CN111499690B

CN111499690B - Novel antigenic peptide aiming at CLDN18-ARHGAP fusion mutation and application thereof

Info

Publication number: CN111499690B
Application number: CN202010327892.1A
Authority: CN
Inventors: 魏嘉; 汪越; 刘宝瑞
Original assignee: Nanjing Drum Tower Hospital
Current assignee: Nanjing Drum Tower Hospital
Priority date: 2020-04-23
Filing date: 2020-04-23
Publication date: 2020-11-24
Anticipated expiration: 2040-04-23
Also published as: CN111499690A

Abstract

The invention relates to a novel antigenic peptide aiming at CLDN18-ARHGAP fusion mutation and application thereof. Based on the fusion of CLDN18(e5) -ARHGAP26(e12), i.e., exon 5 of CLDN18 gene with exon 12 of ARHGAP26 gene, the antigen peptide was synthesized by binding HLA-A1101 for the amino acid sequences at both ends of the fusion site of CLDN18(e5) -ARHGAP26(e12) and using the T cell epitope prediction tool NetMHCpan 4.0. The invention fills the blank of the treatment strategy of the CLDN18-ARHGAP fusion mutation in gastric cancer, can obviously activate T cells, obviously increase the killing capacity of the T cells to tumors, and can be used for preparing vaccines and engineered T cells.

Description

Novel antigenic peptide aiming at CLDN18-ARHGAP fusion mutation and application thereof

Technical Field

The invention relates to the field of targeted drugs and immunotherapy, in particular to a novel antigenic peptide aiming at CLDN18-ARHGAP fusion mutation and application thereof.

Background

Signet Ring Cell Carcinoma (SRCC) is a diffuse type of gastric cancer with high malignancy, accounting for about 17% of all types of gastric cancer. The gastric SRCC focus grows in a diffuse infiltration manner, is easy to rapidly generate abdominal cavity transfer, is not sensitive to chemotherapy, is not a population with the advantages of the existing targeted drugs, and has extremely poor prognosis. Finding an effective treatment method for the subtype gastric cancer has urgent clinical requirements.

The rapid development and breakthrough of tumor immunotherapy have been achieved in recent years, and the key to the success of immunotherapy is whether the specific immune response of targeted tumor cells can be activated. More and more researches show that the tumor neoantigen (neoantigen) is the key point for inducing the specific response, and the neoantigen reactive T cells (NRT cells) are the cornerstone for obtaining clinical response of a plurality of immunotherapy strategies including immune checkpoint inhibitors, engineered immune cells, tumor vaccines and the like. Through high-throughput sequencing and big data analysis and by utilizing bioinformatics, specific new antigens generated by cancer cell gene mutation are screened out, NRT cells are sorted and amplified and returned to patients for carrying out accurate biological immunotherapy, and the method is a new generation of immunotherapy strategy.

However, both the currently reported treatments for NRT and the clinical trials for NRT performed by the research team in the hands of the Applicant have focused on new antigens generated by point mutations in the genes. In our earlier work we found: the load of point mutation of gastric SRCC patients is low, hot point mutation is absent, partial new antigens corresponding to the point mutation have weak immunogenicity, and NRT cell therapy of the targeted point mutation new antigens is difficult to be successfully applied to the treatment of gastric SRCC.

In recent years, with the development of Next Generation Sequencing (NGS), TCGA has completed deep analysis of multiple tumor molecular phenotypes, which lays a foundation for accurate treatment of tumors. The 2014 Nature journal reports the research result of the TCGA plan on the gastric cancer, and based on the second generation sequencing results of 295 cases of primary gastric adenocarcinoma tissues, the genome variation situation is comprehensively analyzed, the gastric cancer is divided into four subtypes, and most of gastric signet ring cell cancers belong to Genome Stable (GS). The histological phenotype of the GS type gastric cancer is mainly diffuse type, the incidence rate of CDH1 (37%), RHOA (15%) and CLDN18-ARHGAP fusion mutation (15%) on the molecular phenotype is high, and the GS type gastric cancer lacks effective response to the existing targeted drugs. Based on the specific molecular characteristics of GS-type gastric cancer, several drugs are currently under development, such as targeting CDH1 and RHOA mutation-associated pathways.

