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Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine

An in vitro detection and specific technology, applied in the field of molecular immunology and cellular immunology, can solve the problems of high uncertainty, high detection cost, low sensitivity, etc., and achieve the effect of low cost, simple detection method and clear results.

Active Publication Date: 2017-05-10
恒瑞源正(上海)生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1) High uncertainty: the length of tumor neoantigen and the corresponding MHC molecule presented are uncertain, so stable MHC-nascent peptide multimers may not be produced;
[0006] 2) Low sensitivity: the ratio of specific T cells that can be effectively detected by the MHC-peptide polymer flow cytometry method is 0.001%, and when it is lower than this value, it will not be detected;
[0007] 3) Function loss: MHC-peptide polymer flow cytometric analysis can only show that specific T cells can specifically bind to MHC-peptide complexes, but cannot detect whether T cells can be effectively stimulated after recognizing antigenic peptides, Secrete functional cytokines or kill tumor cells;
[0008] 4) High cost: Each antigenic peptide needs to be presented by a specific MHC molecule to be recognized by T cells, so the detection cost is high

Method used

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  • Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine
  • Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine
  • Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine

Examples

Experimental program
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Effect test

Embodiment 1

[0064] Example 1. Prediction and synthesis of tumor neoantigens

[0065] Take the isolated tumor tissue for genome analysis to find out non-synonymous gene mutation sites (including new amino acid sequences formed by nucleic acid point mutations, insertions or deletions), and use the antigen prediction software netMHC4 based on the inclusion of the non-synonymous The binding ability of the antigen at the gene mutation site to the corresponding major histocompatibility molecule is to select the antigen peptide sequence with the best binding ability as the candidate tumor neoantigen peptide. The ability of a candidate tumor neoantigen peptide to bind to the corresponding MHC is indicated by the minimum concentration of the antigen peptide required to achieve stable MHC-antigen peptide binding. The lower the concentration, the more stable the binding of the antigen peptide to the MHC molecule, the stronger its antigenicity, and the better it can stimulate the proliferation of spe...

Embodiment 2

[0075] Example 2 Tumor neoantigen peptide pre-stimulation

[0076] In the same case of tumor patient, 10mL of peripheral venous blood was extracted with a heparin sodium anticoagulant tube under informed conditions. Lymphocyte separation medium (Fresenius Kabi Norge AS, Lymphoprep TM ) After the peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation, they were suspended in fresh serum-free medium (AIM-V from Life Technology Company) with a cell density of 1-3x10 6 / mL. In the serum-free medium, add the above tumor neoantigen peptide or the corresponding native peptide (1-10 μg / mL), at 37°C, 5% CO 2Cultivate in the incubator for 36-48h. The purpose of detecting the immune response of the patient's PBMC to the native peptide is to test whether the immune response to the tumor neoantigen is targeting the mutant sequence, so as to determine that the tumor neoantigen-specific T cells only specifically target and recognize the mutant cells, t...

Embodiment 3

[0077] Example 3 Using IFNγ ELISpot method to detect tumor neoantigen-specific T cells that recognize the tumor neoantigen (U-CyTech BV, CT230-PR5)

[0078] For the operation procedure, refer to the manual of the kit. The specific experimental process is as follows:

[0079] Wet the Elispot plate with 70% ethanol aqueous solution at room temperature for 1 hour, then wash it twice with PBS, add IFNγ-coated antibody to coat the Elispot plate, cover the plate and incubate overnight at 4°C, wash it 3 times with PBS, and add blocking solution to block Cover the unbound position of the IFNγ antibody and incubate at 37°C for 1 hour;

[0080] Adjust the pre-stimulated PBMC concentration to 1-3x10 6 / ml, add corresponding 1-10ug / mL tumor neoantigen (neoplastic peptide 1, neonatal peptide 2 in Table 1), tumor native antigen (native peptide 1 and native peptide 2 in Table 1), control peptide ( For example, a peptide of the envelope protein of HIV virus) serum-free cell culture medium ...

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Abstract

The invention relates to a method and kit for in vitro detection of tumor neoantigen specificity T cells and a tumor vaccine. The method for in vitro detection of the tumor neoantigen specificity T cells comprises a step S1 of adding tumor neoantigen stimulation in cell culture fluid of peripheral blood mononuclear cells and performing culture incubation; a step S2 of adopting an IFN gamma enzyme linked immunospot assay method to detect and identify the tumor neoantigen specificity T cells of tumor neoantigens. By implementing the method and kit for in vitro detection of the tumor neoantigen specificity T cells, the tumor neoantigen specificity T cells can be detected in high precision through the IFN gamma enzyme linked immunospot assay method, the detection method is simple, results are clear, and the cost is low. By implementing the tumor vaccine, the tumor neoantigen specificity T cells can be effectively amplified and are effectively used for manufacturing tumor immunotherapy medicines.

Description

technical field [0001] The invention relates to the fields of molecular immunology and cellular immunology, and more specifically relates to a method, a kit, and a tumor vaccine for detecting tumor neoantigen-specific T cells in vitro. Background technique [0002] T cells kill or injure cancer cells by specifically recognizing antigenic peptides (ie, antigenic epitopes) presented by major histocompatibility complex (MHC) of tumor cells through immune receptors on the surface of T cells. CD8+ cytotoxic T cells (Cytotoxic T lymphocytes, CTL) can specifically recognize target cells and secrete an important cytokine interferon gamma (Interferon gamma, IFN gamma), and directly kill cancer cells. CD4+ helper T cells can secrete a variety of cytokines including IFNγ, IL2, IL4 and TNFα after recognizing tumor antigen epitopes, and activate CD8+ CTLs to recognize and kill tumor cells. Currently, finding suitable tumor cell-specific targets, that is, tumor antigens, and expanding im...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/50A61K39/00A61P35/00
CPCA61K39/0011G01N33/505G01N33/53A61K2300/00
Inventor 韩研妍梁小玲陈锡和马民骏唐龙清周向军
Owner 恒瑞源正(上海)生物科技有限公司
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