CN118256604B - Method for screening and identifying new antigen of immunogenicity tumor by using artificial antigen presenting cell - Google Patents
Method for screening and identifying new antigen of immunogenicity tumor by using artificial antigen presenting cell Download PDFInfo
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Abstract
The invention relates to a method for screening and identifying an immunogenic tumor neoantigen by utilizing an artificial antigen presenting cell, belonging to the technical field of tumor immunity. The invention combines tumor whole exon sequencing analysis, HLA and peptide affinity prediction and carrier-based in vitro stimulation of T cell proliferation and anti-tumor cytokine secretion detection, and provides a new method reference for accurately screening new antigens and verifying immunogenicity thereof. The method has the advantages of high screening accuracy of the immunogenic tumor neoantigen, no need of in-vitro and in-vivo verification, and simple operation.
Description
Technical Field
The invention belongs to the technical field of tumor immunity, and in particular relates to a method for screening and identifying an immunogenic tumor neoantigen by utilizing an artificial antigen presenting cell.
Background
The tumor neoantigen is a protein generated by nonsensical mutation of somatic cells in the process of occurrence and development of tumor cells, and the protein is only expressed in tumor tissue cells, but not expressed in normal tissues and cells, and is a tumor immunotherapy specific target. However, not all polypeptides that are tumor mutated can become tumor neoantigens, and only mutant peptides that are effective in stimulating T cell responses can be referred to as tumor neoantigens. It is critical in the early stage how tumors have a high degree of heterogeneity and how to rapidly and accurately predict, screen and identify tumor neoantigens.
At present, the existing new antigen identification method mainly comprises the steps of comparing the exon sequences of tumor tissues and normal tissues of a patient, identifying tumor somatic mutation genes, and then combining the types of main histocompatibility complex molecules of the patient, namely the types of human leukocyte antigen, carrying out tumor mutation polypeptide affinity prediction to obtain candidate new antigens preliminarily. Since the mutant polypeptide is predicted to have a larger number of false positive results, the tumor neoantigen can be screened out by subsequent in vivo and in vitro experiments. By combining the analysis, a tumor neoantigen screening and verifying system can be established to obtain the tumor neoantigen, and a foundation is provided for establishing a precise treatment strategy research based on patient individuation.
Disclosure of Invention
The invention solves the technical problems of the prior art that the method for screening and identifying the tumor neoantigen needs in-vitro verification, has poor accuracy and complex process, provides a method for screening and identifying the high-flux immunogenic tumor neoantigen, the invention combines tumor whole exon sequencing analysis, HLA and peptide affinity prediction and carrier-based in vitro stimulation of T cell proliferation and anti-tumor cytokine secretion detection, and provides a new method reference for accurately screening new antigens and verifying immunogenicity thereof. The method has the advantages of high screening accuracy of the immunogenic tumor neoantigen and simple operation.
According to the object of the present invention, there is provided a method for screening and identifying a high throughput immunogenic tumor neoantigen comprising the steps of:
(1) Performing exon sequencing on a tumor specimen and a non-tumor specimen, wherein the non-tumor specimen is a paracancerous tissue or peripheral blood; taking the exon sequencing result of a non-tumor specimen as a control, and rejecting the germ line mutation to obtain tumor somatic mutation; performing human leukocyte antigen typing on the tumor specimen, and comparing the mutant polypeptide obtained by the tumor somatic mutation with the typing result of the human leukocyte antigen typing to obtain a human leukocyte antigen restrictive new antigen;
(2) Constructing a plasmid, wherein the plasmid contains the heavy chain and light chain genes of the human leukocyte antigen in the step (1) and also contains Flag tags; expressing the plasmid to obtain fusion protein of human leukocyte antigen and Flag label; loading the fusion protein and the antibody of the T cell costimulatory signal on the microsphere to construct an artificial antigen presenting cell;
(3) Incubating the human leukocyte antigen-restricted neoantigen obtained in the step (1) and the artificial antigen-presenting cell obtained in the step (2), so that the human leukocyte antigen-restricted neoantigen is loaded on the human leukocyte antigen of the artificial antigen-presenting cell, and then incubating the human leukocyte antigen with T cells, wherein the surface of the T cells is provided with the human leukocyte antigen identical with the tumor specimen;
If the T cells proliferate and killer cytokine secretion is detected, the human leukocyte antigen-restricted neoantigen is an immunogenic tumor neoantigen;
The human leukocyte antigen-restricted neoantigen is a non-immunogenic tumor neoantigen if proliferation of the T cells does not occur and/or secretion of killer cytokines is not detected.
Preferably, the tumor specimen is a lung adenocarcinoma specimen, a liver cancer specimen or a intestinal cancer specimen.
Preferably, the Flag tag is 3×flag.
