transferring the pMHC compound into a 1.5mL EP tube, then adding 3.96 mu L of streptavidin APC-streptavidin coupled with fluorescence, blowing a liquid transfer gun up and down to mix the mixture, and carrying out ice-bath in a dark place for 30 min;

in the incubation phase of the above step, a blocking solution was prepared: 1.6. mu.L of D-Biotin with a concentration of 50mM and 6. mu.L of NaN with a mass-to-volume ratio of 10%₃Adding into 192.4 μ L PBS, vortexing and shaking to mix well, and collecting the solution as prepared blocking solution. After the incubation of the monomer compound and streptavidin is finished, 2.88 mu L of confining liquid is added into the reaction liquid, and a liquid transfer gun is used for blowing up and down to fully and uniformly mix the solution;

and continuously incubating overnight in the absence of light or ice-bathing for 30min in the absence of light at the temperature of 2-8 ℃, and obtaining the Tetramers after the reaction.

Example 3Tetramer Activity evaluation

In the part, the Tetramer prepared by using the EBV-derived antigen peptide disclosed by the invention and commercial similar EBV-derived antigen active products are dyed on the same batch of DC-CTL, and the two groups of data are compared to evaluate the targeting activity of the self-made Tetramer. After the verification, the respective tetramers were prepared using 22 peptides selected previously, and then the tetramers prepared from the 22 peptides were stained on DC-CTLs prepared by stimulating the antigen peptides, respectively, to evaluate the activity of tetramers prepared from the 22 peptides and the specificity of the peptides after DC-CTL preparation. The specific operation steps are as follows:

reagent, consumable, material and instrument equipment

Reagent: PBS buffer (pH7.4), Peptide sample, and p_sense-MHC、DMSO、50mM D-Biotin、10％(w/v)NaN₃Fluorescent-conjugated Streptavidin, Cell stabilizing Buffer, Streptavidin-conjugated horseradish peroxidase (Avidin HRP), Assay Buffer A, Wash Buffer, Substrate Solution F, Stop Solution, fluorescently labeled membrane protein antibodies (CD3-percP-CY5.5, CD4-APC-CY7, CD8-BV510, CD56-BV 605).

The instrument equipment comprises: ultraviolet lamp (containing 366nm wavelength), water bath, low temperature centrifuge (suitable for 96-well plate and 1.5mL EP tube), pipette (single gun and row gun), shaking table, 37 deg.C incubator, enzyme labeling instrument (containing 450 and 570nm modules), flow cytometer, biological safety cabinet, CO₂Incubator, K2 cell counter.

Experimental procedure

After preparing 37. mu.L of pMHC complex under the experimental conditions of "1. pMHC preparation and peptide replacement" in example 2, 1. mu.L of pMHC complex was taken out and used for ELISA to evaluate the success of pMHC preparation, and the remaining 36. mu.L of pMHC complex was transferred to a 1.5mL EP tube after verification, and tetramers corresponding to the respective antigenic peptides were prepared according to the procedure of "2. Tetramer preparation" in example 2.

Preparing and counting the required DC-CTL cells;

preparation of 1X 10⁶Each cell/sample was placed in a 12X 75mm flow tube and the volume was made up to 200. mu.L with cell staining buffer. The group comprises Blank group, control group, negative control group and experimental group, wherein Blank group is not added with antibody and Tetramer, control group is added with CD3 only&CD4$CD8&CD56 fluorescent antibody, negative control group (Negtive group) and 4 fluorescent antibodies, and the experimental group is prepared by adding the experimental peptide on the basis of 4 fluorescent antibodies. Respectively adding the membrane protein fluorescent antibodies CD3-perCP-CY5.5, CD4-APC-CY7, CD8-BV510 and CD56-BV605 into corresponding cell samples according to the volume of 10 mu L of each 2 mu L, Tetramers reagent amount, blowing and uniformly mixing the solution up and down by using a pipette gun, and incubating the solution at the corresponding temperature in the dark. Wherein, the control group mixes the antibody and adds the antibody into the sample to avoid light and ice bath for 30min to load the sample; and (3) adding four antibodies into the negative control and experimental group, performing ice-bath in a dark place for 20min, performing buffer washing for 2 times, then adding the corresponding Tetramer, performing incubation in a dark place for 10min at room temperature, performing buffer washing for 2 times, loading the machine, performing flow cytometry detection in 2h, and placing the sample without loading the machine in the ice-bath in a dark place. Wherein the sample washing step is as follows: centrifuge at 350g and 4 ℃ for 5min, discard the supernatant and resuspend it in 1mL of staining buffer.

