CN111909992A

CN111909992A - Method for simultaneously detecting immunogenicity of neoantigen and TCR specific to neoantigen

Info

Publication number: CN111909992A
Application number: CN202010811147.4A
Authority: CN
Inventors: 唐云霞; 高志博; 王煜; 张义兴; 王佳茜; 彭厘旻
Original assignee: Shenzhen Yutai Antigen Technology Co ltd
Current assignee: Shenzhen Yutai Antigen Technology Co ltd
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2020-11-10
Anticipated expiration: 2040-08-13
Also published as: CN111909992B

Abstract

The invention discloses a method for simultaneously detecting neoantigen immunogenicity and neoantigen specificity TCR, which obtains antigen-stimulated cells by adding neoantigen polypeptide into Peripheral Blood Mononuclear Cells (PBMC). compared with the traditional method, the method reduces the time for obtaining antigen-specific cells, only needs 13 days in the whole process of verifying the neoantigen immunogenicity and obtaining the TCR CDR3 sequence, and greatly saves the time cost compared with the treatment process of 40 days in the traditional method. Compared with the traditional MHC-peptide tetramer technology, the cost is low, a special tetramer needs to be prepared for one antigen polypeptide in the MHC-peptide tetramer technology, the cost of the sequencing method is obviously reduced, and compared with the traditional ELISPOT method, the testing flux of the method can only reach 10 strips/batch, the sequencing method adopted by the invention is not limited, the efficiency is higher, the accuracy of the testing result is high, and the applicability is wide.

Description

Method for simultaneously detecting immunogenicity of neoantigen and TCR specific to neoantigen

Technical Field

The invention belongs to the technical field of molecular biology and cellular immunology, relates to a method for determining the immunogenicity of a neoantigen, and particularly relates to a method for simultaneously detecting the immunogenicity of the neoantigen and the specificity TCR of the neoantigen.

Background

T lymphocytes (T lymphocytes), abbreviated as T cells, are bone marrow-derived lymphoid stem cells that, after differentiation and maturation in the thymus, are distributed through lymph and blood circulation to immune organs and tissues throughout the body to exert an immune function. The T cell can specifically recognize a neoantigen presented on a Major Histocompatibility Complex (MHC) molecule on the cell surface after being expressed by a tumor cell through a T cell antigen recognition receptor (TCR), generate an antigen-specific T cell, recognize and kill the tumor cell, and complete the anti-tumor cell immunity of an organism. The tumor neoantigen is an ideal target for anti-tumor immunotherapy, so that the search for neoantigens of different types of tumors gradually becomes a research hotspot in the industry to develop new tumor immunotherapy schemes, such as the development of personalized neoantigen vaccines, adoptive neoantigen-specific T cell immunotherapy and the like.

The tumor neoantigen with immunogenicity can activate T cells to generate antigen-specific T cells and exert anti-tumor immune response, so the detection of the immunogenicity of the neoantigen becomes an important part in the development of new anti-tumor immunotherapy methods, and at present, the conventional method for verifying the immunogenicity of the neoantigen comprises: enzyme-linked immunospot assay (ELISPOT) technology, MHC-peptide tetramer flow assay technology, and intracellular factor staining (ICS) technology. The principle of verifying the immunogenicity of the neoantigen by the ELISPOT technology and the ICS technology is similar, and the immunogenicity of the neoantigen is verified by detecting whether T cells can secrete cytokines under the stimulation of the antigen. The tetramer flow analysis technology verifies the immunogenicity of the neoantigen by detecting whether the neoantigen can be combined with TCR, and the analysis technology is specifically as follows: in vitro, MHC polypeptide compound monomer molecules are connected to a base through the interaction of biotin-streptavidin, four monomers are connected to one base to form a tetramer structure, and at most twelve monomers can be connected together to construct a dodecamer. The complex is capable of recognizing TCR of antigen-specific T cells, and T cells bound with MHC-antigen peptide tetramers can be detected and separated by means of a flow cytometer.

Typically, a neoantigen-specific TCR is obtained by: firstly, stimulating the proliferation of T cells by using a neoantigen, obtaining high-purity neoantigen specific T cells by tetramer flow sorting and enrichment when the proportion of the neoantigen specific T cells is increased to about 0.1%, and sequencing the cells to finally obtain various TCR sequences in the neoantigen specific T cells, wherein the TCR sequences of the antigen specific T cells can be applied to the development of tumor diagnosis markers, the dynamic monitoring of tumor immunotherapy and the curative effect evaluation.