The CLDN18-ARHGAP fusion mutation plays a driving role in the early generation and late development processes of the gastric cancer, the epithelial phenotype of the tumor cells with the CLDN18-ARHGAP fusion mutation is lost, epithelial-mesenchymal transition (EMT) occurs, the cells are cancerated, the intercellular adhesion and the cell-extracellular matrix adhesion are reduced, and the tumor cell invasion is easy to occur. Furthermore, patients with gastric SRCC who have a CLDN18-ARHGAP fusion mutation do not benefit from treatment with oxaliplatin/fluorouracil chemotherapeutic drugs and have a poorer prognosis. However, therapeutic strategies against CLDN18-ARHGAP fusion mutations in gastric cancer remain open.

Recent studies have shown that: fusion mutations can also produce novel antigens that are more immunogenic than traditional point mutations. The fusion gene is a chimeric gene formed by connecting the coding regions of two or more genes end to end, and the chimeric amino acid sequences at the two ends of the fusion site have the potential of generating new antigens and have better immunogenicity. The fusion mutation proportion of CLDN18-ARHGAP in gastric SRCC reaches 17 percent, and the fusion mutation proportion is a potential ideal target point for NRT cell treatment. Thus, for gastric SRCC containing CLDN18-ARHGAP fusion mutation, which lacks therapeutic means and has a poor prognosis, treatment of NRT cells with CLDN18-ARHGAP fusion mutation may be a potentially effective therapy.

Disclosure of Invention

The invention aims to provide a novel antigenic peptide aiming at CLDN18-ARHGAP fusion mutation, which is applied to the treatment of the CLDN18-ARHGAP fusion mutation in gastric cancer, in particular to Signet Ring Cell Carcinoma (SRCC).

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a novel antigenic peptide directed against the fusion mutation of CLDN18-ARHGAP, based on the fusion of exon 5 of CLDN18(e5) -ARHGAP26(e12), i.e., CLDN18 gene, with exon 12 of ARHGAP26 gene, and the amino acid sequences at both ends of the fusion site of CLDN18(e5) -ARHGAP26(e12), binding HLA-typing HLA-A1101, using the T cell epitope prediction tool NetMHCpan4.0, to synthesize antigenic peptides, see Table 1.

Synthesizing a plurality of epitope peptides with the amino acid length of 9-11, wherein the sequences of the epitope peptides comprise SEQ ID NO. 1-10.

A long peptide L1 of 26 amino acids in length was synthesized, the sequence of which is SEQ ID NO. 11.

The invention protects the application of the novel antigenic peptide aiming at the CLDN18-ARHGAP fusion mutation in the preparation of drugs for treating gastric cancer targeting and immunotherapy.

The application of the medicine in preparing the medicine for treating gastric cancer targeting and immunotherapy comprises new antigen reactive T cell therapy, engineered T cell therapy, vaccine and the like.

TABLE 1

Compared with the prior art, the invention has the beneficial effects that:

the invention fills the blank of the treatment strategy of the CLDN18-ARHGAP fusion mutation in gastric cancer, can obviously activate T cells and obviously increase the killing capacity of the T cells to tumors. The invention has better immunogenicity, and the induced NRT cells have strong targeted killing capability, can be used for NRT cell immunotherapy of patients with fusion mutation of CLDN18(e5) -ARHGAP26(e12), and can be subsequently used for preparing vaccines and engineered T cells.

Drawings

FIG. 1: peripheral blood mononuclear cells from gastric cancer patients were analyzed for in vitro reactivity to CLDN18-ARHGAP fusion neoantigenic peptide by IFN- γ ELISPOT assay. Positive control: phytohemagglutinin PHA; negative control: no peptide was added.

FIG. 2: FIG. 1 statistical histogram of ELISPOT results.

FIG. 3: dendritic Cells (DCs) were loaded with the antigenic peptide P1 and after stimulation of activated T cells, in vitro reactivity of T cells from gastric cancer patients to CLDN18-ARHGAP fusion neoantigenic peptide P1 and statistical histograms of results were analyzed by IFN- γ ELISPOT assay. Positive control: phytohemagglutinin PHA; negative control: no peptide was added.

