Engineering the binding properties of the T cell receptor:peptide:MHC ternary complex that governs T cell activity

The potency of a T cell is determined in large part by two interactions, binding of a cognate peptide to the MHC, and binding of the T cell receptor (TCR) to this pepMHC. Various studies have attempted to assess the relative importance of these interactions, and to correlate the corresponding binding parameters with the level of T cell activity mediated by the peptide. To further examine the properties that govern optimal T cell activity, here we engineered both the peptide:MHC interaction and the TCR:pepMHC interaction to generate improved T cell activity. Using a system involving the 2C TCR and its allogeneic pepMHC ligand, QL9-L^d, we show that a peptide substitution of QL9 (F5R), increased the affinity and stability of the pep-L^d complex (e.g. cell surface t_1/2-values of 13 min for QL9-L^d versus 87 min for F5R-L^d). However, activity of peptide F5R for 2C T cells was not enhanced because the 2C TCR bound with very low affinity to F5R-L^d compared to QL9-L^d (K_D = 300 μM and K_D = 1.6 μM, respectively). To improve the affinity, yeast display of the 2C TCR was used to engineer two mutant TCRs that exhibited higher affinity for F5R-L^d (K_D = 1.2 and 6.3 μM). T cells that expressed these higher affinity TCRs were stimulated by F5R-L^d in the absence of CD8, and the highest affinity TCR exhibited enhanced activity for F5R compared to QL9. The results provide a guide to designing the explicit binding parameters that govern optimal T cell activities.