In order to detect the immunogenicity of a neoantigen while knowing its corresponding TCR sequence, the traditional approach is: stimulating T cells by using a neoantigen for 3 times, wherein the stimulation time of each time is 10 days, synthesizing polypeptide to prepare tetramer, obtaining the specific T cells of the neoantigen by tetramer flow sorting, and obtaining a TCR sequence by sequencing, wherein the process needs at least 30 days. Before preparing the tetramer, it is necessary to know which type of HLA molecule is presented for the neoantigen polypeptide, usually, whether affinity exists between the neoantigen polypeptide and HLA is a result predicted by a confidence algorithm, which is often deviated from the actual situation, and not every type of HLA molecule can be prepared, in addition, the cost for preparing the tetramer is high, and the tetramer is easy to degrade and cannot be stored for a long time, so that the tetramer needs to be prepared at present.

Therefore, the traditional MHC-peptide tetramer flow analysis technology has the following technical problems: (1) the pre-stimulation time is long, the content of the T cells specific to the neoantigen in Peripheral Blood Mononuclear Cells (PBMC) is low, and the time for stimulating the T cells by the neoantigen is required to be more than 30 days; (2) dendritic (DC) cells are used for pre-stimulation, the DC cells are formed by differentiating monocytes separated from PBMC under the induction of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4(IL-4), and the culture of the monocytes into mature DC cells generally requires 7 days and has a long time period. A certain amount of PBMC is consumed to be specially used for obtaining DC cells; (3) the tetramer is difficult to prepare, expensive in cost, required to be prepared and used on site and difficult to store; (4) the HLA restriction of the polypeptide must be known before the experiment, and the binding of the secondarily prepared MHC molecule to the polypeptide is uncertain, and a stable MHC-peptide tetramer cannot be generated. The above problems lead to the time consuming and economic cost of the conventional MHC-peptide tetramer technology, which is not conducive to the scale-up and standardization of the verification of the immunogenicity of neoantigens.

Disclosure of Invention

Therefore, the present invention is directed to solving the above technical problems, and provides a method for simultaneously detecting the immunogenicity of a neoantigen and the specificity of the neoantigen TCR, which is short in time consumption and low in cost.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the invention provides a method for simultaneously detecting immunogenicity of a neoantigen and specificity of the neoantigen TCR, which comprises the following steps:

s1, separating peripheral blood mononuclear cells from peripheral blood;

s2, adding a neoantigen into the culture solution of peripheral blood mononuclear cells to start co-incubation, and collecting pre-stimulated cells on the tenth incubation day;

s3, collecting the pre-stimulated T cells, extracting RNA of the T cells, and performing reverse transcription to obtain cDNA;

s4, obtaining a TCR CDR3 sequence of the T cell after pre-stimulation through TCR sequencing, comparing the CDR3 sequence obtained through sequencing with the sequencing result of the T cell without antigen stimulation, and determining the TCR with increased frequency.

Preferably, the step S1 further comprises the step of replacing the culture medium and adding IL-2, IL-7 and IL-15 to stimulate the culture on the 3 rd and 7 th days of co-incubation.

Preferably, before step S1, the method further includes: and predicting to obtain a neoantigen with the strongest binding capacity based on the binding capacity of the antigen of the non-synonymous mutation site and the corresponding major histocompatibility molecule, and preparing the neoantigen into an antigen polypeptide.

Preferably, the neoantigen with the strongest binding capacity comprises the sequence: ISFDTHLLY, VEWLGRCIL, SEIISFKSL, CSTSISNFK, SEHGFGPSL, ATSPASASK, MLICCCCTL, SYFRGSYSY, AAASATLAL, YSLPNAPTV, CWISFDTHLLY, ARPVEWLGRCILDA, LCTDVALPLIVHNIQ, EFTDLLSFIGRIR, HGFGPSLPTSGRDRL, VGLAVNSAVLYVLL, LCKMINLSKPDTI.

Preferably, the sequencing of step S4 includes: the cDNA was used as a sample, and the recombinant VDJ gene expressed in the sample was amplified by multiplex PCR technique and then sequenced.

Preferably, after step S4, the method further includes:

s5, detecting the immunogenicity of the neoantigen corresponding to the TCR with the obviously increased TCR number by adopting an IFN-gamma immune spot method.