FIG. 4: dendritic Cells (DCs) were loaded with the antigenic peptide P1 and after stimulation of activated T cells, changes in CD8+ T cell CD137 expression from gastric cancer patients were detected by flow cytometry in response to T cell activation status. Positive control: phytohemagglutinin PHA; negative control: no peptide was added.

FIG. 5: dendritic Cells (DC) of a gastric cancer patient are loaded with the antigen peptide P1, and autologous T cells are stimulated and activated in vitro for two rounds of parallel expansion. Killing experiments were performed on T cells and tumor cells at different potency-to-target ratios against SNU-601 gastric cancer cell line transfected with CLDN18-ARHGAP fusion gene. Negative control: patient T cells induced without peptide addition.

Detailed Description

The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention. The conditions not specified in the examples are generally those in routine experiments.

Prediction of fusion mutant neoantigens by CLDN18(e5) -ARHGAP26(e12)

The most common type of fusion mutation of CLDN18-ARHGAP is CLDN18(e5) -ARHGAP26(e12), i.e., fusion of exon 5 of CLDN18 gene with exon 12 of ARHGAP26 gene, accounts for about 80% of all CLDN18-ARHGAP fusion mutations. Aiming at amino acid sequences at two ends of a CLDN18(e5) -ARHGAP26(e12) fusion site, the epitope peptide synthesis method combines the most common HLA typing (HLA-A1101) of Chinese population, uses a T cell epitope prediction tool NetMHCpan4.0, preferably 9-11 amino acids in length, and synthesizes a long peptide L1 with the length of 26 amino acids. Epitope peptide synthesis, preferably 9-11 amino acids long, was performed for comparison against the amino acid sequence of CLDN18 at both ends of the fusion site with the ARHGAP26 gene.

2. Patient HLA typing detection

2ml of peripheral blood of a patient is extracted, EDTA or sodium citrate is adopted for anticoagulation, four-digit high-resolution typing of an HLA-A locus is detected by using a PCR-SBT technology, and the patient with HLA-A1101 is selected.

3. Peripheral blood PBMC acquisition

(1) Sterilizing cell bags collected by a COBE Spectra MNC system machine (or heparin anticoagulation extracted by an injector), placing the sterilized cell bags into a super clean bench, transferring cells into a 50ml centrifugal tube by using a 20ml injector, and diluting the cells by using normal saline 1: 1;

(2) 20ml of human lymphocyte separation liquid is added into a centrifugal tube;

(3) slowly superposing the cell sap diluted by the normal saline on the human lymphocyte separation fluid along the tube wall by using a Pasteur pipette;

(4) horizontally centrifuging at 800g for 20 minutes;

(5) the tube is divided into three layers after centrifugation, the upper layer is plasma, the lower layer is mainly red blood cells and granulocytes, the middle layer is lymphocyte separation liquid, and a leucocyte layer which mainly comprises mononuclear cells is arranged at the interface of the upper layer and the middle layer, namely a peripheral blood mononuclear cell layer;

(6) inserting into the tunica albuginea layer with a suction tube, sucking mononuclear cells, placing into another 50ml centrifuge tube, supplementing normal saline, horizontally centrifuging for 300g, and 10 min;

(7) discarding the supernatant, adding normal saline, mixing uniformly, horizontally centrifuging for 300g, and 10 min;

(8) discarding the supernatant, supplementing a cell culture medium, uniformly mixing, horizontally centrifuging for 300g, and 5 min;

(9) discarding supernatant, diluting with AIM-V culture solution, counting cells, collecting appropriate amount of frozen cell specimen

4. Dendritic Cell (DC) culture:

(1) according to the method for obtaining the PBMCs, the obtained PBMCs are placed in a plurality of six-hole plate culture flasks and cultured in AIM-V culture medium;

(2)37℃，5％CO₂incubating for 2h in the incubator to allow the monocytes to adhere to the walls;

(3) adherent cells were isolated and 20ml AIM-V medium was added while supplementing recombinant human GM-CSF (800U/ml) and recombinant human IL-4(1000U/ml), 37 ℃ with 5% CO₂Culturing in an incubator, and inducing the monocyte to differentiate to DC;

(4) determining whether half amount of liquid is changed according to the cell amount on the third day, and if the liquid is changed, complementing GM-CSF and IL-4;

(5) adding DC to promote maturation factor LPS (10ng/ml) and IFN-gamma (100IU/ml) on the sixth day to induce DC maturation for 16-48 h;

(6) on day seven, mature dendritic cells were obtained.

