Colon cancer tumor specific TCR sequence and application thereof
阅读说明：本技术 结肠癌肿瘤特异tcr序列及其应用 (Colon cancer tumor specific TCR sequence and application thereof ) 是由 罗微 毛晓帆 余思菲 官展文 张贝莹 林凯容 金亚彬 于 2020-12-03 设计创作，主要内容包括：本发明提供了结肠癌肿瘤特异TCR序列及其应用,所述结肠癌肿瘤特异TCR序列包括9个序列。结肠癌肿瘤特异TCR序列在制备辅助诊断或结肠癌治疗或预后评估结肠癌的试剂或试剂盒中的应用,所述结肠癌肿瘤特异TCR去识别结肠癌的共同抗原后,T细胞增殖且针对结肠癌肿瘤发挥特异性杀伤作用,能够准确的识别结肠癌肿瘤,从而达到治疗或诊断的效果。(The invention provides a colon cancer tumor specific TCR sequence and application thereof, wherein the colon cancer tumor specific TCR sequence comprises 9 sequences. After the colon cancer tumor specific TCR identifies common antigens of colon cancer, T cells proliferate and play a specific killing role aiming at the colon cancer tumor, and the colon cancer tumor can be accurately identified, thereby achieving the effect of treatment or diagnosis.)
1. The application of the colon cancer tumor specific TCR sequence in the preparation of a reagent or a kit for auxiliary diagnosis or colon cancer treatment or prognosis evaluation of colon cancer.
2. The use according to claim 1, wherein the colon cancer tumor-specific TCR sequence is as set forth in SEQ ID No. 1.
3. The use according to claim 1, wherein the colon cancer tumor-specific TCR sequence comprises two sequences TRA2 and TRB2, wherein TRA2 is as set forth in SEQ ID No.2 and TRB2 is as set forth in SEQ ID No. 3.
4. The use according to claim 1, wherein the colon cancer tumor-specific TCR sequence comprises two sequences TRA3 and TRB3, wherein TRA3 is as set forth in SEQ ID No.4 and TRB3 is as set forth in SEQ ID No. 5.
5. The use according to claim 1, wherein the colon cancer tumor-specific TCR sequence comprises two sequences TRA4 and TRB4, wherein TRA4 is as set forth in SEQ ID No.6 and TRB4 is as set forth in SEQ ID No. 7.
6. The use according to claim 1, wherein the colon cancer tumor-specific TCR sequence comprises two sequences TRA5 and TRB5, wherein TRA5 is as set forth in SEQ ID No.8 and TRB5 is as set forth in SEQ ID No. 9.
7. The use according to claim 1, wherein the colon cancer tumor-specific TCR sequence is as set forth in SEQ ID No. 10.
8. The use according to claim 1, wherein the colon cancer tumor-specific TCR sequence is as set forth in SEQ ID No. 11.
9. The use according to claim 1, wherein the colon cancer tumor-specific TCR sequence comprises two sequences TRA8 and TRB8, wherein TRA8 is as set forth in SEQ ID No.12 and TRB8 is as set forth in SEQ ID No. 13.
10. The use according to claim 1, wherein the colon cancer tumor-specific TCR sequence comprises two sequences TRA9 and TRB9, wherein TRA9 is as set forth in SEQ ID No.14 and TRB9 is as set forth in SEQ ID No. 15.
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a colon cancer tumor specific TCR sequence and application thereof.
Background
Colon cancer takes about 70 million people each year and is the fourth most lethal cancer in the world. With the change of dietary structure and life style, the number of colon cancer patients is increasing year by year at a rate of 4-5%, becoming malignant tumor with the fastest incidence, about 40 ten thousand new cases each year, and the mortality rate is close to 60%, which seriously threatens the health and life of people. The tumor immunotherapy is developed rapidly, and the use of targeted PD-1/PD-L1 or CTLA-4 medicaments has proved to have obvious curative effect on various malignant tumors, but has little curative effect on colon cancer. 50% objective remission rate on PD-1 antibody in MSI-H/dMMR colorectal cancer patients. Unfortunately, however, about 95% of patients are of the MSI-L or MSS/pMMR type and are completely unresponsive to drugs such as targeted PD-1. Therefore, there is a great need to develop new immunotherapeutic strategies to improve the survival of colon cancer patients.
