A comparative study elucidating the substantial functional defects of electroporated T cells relative to a mechanical SQZ-based approach

Abstract

where the costimulatory signal is provided in trans on accessory proteins that associate with the antigen binding chain via transmembrane interactions. Methods: Several CD3f-containing CAR chains were designed using the transmembrane and cytoplasmic domains of NKG2D or NKp44, associating with DAP10 and DAP12 respectively. Each CAR contained a B7H6-targeting scFv and was co-expressed with corresponding accessory protein using a 2A site. Primary human T cells engineered with the diverse constructs were screened for CAR expression, phenotype and in vitro function. Results: NKG2D-based CAR complexes were moderately expressed at the cell surface but bound B7H6 and led to potent cells. Modification of the position of the charged residue within the transmembrane domain of the CAR is being used to modulate the surface expression and thus their potency. NKp44-based CAR complexes were more frequently expressed on primary T cells and bound B7H6, though functionality appears to be dependent on the nature of the extracellular spacer. Conclusions: These studies provide proof-of-concept for a novel CAR design where it is possible to incorporate or interchange costimulatory domain(s) in a stoichiometrically controlled way. Recapitulating physiological TCR activation by providing costimulation in trans within the CARpool may result in optimal downstream signaling, thereby enhancing anti-tumoral activity.Legal entity responsible for the study: Celyad SA.