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A method for screening tumor-specific TCRs

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

Active Publication Date: 2022-04-08
BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A method for screening tumor-specific TCRs
  • A method for screening tumor-specific TCRs
  • A method for screening tumor-specific TCRs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 TILs single cell transcriptome and TCR group sequencing

[0032] TILs culture

[0033] After the tumor tissue of the tumor patient is surgically removed, the tumor tissue is chopped into small pieces of 1-2 mm, and each small piece of tumor tissue is placed in a well of a 24-well cell culture plate, and then T cell culture medium is added. T cell medium contains 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), 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 culture incubator until final TILs were obtained.

[0034] Co-incubation with corresponding tumor cells

[0035] TILs and corresponding autologous tumor cells were co-incubated in X-VIVO15 serum-free medium for 12 hours, then washed with PBS, according to 10Xgenomic requirements, cell size: ≤30 μm; cell viabili...

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 applicant synthesized the above-mentioned 9 non-repetitive TCRs respectively, and in each TCR core Add XbaI and SalI restriction sites to both ends of the nucleotide sequence, and clone into the pUC57 vector;

[0058] (2) Digest the pUC57 vector containing the target gene with XbaI and SalI, cut the gel and recover the target gene fragment;

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

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

Embodiment 3

[0061] Example 3 Preparation of TCR lentivirus

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

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Abstract

The invention relates to a method for screening tumor-specific TCR, which belongs to the technical field of tumor immunity. In the present invention, after co-incubating tumor cells of tumor patients and tumor infiltrating T cells (TILs) corresponding to autologous tumor cells in vitro, these cells are subjected to single-cell transcriptome and TCR group sequencing to obtain the TCR sequence of each TILs cell, for For all TILs cells expressing the same TCR, the average value of the expression values ​​of the 10 T cell markers was used as the activation score of the TCR marker. According to the activation score, the one with the higher score was selected as the TCR corresponding to the tumor-specific T cell.

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 the cell surface. TCR is a receptor molecule on the surface of T cells, which specifically recognizes the antigen peptide-MHC complex on the antigen-presenting cell, and then Stimulate T cell immune response. Since the TCR on the surface of most T cells cannot recognize tumor cells, T cells cannot effectively kill tumor cells, resulting in the rapid expansion of tumor cells. If T cells that specifically recognize tumor cells are found, the corresponding TCR is cloned, and the TCR is introduced 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 abili...

Claims

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Application Information

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Patent Type & Authority Patents(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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