阅读说明：本技术 Hla-a2限制性膀胱癌肿瘤新抗原肽序列及其应用 (HLA-A2 restricted bladder cancer tumor neoantigen peptide sequence and application thereof ) 是由 沈海波 王颖 王晨 段黄琪 曹志伟 于 2019-03-12 设计创作，主要内容包括：本发明公开了HLA-A2限制性膀胱癌肿瘤新抗原肽序列,选自以下12个基因的抗原序列的任意一种或任意多种组合：CDKN1A基因,RHOB基因,CDC42基因,DDB1基因,AHNAK基因,ANP32A基因,ALDH16A1基因,MET基因,PRDX6基因,MKI67基因,GAK基因,DSG3基因,其抗原序列分别为如SEQ ID NO:1-12所示。此外,本发明公开了上述抗原序列在制备膀胱癌诊断试剂和膀胱癌治疗药物中的应用。(The invention discloses an HLA-A2 restricted bladder cancer tumor neoantigen peptide sequence, which is selected from any one or any combination of antigen sequences of the following 12 genes: CDKN1A gene, RHOB gene, CDC42 gene, DDB1 gene, AHNAK gene, ANP32A gene, ALDH16A1 gene, MET gene, PRDX6 gene, MKI67 gene, GAK gene and DSG3 gene, and the antigen sequences are respectively shown in SEQ ID NO. 1-12. In addition, the invention discloses application of the antigen sequence in preparation of a bladder cancer diagnostic reagent and a bladder cancer treatment drug.)

An HLA-A2-restricted bladder cancer tumor neoantigen peptide sequence characterized by: any one or any combination of more than one of the following antigen sequences:

CDKN1A gene, mutant peptide fragment sequence: FVTETPLEV, as shown in SEQ ID NO: 1;

RHOB gene, mutant peptide fragment sequence: YLDTDVILM, as shown in SEQ ID NO: 2;

CDC42 gene, mutant peptide sequence: YLQTDVFLV, as shown in SEQ ID NO. 3;

DDB1 gene, mutant peptide segment sequence: TLAEDLNLL, as shown in SEQ ID NO. 4;

AHNAK gene, mutant peptide sequence: YLDLKGPKV, as shown in SEQ ID NO: 5;

ANP32A gene, mutant peptide segment sequence: TLIANLPKL, as shown in SEQ ID NO: 6;

ALDH16A1 gene, mutant peptide segment sequence: GLDGAVDMV, as shown in SEQ ID NO: 7;

MET gene, mutant peptide fragment sequence: LQSEGSPLV, as shown in SEQ ID NO: 8;

PRDX6 gene, mutant peptide segment sequence: IIDDRNWEL, as shown in SEQ ID NO: 9;

MKI67 gene, mutant peptide fragment sequence: KLGDVITII, as shown in SEQ ID NO: 10;

GAK gene, mutant peptide fragment sequence: VLDEGGSPI, as shown in SEQ ID NO: 11;

DSG3 gene, mutant peptide segment sequence: YLARIEENI, as shown in SEQ ID NO: 12.

2. The HLA-a 2-restricted bladder cancer tumor neoantigen peptide sequence of claim 1, wherein:

the amino acid mutation sites of the CDKN1A gene are as follows: G61V;

the amino acid mutation sites of the RHOB gene are as follows: P75L;

the amino acid mutation sites of the CDC42 gene are as follows: P73L;

the amino acid mutation sites of the DDB1 gene are as follows: S25L;

the amino acid mutation sites of the AHNAK gene are: D4855Y;

the amino acid mutation sites of the ANP32A gene are as follows: S56L;

the amino acid mutation sites of the ALDH16A1 gene are as follows: G343V;

the amino acid mutation sites of the MET gene are as follows: R1148Q;

the amino acid mutation sites of the PRDX6 gene are as follows: R108W;

the amino acid mutation sites of the MKI67 gene are as follows: H84L;

the amino acid mutation sites of the GAK gene are as follows: S829L;

the amino acid mutation sites of the DSG3 gene are as follows: S273L.

3. The HLA-a 2-restricted bladder cancer tumor neoantigen peptide sequence of claim 1 or 2, wherein: the mutation type of the antigen sequence is missense mutation.