In recent years, immunotherapy of tumors has been greatly advanced, which aims to activate the human immune system, and it is desired to kill cancer cells and tumor tissues by means of autoimmune functions. Adoptive Cellular Therapy (ACT), an important component of tumor immunotherapy, achieves anti-tumor purposes by reinfusing self or allogeneic (specific or non-specific) immune cells that have been expanded in vitro back into the patient.
A T Cell Receptor (TCR) is a receptor molecule on the surface of a T cell that specifically recognizes an antigenic peptide-MHC complex on an antigen presenting cell, thereby triggering a T cell immune response. Since TCR molecules determine the antigen recognition specificity of T cells, if TCR specific to tumor antigens is transferred into common T cells, the T cells can be endowed with the recognition capability of the tumor antigens, and the T cells can be transferred into patients after being activated and proliferated in vitro, so that the anti-tumor effect can be exerted. Therefore, a large number of T cells for identifying specific antigens can be conveniently obtained by utilizing a TCR gene introduction method, the T cells modified by the TCR gene are called TCR-T, and the TCR-T becomes a research hotspot in tumor immunotherapy in recent years and shows good treatment effect in clinical experiments.
The TCR molecule mainly consists of two chains of alpha and beta, and encoding genes V, (D) J and C of the TCR molecule have MHC recognition restriction through germline rearrangement in the process of T cell development and through positive selection and negative selection processes in thymus. The TCR α β produced by the mature T cells of the body constitutes a Repertoire of antigen recognition receptors (reportire) capable of binding tens of millions of antigens, and theoretically, the capacity of the TCR α β receptor Repertoire is estimated to be more than 15 times of 10. The successful acquisition of tumor antigen-specific TCR is an important prerequisite for tumor TCR-T cell therapy, and currently, the screening of tumor-specific TCR genes is mainly performed by obtaining tumor antigen-specific recognized T cells and then cloning their TCR genes.
The occurrence and development of colon cancer are closely related to T cell immunity, and a large number of researches report that T cell infiltration in cancer tissues is related to prognosis. Early studies found that increased T cell abundance after treatment was associated with remission from colorectal cancer. T cell mediated tumor clearance requires three conditions to be met: identifying and activating tumors; relieving immunosuppression; can home to tumor tissue. In order to allow T cells to recognize tumors, scientists such as Eshhar, Carl June and Rosenberg develop a T cell receptor redirection technology (T cell receptor redirection), and chimeric antigen-receptor T cells (CAR-T) or T cell receptor gene modified T cells (TCR-T) are prepared by a genetic engineering means to improve the recognition and killing of tumors. CAR-T and TCR-T are taken as adoptive reinfusion therapy frontage technology of immune cells, so that the acquisition of function-enhanced T cells based on synthetic biology, immunology and genetic modification technology becomes possible.
Disclosure of Invention
The invention aims to provide a colon cancer tumor specific TCR sequence and application thereof, which aims to solve one or more technical problems in the prior art and at least provide a beneficial selection or creation condition.
The technical scheme adopted by the invention is as follows:
screening out a colon cancer tumor specific TCR sequence by using a specific TCR sequence screening method, wherein the colon cancer tumor specific TCR sequence comprises 9 types of TCR sequences which are respectively:
clonotype1, comprising a TRB1 sequence as shown in SEQ ID NO. 1;
clonotype2 comprising two sequences of TRA2 and TRB2, wherein TRA2 is shown as SEQ ID NO.2, and TRB2 is shown as SEQ ID NO. 3;
clonotype3 comprising two sequences of TRA3 and TRB3, wherein TRA3 is shown as SEQ ID No.4 and TRB3 is shown as SEQ ID No. 5;
clonotype4 comprising two sequences of TRA4 and TRB4, wherein TRA4 is shown as SEQ ID NO.6 and TRB4 is shown as SEQ ID NO. 7;
clonotype5 comprising two sequences of TRA5 and TRB5, wherein TRA5 is shown as SEQ ID NO.8 and TRB5 is shown as SEQ ID NO. 9;
clonotype6, comprising a TRB6 sequence as shown in SEQ ID NO. 10;
clonotype7 comprising a TRB7 sequence as shown in SEQ ID NO. 11;
clonotype8 comprising two sequences of TRA8 and TRB8, wherein TRA8 is shown as SEQ ID No.12 and TRB8 is shown as SEQ ID No. 13;
clonotype9 comprising two sequences, TRA9 and TRB9, wherein TRA9 is shown in SEQ ID NO.14 and TRB9 is shown in SEQ ID NO. 15.