Preferably, the step S5 includes:

s501, obtaining PBMCs and culturing mature DC cells;

s502, obtaining CD8+ T cells: screening suspended non-adherent PBMCs through CD8 magnetic beads to obtain CD8+ T cells, and freezing and storing the CD8+ T cells;

s503, first pre-stimulation of T cells: collecting the mature DCs, dividing the mature DCs into 3 parts, freezing and storing two parts of the mature DCs, and adding the neoantigen polypeptide into the rest part of the mature DCs for co-loading; (ii) resuscitating the CD8+ T cells and co-culturing with DC cells loaded with neoantigen polypeptide;

s504, second pre-stimulation of T cells: resuscitating a part of DC cells, and adding a neoantigen polypeptide for co-loading; collecting the first pre-stimulated T cells, and co-culturing with the DC cells loaded with the neoantigen polypeptide;

s505, processing an ELISPOT plate;

s506, collecting the T cells pre-stimulated for the second time;

s507, recovering the residual frozen DC cells and loading neoantigen polypeptides;

s508, paving the DC cells on the ELISPOT plate;

s509, preparing an antibody Anti-IFN-gamma, and culturing the antibody;

s510, removing the antibody in the ELISPOT plate, and cleaning the ELISPOT plate;

and S511, adding a developing solution to the ELISPOT plate in a dark condition, observing the spots in the plate holes at intervals of 2-3min, and reading the number of the spots.

Preferably, the step S501 of culturing mature DC cells specifically includes: adherent monocytes were added to X-VIVO medium containing 5% FBS, and GM-CSF and IL-4 were added for 3 days of culture, GM-CSF, IL-4, TNF- α, IL- β, IL-6, PGE2 were added after the fluid change on day 4 to continue culturing for 48h, and mature DCs were collected on day 7 of culture.

Preferably, the recovering the CD8+ T cells and co-culturing with the DC cells loaded with neoantigen polypeptide in step S503 comprises: CD8+ T cells were thawed and resuspended in AIM-V medium containing 10% FBS, following DC: t is 1: 2-4 ratio, CD8+ T cells were co-cultured with polypeptide-loaded DCs and IL-21 was added, and on day 4, the fluid was changed to add cytokines: IL-2, IL-7 and IL-15 are cultured, then three kinds of cytogenes are changed and supplemented every 1-2 days, and the cytogenes are collected on the 12 th day.

Preferably, the step S504 of collecting the first pre-stimulated T cells and co-culturing with the DC cells loaded with the neoantigen polypeptide comprises: first pre-stimulated T cells were harvested in AIM-V (+ 10% FBS) medium, following DC: t is 1: 2-4, co-cultured with polypeptide-loaded DC cells and IL-21 was added, and on day 4, cytokine: IL-2, IL-7, IL-15 were cultured, followed by fluid changes and supplementation with three cytokines every 1-2 days, collected on day 7 with AIM-V (+ 2% FBS) medium, and cultured for 24 h.

Compared with the prior art, the technical scheme of the invention has the following advantages:

compared with the traditional method, the method for simultaneously detecting the immunogenicity of the neoantigen and the specificity of the neoantigen TCR obtains the cells stimulated by the antigen by adding the neoantigen polypeptide into Peripheral Blood Mononuclear Cells (PBMCs), reduces the time required for obtaining the cells stimulated by the antigen, only needs 13 days in the whole process of verifying the immunogenicity of the neoantigen and obtaining the CDR3 sequence of the TCR, greatly saves the time cost compared with the traditional method which has a treatment process of up to 40 days, and has high accuracy, and the results are consistent when the method is verified by ELISPOT. Compared with the traditional MHC-peptide tetramer technology, the method has low cost, one antigen polypeptide in the MHC-peptide tetramer technology needs to prepare a special tetramer, the quantity of PBMC needed by the sequencing method is half or less than that of an ELISPOT method, the cost is obviously reduced, and compared with the traditional ELISPOT method, the testing flux of the sequencing method is not limited, the efficiency is higher, the testing result is high in accuracy and the applicability is wide.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a flow chart of a detection method according to an embodiment of the present invention;

FIG. 2 is a graph showing the number of spots in wells of an ELISPOT plate according to an embodiment of the present invention.

Detailed Description

Examples

This example provides a method for simultaneously detecting neoantigen immunogenicity and a neoantigen-specific TCR, as shown in figure 1, comprising the steps of:

s1, collecting peripheral blood of the patient, and separating Peripheral Blood Mononuclear Cells (PBMC) from the peripheral blood.

