CN109021062B

CN109021062B - Screening method of tumor neoantigen

Info

Publication number: CN109021062B
Application number: CN201810885573.5A
Authority: CN
Inventors: 张崇骞; 赵永浩; 马赛; 闫成海; 张晓霞; 彭继荣; 张晓东
Original assignee: Beierda Pharmacy Suzhou Co ltd
Current assignee: Beierda Pharmacy Suzhou Co ltd
Priority date: 2018-08-06
Filing date: 2018-08-06
Publication date: 2021-08-20
Anticipated expiration: 2038-08-06
Also published as: CN109021062A

Abstract

The invention provides a method for screening a tumor neoantigen, which comprises the following steps: A. obtaining a polypeptide sequence corresponding to a mutant gene of a tumor tissue cell; B. respectively carrying out affinity prediction on the polypeptide sequences and a Main Histocompatibility Complex (MHCI), and screening out polypeptide sequences with the affinity exceeding a specified threshold; C. and predicting the polypeptide enzyme cutting site of the polypeptide sequence corresponding to the mutant gene, reserving the polypeptide sequence which is not positioned in the middle of the polypeptide sequence with the affinity exceeding the specified threshold value and can be independently cut into peptide segments without enzyme cutting of the enzyme cutting site, and taking the polypeptide sequence as a candidate polypeptide sequence of the tumor neoantigen. Therefore, the screening method of the tumor neoantigen facilitates subsequent further targeted experiments, can greatly reduce the times of the experiments, and realizes time saving, labor saving and cost saving.

Description

Screening method of tumor neoantigen

Technical Field

The invention relates to the field of antigen screening, in particular to a screening method of a tumor neoantigen.

Background

Tumor vaccines (tumor vaccines) are one of the hot spots in recent years of research, whose principle takes tumor antigens in various forms such as: tumor cells, tumor-related proteins or polypeptides, genes for expressing tumor antigens, and the like are introduced into a patient body, so that the immunosuppression state caused by tumors is overcome, the immunogenicity is enhanced, the immune system of the patient is activated, and the cellular immunity and humoral immunity response of the organism are induced, thereby achieving the purpose of controlling or eliminating the tumors. In 4 months 2010, the Food and Drug Administration (FDA) approved Provenge/sipuleucel-T for treating advanced prostate cancer, making it the first autologous active immunotherapy drug and the first true therapeutic cancer vaccine, paving the way for the development of other similar products (1, 2).

In 2017, 2 technical teams have achieved favorable results in the personalized tumor vaccine clinical trial based on the NGS, and the clinical trial results of the U.S. team: of 6 melanoma patients vaccinated with the vaccine, 4 tumors completely disappeared and no recurrence within 32 months, and 2 tumors remained and completely disappeared after receiving adjuvant therapy; results of clinical trials on the german team: of the 13 vaccinated patients, 8 had completely disappeared tumors and no recurrence within 23 months, and the remaining 5 patients had 2 had developed tumor shrinkage due to the tumor spread at the time of vaccination, of which 1 had completely regressed 1,2 after receiving adjuvant therapy. The technology or the treatment method utilizes the individualized tumor neogenesis antigen to regulate or activate the immune system to kill the tumor, and is combined with other tumor treatment methods in principle to possibly change the tumor into the chronic disease, and the future market potential is huge if large-scale clinical verification is successful (3, 4).

However, the research and development of tumor vaccines are carried out one by one through experimental means, the process is time-consuming, labor-consuming and expensive, and suitable tumor vaccines (tumor antigens) are not easy to find, so that a method for screening tumor neoantigens is urgently needed at present, and suitable tumor vaccines are obtained through screening tumor neoantigens, so that the targeted experiments can be carried out conveniently in the future, the times of the experiments are greatly reduced, and time, labor and cost are saved.

Disclosure of Invention

In view of the above, the main objective of the present invention is to provide a method for screening tumor neoantigens, which facilitates further experiments with pertinence in the following, greatly reduces the number of experiments, and saves time, labor and expenses.

