Dual- inhibitory domain iCARs improve the efficiency of the AND- NOT gate CAR T strategy

CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on- target, off -tumor toxicity, where CAR T cells also damage nor-mal tissues that express the targeted antigen. To reduce this detrimental side- effect, Boolean -logic gates like AND -NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2- targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5- targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to signif-icant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR -1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR -1 or SIGLEC-9 were combined with PD -1 into a single dual- inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND -NOT gate CAR T cell selectivity.