The colon cancer tumor specific TCR sequence is applied to the preparation of a reagent or a kit for colon cancer auxiliary diagnosis or colon cancer treatment/prognosis evaluation.
The specific TCR sequence of the colon cancer provided by the invention is applied to the preparation of a reagent or a kit for auxiliary diagnosis or colon cancer treatment or prognosis evaluation of colon cancer, and after the common antigen of colon cancer is identified by the specific TCR of the colon cancer, T cells proliferate and play a specific killing role aiming at the colon cancer tumor, so that the colon cancer tumor can be accurately identified, and the treatment or diagnosis effect is achieved.
Drawings
FIG. 1 is a graph showing the results of first-step cell clustering using Seurat.
FIG. 2 is a graph of 31 subpopulations of cells annotated for reference according to the ENCODE and BLUE PRINT subpopulations.
FIG. 3 is a graph of the final cell clustering results, wherein FIG. 3-a is a graph of the levels of cells expressing CD103(ITGAE), CD159(KLRC2), PD-1(PDCD1), CTLA4, CD39(ENTPD1), TIM-3(HAVCR2), CD8A and CD 4; FIG. 3-b is a graph defining T cell subpopulations, using each of the 29 published cell subpopulations as a reference; FIG. 3-c is a TSNE visualization display final cell clustering result chart.
Detailed Description
The following steps are only used for illustrating the technical scheme of the invention and are not limited; although the present invention has been described in detail with reference to the foregoing steps, it will be understood by those of ordinary skill in the art that: the technical solutions recorded in the foregoing steps may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the present invention in its various steps.
Example 1: screening of colon cancer specific TCR sequences
1. Isolation of colon cancer pathological tissue infiltrating mononuclear cells
The method for separating the mononuclear cells infiltrated by the pathological tissues of the colon cancer comprises the following steps:
(1) placing isolated fresh colon cancer pathological tissue excised in operation into a sterile plate, repeatedly washing with sterile PBS or Hank's, and subtracting surrounding connective tissue and adipose tissue, shearing the tissue into pieces as much as 1-2 mm in the plate with ophthalmologic forceps3Small pieces of size;
(2) pouring a freshly prepared tissue digestive juice (20mg of collagenase I +10 mu L of DNase (10mg/mL) +10mL of incomplete RPMI1640), uniformly mixing, and digesting in a shaker at 37 ℃ for 1-2 hours;
(3) adding 10mL HBSS to terminate digestion, transferring digested tissue fluid to a 70-micron screen by using a sterile Pasteur pipette, filtering to a new 50-mL centrifuge tube, centrifuging (1800rpm, 8min, normal temperature), and slowly increasing and decreasing the rotation speed during centrifugation;
(4) discarding the supernatant, washing for 2 times by Hank's, discarding the supernatant if the cell mass has red blood cells visible to naked eyes, adding 5mL of erythrocyte lysate, blowing off the cell mass slightly, standing for 5-10 min by RT, observing the color of the liquid at any time, stopping when the liquid becomes clear and transparent, adding 20-30 mLHank's liquid to stop, and centrifuging and washing for two times;
(5) after the final centrifugation, the supernatant was discarded, and the cells were resuspended in complete RPMI1640 medium (500mL incomplete RPMI1640 solution +50mL inactivated fetal bovine serum + 100. mu.g/mL penicillin + 100. mu.g/mL streptomycin) and counted.
2. Flow sorting CD3+T cells
Separating the freshly separated particles by 10-50X 106The tissue infiltrates single nuclear cells, the supernatant is discarded after centrifugation, and the tissue is washed twice with sterile purification buffer (1L 1 XPBS +5g BSA +2mM EDTA-Na2, pH7.2-7.4), about 4 mL/tube, centrifuged at 1800rpm and 4 ℃ for 8 min;
discarding the supernatant, resuspending with 200. mu.L of purified buffer, adding fluorescent antibody (20. mu.L each of anti-CD 45-V450 and anti-CD 3-PE-CF 594), incubating at 4 ℃ for 30min in the absence of light; washing twice with sterile purified buffer solution, 4 mL/tube, centrifuging at 1800rpm and 4 deg.C for 8min, discarding supernatant, and resuspending with 500 μ L purified buffer solution;
filtering the cell suspension through a 40-micron screen to remove adhered cell masses;
according to BD Asia II operating manual, sterile processing and sorting system, setting sorting parameters, adding 10 μ L7-AAD dye into cell suspension after surface molecule staining before machine sorting, incubating for 10min in dark, machine sorting, and shooting to 7-AAD in single cell-CD45-V450+CD3-PE-CF594+And collecting the shot cell population.