First, 20ml of peripheral blood of a patient was taken, PBMC was obtained by density gradient centrifugation using a lymphocyte separation medium, serum-free AIM-V medium was added thereto, and PBMC was resuspended at 1X10⁶One/ml, and then pressPBMCs were plated in 18 wells of 24-well plates at 1 ml/well.

S2, adding the neoantigen into the PBMC to start co-incubation, performing pre-stimulation, and collecting pre-stimulated cells on the tenth incubation day.

17 neoantigens were added to 17 wells plated with PBMC and medium for CO-incubation, and no neoantigen was added to 18 th well as a control, which was prepared by placing the well plate at 37 ℃ with 5% CO₂The incubator of (1), culturing for 10 days, and collecting the pre-stimulated cells on the tenth day.

Wherein, on the 3 rd and 7 th days of the culture, the culture medium is changed by half, and then IL-2, IL-7 and IL-15 are added for culture.

S3, collecting the pre-stimulated T cells, extracting RNA, and performing reverse transcription to obtain cDNA.

The cells after pre-stimulation were collected in 18 aliquots of AIM-V medium, 1 aliquot of which was not pre-stimulated with antigen, and the remaining 17 aliquots were pre-stimulated with neoantigen, and used as experimental groups.

S4, TCR sequencing to obtain a TCR CDR3 sequence of the T cell after pre-stimulation, and comparing the CDR3 sequence obtained by sequencing with the sequencing result of the T cell without antigen stimulation to determine the TCR with increased frequency.

The cDNA was used as a sample, all VDJ genes expressed in the sample were amplified by multiplex PCR, and then sequenced by a next-generation sequencing technique.

S5, detecting the immunogenicity of the neoantigen corresponding to the TCR with the obviously increased TCR number by adopting an IFN-gamma immune spot method. This step is used to demonstrate the consistency between the immunogenicity of the neoantigen demonstrated in this example and the immunogenicity of the neoantigen demonstrated by the gold standard ELISPOT method.

Before step S1, the method further comprises the step of determining the sequence of the neoantigen:

through a second-generation sequencing technology, non-synonymous mutation sites are found out, and based on the binding capacity of antigens of the non-synonymous mutation sites and corresponding major histocompatibility molecules, 17 neoantigens with the strongest binding capacity are predicted and obtained by using antigen prediction software, wherein the sequences of the 17 neoantigens are shown in table 1:

TABLE 1

Wherein the site of the non-synonymous mutation is determined by: the mutation site is located on the tumor cell genome, the tumor cell genome is compared with the normal cell genome (PBMC cells are normal cells), the mutation site is found, and if the mutation base causes the change of the amino acid sequence, the mutation site is the non-synonymous mutation site.

And synthesizing the neoantigen into an antigen polypeptide with the purity higher than 95% for subsequent experiments.

In this embodiment, in step S4, after TCR sequencing, the sequencing result is analyzed as follows: data quality control, frequency analysis after V-D-J gene rearrangement, CDR3 recognition and length distribution, clone analysis and diversity analysis, difference analysis, sequence network construction or evolution analysis.

After comparing the sequencing results of the TCRs of the T cells which are not stimulated by the antigen and the T cells which are stimulated by the DC loaded with the unloaded polypeptide, the TCRs with obviously increased quantity in 11T cells are finally found out, and the immunogenicity of the 11 neoantigens corresponding to the TCRs is preliminarily confirmed. The TCR assay results are specifically shown in table 2:

TABLE 2

Namely, 11 of the 17 neoantigens corresponding to the numbers were immunogenic.

The step S5 is used to further verify the sequencing result, and specifically uses IFN- γ immunoblotch to detect the immunogenicity of the 11 neoantigens obtained above, and the experimental steps specifically include:

s501, PBMC is obtained, and mature DC cells are cultured.

Collecting 100ml of peripheral blood of patient, separating with lymphocyte separation solution by density gradient centrifugation to obtain PBMC, adding AIM-V culture medium containing 10% serum, and resuspending PBMC to 5 × 10⁶And (4) paving the PBMCs in a six-well plate according to the concentration per ml/well of the PBMCs and culturing for 2h, and taking out the suspension cells to obtain adherent monocytes.