4. The HLA-a 2-restricted bladder cancer tumor neoantigen peptide sequence of claim 1, wherein: the antigen sequence is obtained by screening through the following steps:

(1) bioinformatic predictions of HLA-A2 restricted wild-type and mutant bladder cancer antigen peptides;

(2) detecting the binding force of HLA-A2 restricted wild type and mutant bladder cancer antigen peptides;

(3) determination of affinity constants of HLA-A2-restricted bladder cancer mutant antigen peptides.

5. The HLA-A2-restricted bladder cancer tumor neoantigen peptide sequence of claim 4, wherein the step (1) is specifically: after the separation of peripheral blood mononuclear cells of HLA-A2 positive bladder cancer patients, an enzyme-linked immunospot assay was performed using tumor neoantigens.

6. The HLA-A2-restricted bladder cancer tumor neoantigen peptide sequence of claim 4, wherein the step (2) is specifically: and (3) carrying out statistical analysis on immune reaction generated after the stimulation of the tumor neoantigen, selecting a predicted peptide segment with a ratio above a threshold value and a mutant average fluorescence intensity in the front row for repeated experiments, and screening out a peptide segment which can stably express a main histocompatibility complex molecule in the repeated experiments, wherein the average fluorescence intensity is higher than that of a negative control and has a larger difference with a wild type peptide segment as a candidate peptide segment of the tumor neoantigen.

7. Use of the HLA-a 2-restricted bladder cancer tumor neoantigen peptide sequence of any one of claims 1 to 5 for the preparation of a medicament for the treatment of bladder cancer.

8. The use of claim 6, wherein the bladder cancer therapeutic comprises a tumor vaccine and antigen-specific T cells for bladder cancer.

9. Use of the HLA-a 2-restricted bladder cancer tumor neoantigen peptide sequence of any one of claims 1 to 5 for the preparation of a diagnostic reagent for bladder cancer.

10. The use of claim 9, wherein the bladder cancer diagnostic reagent comprises a non-invasive urine diagnostic kit.

Technical Field

The invention belongs to the field of tumor immunotherapy, and particularly relates to an HLA-A2 restrictive bladder cancer tumor neoantigen peptide sequence; in addition, the invention also relates to the application of the HLA-A2 restricted bladder cancer tumor neoantigen peptide sequence in the preparation of bladder cancer diagnostic reagents and bladder cancer treatment drugs.

Background

Tumor neoantigen (neoantigen) refers to an epitope produced by somatic genetic mutation during tumorigenesis and development. Thanks to The implementation of The tumor genome project and The TCGA (The cancer genome atlas) public database, information on mutated genes in tumor cells can be obtained by bioinformatic analysis, thus forming The concept of tumor neoantigen profiling based on tumor genome sequences. The mutation state of the tumor cells is reflected on one hand, and the biological behavior of the tumor cells is further influenced; meanwhile, the antigen (peptide) with strong immunogenicity becomes an important candidate antigen for starting specific cellular immune response, and is expected to promote the application of immunotherapy based on tumor neoantigens in tumor immunotherapy (Carreno BM et al, Science,2015,348(6236): 803-8; Ott PA et al, Nature,2017,547(7662): 217). The application of the tumor neoantigen in tumor Immunotherapy can be used for preparing T cell receptor chimeric T cells, in vitro induction of antigen-specific T cells and preparation of tumor neoantigen vaccines (Spear TT et al, cancer immunology, 2016,65(3):293-304), wherein the tumor neoantigen vaccines have been successfully applied to melanoma (Sahin U et al, Nature,2017,547(7662): 222).

Bladder cancer is a tumor with a high tumor mutation load (Schumacher T N et al, Science,2015,348 (6230):69-74), but no relevant reports of bladder cancer tumor antigen peptides have been reported so far. The existing TCGA data contains sequencing data of all exons of bladder cancer and peripheral blood from different populations, and provides original data for predicting HLA-A2-restricted mutant antigen peptide by using a bioinformatics method. At present, the prediction methods of tumor antigen candidate peptides based on large samples are numerous, and the predicted mutant peptides can be verified after in vitro affinity determination and immunoreactivity determination in tumor patients.

Meanwhile, because the mutation rate of the tumor neoantigen peptide in a tumor patient is low at present and the tumor neoantigen peptide has obvious individuation characteristics, the individuation degree of tumor immunotherapy based on the tumor antigen peptide is extremely high, the treatment cost is high, and the tumor antigen peptide obtained by screening can not cover more people, so that a tumor antigen peptide library is necessary to be established, the coverage rate of the tumor antigen peptide library in the patient reaches a higher level, and the tumor neoantigen obtained by screening is used for tumor vaccines or is used for establishing antigen-specific T cells for cell therapy.