The application provides a method for screening a tumor neoantigen, which comprises the following steps:

A. obtaining a polypeptide sequence corresponding to a mutant gene of a tumor tissue cell;

B. respectively carrying out affinity prediction on the polypeptide sequences and a Main Histocompatibility Complex (MHCI), and screening out the polypeptide sequences with the affinity exceeding a specified threshold;

C. and predicting the polypeptide enzyme cutting site of the polypeptide sequence corresponding to the mutant gene, reserving the polypeptide sequence which is not positioned in the middle of the polypeptide sequence with the affinity exceeding the specified threshold value and can be independently cut into peptide segments without enzyme cutting of the enzyme cutting site, and taking the polypeptide sequence as a candidate polypeptide sequence of the tumor neoantigen.

In view of the above, the screening method of tumor neoantigens provided by the present application comprises the following steps: the polypeptide sequences are respectively subjected to affinity prediction with a Main Histocompatibility Complex (MHCI) for screening (the affinity of the polypeptide and the MHCI ensures that the polypeptide MHCI complex can be successfully identified by a T cell surface receptor (TCR), so that T cells are activated, and relevant cellular immune response is triggered), and the prediction of a polypeptide restriction enzyme site is screened (the prediction of the polypeptide restriction enzyme site can reflect whether the polypeptide is easily cut and presented on MHC, and the prediction of the polypeptide restriction enzyme site can assist in predicting whether core polypeptide can be correctly cut).

Preferably, the tumor neoantigen of step C further comprises: the tumor tissue, the tumor tissue related protein, the mutant DNA sequence or the mutant RNA sequence of the tumor tissue cell.

Preferably, step C is followed by:

D. and performing affinity prediction on the core polypeptide sequence and an antigen processing related transporter, and taking the core polypeptide sequence with the predicted result showing that the affinity is within a specified range as a tumor neoantigen.

Therefore, the affinity of the polypeptide and the transporter related to antigen processing reflects whether the polypeptide can be successfully carried out in the presentation process, so that the better affinity can ensure that the polypeptide can be successfully presented to MHCI molecules to a certain extent, and the affinity of the polypeptide and the MHCI can be predicted as an auxiliary means when the affinity of the polypeptide and the MHCI is predicted.

Preferably, the step C or D further comprises:

E. the core polypeptide is subjected to a structural docking test with the major histocompatibility complex MHCI, and the structure of the docking conformation of the resulting complex is scored by an empirical scoring function of free energy, and the highest scoring docking conformation is retained.

Therefore, the docking of the polypeptide based on the structure and the MHCI molecule is an effective means for researching the interaction rule between the polypeptide ligand and the receptor biomacromolecule and predicting the binding mode and the affinity of the polypeptide ligand and the receptor biomacromolecule. The objective of docking here is to obtain a reliable and reasonable molecular binding conformation of the epitope polypeptide to MHCI. And then scoring by an empirical scoring function of the binding free energy, and quantitatively estimating the size of the affinity of the epitope polypeptide and the MHCI molecule.

Preferably, after the step E, the method further comprises:

F. simulating by molecular dynamics the interaction and kinematic changes between the highest scoring docked conformation and the major histocompatibility complex, MHCI; and analyzing the sequence composition of the binding site of the core polypeptide sequence and the MHC (major histocompatibility complex) MHCI.

From the above, the molecular dynamics simulation is to simulate the interaction and movement change of macromolecules and polypeptides according to the basic principle of Newton mechanics, so as to explore the rules behind the life phenomena which cannot be solved by experimental means. The interaction rule and the movement change between the MHCI and the polypeptide complex are discussed by a molecular dynamics simulation means, the interaction and the affinity between the polypeptide and the MHCI in a stable state can be visually shown, and whether the polypeptide can be stably combined with the MHCI or not can be accurately predicted.

Preferably, after the step F, the method further comprises:

G. when a mutant amino acid is judged to be present in the sequence composition of the junction and the mutant amino acid is judged to be tightly bound to the MHC; and taking the candidate polypeptide sequence as the sequence of the screened tumor neoantigen.

From the above, it was demonstrated that the polypeptide stably binds to MHCI, and it is the generation of this mutated amino acid that makes it possible to use the candidate polypeptide sequence as a sequence for a new tumor antigen to be screened.