(7) Harvesting mature DC, adding antigen peptide 10ug/ml into DC culture solution, standing at 37 deg.C and 5% CO₂And (3) incubating in an incubator for 4-6 hours, centrifuging cells, using preheated PBS for resuspension, and freezing and storing a back shadow after 30Gy radiation irradiation.

5. Enzyme-linked immunosorbent assay (ELISPOT method)

(1) Activating the pre-coated plate, adding 200 mu L of AIM-V serum-free culture medium, standing at room temperature for 10 minutes, and pouring;

(2) adding T cells and polypeptides according to the designed groups, wherein the concentration of the T cells and the polypeptides is 10-50 mu g/ml, and each group has 3 multiple pores;

(3) after all samples were added, the plate was covered, marked, and placed at 37 ℃ with 5% CO₂The incubator was incubated for 20 hours.

(4) Pouring cells and culture medium in the holes;

(5) cell lysis: add 200. mu.L ice-cold deionized water to each well, ice-cool for 10min in a refrigerator at 4 ℃ (cell lysis by hypotonic method);

(6) washing the plate: washing each well with 260. mu.L of 1XWashing buffer for 6 times, each time for 60 seconds, and drying on absorbent paper after each washing;

(7) and (3) incubation of the detection antibody: mu.L of diluted biotin-labeled antibody was added to each well at 37 ℃ with 5% CO₂Incubating in an incubator for 1 hour;

(8) washing the plate: washing each well with 260. mu.L of 1XWashing buffer for 6 times, each time for 60 seconds, and drying on absorbent paper after each washing;

(9) and (3) avidin incubation: adding 100 mu L of diluted enzyme-labeled avidin into each hole, and incubating for 1 hour at 37 ℃;

(10) washing the plate: washing each well with 260. mu.L of 1XWashing buffer for 6 times, each time for 60 seconds, and drying on absorbent paper after each washing;

(11) color development: according to the reagent configuration, AEC color developing solution is prepared, 100 μ L of color developing solution is added into each well, the temperature is 37 ℃, and 5% CO is added₂Developing color in the incubator, and checking once every 5 minutes;

(12) after the spots have grown to a suitable size, they are washed 2 times with deionized water and the color development is terminated. Reversely buckling the plate on absorbent paper, patting to dry fine water drops, taking down the protective layer, placing the protective layer in a ventilated place, standing at room temperature, and naturally drying the film;

(13) the ELISPOT plate spots were counted and various parameters of the spots were recorded and analyzed.

6. Flow cytometry to determine markers of T cell activation

(1) Modulating the concentration of activated PBMCs or T lymphocytes to 1X 10⁶About/ml. Blowing the cell suspension evenly, centrifuging at 1500rpm for 5min, discarding the supernatant, adding PBS, centrifuging at 1500rpm for 5min, discarding the supernatant, then re-suspending the cells with 100 μ l PBS to a flow tube, and marking each group of cells on the tube wall;

(2) adding fluorescent antibody labeled cells such as anti-human CD8 and CD137, and setting a negative control group, a single-standard group and an isotype control group;

(3) dyeing at 4 ℃ in dark for 30 min;

(4) adding 1ml of PBS, and gently blowing and beating by a pipette gun to be uniformly mixed;

(5) centrifuging at 1500rpm for 5min, and discarding the supernatant;

(6) add 100. mu.l PBS to resuspend the cells and test on the machine. BD software collected the data and analyzed the streaming results with Flowjo software.

7. Induction of PBMC activation in vitro

(1) Heparinized anticoagulated PBMC were isolated by the method described above and suspended in AIM-V medium (Gibico, USA) and counted.

(2) 96-well plate with U-shaped bottom, 1X 10 per well⁵Each PBMC was diluted in 200ul of medium and incubated with 10. mu.M of antigenic peptide, with the U-shaped bottom being used to promote better cell-to-cell contact. The medium was composed of AIM-V medium (Gibco) containing a small amount of interleukin-2 (IL-2,100U/mL, Peprotech) and 10% fetal calf serum (FCS, Gibco).