3. Removal of dead cells
Centrifuging the flow-sorted cells, washing the cells for 2 times by using sterile 1 XPBS, resuspending the cells by using a proper amount of sterile 1 XPBS, taking 10 mu L of cell suspension, mixing the cell suspension with 10 mu L of trypan blue, counting the cells and the cell survival rate by using a Life Count cell counter, and when the cell survival rate is less than 80%, carrying out cell death removal treatment by using magnetic beads for removing the dead cells by using Meitian and gentle magnetic beads.
4. Single cell transcriptome library construction, VDJ library construction and sequencing
Marking single cells of the cells after dead cell removal treatment by using a magnetic bead labeling technology, marking the same label on each RNA of the same cell, carrying out reverse transcription and enzyme digestion, respectively amplifying a transcriptome and a VDJ, finally carrying out sequencing by using 10 multiplied single cell library construction and Illumina Hiseq4000 double end 150, and finally obtaining a gene expression profile and a TCR sequence of 22741 cells in total.
5. Data analysis and screening of colon cancer tumor specific CD8+TCR
Converting the fastq file obtained after sequencing into an expression matrix of cell gene and obtaining TCR of each cell by using a data processing method corresponding to single cell sequencing;
reading in an expression matrix by using Seurat software, removing cells with over-low or over-high UMI, and intercepting by taking 3% of cells in front and behind as a threshold;
integrating data of each sample by using a CCA method provided in Seurat;
dividing the cells into groups by using a cell clustering method provided in Seurat, adjusting the resolution to make the cell groups reach 30 to 35 groups, and calculating a mean expression profile of each cell group, wherein the result is divided into 31 groups as shown in FIG. 1;
reading in a mean expression profile of 32 cell populations by using SingleR software, preliminarily defining the cell populations according to ENCODE and BLUEPRINT databases, and filtering non-T cells in the data, wherein the results are shown in fig. 2, and the 30 th and 31 th populations are non-T cells and are filtered;
after filtration, in the CD8 positive cell population, looking at the expression level of ITGAE gene, select ITGAE high expressing cell population, among which KLRC2 expression level is observed, KLRC2 high expressing cell population is defined as IEL, and ENTPD1 expression level is observed in the remaining cell population, ENTPD1 high expressing cell population is CD8Trm Ex, and the remaining cell population is CD8Trm, downloading GSE107011 data in GEO database, which contains the expression profile of 29 immune cells in blood, and using singleR to match the cell population with the GSE107011 data, to obtain definition and annotation of the remaining cell population, which can be defined as: CD8EM, CD8EM Ex promoter, CD8Naive, CD8Tcm, CD8TE, MAIT, CD4Naive, CD4Tcm, Th1, Th17, Th1.Th17, Th2, TFH and Treg, the results are shown in FIG. 3, wherein FIG. 3-a is a level diagram of cells expressing CD103(ITGAE), CD159(KLRC2), PD-1(PDCD1), CTLA4, CD39(ENTPD1), TIM-3(HAVCR2), CD8A and CD 4; FIG. 3-b is a graph defining T cell subpopulations, using each of the 29 published cell subpopulations as a reference; FIG. 3-c is a TSNE visualization display final cell clustering result chart;
screening tumor specific TCR, wherein the CD8 cell with the tumor specific TCR is a cell which is highly expanded in CD8Tem and CD8Trm Ex cells in tumor tissues, and a plurality of sequences with the highest TCR frequency are searched in the CD8Tem and CD8Trm Ex cells respectively, and can be used for forming tumor specific TCR sequences, namely CD8EM and CD8Trm Ex high-frequency CDR3 sequences in tumors, and the result is shown in table 1, and 9 colon cancer specific TCR sequences can be screened out.
The colon cancer tumor specific TCR sequences are applied to preparation of a reagent or a kit for auxiliary diagnosis or colon cancer treatment or prognosis evaluation of colon cancer, and after the screened 9 colon cancer specific TCR sequences are used together to identify common antigens of colon cancer in application, T cells proliferate, thereby playing a specific killing role on colon cancer tumors, accurately identifying the colon cancer tumors and achieving the effect of treatment or diagnosis.
TABLE 1 Colon cancer specific TCR sequences
SEQUENCE LISTING
<110> first-person hospital in Foshan City
<120> colon cancer tumor specific TCR sequence and application thereof
<130> 2020.11.09
<160> 15
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