Disclosure of Invention

The invention aims to solve the technical problem of providing an HLA-A2 restrictive bladder cancer tumor neoantigen peptide sequence, predicting high-affinity HLA-A2 restrictive wild-type and mutant type antigen by utilizing bladder cancer exon sequencing and bioinformatics methods in a TCGA database, and obtaining the bladder cancer tumor neoantigen with certain coverage rate and high immunoreactivity through in vitro affinity determination and bladder cancer population peripheral immunoreactivity analysis.

The second technical problem to be solved by the invention is to provide the application of the HLA-A2 restrictive bladder cancer tumor neoantigen peptide sequence in the preparation of bladder cancer diagnostic reagents and bladder cancer treatment drugs. Including the application in preparing tumor vaccine and antigen specific T cell of bladder cancer.

In order to solve the technical problems, the invention adopts the following technical scheme:

in one aspect of the invention, HLA-A2 restricted bladder cancer tumor neoantigen peptide sequences are provided, selected from any one or any combination of the following antigen sequences:

CDKN1A gene, mutant peptide fragment sequence: FVTETPLEV, as shown in SEQ ID NO: 1;

RHOB gene, mutant peptide fragment sequence: YLDTDVILM, as shown in SEQ ID NO: 2;

CDC42 gene, mutant peptide sequence: YLQTDVFLV, as shown in SEQ ID NO. 3;

DDB1 gene, mutant peptide segment sequence: TLAEDLNLL, as shown in SEQ ID NO. 4;

AHNAK gene, mutant peptide sequence: YLDLKGPKV, as shown in SEQ ID NO: 5;

ANP32A gene, mutant peptide segment sequence: TLIANLPKL, as shown in SEQ ID NO: 6;

ALDH16A1 gene, mutant peptide segment sequence: GLDGAVDMV, as shown in SEQ ID NO: 7;

MET gene, mutant peptide fragment sequence: LQSEGSPLV, as shown in SEQ ID NO: 8;

PRDX6 gene, mutant peptide segment sequence: IIDDRNWEL, as shown in SEQ ID NO: 9;

MKI67 gene, mutant peptide fragment sequence: KLGDVITII, as shown in SEQ ID NO: 10;

GAK gene, mutant peptide fragment sequence: VLDEGGSPI, as shown in SEQ ID NO: 11;

DSG3 gene, mutant peptide segment sequence: YLARIEENI, as shown in SEQ ID NO: 12.

As a preferred technical scheme of the invention:

the amino acid mutation sites of the CDKN1A gene are as follows: G61V;

the amino acid mutation sites of the RHOB gene are as follows: P75L;

the amino acid mutation sites of the CDC42 gene are as follows: P73L;

the amino acid mutation sites of the DDB1 gene are as follows: S25L;

the amino acid mutation sites of the AHNAK gene are: D4855Y;

the amino acid mutation sites of the ANP32A gene are as follows: S56L;

the amino acid mutation sites of the ALDH16A1 gene are as follows: G343V;

the amino acid mutation sites of the MET gene are as follows: R1148Q;

the amino acid mutation sites of the PRDX6 gene are as follows: R108W;

the amino acid mutation sites of the MKI67 gene are as follows: H84L;

the amino acid mutation sites of the GAK gene are as follows: S829L;

the amino acid mutation sites of the DSG3 gene are as follows: S273L.

As a preferred technical scheme of the invention, the mutation type of the antigen sequence is missense mutation.

As a preferred technical scheme of the invention, the antigen sequence is obtained by screening through the following steps:

(1) prediction of HLA-A2 restricted wild type and mutant bladder cancer antigen peptides;

(2) detecting the binding force of HLA-A2 restricted wild type and mutant bladder cancer antigen peptides;

(3) determination of affinity constants of HLA-A2-restricted bladder cancer mutant antigen peptides.

As a preferred technical scheme of the invention, the step (1) is specifically as follows: after the separation of peripheral blood mononuclear cells of HLA-A2 positive bladder cancer patients, an enzyme-linked immunospot assay was performed using tumor neoantigens.