(3) PBMC were cultured for 3 days per round of stimulation with medium exchange half-way, supplemented with phase antigen peptide (10uM) and IL-2 (100U/mL).

(4) After 2-3 weeks of peptide in vitro stimulation culture, the fresh culture medium is replaced, and 10 μ M of antigenic peptide is added again for stimulation culture overnight. Within 24h (i.e., on day 7 and/or day 10 of culture) of the last round of stimulation, the level of T cell immune response was assessed by ELISPOT to detect IFN- γ expression, identifying the immunogenicity of the different antigenic peptides. PBMC were stimulated as negative control (NS) without peptide (medium) or with irrelevant peptide, and Phytohemagglutinin (PHA) was stimulated as positive control.

8. Stimulation of activated T cells using Dendritic Cells (DCs) loaded with antigenic peptides

Mature DCs (1X 10)⁴Per 100 ul/well) at 37 ℃ 5% CO₂In the incubator, peptide-loaded 10uM pulses were pulsed for 4-6 hours, washed with pre-warmed PBS, and then incubated overnight with T cells in complete AIM-V medium at a ratio of stimulus to effector of 1: 10. The level of activation of T cells was assessed the following day. The number and intensity of activated T cells after stimulated culture were measured by INF- γ ELISPOT and the expression level of the T cell activation marker 4-1BB (CD137) was evaluated by flow cytometry.

9. Preparation of neoantigen reactive T cells (NRT)

(1) Suspension cells transferred to F225 culture flask to adjust cell concentration to 1X 10⁷Each ml, supplemented with IL-2(100U/ml), IL-7(10ng/ml), IL-15 (1)0ng/ml)，37℃，5％CO₂Culturing in an incubator for 7 days.

(2) Mature DC is harvested on day 7, and the antigen peptide 25ug/ml is added to the DC culture medium and placed at 37 deg.C with 5% CO₂And (4) incubating for 4-6 hours in the incubator.

(3) Centrifuging at 1500rpm for 5min, discarding supernatant, mixing and culturing the DC loaded with new antigen and suspension cells, and supplementing part of fresh culture medium containing (IL-2, IL-7 and IL-15). 37 ℃ and 5% CO₂Incubating for 10 days;

(4) supplementing a fresh complete culture medium and corresponding cytokines IL-7, IL-15 and IL-2 by half every 2 to 3 days according to the growth condition of the cells and the color of the culture medium, and then conveying the culture medium to a bottle; depending on the T cell expansion, OKT3 monoclonal antibody was added in appropriate amounts and a second round of stimulation was performed with irradiated ECCE cells or APC loading peptide prepared from thawed PBMC.

(5) The T cells are stimulated by antigen for about 10 days, and the T cells are harvested for detection and function experiments.

Experiment on in vitro killing of tumor cells by NRT cells

CFSE/PI labeling: NRT cells induced by antigen peptides are used as effector cells (E), SNU-601 human gastric cancer cell lines transfected with CLDN18(E5) -ARHGAP26(E12) fusion genes are used as target cells (T), and the antigen-specific killing capacity of the NRT is evaluated.

(1) Labeling of target cells:

a) SNU-601 human gastric cancer cells transfected with CLDN18(e5) -ARHGAP26(e12) fusion gene collected in the logarithmic growth phase were cultured in serum-free RPMI 1640 medium at 37 ℃ in 5% CO₂Co-incubating for 2-4 h in the incubator.

b) Then, SNU-601 was collected in a centrifuge tube, centrifuged at 1000rpm for 5 minutes to pellet the cells, washed once with PBS, and the concentration of the target cells was adjusted to 10 with PBS⁶Adding CFSE (final concentration of 4nM) into the mixture per ml, uniformly mixing, incubating for 10 minutes at 37 ℃ in the dark, adding PBS with more than ten times of volume, centrifuging again at 1000rpm for 5 minutes, discarding the supernatant, and washing twice. Resuspending target cells in culture medium at a cell concentration of 10⁵Individual cells/ml. Standing at 37 deg.C, CO₂And (5) an incubator for standby.