Adding the adherent mononuclear cells into an X-VIVO culture medium containing 5% FBS, and adding GM-CSF and IL-4 to culture the cells for 3 days; after the fluid change on day 4, GM-CSF, IL-4, TNF-alpha, IL-beta, IL-6, PGE2 were added to continue culturing for 48h, and mature DCs were collected on day 7.

S502, obtaining CD8+ T cells: screening the suspended non-adherent PBMC by CD8 magnetic beads to obtain CD8+ T cells, and freezing and storing the CD8+ T cells.

S503, first pre-stimulation of T cells: collecting the mature DCs, dividing the mature DCs into 3 parts, freezing and storing two parts of the mature DCs, and adding the neoantigen polypeptide into the rest part of the mature DCs for co-loading; the CD8+ T cells were revived and co-cultured with DC cells loaded with neoantigen polypeptide.

Mature DCs were first collected, divided into three portions, one portion of DCs (the remaining two portions were frozen), added to serum-free X-VIVO medium, and resuspended at 1X10⁶The antigen is added into the mixture per ml, the mixture is divided into 12 parts according to 200 ul/part, wherein 11 parts are added with 11 pieces of neoantigens according to the antigen polypeptide concentration of 10ug/ml, and the other 1 part is not added with the neoantigen polypeptide, and the loading time is 2-4 h.

CD8+ T cells were thawed and resuspended in AIM-V medium containing 10% FBS, following DC: t is 1: 2-1: 4, dividing the CD8+ T cells into 12 portions, co-culturing with 11 portions of DC loaded with antigenic polypeptide and 1 portion of DC not loaded with antigenic polypeptide, respectively, and adding IL-21, at day 4, changing the fluid to add three cytokines: culturing IL-2, IL-7 and IL-15, then changing liquid and supplementing three cytokines every 1-2 days, and reaming if the cells are full; collected on day 12.

S504, second pre-stimulation of T cells: resuscitating a part of DC cells, and adding a neoantigen polypeptide for co-loading; the first pre-stimulated T cells were harvested and co-cultured with DC cells loaded with neoantigenic polypeptide.

One part of DC is recovered: resuscitate a aliquot of DCs in X-VIVO (+ 5% FBS) medium, place in low adsorption well plates at 37 deg.C, 5% CO₂After 2h in the incubator, serum-free X-VIVO medium was added and resuspended at 1X10⁶The antigen is added into the mixture per ml, the mixture is divided into 12 parts according to 200 ul/part, 11 parts of the mixture are added with 11 new antigens according to the antigen polypeptide concentration of 10ug/ml, and the other 1 part of the mixture is not added with the antigen polypeptide, and the loading time is 2-4 h.

Collecting the first pre-stimulated T cells: first pre-stimulated T cells were harvested with AIM-V (+ 10% FBS) medium and counted. According to DC: t is 1: 2-1: 4, co-culturing with 11 parts of DC loaded with antigenic polypeptide and 1 part of DC not loaded with antigenic polypeptide, respectively, and adding IL-21, at day 4, changing the medium to add three cytokines: culturing IL-2, IL-7 and IL-15, then changing liquid and supplementing three cytokines every 1-2 days, and reaming if the cells are full; on day 7, harvest was performed on AIM-V (+ 2% FBS) medium and was performed at 5X 10⁶And (4) plating the cells in a 6-well plate for 24 h.

And S505, processing the ELISPOT board.

Before plating, the ELISPOT plate is washed for 5 times by adopting PBS (sterile) according to 200 ul/hole; then, 1640+ 10% FBS medium (200. mu.l/well) was added thereto, and the mixture was placed at 37 ℃ in 5% CO₂The incubator was closed for 30 minutes, after which the medium was removed (the closed medium was spun off).

S506, collecting the T cells pre-stimulated for the second time.

Specifically, the resting second pre-stimulated T cells were collected: collecting T cells by serum-free AIM-V culture, centrifuging for 5min at 400g, adding serum-free AIM-V culture medium, and resuspending to 3 × 10⁵One per ml.

S507, recovering the residual frozen DC cells and loading the neoantigen polypeptide.

Resuscitate a aliquot of DCs in X-VIVO (+ 5% FBS) medium, place in low adsorption well plates at 37 deg.C, 5% CO₂After 2h in the incubator, serum-free X-VIVO medium was collected and added and resuspended at 1X10⁶Taking 12 parts of DC cells per ml according to 200 ul/part, adding 11 neoantigens into 11 parts of DC cells according to the antigen polypeptide concentration of 10ug/ml, wherein the loading time is 2-4h, and the other 1 part of DC cells are not loaded with antigen polypeptide; thereafter, each DC was resuspended 2 x10 in serum-free AIM-V medium⁴One per ml.