As a preferred technical scheme of the invention, the step (2) is specifically as follows: and (3) carrying out statistical analysis on immune reaction generated after the stimulation of the tumor neoantigen, selecting a predicted peptide segment with a ratio above a threshold value and a mutant average fluorescence intensity in the front row for repeated experiments, and screening out a peptide segment which can stably express a main histocompatibility complex molecule in the repeated experiments, wherein the average fluorescence intensity is higher than that of a negative control and has a larger difference with a wild type peptide segment as a candidate peptide segment of the tumor neoantigen.

In one aspect of the invention, the HLA-A2 restricted bladder cancer tumor neoantigen peptide sequence is provided for use in preparation of bladder cancer therapeutic drugs. The bladder cancer therapeutic drug comprises a tumor vaccine and antigen-specific T cells of bladder cancer and the like.

In one aspect of the invention, the invention provides the application of the HLA-A2-restricted bladder cancer tumor neoantigen peptide sequence in the preparation of bladder cancer diagnostic reagents (such as noninvasive urine diagnostic kits). The bladder cancer diagnostic reagent comprises a noninvasive urine diagnostic kit and the like.

The HLA-A2 is the most common HLA genotype (HLA, i.e. human leukocyte antigen, is a complex essential in antigen recognition, A2 is one of the most common subtypes), and the HLA-A2-restricted bladder cancer tumor neoantigen peptide sequence means that the antigen peptide sequence is recognized only by the HLA-A2 complex.

Compared with the prior art, the invention has the beneficial effects that: through the implementation of the technical scheme, 12 HLA-A2 restrictive mutant bladder cancer antigen peptides with immunoreactivity in Chinese bladder cancer patients are obtained by screening, can be used as candidate antigen peptides of bladder cancer tumor vaccines, and are induced in vitro to generate antigen-specific T cells for cell therapy of the bladder cancer patients. After consulting the relevant patents, we found that two patents with application publication numbers CN 105473742 a and CN 105705653A on bladder cancer relate to the two genes CDKN1A (AG1) and MET (AG8) of this patent, but neither relate to tumor neoantigen studies of bladder cancer. Therefore, 12 HLA-A2 restricted bladder cancer tumor neoantigen peptide sequences screened by the invention are brand new in the field, and the reactivity detection (enzyme-linked immunosorbent assay) of the 12 bladder cancer tumor neoantigen peptide sequences in people proves that the 12 bladder cancer tumor neoantigen peptide sequences can be used as candidate antigen peptides of bladder cancer tumor vaccines, and antigen-specific T cells are generated by the in vitro induction of the antigens and are used for cell therapy of bladder cancer patients. Compared with the prior art, the bladder cancer tumor neoantigen peptide library established by the invention can improve the coverage rate of a tumor neoantigen therapy in a patient, and can reduce the treatment cost of the patient and improve the treatment effect of the patient as an accurate treatment therapy.

Drawings

FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D are schematic diagrams showing the results of binding force detection of the tumor neoantigen predicted peptide fragment of the present invention. In FIG. 1A, the average fluorescence intensity of 57 predicted peptides detected by flow-type analysis is ranked according to the average fluorescence intensity of mutant peptides from high to low. FIG. 1B shows the mean fluorescence intensity ratio of 57 pairs of predicted peptide mutants to wild type, ranked from high to low, with a threshold of 1.5. Fig. 1C and 1D are repeated experiments with 18 pairs of predicted peptides, with the first 12 pairs renumbered. Genes is the gene name; MFI: average fluorescence intensity; ratio: average fluorescence intensity ratio; wild-type peptide fragment; mutant-type Mutant peptide fragment. FIG. 1C is a graph showing the mean fluorescence intensity of wild-type peptide fragments and mutant peptide fragments; FIG. 1D is a graph showing the ratio of the mean fluorescence intensity of the wild-type peptide fragments.

FIG. 2 is a schematic diagram showing the affinity detection result of the candidate peptide fragment of the tumor neoantigen of the present invention. Wherein the mean fluorescence intensity of each peptide fragment was deducted from the mean fluorescence intensity of the negative control. Concentration, peptide fragment Concentration; PositiveControl, positive control; AG: numbering antigen peptides; mt: a mutant peptide fragment; MFI: mean fluorescence intensity.

FIGS. 3A and 3B are schematic diagrams showing the results of typing of HLA-A2 of a patient by the flow assay of the present invention. Among them, FIG. 3A represents a patient positive for HLA-A2, and FIG. 3B represents a patient negative for HLA-A2.

