Hello everyone, I plan to begin a project where I want to analyze T-cells transduced with a TCR specific for HER2 after co-incubation with macrophages that present a HER2-derived peptide. After searching for a predesigned vector with a HER2-specific TCR, I have found several databases that show CDR3 regions that are specific for HER2. Can I can clone the cDNA that encodes a HER2-specific CDR3 into a TCR alpha chain and beta chain framework specific for the HLA-A2 MHC haplotype and then clone this into a lentivirus-producing plasmid for T-cell transduction? If not, what is the best way to conduct a TCR transfer short of isolating HER2-specific T cells and sequencing the TCR?
You can reconstruct any TCR clonotypes you find in databases using commercial gene synthesis pretty easily, but I would recommend you reconstitute the whole VDJ sequence of the clonotypes-of-interest instead of transplanting only the CDR3 portion into the V and J genes of another clonotype. The resulting antigen-specificity would be unpredictable and the TCR may not even actually react to your HER2 peptide anymore.
Most literature/repository sources should include the associated V and J gene usage with any CDR3 you find. You can put together the whole chains by taking the reference V/J gene sequences from the IMGT database and inserting the CDR3 in between as appropriate.
Hey,
1-You should first check if your TCR is really specific for HER-2 in TCR free cells as TG40 cell line that once you transduced with lentivirus-TCR-HER-2 and in presence of HER-2 coculture with solenocytes irradiated you should see an increase in IL-2 secretion by ELISA. Then you can pass to transduction with T cells where you have competition between present TCR and transduced TCR.
2-Regarding the sequence, you can simply add CDR3 sequence to TCR alfa and beta chain by synthetize it in a company then cloned in your vector of interest. But also you can forget about CDR3 and focus on TCR alfa and beta specificity for your HER-2 antigen that you should define. You can sequence TCR in company as:
https://www.creative-biolabs.com/magic-tcr-repertoire-sequencing-service.html?gclid=CjwKCAiA2O39BRBjEiwApB2IkrMnq2EjlwTUjeUf1VjzqPY_xWZiu6u-3CW5hvY6KjPFXQe7DB4zJhoCK5MQAvD_BwE
Good luck
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