(2) Preparation of Effector cells

Referring to the NRT cell preparation method in the experiment, different groups of effector cells were collected into each centrifuge tube, centrifuged at 1000rpm for 5 minutes to pellet the cells, and the supernatant was discarded. Resuspending the cells in culture medium at a cell concentration of 5X 10⁶Individual cells/ml. Effector cells and CFSE labeled target cells were added at a predetermined effective-to-target ratio. In CO₂Cells were incubated in an incubator at 37 ℃ for 4-16 hours.

(3) PI staining

The cell mixture was centrifuged to pellet the cells, and the supernatant was discarded. Each group of cells was resuspended in 100. mu.l PBS and PI was added to a final concentration of 50. mu.g/ml. Incubate at 4 ℃ for 15 minutes in the dark. The cell mixture was centrifuged to pellet the cells, after which the cells were resuspended in 400. mu.l PBS. The results were detected and analyzed by flow cytometry.

And (3) analyzing an experimental result:

prediction of fusion mutant neoantigens by CLDN18(e5) -ARHGAP26(e12)

Prediction of results by netmhcpana 4.0 software positive interpretation of the two above predictions: IC50<500nM or% Rank <2.0 is considered capable of binding to HLA molecules; IC50<50nM or% Rank <0.5 is considered to be capable of strongly binding to HLA molecules. However, the prediction accuracy of NetMHCpan4.0 is limited, so that the method is not limited to selecting a peptide sequence with the% Rank prediction result of <2.0 for synthesis, but expands the standard and selects a peptide sequence with smaller% Rank score for synthesis. The table shows 10 synthetic short peptide sequences and 1 long peptide sequence. Wherein CLDN18(e5) -ARHGAP26(e12) related peptide P1-4, L1; CLDN 18-related peptide P5-7; ARHGAP26 related peptide P8-10.

2. PBMC activation was induced in vitro with P1-10 and L1, respectively, and IFN-. gamma.secretion levels in the supernatant were measured by ELISPOT, and patient T cell reactivity to each antigenic peptide was compared, as a result: the level of IFN-gamma secreted by PBMC can be improved by more than 3 times compared with a negative control group by fusing neoantigen peptides P1, P4 and L1 and CLDN 18-related peptide P6.

3. Dendritic Cells (DC) are used for loading the antigen peptide P1, after activated T cells are stimulated, the IFN-gamma secretion level in the supernatant is detected through ELISPOT, and the result shows that the fused new antigen peptide P1 can stimulate PBMC to secrete the IFN-gamma level which is improved by more than 60 times compared with a negative control group; the CD137 of the CD8+ T cells is 5 times higher than that of a control group after being stimulated by P1 through flow cytometry detection, which indicates that P1 can obviously activate the T cells and induce P1 specific NRT cells.

4. The efficient target cell killing experiment shows that P1 specific NRT cells can obviously increase the killing capacity of T cells to tumors.

Therefore, the CLDN18(e5) -ARHGAP26(e12) fusion mutation related peptide P1(RTEDEVYNSNK) has better immunogenicity, and the induced NRT cells have strong targeted killing capability, and can be used for the NRT cell immunotherapy of patients with CLDN18(e5) -ARHGAP26(e12) fusion mutation. Can be used for preparing vaccines and engineered T cells.

The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.

Sequence listing

<110> affiliated drum building hospital of Nanjing university college of medicine

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Claims

1. A neoantigenic peptide directed to a CLDN18-ARHGAP fusion mutation, wherein: based on the fusion of CLDN18(e5) -ARHGAP26(e12), namely CLDN18 gene exon 5 and ARHGAP26 gene exon 12, aiming at the amino acid sequences at two ends of the fusion site of CLDN18(e5) -ARHGAP26(e12), HLA-A1101 is combined, and the T cell epitope prediction tool NetMHCpan4.0 is used for synthesizing antigen peptide with the sequence of SEQ ID NO. 1.

2. Use of the neoantigenic peptide of claim 1 directed against a CLDN18-ARHGAP fusion mutation for the preparation of a medicament for targeted therapy and immunotherapy of gastric cancer.

3. The use of the neoantigenic peptide of CLDN18-ARHGAP fusion mutation according to claim 2 in the preparation of a medicament for targeted therapy and immunotherapy of gastric cancer, wherein: the treatment includes neoantigen-reactive T cell therapy, engineered T cell therapy, vaccine therapy.