FIG. 4A, FIG. 4B and FIG. 4C are schematic diagrams of immunoreactivity detection results of the candidate peptide fragment of the bladder cancer tumor neoantigen in bladder cancer patients of Chinese population. FIG. 4A shows that 12 pairs of candidate peptides were tested for immunoreactivity in peripheral blood of 26 patients with bladder cancer (sample No. P1-P26), and secretion of interferon- γ was detected by ELISA, wherein a secretion amount of greater than 5 indicates a response. FIG. 4B shows the results of an ELISA spot test on patient # 26 (sample # P26). FIG. 4C is the average reactivity of 26 patients with bladder cancer to wild-type and mutant peptide fragments. In the figure, Patients (P) patient number; wild-type (wt): a wild-type peptide fragment; mutant-type (mt) Mutant peptide fragments; AG: numbering antigen peptides; BLANK: blank control; PHA: a positive control; total points/num of tested peptides is the ratio of the Total number of reaction points to the number of test peptide segments; Lg-Reaction is logarithm of ratio of total Reaction point number to test peptide segment number.

FIG. 5 is a statistical chart of the matched sample T test of the mutant and wild type peptide fragments of the present invention; in FIG. 5, Lg-Reaction is the logarithm of the ratio of the number of total Reaction spots to the number of test peptide fragments; wild-type (wt): a wild-type peptide fragment; mutant-type (mt) Mutant peptide fragment.

Detailed Description

The present invention will be described more specifically with reference to examples. It should be understood that the embodiments described herein are intended to illustrate, but not limit the invention.

Firstly, experimental materials:

t2 cell line

The T2 cell is a B lymphoblast immortalized cell line transfected with human leukocyte antigen chimeric molecules, which cannot synthesize beta 2 microglobulin, the expression of human leukocyte antigen on the cell surface is unstable, and exogenous antigen peptide can be presented on the cell surface after exogenous beta 2 microglobulin is added to form a stable human leukocyte antigen-antigen peptide compound. Introduced at the university of Stanford, USA.

2. Reagent

Antigen peptide: shanghai worker

Ficoll separation liquid: alere Technologies AS 1114547

1640 medium: gibco

Fetal bovine serum: gibco

β 2 microglobulin: sigma M4890-250UG

FITC-anti-HLA-a 2 antibody: clone No. BB7.2, product of abcam

ELISPOT kit: U-CyTech biosciences CT 230-PR20

II, an experimental method:

prediction of HLA-A2-restricted wild-type and mutant bladder cancer antigen peptides

Bladder cancer samples were retrieved from the TCGA database, each sample comprising a cancer sample and a normal sample, and mean values of gene expression levels were obtained from TCGA level 3. Obtaining mutation site information from a Firehose database in bladder cancer samples, wherein the mutation only comprises the selected missense mutation. And downloading the corresponding transcript information of the gene to obtain the protein sequence and the corresponding mutation. The method comprises the steps of calculating the affinity of high-frequency Human Leukocyte Antigen (HLA) subtypes (including type I and type II) and all peptide fragments of Chinese Han population in a traversal way, and classifying (1)0-50 as strong binding according to the affinity result; (2)50-500 ═ weak binding; (3) no binding, and selecting the human leukocyte antigen and peptide fragment pairs with strong binding of cancer samples and no binding of normal samples according to affinity classification. Making a corresponding list of all pairs according to different subtypes, and obtaining the score of each gene mutation after calculating the product of the affinity difference value and the expression quantity of the wild type peptide fragment and the mutant type peptide fragment, wherein the larger the difference value is, the higher the gene expression quantity is, and the larger the score is. The pairing of the list which is ranked according to the scores and is the pairing with high binding capacity and high expression quantity is required to be found, and the pairing which is ranked at the top can be taken according to the quantity requirement by using the experimental data.

