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.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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.
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com