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Method for screening tumor specific TCR

A TCR-T, tumor technology, applied in the field of tumor immunity, can solve the problems of large differences in expression levels, missing tumor-specific T cells, false negatives, etc.

Active Publication Date: 2022-03-04
BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, different types of T cells (such as naive T cells, effector T cells, and memory T cells) and different times after antigen stimulation will affect the expression of T cell markers, that is, the expression levels of different markers vary greatly, so using a single Marker screening of tumor-specific T cells may miss some tumor-specific T cells that do not express a certain marker, resulting in false negatives

Method used

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  • Method for screening tumor specific TCR
  • Method for screening tumor specific TCR
  • Method for screening tumor specific TCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Sequencing of single-cell transcriptome and TCR group of TILs

[0032] Culture of TILs

[0033] After the tumor tissue of tumor patients was surgically removed, the tumor tissue was minced into small pieces of 1-2 mm, and each small piece of tumor tissue was placed in one well of a 24-well cell culture plate, and then T cell culture medium was added. T cell culture medium containing X-VIVO 15 serum-free medium (Lonza, USA) and IL2 (50U / ml; Peprotech, USA), IL-7 (10 ng / mL; Peprotech, USA), IL-15 (10 ng / ml; Peprotech, USA) / mL, Peprotech, USA), OKT3 antibody (50ng / ml; ACRO, USA) and anti-CD28 antibody (1ug / ml; T&L Biotechnology, China). The small pieces of tumor tissue were cultured in a cell incubator until final TILs were obtained.

[0034] Co-incubated with corresponding tumor cells

[0035] TILs and corresponding autologous tumor cells were co-incubated in X-VIVO15 serum-free medium for 12 hours, and then washed with PBS. According to 10Xgenomic requireme...

Embodiment 2

[0056] Example 2 Construction of TCR-T lentiviral vector

[0057] (1) In Example 1, a total of 21 TCRs were found, but since the partial TCRs of TCR1-3 obtained by different test groups were the same, the above 9 non-repetitive TCRs were synthesized in the present application, and each TCR core was used in each TCR core. The two ends of the nucleotide sequence were added with XbaI and SalI restriction sites respectively, and cloned into the pUC57 vector;

[0058] (2) The pUC57 vector containing the target gene was digested with XbaI and SalI double enzymes, and the target gene fragment was recovered by cutting gel;

[0059] (3) The original vector pCDH-EF1-Luc2-T2A-tdTomato was digested with XbaI and SalI double enzymes, and the vector fragment of about 6.5kb was recovered by cutting gel;

[0060] (4) Use DNA ligase to connect the recovered target gene and vector fragment to obtain a recombinant lentiviral vector carrying each TCR.

Embodiment 3

[0061] Example 3 Preparation of TCR lentivirus

[0062] The 9 recombinant lentiviral vectors of Example 2 were transfected into 293ft cells by transfection reagent (PEI) to produce lentivirus. The specific method includes: packaging plasmid mixture (pMDL:VSV-G:REV=5:3:2, mass ratio) and TCR1 lentiviral vector in 500 μL serum-free medium Opti-MEM at a mass ratio of 1:1, vortex Swirl until well mixed. Add 32 g PEI to 500 μL of serum-free medium Opti-MEM and vortex to mix well. Then, 500ul of the plasmid mixture was mixed with 500ul of PEI, and it was added to 293ft cells with a confluence of about 90%. After 48 hours, the virus supernatant was collected, and after ultracentrifugation, the virus was concentrated 100 times to obtain the concentrated virus.

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Abstract

The invention relates to a method for screening tumor specific TCR, and belongs to the technical field of tumor immunity. According to the method, after tumor cells of a tumor patient and tumor infiltration T cells (TILs) corresponding to autologous tumor cells are co-incubated in vitro, the cells are subjected to single cell transcriptome and TCR group sequencing to obtain a TCR sequence of each TILs cell, and for all the TILs cells expressing the same TCR, the TCR sequence of each TILs cell is subjected to single cell transcriptome and TCR group sequencing to obtain the TCR sequence of each TILs cell. Taking the average value of the expression values of the 10 T cell markers as the activation score of the TCR marker, and screening the TCR marker with the higher score as the TCR of the corresponding tumor specific T cell according to the activation score.

Description

technical field [0001] The invention relates to the technical field of tumor immunity, in particular to a method for screening tumor-specific TCRs. Background technique [0002] T cells recognize the corresponding antigen through the T cell receptor (TCR) on their cell surface. TCR is a receptor molecule on the surface of T cells, which specifically recognizes the antigen peptide-MHC complex on antigen presenting cells, and then Stimulates T-cell immune responses. Since most TCRs on the surface of T cells cannot recognize tumor cells, T cells cannot effectively kill tumor cells, resulting in rapid expansion of tumor cells. If you find a T cell that specifically recognizes tumor cells, clone its corresponding TCR, and then introduce the TCR into T cells through gene editing (such as lentivirus), T cell receptor gene-modified T cells (TCR-T) will be generated. After the antigen-specific TCR is transferred into ordinary T cells, it can endow the T cells with the ability to re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6881C12N15/12C12N15/867C12N5/10A61K39/00A61P35/00G16B50/00
CPCC12Q1/6881C07K14/7051C12N15/86C12N5/0636A61K39/0011A61P35/00G16B50/00C12Q2600/158C12N2740/15043C12N2800/107C12N2510/00A61K2039/5158A61K2039/5156
Inventor 张超亭陆哲明
Owner BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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