Binding assays for HLA-A2-restricted wild-type and mutant bladder cancer antigen peptides

To confirm whether tumor neoantigen predicted peptides could cause expression of major histocompatibility complex on T cell surface, we used T2 cells (0.5 × 10)⁶The method comprises the steps of incubating for 4 hours in a 37 ℃ incubator, adding known positive OVA66 antigen peptide L235 (with the sequence being FLPDHINIV) into a positive control group, adding no antigen peptide into a negative control group, and setting a T2 cell control group to add β microglobulin and antigen peptide into the negative control group, after incubation is finished, marking the expression of major histocompatibility complex molecules on the surface of a T2 cell by using a direct immunofluorescence method, measuring the average fluorescence intensity of the major histocompatibility complex molecules on the surface of the T2 cell by using flow cytometry to determine the binding capacity of the candidate antigen peptide of the bladder tumor, after the average fluorescence intensity corresponding to each pair of peptide segments is obtained, sorting according to the average fluorescence intensity of the mutant peptide segments, and simultaneously selecting the peptide segment with the average fluorescence intensity ratio of the mutant peptide segment to the wild type (taking the average or median as a threshold value) to obtain the peptide segment with the average fluorescence intensity close to the front, so as to stimulate the T2 cell to generate high expression quantity, and simultaneously remove the complex with the difference of the major histocompatibility of the wild type complex expressed by repeated peptide segments.

Determination of affinity constant of HLA-A2-restricted bladder cancer mutant antigen peptide

T2 cells (0.5 × 10)⁶The affinity level was expressed by the concentration of candidate peptide fragments exhibiting 50% of the strongest mean fluorescence intensity, which was selected after co-incubation of β 2 microglobulin (Sigma,3ug/mL) and various concentrations (0, 0.4, 2, 10, 20ug/mL) of tumor neoantigen candidate peptide fragments for 4 hours, labeling of T2 cells by immunofluorescence, harvesting of T2 cells by BDFACS CantoII and detection of the mean fluorescence intensity of major histocompatibility complex molecules on the cell surface.

4. Isolation of peripheral blood mononuclear cells

Taking 15mL of peripheral blood of a bladder cancer patient, anticoagulating heparin, adding the anticoagulated heparin into 5mL of Ficoll separating medium according to the volume ratio of 1:1, and horizontally centrifuging for 22min at normal temperature 2300rpm by adopting a slow lifting mode. Gently sucking the middle PBMCs layer, transferring the PBMCs layer into a centrifuge tube containing 10mL 1640 culture medium, centrifuging at 1500rpm for 10min, and discarding the supernatant; the PBMCs were resuspended in 10mL 1640 medium, centrifuged at 1500rpm for 10min, the supernatant discarded, and the cells were finally resuspended in 1mL 1640 complete medium (10% FBS). And (6) counting the cells.

5. Flow assay for HLA-A2 typing of patients

Flow assay is used to analyze the expression of major histocompatibility complex molecules on the cell surface. The cells after completion of incubation were incubated with PE-mouse anti-human-HLA-A2 (clone No. BB7.2, product of Sigma) at 4 ℃ for 40 minutes in the absence of light for surface marker staining. Cells were collected on a FACS Canto II flow cytometer and analyzed using FlowJo software (Treestar, Inc.).

6. Enzyme-linked immunospot assay

Adding 25 μ l 70% ethanol into each well of ELISPOT culture plate, standing at room temperature for 1min, washing with 200 μ l endotoxin-free PBS (10010, invitrogen) for 2 times, patting the plate on sterilized filter paper, adding 50 μ l anti-IFN- γ Coating Antibody into each well, removing liquid after overnight at 4 deg.C, adding 200 μ l PBS to each well, washing for 3 times, adding 200 μ l blocking Buffer into each well, blocking at 37 deg.C for 1h, removing blocking solution, adding 100 μ l 2.5 × 10 into each well⁶PBMC cell suspension/ml and corresponding stimuli. Wherein 2 mug/ml antigen peptide is added into the experimental hole, and 2.5 mug/ml PHA is used as positive control; culturing at 37 deg.C with 5% CO2 and 100% humidity in cell culture box for 20 h; the cell fluid was removed and washed 2 times with 200. mu.l of room temperature PBS per well. Followed by 5 washes with 250 μ l PBST (PBS containing 0.05% Tween-20); adding 100 μ l of anti-IFN-gamma Detection Antibody into each well, sealing with sealing film, and combining at 37 deg.C for 1h or 4 deg.C overnight; the supernatant was discarded and 5 washes were performed by adding 250. mu.l PBST per well. Adding 100 mu l of Streptavidin-HRP solution into each hole, pasting and sealing by using a sealing film, and combining for 1h at 37 ℃; discard the supernatant, add 250. mu.l PBST per well and wash 5 times; adding 100 μ l of AEC substrate into each well, and developing in dark at room temperature for 30 min; removing the plastic plate at the bottom of the ELISPOT plate, and distillingThoroughly washing the front and back sides of the PVDF membrane by water to terminate the reaction; drying overnight at room temperature in the dark, and reading the number of spots with an enzyme-linked spot counter.