Preferably, the step a includes:

A. extracting DNA of tumor tissue cells, and performing DNA sequencing on the tumor tissue cells;

B. comparing the sequenced DNA sequence with the normal DNA sequence of the tissue cell to obtain a mutant DNA sequence;

C. and acquiring a corresponding encoded polypeptide sequence according to the mutated DNA sequence through biological software.

Thus, the polypeptide sequence encoded by the mutant gene of the tumor tissue cell can be obtained through the steps.

Preferably, in step a, the polypeptide sequence is: polypeptide sequences containing 8-30 amino acid residues.

From the above, a polypeptide sequence having a length of 8 to 30 amino acid residues is preferred in affinity, and too long affects the affinity of the polypeptide sequence, while too short affects the efficacy of the polypeptide.

In summary, the screening method of the tumor neoantigen provided by the present application comprises the following steps: predicting the affinity of the polypeptide and MHCI, simultaneously assisting with proteasome cleavage site prediction, TAP transport prediction, structure-based MHC and polypeptide docking tests, and performing molecular dynamics simulation between the docking conformation with the highest molecular dynamics simulation score and the MHCI; the screened tumor neoantigen is convenient for subsequent further targeted experiments, the times of the experiments can be greatly reduced, and time, labor and cost can be saved.

Drawings

FIG. 1 is a flow chart of a method for screening tumor neoantigens according to the present embodiment;

FIG. 2 is a schematic representation of several docking conformations resulting from docking of a polypeptide of the embodiments of the present application with MHCI;

fig. 3 is a schematic representation of the docking conformation of core polypeptide VLAKKLKFV of the present example with the highest score after flexible docking with MHCI (specifically HLA-a 0201);

FIG. 4 is a schematic representation of the conformation of core polypeptide VLAKKLKFV after conformation of MHCI (specifically HLA-A0201) according to the present embodiment to molecular dynamics simulation;

fig. 5 is a schematic representation of the binding image of the mutant residue Lys4 in core polypeptide VLAKKLKFV of an embodiment of the present application to MHCI (specifically HLA-a 0201) binding groove;

FIG. 6 shows the results of molecular docking scores for 4 polypeptides designed in the examples of this application;

FIG. 7 shows molecular docking conformations of 4 polypeptides with HLA-A0201 designed in the examples of the present application;

fig. 8 shows the results of the affinity assay for HLA-a 0201T 2 cells in the 4 polypeptides designed in this example of the present application.

Detailed Description

The present application will be described below with reference to the drawings in the embodiments of the present application.

Example one

As shown in fig. 1, this embodiment provides a method for screening tumor neoantigens, comprising the steps of:

s101, obtaining a polypeptide sequence corresponding to the mutant gene of the tumor tissue cell of the patient. Specifically, the method comprises the following steps:

A. extracting DNA of tumor tissue cells by an SDS method, and carrying out DNA sequencing on the DNA;

B. and comparing the sequenced DNA sequence with the DNA sequence of the normal wild tissue cell to obtain a mutant DNA sequence different from the DNA sequence of the normal wild tissue cell. The normal DNA sequence of the tissue cell can be obtained by the existing database. Wherein the database may be: COSMIC, NCBI, UCSC, Ensembl, TCGA, etc.

C. And obtaining the polypeptide sequence correspondingly coded by the mutant DNA sequence through biological software. The biological software may be DNA-man, or other software capable of translating DNA sequences into amino acid sequences. Wherein the polypeptide sequence is: a polypeptide sequence comprising at least 8-30 amino acid residues.

The present embodiment of the present application takes two peptide fragments obtained from the following two tumor tissue cells as examples:

>s1_ACPP_E34K

DRSVLAKKLKFVTLVFRHGDRSPID

>s2_MECOM_Q216K

EDSDKLFESKAELADHQKF

s102, respectively carrying out affinity prediction on the polypeptide sequences and the MHC (major histocompatibility complex) MHCI, and screening out the polypeptide sequences with the affinity exceeding a specified threshold value.