S508, paving the DC cells on the ELISPOT plate.

The DC cells were added to the wells at 100 ul/well and 100ul of AIM-V (plasma free) medium was added to each of the remaining wells. Then, DC cells (loaded with polypeptide and unloaded DC) were added to the corresponding wells at 50 ul/well, leaving wells without DC cells, 50 ul/well of AIM-V (plasma-free) medium was added, and the well-plated ELISPOT plates were placed in CO at 37 ℃₂Culturing in an incubator for 24h, and throwing off the culture medium in the holes after 24 h; the well plate was washed 5 times with PBS (sterile) at 200 ul/well.

S509, preparing an antibody Anti-IFN-gamma and culturing the antibody.

Preparing primary antibody (Anti-IFN-gamma) by using PBS (0.5% FBS) solution (required to be filtered by a 0.22 mu m filter head) (diluted according to the proportion of 1: 200), and then adding the prepared primary antibody into the holes according to 100 ul/hole; then put into CO at 37 DEG C₂And (5) in an incubator for 2 h.

S510, removing the antibody in the ELISPOT plate, and cleaning the ELISPOT plate.

After 2h, throwing off primary antibodies in the holes; the well plate was washed 5 times with PBS (sterile) solution at 200 ul/well, and the PBS solution in the well was dried.

Adding color developing solution (used after filtering with 0.22 μm filter head) at a dark place according to 100 ul/hole, and observing the spots in the hole every 2-3 min; the color development time is generally 10-15 min. And when the spots in the pore plate are not obviously increased, washing the front side and the back side of the ELISPOT plate by using water, drying the water in the pore plate by using a drying method, placing the ELISPOT plate in a drying and light-proof position, drying in the air, and reading the plate to obtain the number of the spots in the pores of the ELISPOT plate.

Comparing with the number of spots of the blank control group, obtaining the number of the antigen specific T cells: of these 11 polypeptides were able to stimulate the production of neoantigen-specific T cells, and the plate reading results are shown in FIG. 2.

The test result shows that the result obtained by TCR sequencing is consistent with the result obtained by an ELISPOT experiment, which indicates that the immunogenicity of the neoantigen determined by TCR sequencing has high reliability.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

SEQUENCE LISTING

<110> Shenzhen Yutai antigen science and technology Limited

<120> method for simultaneously detecting neoantigen immunogenicity and neoantigen-specific TCR