7. Statistical analysis

We performed statistical analysis using SPSS (IBM, SPSS, Statistics version21) software, and P <0.05 was considered meaningful.

Third, experimental results

Prediction of HLA-A2-restricted wild-type and mutant bladder cancer antigen peptides

There were 412 bladder cancer samples in TCGA, each sample comprising cancer and normal samples, respectively. Number of genes in bladder cancer samples obtained from Firehose: 16195, number of mutations: 84719, the mutation only comprises the missense mutation selected. The transcript information corresponding to the gene was downloaded to obtain 13718 protein sequences corresponding to the number of mutations 64134. After the affinity of the high-frequency human leukocyte antigen subtypes of Chinese Han people is calculated in a traversal way, the affinity of traversal pairing of 34 human leukocyte antigen subtypes and 128268 peptide fragments (including 64134 cancer sample peptide fragments and 64134 normal sample peptide fragments) is obtained. Pairs with strong binding to cancer samples and no binding to normal samples were selected according to affinity classification, and 8827 pairs were selected from 2180556 pairs, which contained 30 subtypes (the remaining 4 subtypes did not satisfy the conditions). We selected a consensus 920 for human leukocyte antigen-a x 02: the first 57 pair of the 01 restriction peptide fragment pair was chemically synthesized. The name of each gene, the amino acid mutation site, the peptide sequences of the wild type and the mutant type, the affinity, the gene expression level and the score are included in table 1.

TABLE 1

Table 1 shows the peptide fragment information of 57 restriction prediction for human leukocyte antigen-A2. The list is sorted by score. The gene subscript is the amino acid mutation site, and the number (N) indicates that the N-th amino acid is mutated. The affinity represents the binding force of the corresponding 9 peptides, and the smaller the numerical value, the stronger the binding force; the expression level represents the mean of the gene expression levels obtained from TCGA level 3; the score represents the product of the difference in affinity between the wild-type and mutant peptide fragments multiplied by the amount of expression.

Binding assays for HLA-A2-restricted wild-type and mutant bladder cancer antigen peptides

In the first experiment, the average fluorescence intensity of mutant type of almost all peptide fragments is higher than that of negative control (4665), and the average fluorescence intensity of wild type peptide fragments is basically equal to that of the negative control. Wherein CDKN1A_G61V、RHOB_P75L、AHNAK_D4855YThe average fluorescence intensity of the mutant type of the predicted peptide fragment encoded by the three genes is higher than that of the positive control (10400), and the ratio of the average fluorescence intensity of the mutant type to the wild type of the three genes is positioned in the front of 57 pairs of peptide fragments (see fig. 1A and fig. 1B). We selected the predicted peptide with the ratio above the threshold (mean 1.5) and the mean fluorescence intensity of the mutant at the first 18 positions for repeat experiments. In repeated experiments, the average fluorescence intensity of 6 mutant types in the 18 pairs of peptide fragments is lower than that of a negative control, and the difference from the wild type is not large, so the peptide fragments cannot be used as tumor neoantigen candidate peptide fragments. The remaining 12 mutant versions of the peptides still stably expressed major histocompatibility complex molecules in the repeated experiments, and the mean fluorescence intensity was higher than that of the negative control and was much different from that of the wild-type peptide (see fig. 1C and fig. 1D).

Determination of affinity constant of HLA-A2-restricted bladder cancer mutant antigen peptide

At different concentrations, 12 showed different affinities for mutant forms of tumor neoantigen peptide candidates, increasing with increasing concentration. The level of affinity of the mutant peptide fragment was in the range of 0.6-11ug/ml, with the highest affinity being AG2-Mt and the lowest being AG8-Mt (see FIG. 2).

4. Isolation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells were well isolated from all of 26 patients with HLA-A2 positive bladder cancer.

5. Flow assay for HLA-A2 typing of patients

The fluorescence expression level of cell surface PE of HLA-A2-positive bladder cancer patients is obviously higher than that of negative patients, and the patients can be confirmed to be HLA-A2-positive (see FIG. 3A and FIG. 3B). Wherein FIG. 3A is a patient positive for HLA-A2 and FIG. 3B is a patient negative for HLA-A2.