Preferably, the antibody to the T cell costimulatory signal is a murine anti-human CD28 antibody.
Preferably, the microspheres are sulfate latex microspheres or phospholipid microspheres.
In general, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) The multiple screening and verification system ensures the stability and accuracy of experimental results: the research combines tumor whole exon sequencing analysis, HLA and peptide affinity prediction and detection based on HLA-A2/aAPCs in vitro stimulated T cell proliferation and anti-tumor cytokine secretion, and provides a new method reference for accurately screening new antigens and verifying immunogenicity thereof.
(2) The HLA-A2/aAPCs of the invention are convenient to use: HLA-A2 is a human leukocyte antigen type with a crowd covering about 50%, and is loaded on sulfate latex microspheres to form artificial antigen presenting cells, namely aAPCs, and the product can be prepared in advance, is convenient to store and transport, and can be used for in vitro verification experiments of new antigens of different individuals.
(3) The invention establishes accurate treatment strategy research based on patient individuation: and (3) analyzing the individual exon data and a new antigen polypeptide library so as to screen new antigen polypeptides with specific individual immunogenicity.
(4) The invention has expandability: the system has low requirement on HLA molecular expression quantity and purity, is easy to expand from HLA-A2 to other HLA types, and provides reference for preparing aaPCs with various HLA types and establishing a new antigen immunogenicity identification system.
(5) The protein purity requirement of the invention on protein molecules is low: the HLA-A2 expression protein yield obtained by the insect expression system established in the research is not very high, and enough fusion proteins cannot be provided for mass preparation and purification, but the preparation mode of the HLA-A2/aAPCs established in the research does not need protein purification, and the enrichment of the HLA-A2 fusion proteins on the surfaces of the microbeads can be promoted by specifically combining the Flag tag of the HLA-A2 fusion proteins with the microbeads coated with the anti-Flag antibody.
Drawings
FIG. 1 shows the somatic mutation characteristics of cancer tissues and the tumor clone heterogeneity results: wherein A is a mutation type spectrum; b is a mutation spectrum heat map.
FIG. 2 is a screen for HLA-A2 restricted neoantigens according to the invention.
FIG. 3 is a schematic diagram showing the construction of the pFastBac TM Dual+ [ β2m+HLA-A2-3 XFlag ] expression plasmid of the present invention.
FIG. 4 is a flow chart showing the establishment of an in vitro stimulation system for HLA-A2/aAPCs of the present invention.
FIG. 5 shows the results of in vitro stimulation experiments of the immunogenicity of the novel antigens of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The invention discloses a method for screening and identifying an immunogenic tumor neoantigen by utilizing an artificial antigen presenting cell, which comprises the following steps:
1. Tumor specimen and peripheral blood specimen exon sequencing
Collecting lung adenocarcinoma samples of different positions of the same patient, collecting the tissues beside the cancer or peripheral blood of the corresponding patient as a control for eliminating the germ line mutation to obtain tumor somatic mutation, obtaining a tumor exon sequencing result through whole exon sequencing, and carrying out library building and belief generation analysis;
1.1 library construction and library establishment;
1.2 quality control of whole exon sequencing data;
1.3, counting cancer tissue somatic mutation characteristics and comparing tumor clone heterogeneity;
1.4 screening drug treatment targets, driving genes, susceptibility genes and oncogenic signal paths;
1.5 HLA typing results of human leukocyte antigens of the specimens;
1.6HLA-A2 human leukocyte restriction neoantigen screening;
2. expression of Flag-tagged HLA-A2 fusion protein and construction of HLA-A2/aAPCs
2.1PFastBac TM Dual+ [ β2m+HLA-A2-3 XFlag ] plasmid construction;
2.2 identification of recombinant bacmid;
2.3 extraction and transfection of recombinant bacmid;
2.4 ELISA identification of HLA-A2 fusion proteins;
2.5HLA-A2 fusion protein flow identification;
2.6HLA-A2/aAPCs construction;
3. in vitro stimulation experiment of immunogenicity of novel antigen
3.1 Human peripheral blood mononuclear cell separation;
3.2 human peripheral blood HLA-A2 flow typing detection;
3.3 staining of effector cell CFSE markers;
3.4 in vitro stimulation of candidate neoantigens HLA-A2+ healthy individual lymphocyte experiments;
3.5CBA detection of IFN-gamma cytokine secretion levels.
The following are specific examples
Example 1
1. Tumor specimen and paracancerous specimen exon sequencing
1.1 Library construction and library construction
The genome DNA of the sample to be tested is broken randomly by a breaking instrument, so that the genome DNA is divided into a plurality of fragments with the length of 180-280 bp. Next, libraries were constructed using the Agilent human exon kits, and these fragments were subjected to end repair, phosphorylation, and A-tailed treatment. Then, each fragment was ligated with an upper adaptor at both ends to prepare a DNA library and the effective concentration value in the library was precisely measured using the Q-PCR method.