<130> 20200730

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<211> 17

<212> PRT

<213> Artificial

<400> 34

Cys Ala Ser Ser Ile Pro Phe Ala Phe Ser Ser Tyr Asn Glu Gln Phe

1 5 10 15

Phe

<210> 35

<211> 15

<212> PRT

<213> Artificial

<400> 35

Cys Ala Ser Ser Pro Arg Thr Gly Glu Val Glu Thr Gln Tyr Phe

1 5 10 15

<210> 36

<211> 15

<212> PRT

<213> Artificial

<400> 36

Cys Ala Ser Ser Gln Asp Glu Gly Leu Tyr Glu Lys Leu Phe Phe

1 5 10 15

<210> 37

<211> 16

<212> PRT

<213> Artificial

<400> 37

Cys Ala Ser Ser Tyr Gly Pro Gly Gly Ser Thr Asp Thr Gln Tyr Phe

1 5 10 15

<210> 38

<211> 14

<212> PRT

<213> Artificial

<400> 38

Cys Ala Ser Ser Leu Leu Thr Gly Thr Thr Glu Ala Phe Phe

1 5 10

<210> 39

<211> 16

<212> PRT

<213> Artificial

<400> 39

Cys Ala Ser Ser Leu Val Met Arg Arg Leu Gly Thr Glu Ala Phe Phe

1 5 10 15

<210> 40

<211> 17

<212> PRT

<213> Artificial

<400> 40

Cys Ser Ala Arg Ala Phe Ser Gln Ala Gly Arg Glu Asp Ile Gln Tyr

1 5 10 15

Phe

<210> 41

<211> 13

<212> PRT

<213> Artificial

<400> 41

Cys Ser Ala Arg Gly Thr Arg Ser Tyr Glu Gln Tyr Phe

1 5 10

<210> 42

<211> 14

<212> PRT

<213> Artificial

<400> 42

Cys Ala Ser Ser Leu Leu Asp Leu Tyr Asn Glu Gln Phe Phe

1 5 10

<210> 43

<211> 14

<212> PRT

<213> Artificial

<400> 43

Cys Ala Ser Ser Leu Ser Gly Trp Glu Phe Glu Gln Tyr Phe

1 5 10

<210> 44

<211> 13

<212> PRT

<213> Artificial

<400> 44

Cys Ala Ser Ser Gln Ile Ser Asn Tyr Gly Tyr Thr Phe

1 5 10

<210> 45

<211> 13

<212> PRT

<213> Artificial

<400> 45

Cys Ser Ala Ala Pro Gln Met Asn Thr Glu Ala Phe Phe

1 5 10

<210> 46

<211> 15

<212> PRT

<213> Artificial

<400> 46

Cys Ala Ser Arg Asp Asn Arg Val Trp Asn Gln Pro Gln His Phe

1 5 10 15

<210> 47

<211> 13

<212> PRT

<213> Artificial

<400> 47

Cys Ala Ser Ser Pro Gly Phe Ser Gly Glu Leu Phe Phe

1 5 10

<210> 48

<211> 16

<212> PRT

<213> Artificial

<400> 48

Cys Ala Ile Ser Ala Pro Gln Gly Val Thr Thr Tyr Glu Gln Tyr Phe

1 5 10 15

<210> 49

<211> 16

<212> PRT

<213> Artificial

<400> 49

Cys Ala Ser Asn Leu Arg Ala Glu Leu Asp Ser Tyr Glu Gln Tyr Phe

1 5 10 15

<210> 50

<211> 15

<212> PRT

<213> Artificial

<400> 50

Cys Ala Ser Ser His Pro Gly Ser Met Asn Thr Glu Ala Phe Phe

1 5 10 15

<210> 51

<211> 14

<212> PRT

<213> Artificial

<400> 51

Cys Ala Ser Ser Leu Ser Pro Val Asn Thr Glu Ala Phe Phe

1 5 10

<210> 52

<211> 13

<212> PRT

<213> Artificial

<400> 52

Cys Ala Ser Ser Leu Ser Ser Gly Tyr Glu Ala Phe Phe

1 5 10

<210> 53

<211> 14

<212> PRT

<213> Artificial

<400> 53

Cys Ala Ser Ser Thr Gly Leu Ser Asn Thr Glu Ala Phe Phe

1 5 10

<210> 54

<211> 14

<212> PRT

<213> Artificial

<400> 54

Cys Ala Ser Ser Thr Leu Gly Leu Ala Asp Glu Gln Phe Phe

1 5 10

<210> 55

<211> 17

<212> PRT

<213> Artificial

<400> 55

Cys Ala Ser Ser Thr Arg Gln Gly Val Ile Ser Asn Gln Pro Gln His

1 5 10 15

Phe

<210> 56

<211> 11

<212> PRT

<213> Artificial

<400> 56

Cys Ala Ser Thr Pro Gly Asp Glu Gln Phe Phe

1 5 10

<210> 57

<211> 13

<212> PRT

<213> Artificial

<400> 57

Cys Ser Ala Arg Asp Leu Phe Gly Gln Pro Gln His Phe

1 5 10

<210> 58

<211> 13

<212> PRT

<213> Artificial

<400> 58

Cys Ser Ala Arg Asp Leu Phe Ser Gln Pro Gln His Phe

1 5 10

<210> 59

<211> 13

<212> PRT

<213> Artificial

<400> 59

Cys Ser Ala Arg Asp Leu Tyr Gly Gln Pro Gln His Phe

1 5 10

<210> 60

<211> 15

<212> PRT

<213> Artificial

<400> 60

Cys Ala Ser Ser Gln Gly Gln Ser Pro Asn Glu Lys Leu Phe Phe

1 5 10 15

<210> 61

<211> 14

<212> PRT

<213> Artificial

<400> 61

Cys Ser Ala Arg Asp Leu Gly Gln Thr Tyr Gly Tyr Thr Phe

1 5 10

<210> 62

<211> 14

<212> PRT

<213> Artificial

<400> 62

Cys Ala Ser Arg Arg Thr Gly Phe Arg Thr Glu Ala Phe Phe

1 5 10

<210> 63

<211> 13

<212> PRT

<213> Artificial

<400> 63

Cys Ala Ser Ser Ser Thr Gly Phe Ala Glu Ala Phe Phe

1 5 10

Claims

1. A method for simultaneously detecting neoantigen immunogenicity and neoantigen-specific TCR, comprising the steps of:

s1, separating peripheral blood mononuclear cells from peripheral blood;