6. Enzyme-linked immunospot assay

The mononuclear cells in the peripheral blood of the bladder cancer patient show different immunoreactivity after being stimulated by 12 tumor neoantigen candidate peptides. The reactivity of different patients to different candidate peptides was different, but the reactivity of the mutant peptide was higher than that of the wild type as a whole (see FIG. 4A, FIG. 4B, FIG. 4C and Table 2). FIG. 4A, FIG. 4B and FIG. 4C are schematic diagrams of immunoreactivity detection results of the candidate peptide fragment of the bladder cancer tumor neoantigen in bladder cancer patients of Chinese population. FIG. 4A shows that 12 pairs of candidate peptides were tested for immunoreactivity in peripheral blood of 26 patients with bladder cancer (sample No. P1-P26), and secretion of interferon- γ was detected by ELISA, wherein a secretion amount of greater than 5 indicates a response. FIG. 4B shows the results of an ELISA spot test on patient # 26 (sample # P26). FIG. 4C is the average reactivity of 26 patients with bladder cancer to wild-type and mutant peptide fragments. Taking P26 as an example, it can be seen that AG2, AG6, AG9 and AG12 peptide fragments stimulate the peripheral blood mononuclear cells to cause a certain degree of immune response, while the corresponding wild-type peptide fragments do not cause enough immune response, which indicates that the peripheral blood mononuclear cells of the patient can recognize and generate immune killing effect against the four tumor neoantigen peptide fragments. In the figure, Patients (P) patient number; wild-type (wt): a wild-type peptide fragment; mutant-type (mt) Mutant peptide fragments; AG: numbering antigen peptides; BLANK: blank control; PHA: a positive control; total points/num of testepeps the ratio of the number of Total reaction points to the number of test peptide segments.

7. Statistical analysis

Matched sample T-test of mutant and wild-type peptides, P <0.05, the difference was statistically significant, i.e. at the global level, the pool of peptides consisting of these 12 tumor neoantigen peptides could elicit specific recognition in bladder cancer patients (see figure 5). In FIG. 5, Lg-Reaction is the logarithm of the ratio of the number of total Reaction spots to the number of test peptide fragments; wild-type (wt): a wild-type peptide fragment; mutant-type (mt) Mutant peptide fragment.

Through the implementation of the technical scheme, the following 12 HLA-A2 restriction mutant bladder cancer antigen peptides with immunoreactivity in Chinese human bladder cancer patients are obtained through screening (see table 2). The 12 peptide fragments can show stronger affinity in virtual screening and in-vitro screening of the peptide fragments (see the experimental results 1, 2 and 3), and a peptide fragment library consisting of the 12 peptide fragments can also stimulate enough immune killing effect in peripheral blood mononuclear cells of bladder cancer patients (see the experimental results 4, 5, 6 and 7), so the peptide fragments can be used as candidate antigen peptides of bladder cancer tumor vaccines, and antigen-specific T cells are generated by in-vitro induction of the antigens, and are used for cell therapy of the bladder cancer patients.

TABLE 212 basic information of HLA-A2 restriction bladder cancer tumor neoantigen peptide

The above is only a specific application example of the present invention, and the protection scope of the present invention is not limited in any way; other variations and modifications will be apparent to persons skilled in the art in light of the above description. All embodiments need not be described or illustrated herein. The technical solutions similar to the above embodiments formed by equivalent transformation or equivalent replacement fall within the scope of the claims of the present invention.

Sequence listing

<110> Xinhua hospital affiliated to Shanghai university of traffic medical school

<120> HLA-A2 restricted bladder cancer tumor neoantigen peptide sequence and application thereof

<130>WH-NP-19-100150

<160>12

<170>PatentIn version 3.5

<210>1

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>1

FVTETPLEV 9

<210>2

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>2

YLDTDVILM 9

<210>3

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>3

YLQTDVFLV 9

<210>4

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>4

TLAEDLNLL 9

<210>5

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>5

YLDLKGPKV 9

<210>6

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>6

TLIANLPKL 9

<210>7

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>7

GLDGAVDMV 9

<210>8

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>8

LQSEGSPLV 9

<210>9

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>9

IIDDRNWEL 9

<210>10

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>10

KLGDVITII 9

<210>11

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>11

VLDEGGSPI 9

<210>12

<211>9

<212>PRT

<213> Artificial sequence (unknown)

<400>12

YLARIEENI 9