Specifically, the affinity of the polypeptide to MHCI ensures that the polypeptide MHCI complex can be successfully recognized by a T cell surface receptor (TCR), thereby activating T cells and initiating relevant cellular immune responses. Therefore, it is important to predict the affinity of the polypeptide for MHCI, and the degree of affinity of the polypeptide for MHCI is critical to whether the polypeptide can be successfully used as a tumor neoantigen vaccine.

The MHCI sources of the application are four hybrid MHCI types of HLA-A2402, HLA-A0201, HLA-B1501 and HLA-B4402. Respectively carrying out artificial neural network-based affinity prediction on the two polypeptides in the step S101 (wherein, the affinity prediction methods include but are not limited to artificial neural network, deep learning, support vector machine, specificity score matrix, 3D-QSAR (ComaFA, CoMSIA), hidden Markov model and other knowledge base-based prediction methods):

the results are as follows:

table one: and (3) predicting the affinity of the sequence DRSVLAKKLKFVTLVFRHGDRSPID with HLA-A2402.

Table two: sequence DRSVLAKKLKFVTLVFRHGDRSPID prediction of HLA-A0201 affinity.

Table three: sequence DRSVLAKKLKFVTLVFRHGDRSPID prediction of HLA-B1501 affinity.

Table four: the sequence DRSVLAKKLKFVTLVFRHGDRSPID and HLA-B4402 affinity prediction result.

Table five: and (3) predicting the affinity of the sequence EDSDKLFESKAELADHQKF with HLA-A2402.

Table six: sequence EDSDKLFESKAELADHQKF prediction of HLA-A0201 affinity.

TABLE VII: sequence EDSDKLFESKAELADHQKF prediction of HLA-B1501 affinity.

Table eight: the sequence EDSDKLFESKAELADHQKF and HLA-B4402 affinity prediction result.

The affinity of VLAKKLKFV _ ACPP to HLA-A0201 was found to be 59.32Nm by analysis of the results as above; KLFESKAEL _ MECOM has 19.05Nm affinity for HLA-A0201; KLKFVTLVF _ ACPP has an affinity for HLA-A1501 of 13.34 Nm; AELADHQKF _ MECOM has an affinity for HLA-B4402 of 15.25 Nm. The above polypeptides have excellent affinity to the relevant MHCI types. In the implementation case, the affinity less than or equal to 500Nm is used as an acceptable predicted value, and the affinity is strong in the range of 0-150 Nm. HLA-a0201 typing is very widespread among asian populations, so we chose the polypeptide sequence VLAKKLKFV _ ACPP with affinity above a specified threshold as the core 9 peptide of DRSVLAKKLKFVTLVFRHGDRSPID and KLFESKAEL _ MECOM as the core 9 peptide of EDSDKLFESKAELADHQKF.

S103, predicting the polypeptide enzyme cutting sites of the polypeptide sequence in the S101, reserving the polypeptide sequence which is not positioned in the middle of the polypeptide sequence with the affinity exceeding the specified threshold value and can be independent of the enzyme cutting sites, and taking the polypeptide sequence as a candidate polypeptide sequence of the tumor neoantigen.

Specifically, the prediction of the cleavage site of the polypeptide can reflect whether the polypeptide is easily cleaved and presented on MHC, and the prediction of the cleavage site of the polypeptide can assist in predicting whether the core polypeptide can be correctly cleaved. The method is mixed with a prediction algorithm for predicting the multi-enzyme body shearing capability, and can accurately predict the shearing sites of the polypeptide in the protein, thereby assisting in predicting the antigen forming potential of the new antigen polypeptide. Respectively predicting > s1_ ACPP _ E34K by means of an artificial neural network or correlation regression analysis and the like: DRSVLAKKLKFVTLVFRHGDRSPID and > s2_ MECOM _ Q216K: EDSDKLFESKAELADHQKF cleavage sites in the proteasome.

The results are as follows:

table nine: sequence DRSVLAKKLKFVTLVFRHGDRSPID predicted by a multienzyme cleavage site.

By predicting the sequence DRSVLAKKLKFVTLVFRHGDRSPID as a multienzyme cleavage site, the two ends of the core polypeptide VLAKKLKFV can be accurately cleaved in the multienzyme.