2. The method for simultaneously detecting both neoantigen immunogenicity and neoantigen-specific TCR according to claim 1, wherein step S1 further comprises the steps of changing the culture medium and adding IL-2, IL-7, and IL-15 to stimulate the culture at the time of co-incubation days 3 and 7.

3. The method for simultaneously detecting neoantigen immunogenicity and neoantigen-specific TCR according to claim 1 or 2, wherein step S1 is preceded by: and predicting to obtain a neoantigen with the strongest binding capacity based on the binding capacity of the antigen of the non-synonymous mutation site and the corresponding major histocompatibility molecule, and preparing the neoantigen into an antigen polypeptide.

4. The method for simultaneously detecting neoantigen immunogenicity and neoantigen-specific TCR according to claim 3, wherein the most potent neoantigen binding comprises the sequence:

ISFDTHLLY, VEWLGRCIL, SEIISFKSL, CSTSISNFK, SEHGFGPSL, ATSPASASK, MLICCCCTL, SYFRGSYSY, AAASATLAL, YSLPNAPTV, CWISFDTHLLY, ARPVEWLGRCILDA, LCTDVALPLIVHNIQ, EFTDLLSFIGRIR, HGFGPSLPTSGRDRL, VGLAVNSAVLYVLL, LCKMINLSKPDTI.

5. The method for simultaneously detecting neoantigen immunogenicity and neoantigen-specific TCR according to claim 4, wherein the sequencing of step S4 comprises: the cDNA was used as a sample, and the recombinant VDJ gene expressed in the sample was amplified by multiplex PCR technique and then sequenced.

6. The method for simultaneously detecting neoantigen immunogenicity and neoantigen-specific TCR according to claim 5, wherein step S4 is further followed by:

7. The method for simultaneously detecting neoantigen immunogenicity and neoantigen-specific TCR according to claim 6, wherein step S5 comprises:

s501, obtaining PBMCs and culturing mature DC cells;

s505, processing an ELISPOT plate;

s506, collecting the T cells pre-stimulated for the second time;

s508, paving the DC cells on the ELISPOT plate;

s509, preparing an antibody Anti-IFN-gamma, and culturing the antibody;

8. The method for simultaneous detection of neoantigen immunogenicity and a neoantigen-specific TCR according to claim 7, wherein the step S501 of culturing mature DC cells is specifically: adherent monocytes were added to X-VIVO medium containing 5% FBS, and GM-CSF and IL-4 were added for 3 days of culture, GM-CSF, IL-4, TNF- α, IL- β, IL-6, PGE2 were added after the fluid change on day 4 to continue culturing for 48h, and mature DCs were collected on day 7 of culture.

9. The method for simultaneously detecting neoantigen immunogenicity and neoantigen-specific TCR according to claim 8, wherein the recovering the CD8+ T cells and co-culturing with the neoantigen polypeptide-loaded DC cells in step S503 comprises: CD8+ T cells were thawed and resuspended in AIM-V medium containing 10% FBS, following DC: t is 1: 2-4 ratio, CD8+ T cells were co-cultured with polypeptide-loaded DCs and IL-21 was added, and on day 4, the fluid was changed to add cytokines: IL-2, IL-7 and IL-15 are cultured, then three kinds of cytogenes are changed and supplemented every 1-2 days, and the cytogenes are collected on the 12 th day.

10. The method for simultaneously detecting neoantigen immunogenicity and neoantigen-specific TCR according to claim 9, wherein the step S504 of collecting and co-culturing first pre-stimulated T cells with the neoantigen polypeptide-loaded DC cells comprises: first pre-stimulated T cells were harvested in AIM-V (+ 10% FBS) medium, following DC: t is 1: 2-4, co-cultured with polypeptide-loaded DC cells and IL-21 was added, and on day 4, cytokine: IL-2, IL-7, IL-15 were cultured, followed by fluid changes and supplementation with three cytokines every 1-2 days, collected on day 7 with AIM-V (+ 2% FBS) medium, and cultured for 24 h.