1.2 Quality control of Whole exon sequencing data
The base mass value distribution, the total mass value distribution and the base distribution proportion of each position of the read are compared, so that the separation degree of the bases, the GC content distribution, the N content of each position of the read, whether the read further comprises a sequenced linker sequence and the read repetition rate introduced in the amplification process of the experiment are analyzed.
1.3 Statistical cancer tissue somatic mutation characterization and tumor clone heterogeneity comparison
And comparing the sequences with high quality obtained by sequencing to a ginseng genome, detecting mutation information of the germ line gene in a tumor sample, eliminating sample background mutation through sample germ line mutation to reduce the existence of false positive data, detecting somatic mutation of a paired cancer sample, and finally analyzing the detected mutation. This example includes 4 tumor samples from the same patient, 4 samples having different infiltration characteristics, so that subsequent tumor clone heterogeneity comparisons are required. And MuTect, comparing tumor tissue with adjacent tissue or peripheral blood by using MuTect < 20 > software, filtering the germ line mutation to obtain mutation information of somatic cells of the tumor tissue, obtaining a vcf result file, and carrying out sample annotation by using vep < 21 > software.
FIG. 1 shows the somatic mutation characteristics of cancer tissues and the tumor clone heterogeneity results: wherein A is a mutation type spectrum; b is a mutation spectrum heat map.
1.4 Screening of drug therapeutic target, driver Gene, susceptibility Gene and oncogenic Signal pathway
The genome or exome of the tumor was analyzed to find new driver mutations. Several genes associated with susceptibility to cancer were examined and individuals were found to be sensitive to cancer. Detecting a gene expression profile or genotyping associated with prognosis in a particular cancer, and assessing recurrence and metastasis risk. The screened drug treatment targets, driving genes, susceptibility genes and oncogenic signal paths can provide theoretical basis for personalized treatment of the subsequently screened mutant genes.
1.5 HLA typing results of specimens
The main chromosome is selected for genome alignment to avoid the influence of polymorphism and high complexity of an HLA region on sequencing result analysis.
1.6 Tumor mutation load, analysis of number of neoantigens, and HLA-A2 restriction neoantigen screening
The number of somatic mutations after germ line removal of tumor genome, i.e., tumor mutation load. About 10% of the nonsensical mutations can result in mutated peptide fragments that bind with high affinity to MHC, and 1% of these are screened for novel antigen polypeptides that are recognized by T cells in tumor patients.
FIG. 2 is a screen for HLA-A2 restricted neoantigens according to the invention. 7 candidate neoantigen polypeptides with the strongest binding capacity are screened out of 30 mutant polypeptides by predicting the affinity of the restriction neoantigen to the human leukocyte antigen.
2. Expression of Flag-tagged HLA-A2 fusion protein and construction of HLA-A2/aAPCs
Construction of the 2.1pFastBac TM Dual+ [ beta.2m+HLA-A 2-3 XFlag ] plasmid
Digestion and ligation of the pFastBac TM Dual plasmid and the beta 2m Gene, extraction of the pFastBac TM Dual + [ beta 2m ] plasmid, digestion and ligation of the pFastBac TM Dual + [ beta 2m ] and [ HLA-A2-3 xFlag ] genes, and extraction of the pFastBac TM Dual + [ beta 2m ] + [ HLA-A2-3 xFlag ] plasmid.
FIG. 3 is a schematic diagram showing the construction of pFastBac TM Dual+ [ β2m+HLA-A2-3 XFlag ] expression plasmid. As shown in FIG. 3, the light chain gene β2m and the heavy chain fusion gene HLA-A2-3 xFlag were cloned into the Pp10 and Pph promoters of the insect expression vector pFastBac TM Dual, respectively, to form pFastBac TM Dual+ [ β2m+HLA-A2-3 xFlag ] recombinant plasmids.
2.2 Identification of recombinant bacmid
PFastBac TM Dual+ [ β2m+HLA-A2-3 XFlag ] was added to DH10Bac competent, the bacterial culture dishes were placed in a 37℃bacterial incubator, after 3 days, single white colonies were picked, and the picked single colonies were added to liquid LB medium containing tetracycline (10. Mu.g/ml), kanamycin (50. Mu.g/ml) and gentamicin (7. Mu.g/ml), and the bacteria were shake cultivated at 37℃and 250rpm overnight. And (3) repeating the colony shaking step, and taking bacterial liquid and using M13 primer PCR to identify whether the target gene is successfully inserted.
