Kramer, AM;
(2017)
Delineating the impact of binding-domain affinity and kinetic properties on Chimeric Antigen Receptor T-cell function.
Doctoral thesis , UCL (University College London).
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Abstract
CD19 Chimeric Antigen Receptor (CAR) therapy represents a breakthrough in the treatment of relapsed/refractory acute lymphoblastic leukaemia and early-phase clinical trials with CAR-modified-T cells have shown unprecedented responses. A CAR is a recombinant receptor that combines a single chain variable fragment (scFv) against a tumour-associated antigen and an intracellular activation domain of the T cell receptor (TCR), recognizing membrane-bound antigen, typically with a higher affinity compared to TCR-pMHC affinity. We compared the influences of differences in on- and off-rates underlying the binding kinetics of CD19 CARs on downstream CAR T cell responses and constructed a novel CAR derived from the CAT hybridoma demonstrating a lower affinity as a result of a greater off-rate, whilst maintaining an on-rate nearly identical to FMC63, the scFv most-used in clinical trials. Using in vitro pre-clinical models we demonstrated that CAT-CAR+ T cells showed increased proliferation, higher cytotoxic responses, and increased cytokine production, as well as a greater number of interactions between CAT-CAR+ T cells and target cells and a greater motility in comparison to FMC63. In vivo CAT-CAR+ T cells showed an enhanced ability to clear disease, proliferate and produce cytokines, displaying markers characteristic for improved T cell fitness. We believe that, analogous to the natural TCR, a lower overall binding affinity might be mitigated by a relatively faster off-rate in the setting of a constant on-rate and propose that this may enhance CAR T cell function through serial triggering, where increased target:effector interaction time, may lead to exhaustion and activation induced cell death. These data have important implications for the development of future CARs.
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