DataSheet_1_Modifications outside CDR1, 2 and 3 of the TCR variable β domain increase TCR expression and antigen-specific function.pdf

T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy 30 against human malignancies and viral infections. Since the first human clinical trial was carried 31 out in 2006, several strategies have been developed to improve the efficacy and safety of TCR 32 engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs 33 whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how 34 modifications of framework residues in the TCR variable domains affect TCR expression and 35 function. We used bioinformatic and protein structural analyses to identify candidate amino 36 acid residues in the framework of the variable β domain predicted to drive high TCR surface 37 expression. Changes of these residues in poorly expressed TCRs resulted in improved surface 38 expression and boosted target cell specific killing by engineered T cells expressing the 39 modified TCRs. Overall, these results indicate that small changes in the framework of the TCR 40 variable domains can result in improved expression and functionality, while at the same time 41 reducing the risk of toxicity associated with TCR mis-pairing.