Effect of the killing style of individual CAR T cells on the clinicopathological behavior of therapeutic products in the clinic

2548 Background: CAR T cell products that signal through CD28 and 41BB are highly effective against B lineage leukemia, despite exhibiting distinct efficacy and toxicity profiles in patients. Specifically, CD28 has been linked to higher upfront lytic activity and toxicity, while 41BB induces longer CAR T cell persistence and more durable progression free survival. We reasoned that the killing style of individual CAR T cells can explain the clinicopathological behavior of CAR T cell products in the clinic. Methods: We studied the cellular kinetics of CD28 and 41BB signaling CD19 CAR T cell products approved in the clinic, their single cell killing behavior in high throughput, the molecular composition of the lipid rafts inside the CAR immune synapse (CARIS) they form with their target cells, and their CARIS mechanical properties, then corroborated these findings in one kinetic model to explain their distinct clinicopathological behavior in the clinic. Results: We discovered that tumor encounter polarizes CD28 CAR T cell products to a predominance of more differentiated CD8 effectors that rely on short-lived highly lethal synapses with tumor cells. CD28 CAR T cell mediated synapses are characterized by fast mechanical buildup and resolution and brisk shuttling of CAR and other associated molecules into the CARIS and rapid polarization of the killing machinery in a manner that supports a mastery of serial killing mediated by perforin and granzymes. In contrast, 41BB responds to tumor stimulation by a T h2 -polarizing cytokine milieu that robustly expands a balanced CD4/CD8 population of effectors preserving a central memory phenotype. 41BB CAR T cells engage in lengthier synapses characterized by slower accumulation of CAR molecules in a mechanically tonic CARIS and rather collaborative yet lengthy Fas ligand-mediated killing process. Conclusions: We show here that CAR T cell signaling domains mediate distinct killing dynamics that can explain their behavior in patients. By understanding and potentially modulating these dynamics, the CAR T cell functionality and toxicity can be rationally optimized.