TABLE Ten: sequence EDSDKLFESKAELADHQKF predicted by a multienzyme cleavage site. By predicting the EDSDKLFESKAELADHQKF proteasome cleavage site, it was found that a strong cleavage site was found at the ninth position against EDSDKLFESKAELADHQKF, and it was difficult to generate a core polypeptide of KLFESKAEL in a human state, and thus the polypeptide was filtered out. VLAKKLKFV is a peptide fragment capable of being separated, and it is reserved as a candidate polypeptide sequence of tumor neoantigen.

S104, performing affinity prediction on the candidate polypeptide sequence (the core polypeptide VLAKKLKFV) and an antigen processing related transporter, and taking the core polypeptide sequence with the predicted result showing that the affinity is within a specified range as a tumor neoantigen.

Specifically, the affinity of the polypeptide and an antigen processing-related transporter reflects whether the polypeptide can be successfully carried out in the presentation process, so that the better affinity can ensure that the polypeptide can be successfully presented to MHCI molecules to a certain extent, and the affinity of the polypeptide and the MHCI can be predicted as an auxiliary means when the affinity of the polypeptide and the MHCI is predicted. An algorithm trained on a laminated support vector machine method is used for predicting the affinity of the polypeptide and an antigen processing related transporter, and 88% of correlation is verified by a jack-knit method.

TABLE eleven core polypeptide VLAKKLKFV prediction of affinity for antigen processing-associated transporters

Affinity analysis with polypeptides found that both polypeptides showed moderate affinity to TAP, and that we set polypeptides of moderate and high affinity in the SOP as potential candidate polypeptides. The core polypeptide VLAKKLKFV is predicted to have higher affinity for antigen processing-associated transporters.

S105, performing a structural docking test of the candidate polypeptide sequence (core polypeptide VLAKKLKFV) with mhc i, and scoring the structure of the docked conformation of the resulting complex by an empirical free energy scoring function, and retaining the highest scored docked conformation.

Specifically, the docking of the polypeptide based on the structure and the MHCI molecule is an effective means for researching the interaction rule between the polypeptide ligand and the receptor biomacromolecule and predicting the binding mode and the affinity of the polypeptide ligand and the receptor biomacromolecule. The objective of docking here is to obtain a reliable and rational binding conformation of the epitope polypeptide to the MHCI (here specifically HLA-a 0201) molecule. The method comprises the steps of structural-based MHCI and polypeptide docking modes, wherein the methods include but are not limited to peptide binding groove electrostatic distribution, energy matching, space matching, lattice point calculation, fragment growth, simulated annealing, genetic algorithm and the like; the docking mode classification includes rigid docking, semi-flexible docking, and flexible docking. In the docking process, the quality of the interaction between the epitope polypeptide molecule and the MHCI molecule is evaluated in real time according to the principles of geometric complementation, energy complementation and chemical environment complementation, and the optimal binding mode of the epitope polypeptide molecule and the MHCI molecule is found. And then scoring by an empirical scoring function of the binding free energy, and quantitatively estimating the size of the affinity of the epitope polypeptide and the MHCI molecule.

We flexibly docked the polypeptide to MHCI by the rosetta docking procedure, resulting in 200 docked conformations. FIG. 2 is a schematic representation of several docking conformations generated after flexible docking of polypeptides of the embodiments of the present application with MHCI.

And the structure of the docking conformation of the resulting complex is scored by an empirical scoring function of free energy, the docking score being as follows:

total_score	rm sBB	descrip tion
			-574.774	13.556	top_1.pdb
-574.001	9.972	top_2.pdb
			-573.248	2.625	top_3.pdb
-572.879	2.534	top_4.pdb
			-572.448	1.461	top_5.pdb
-572.329	4.19	top_6.pdb
			-572.216	1.742	top_7.pdb
-572.139	1.804	top_8.pdb
			-572.052	6.753	top_9.pdb
-571.849	2.073	top_10.pdb

prediction of affinity of the twelve core polypeptide VLAKKLKFV for transporters involved in antigen processing

As shown in fig. 3, we selected the highest scoring docked conformation as the conformation for subsequent analysis.

