Description

A comparison of T cell sensitivity using TCR and CAR constructs with the same pepMHC binding domain

Daniel Harris¹, Sheena Smith¹, Jennifer Stone¹, Qi Cai¹, Philip Kruger², Melissa Lever², Thomas Schmitt³, Philip Greenberg³, Omer Dushek², David Kranz¹

¹Department of Biochemistry, University of Illinois, Urbana-Champaign, Illinois, USA ²Department of Pathology, University of Oxford, Oxford, UK ³Fred Hutchinson Cancer Center, Seattle, Washington, USA

T cell receptors (TCRs) and chimeric antigen receptors (CARs) have been used successfully to redirect T cells to cancer antigens. Comparisons between the two receptors have been challenging due to differences in target antigens and affinities. Using yeast-display, we engineered single-chain, high-affinity TCRs (in scFv format) against WT1/HLA-A2 and MART1/HLA-A2 as models to compare the sensitivity of TCRs and CARs. The variable domains were expressed both as full-length αβ heterodimer TCRs as well as CAR constructs. These two formats allowed us to compare the mechanistic properties of TCRs and CARs, using receptors that shared the same affinity for the same ligand, WT1/HLA-A2 or MART1/HLA-A2. We observed that despite the CAR being expressed at 10-fold higher levels on the surface of T cells, the full-length TCR endowed T cells with nearly 100-fold greater sensitivity for the HLA-A2 complexes. A mathematical model for receptor triggering was applied, taking into account the observed TCR and CAR surface levels and sensitivities. This model suggests that the differences in sensitivities are likely due to more efficient signaling kinetics initiated by TCRs as compared to CARs. To compare the quantitative responses, we also measured a panel of cytokines released from activated CD8 and CD4 T cells transduced with the TCR and CAR at different antigen densities.