Results and analysis of the experiments

As shown in fig. 8-9, from the results: 1. the existing commercial EBV source antigen peptide and the antigen peptide of the invention both effectively generate pMHC; 2. the positive rate of the Tetramer _ EBV prepared by the method is slightly higher than that of the same commercial product, which indicates that the self-made Tetramer is successful; 3. the antigen peptide prepared by the invention can effectively target and mark DC-CTL prepared by stimulating the peptide corresponding to the Tetramer, which shows that the Tetramer can be used for evaluating the quality of DC-CTL medicaments.

Sequence listing

<110> Beijing Zhenzhi medical science and technology Limited liability company

<120> tumor antigen epitope peptide, polymer and application thereof

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Claims

1. A tumor epitope peptide, which comprises a tumor neogenesis epitope peptide or a tumor-associated epitope peptide; wherein the content of the first and second substances,

the amino acid sequence of the tumor neogenesis antigen epitope peptide is shown in any one of SEQ ID NO.1, 3-10 and 12; or the amino acid sequence of the tumor neoantigen epitope peptide has more than 90 percent of similarity with the sequence shown in any one of SEQ ID NO.1, 3-10 and 12 through amino acid substitution, deletion or addition; or the amino acid sequence of the tumor neoantigen peptide comprises a sequence shown in any one of SEQ ID NO.1, 3-10 and 12 or a sequence which has more than 90% similarity with the sequence shown in any one of SEQ ID NO.1, 3-10 and 12 through amino acid substitution, deletion or addition;

the amino acid sequence of the tumor-associated epitope peptide is shown in any one of SEQ ID NO.13, 16-18 and 20-27; or the amino acid sequence of the tumor-associated epitope peptide has more than 90 percent of similarity with the sequence shown in any one of SEQ ID NO.13, 16-18 and 20-27 through amino acid substitution, deletion or addition; or the amino acid sequence of the tumor-associated antigenic peptide comprises a sequence shown in any one of SEQ ID NO.13, 16-18 and 20-27 or a sequence which has more than 90% similarity with the sequence shown in any one of SEQ ID NO.13, 16-18 and 20-27 through amino acid substitution, deletion or addition.

2. A method for screening the tumor epitope peptide according to claim 1, comprising the steps of:

1) carrying out newborn and related antigen epitope prediction on mutation sites and related antigens which occur at high frequency in tumors by adopting an antigen epitope prediction algorithm to obtain candidate antigen epitope polypeptide sequences, and screening the predicted affinity scores of epitope peptides and HLA-A1101 MHC compounds to obtain a first batch of candidate high-affinity epitope peptides;

3) detecting whether the second batch of candidate high-affinity epitope peptides screened in the step 2) have immunogenicity by using a PBMC sample of a tumor patient of HLA-A1101 type by adopting an enzyme-linked immunosorbent assay, stimulating PBMC to secrete immune effector factor IFN gamma, and screening out the neonatal antigen epitope peptide or related antigen epitope peptide with the immunogenicity so as to obtain the tumor antigen epitope peptide.

3. A multimer, wherein the antigenic peptide of said multimer is the tumor epitope peptide of claim 1.

4. The multimer of claim 3, wherein the multimer is a tetramer, pentamer, hexamer, or a multimer prepared by polymerizing pMHC with another vector.

5. The multimer of claim 4, wherein said multimer is a tetramer comprising streptavidin that binds to four sets of biotin attached to the MHC α 3 heavy chain, each MHC having specifically attached thereto a tumor epitope peptide.

6. A method of preparing the multimer of claim 5, comprising the steps of:

1) pMHC preparation and peptide replacement:

preparation of biotin-carrying sensitive peptide p by pMHC monomer preparation method_senseMHC, to prepare the resulting sensitive peptide p_senseBased on MHC, byIncubating the mixture with tumor epitope peptide serving as target peptide after ultraviolet irradiation to prepare a target peptide pMHC monomer compound;

2) co-incubating the target peptide pMHC monomer compound prepared in the step 1) with streptavidin, and combining the streptavidin with biotin connected to an MHC alpha 3 heavy chain to prepare a tetramer.

7. The method of claim 6, wherein the sensitive peptide sequence is KILGFVVFJV.

8. The preparation method of claim 6, wherein before adding avidin in step 1), the target peptide pMHC is incubated with an ELISA plate coated with β 2M antibody to capture the target peptide pMHC on the ELISA plate, the prepared target peptide pMHC is detected by ELISA method, and a tetramer is further prepared after the success of the preparation of the target peptide pMHC is confirmed;

and 2) the streptavidin is streptavidin with fluorescence.

9. Use of the tumor epitope peptide of claim 1 for detecting the antigen specificity of DC-CTL or TCR-T.

10. Use of a multimer according to any of claims 3-5 for detecting the antigen specificity of a DC-CTL or a TCR-T.