A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function.

ABSTRACT: The efficacy of chimeric antigen receptor T cells (CART) in solid tumors is limited by immune inhibition. In our study, we observed that effector cytokines mediated the upregulation of the PD-L1 immune checkpoint in primary glioblastoma. To offset the PD-L1 inhibitory signal, we engineered PD-1 checkpoint reversal receptors (CPR) with a CD28 or 41BB costimulatory endodomain and coexpressed them with a first-generation or a CD28-containing second-generation HER2-specific CAR (CPR/CART) using bicistronic vectors. We found that bipartite T-cell activation, by CAR-generated signal 1 and CPR costimulation (signal 2), fine-tuned proinflammatory cytokine release and sustained antitumor activity. Whereas both CPR28 and CPR41BB effectively counteracted the PD-1 signal in vitro, CPR41BB, when coexpressed with a first-generation CAR (CARζ/CPR41BB), promoted central memory differentiation following repeat antigenic stimulation. CARζ/CPR41BB T cells formed a robust immune synapse with tumor targets, similar to a 41BB-containing second-generation CART, maintained the favorable metabolic parameters associated with 41BB costimulation, and demonstrated superior antitumor function after adoptive transfer in xenograft models of gioblastoma and metastatic osteosarcoma. Thus, a CPR molecule with 41BB costimulation that curtails PD-1 inhibition and complements CAR signaling to optimize T-cell activation could enhance CART efficacy against solid tumors. SIGNIFICANCE: Enhancing CART function and persistence while balancing immune effector-mediated inflammation is crucial. Using our clinically relevant HER2-CAR platform, we demonstrate that tumor-intrinsic signals like the PD-1/PD-L1 immune checkpoint can be leveraged in CART design to modulate immune synapse and metabolic parameters, improving antitumor function without increasing cytokine production.