Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies by redirecting patient T cells to destroy cancer cells using engineered receptors. However, CAR therapies carry significant risk of inducing cytokine release syndrome (CRS), a potentially deadly toxicity caused by excessive release of inflammatory cytokines. The ability to minimize CRS toxicity whilst guaranteeing adequate tumour cell-killing is therefore vital to the continued improvement of CAR therapies. There is a growing appreciation that CAR hinge and transmembrane (TM) domains influence CAR potency, however our understanding of the structure function relationship of these regions remains ill-defined. I aimed to investigate the functional relevance of a highly conserved motif within the CD28 TM sequence used in clinical CARs, with the aim of leveraging this knowledge to predictably modulate CAR activity.
Hypothesizing that the polar residues within this conserved motif (YSxxxT) may facilitate co-association of CARs with endogenous CD28, I disrupted this motif by generating a CAR construct possessing a CD28TM sequence with hydrophobic substitutions (FAxxxV). This mutant maintained robust surface expression in primary mouse T cells, however significantly reduced activation-induced co-association of endogenous CD28 and CARs as visualised by fluorescence microscopy. Furthermore, these mutations resulted in a dramatic reduction in CAR T cell cytokine secretion in response to target cells, despite demonstrating no significant impact on CAR T cell cytotoxicity.
These findings demonstrate a previously unappreciated role for the CD28 TM in CAR effector function and highlight the importance of systematically investigating the functional contributions of CAR framework regions. The mutations described in this work may represent a simple means of reducing CAR T cell cytokine secretion without sacrificing cytotoxicity in any CAR construct possessing the CD28 TM sequence. In vivo experiments are currently underway to validate tumour control and T cell persistence in mouse models of disease.