T cells engineered to express artificial chimeric antigen receptors (CARs) that selectively target tumor-specific antigens or deleterious cell types offer transformative therapeutic possibilities. CARs contain an N-terminal extracellular antigen recognition domain, C-terminal intracellular signal transduction domains, and connecting hinge and transmembrane regions, each of which have been varied to optimize targeting and minimize toxicity. We find that a CD22-targeting CAR harboring a CD8α hinge (H) exhibits greater cytotoxicity against a low antigen density CD22+ leukemia as compared to an equivalent CAR with a CD28 H. We therefore studied the biophysical and dynamic properties of the CD8α H by nuclear magnetic resonance (NMR) spectroscopy. We find that a large region of the CD8α H undergoes dynamic chemical exchange between distinct and observable states. This exchanging region contains proline residues dispersed throughout the sequence that undergo cis-trans isomerization. Up to four signals of differing intensity are observed, with the most abundantly populated being intrinsically disordered and with all prolines in the trans isomerization state. The lesser populated states all contain cis prolines and evidence of local structural motifs. Altogether, our data suggest that the CD8α H lacks long-range structural order but has local structural motifs that transiently exchange with a dominant disordered state. We propose that structural plasticity and local structural motifs promoted by cis proline states within the CD8α H are important for relaying and amplifying antigen-binding effects to the transmembrane and signal transduction domains.
Keywords: CAR T-cell; CD8; Intrinsically disordered proteins; Linker; Proline isomerization.
Published by Elsevier Inc.