2.3 Recombinant bacmid extraction and transfection
And (3) extracting recombinant stem grains from the overnight culture fungus liquid, detecting the liquid by a Nano drop instrument, and marking the concentration. SF9 cells of 1X 10 6 were inoculated into a six-well plate, 16. Mu.g of Bacmid and 8. Mu.l of a Transfection reagent were added, and the culture broth was incubated at 27℃for 5 days, after which the baculovirus first-generation virus P1 was harvested. The infection of SF9 cells is continued as described above until the maximum yield of expressed protein of interest is detected.
2.4 ELISA identification of HLA-A2 fusion proteins
The capture antibody (W6/32 antibody) was coated overnight in a well plate at 4℃and blocked, 100. Mu.l of sample was added to each well, HRP-. Beta.2m antibody was added, washed, and TMB was added for color development, and OD was measured. The highest OD value was detected at the highest yield of the target protein.
2.5HLA-A2 fusion protein flow assay
The mouse-derived anti-Flag antibody was incubated with sulfate latex beads for 24h, 3% BSA was added for blocking 24h after washing, HLA-A2 protein was added for 24h after washing, 1. Mu.l of sulfate latex beads were added to 50. Mu.l of PBS after washing again, and 0.5. Mu.l of FITC-labeled murine anti-human HLA-A2 antibody was added for flow-through detection. The positive rate detected by flow cytometry should be greater than 95%.
2.6HLA-A2/aAPCs construction
The mouse-derived anti-Flag antibody and the mouse anti-human CD28 antibody are incubated with sulfate latex microbeads for 24 hours, 3% BSA is added for blocking 24 hours after washing, HLA-A2 protein is added for incubation for 24 hours after washing, and the mixture is dissolved in PBS for preservation after washing again.
3. In vitro stimulation experiment of immunogenicity of novel antigen
3.1 Isolation of human peripheral blood mononuclear cells to obtain mononuclear cells including T cells
Uniformly mixing a blood sample with PBS1:1 in equal volume, adding lymphocyte separation liquid, performing gradient centrifugation at room temperature for 25min, sucking an intermediate off-white lymphocyte layer, transferring to a test tube, washing twice after the PBS is filled, and adding 1640 culture medium with 10% FBS.
3.2 Human peripheral blood HLA-A2 flow typing detection
Red blood cells are lysed by adding red blood cell lysate into human peripheral blood at room temperature, and after PBS washing, anti-HLA-A 2 antibodies and anti-human CD3 antibodies are added for staining and flow staining identification.
3.3 CFSE marker staining of effector cells
PBMC were stained with CFSE staining solution at a ratio of 1:8000 in a 37℃cell incubator, terminated with half the volume of the suspension in FBS, washed twice with PBS and supplemented with 1640 medium with 10% FBS.
3.4 In vitro stimulation of candidate neoantigens HLA-A2+ (Positive) healthy individual lymphocyte experiments
Adding PBMCs of CFSE-stained HLA-A2+ (positive) healthy individuals and prepared artificial antigen cells 1:1 into each 96-well plate, adding human IL-2 cytokines and neoantigen polypeptides, supplementing IL-2 and polypeptides by half-quantity liquid exchange on the 3 rd and 7 th days, maintaining original concentration culture, taking cytofection anti-human CD3 and anti-human CD8 antibodies on the 10 th day, and detecting on the machine. 7S-MT (candidate neoantigen polypeptide) induced proliferation of CD8 + (positive) T cells with significant differences compared to the control group.
3.5CBA detection of IFN-gamma cytokine secretion levels
12.5 Μl of cell supernatant is incubated with CBA capture microspheres at room temperature and in a dark place, detection antibodies are added after washing, incubation is carried out at room temperature and in a dark place, SA-PE antibodies are added, incubation is carried out at room temperature and in a dark place, PBS is used for resuspension and transfer to a flow tube after washing, and detection is carried out on the flow tube. Stimulation of group cell supernatants by 7S-MT (candidate neoantigen polypeptide) detected secretion of killer cytokines, with significant differences compared to the control group.
FIG. 4 is a flow chart of an in vitro stimulation experiment of neoantigen immunogenicity, in which proliferation of T cells and secretion of cell supernatant killer cytokines were examined after incubating artificial antigen presenting cells with the synthesized antigen polypeptides, PBMC for 10 days.
FIG. 5 shows the results of in vitro stimulation experiments of the immunogenicity of a neoantigen, wherein A is the proliferation level of lymphocytes and B is the secretion level of killer cytokines. The level of lymphocyte proliferation and the level of killer cytokine secretion in an in vitro stimulation experiment can reflect the immunogenicity of the neoantigen, and the higher the level of lymphocyte proliferation, the higher the level of killer cytokine secretion, the stronger the immunogenicity of the neoantigen.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
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