S106, simulating the interaction and movement change between the docking conformation with the highest score and the MHCI through molecular dynamics; and the sequence composition of the binding site of the candidate polypeptide sequence (core polypeptide VLAKKLKFV) to MHC (major histocompatibility Complex) was obtained from this analysis.

Specifically, molecular dynamics simulation is to simulate the interaction and movement change of macromolecules and polypeptides according to the basic principle of Newton mechanics, so as to explore the rules behind the life phenomena which cannot be solved by experimental means. The interaction rule and the movement change between the MHCI and the polypeptide complex are discussed by a molecular dynamics simulation means, the interaction and the affinity between the polypeptide and the MHCI in a stable state can be visually shown, and whether the polypeptide can be stably combined with the MHCI or not can be accurately predicted. Among them, molecular dynamics simulation of MHCI and polypeptide complex includes, but is not limited to, NAMD, Amber, Gromacs, etc. We performed molecular dynamics simulations at 10ps, with the following results:

table thirteen: results of molecular dynamics simulation

As shown in fig. 4, the conformations after molecular dynamics simulation between core polypeptide VLAKKLKFV and MHCI (HLA-a 0201) are shown. Through further analysis of molecular dynamics results, it was found that: the anchor residues in the two segments of the core polypeptide are tightly bound to Glu63, Lys66, Tyr171, Trp123, Lys146, and the like of HLA-a0201, respectively, by hydrogen bonds or the like.

S107, when the amino acid of the candidate polypeptide sequence (core polypeptide VLAKKLKFV) at the position where the candidate polypeptide sequence is combined with the MHCI is judged to be a mutant amino acid, and the mutant amino acid is judged to be tightly combined with the MHCI, the candidate polypeptide sequence is taken as the sequence of the screened tumor neoantigen.

Specifically, the wild-type sequence of the polypeptide VLAKKLKFV is VLAEKLKFV, and is Glu4Lys mutation, and the results of molecular dynamics simulation show that Lys4 is tightly combined with the binding groove of HLA-A0201. The above results were combined to find that VLAKKLKFV has a very strong affinity for HLA-A0201.

To better illustrate the affinity effect of the polypeptide sequences obtained by the screening method of the present application, the present application also performed the following tests:

firstly, docking a polypeptide set with HLA-A0201 molecules by using a molecular docking program ZDCK, and qualitatively analyzing the affinity between the polypeptide and the HLA:

polypeptide molecules were designed as follows:

serial number	Polypeptide name	Polypeptide sequence	netMHC4.0(nM)
				1	positive ctrl	NH2-YLLPAIVHI-CONH2	4.32
2	negative ctrl	NH2-LGKRGSKPK-CONH2	43819.37
				3	core-pep1	NH2-VLAKKLKFV-CONH2	59.32
4	core-pep2	NH2-KLFESKAEL-CONH2	119.05

Table fourteen: designed polypeptide sequence

And constructing a polypeptide molecule small data set according to the polypeptide sequences, performing receptor HLA-A0201 modeling and binding region determination, performing docking of the receptor HLA-A0201 and the polypeptide molecule small data set, and finally, comparing and analyzing docking scores of the polypeptide sequences.

As shown in fig. 6, the results of molecular docking scores for 4 polypeptides designed in the examples of the present application; the higher the score, the higher the affinity of the polypeptide for the corresponding MHCI. It was found by analysis that the polypeptide sequence No. 3 selected by the screening method described above was the highest docking score, followed by the control polypeptide sequence No. 1 (which is a known polypeptide sequence with good affinity), followed by the polypeptide sequence No. 4, and followed by the lowest 1. And the results are substantially in accordance with those predicted by the knowledge base-based software netMHC4.0 (NeMHC4.0 predicts the value of the affinity of the polypeptide to HLA molecules in Nm, the lower the value, the stronger the affinity)

II, polypeptide affinity detection

1. T2 cell culture: t2 cells were purchased from ATCC and cultured in 20% FBS IMDM (Gibco) complete medium;

2. the predicted polypeptide sequence is synthesized by a solid phase, the purity of the polypeptide is more than or equal to 95 percent, and the polypeptide is dissolved by DMSO and then frozen at-80 ℃ for storage;

3. the following raw materials were charged into a 24-well plate: t2 cells, 1X10^6 cells/well; native human β 2 microglobulin (Prospec) at a final concentration of 0.5 μ M; each polypeptide was set to a final concentration gradient of: mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M and 80. mu.M were added to each 24-well plate, and incubated at 37 ℃ in a 5% CO2 incubator for 16 hours. Setting blank group and control group (without polypeptide) in experiment;

4. transferring the cells into a 1.5ml centrifuge tube, washing for 2 times by using 1ml 1XPBS, and discarding the supernatant;

5. adding FITC Mouse Anti-Human HLA-A2(BD Biosciences, Oxford, U.K.) and incubating for 1h at 4 ℃ in the dark;

6. washing with 1ml 1XPBS for 2 times, and discarding the supernatant;

7. resuspend the cells with 500 μ L1 XPBS and transfer to a flow analysis tube;

8. detection with a flow analyzer (BD Biosciences);

9. analyzing the detection result by using Flow-Jo and GraphPad Prism;

10. the detection result is expressed as a Fluorescence Index (FI), and FI is MFI sample/MFI background.

The results are shown in FIG. 8, and it can be seen from FIG. 8 that the affinity results 3 and 1 are the highest, followed by 4 and 2, which are substantially consistent with the molecular docking score results in FIG. 6.

In summary, the screening method of the tumor neoantigen provided by the present application comprises the following steps: predicting the affinity of the polypeptide and MHCI, simultaneously assisting with proteasome cleavage site prediction, TAP transport prediction, structure-based MHC and polypeptide docking tests, and performing molecular dynamics simulation between the docking conformation with the highest molecular dynamics simulation score and the MHCI; the screened tumor neoantigen is obtained, and further targeted experiments are carried out according to the tumor neoantigen, so that the times of the experiments can be greatly reduced, and time, labor and cost are saved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for screening a tumor neoantigen, comprising the steps of:

C. predicting a polypeptide enzyme cutting site of a polypeptide sequence corresponding to the mutant gene, reserving a polypeptide sequence which is not positioned in the middle of the polypeptide sequence with the affinity exceeding a specified threshold value and can be independently cut into a peptide segment without enzyme cutting of the enzyme cutting site, and taking the polypeptide sequence as a candidate polypeptide sequence of the tumor neoantigen;

wherein, said step C is followed by:

D. performing affinity prediction on the candidate polypeptide sequence and an antigen processing related transporter, and taking the candidate polypeptide sequence with the predicted result showing that the affinity is within a specified range as a tumor neoantigen;

wherein, said step C or D is followed by:

E. performing structural docking test on the candidate polypeptide sequence and the Main Histocompatibility Complex (MHCI) by adopting a Rosetta docking program and a ZDCK docking program, scoring the structure of the docking conformation of the generated complex through an empirical scoring function of free energy, and reserving the docking conformation with the highest score;

after the step E, the method further comprises:

F. simulating by molecular dynamics the interaction and kinematic changes between the highest scoring docked conformation and the major histocompatibility complex, MHCI; and analyzing and obtaining the sequence composition of the combination part of the candidate polypeptide sequence and the main histocompatibility complex MHCI;

after the step F, the method further comprises:

G. when a mutant amino acid is judged to be present in the sequence composition of the junction and the mutant amino acid is judged to be tightly bound to MHC class II; and taking the candidate polypeptide sequence as the sequence of the screened tumor neoantigen.

2. The method of claim 1, wherein step a comprises:

a1, extracting DNA of tumor tissue cells, and carrying out DNA sequencing on the tumor tissue cells;

a2, comparing the sequenced DNA sequence with the DNA sequence of the normal wild tissue cell to obtain a mutant DNA sequence;

a3, obtaining the polypeptide sequence correspondingly coded by the mutated DNA sequence through biological software.

3. The method of claim 1, wherein the polypeptide sequence of step a is: a polypeptide sequence comprising 9 amino acid residues.