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

The efficacy of CAR 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 (GBM). To offset the PD-L1 inhibitory signal, we engineered PD-1 checkpoint reversal receptors (CPR) with a CD28 or 41BB co-stimulatory endodomain and co-expressed 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 co-stimulation (signal 2), fine-tuned pro-inflammatory cytokine release and sustained antitumor activity. While both CPR28 and CPR41BB effectively counteracted the PD-1 signal in vitro, CPR41BB, when co-expressed 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 4-1BB-containing second-generation CART, maintained the favorable metabolic parameters associated with 4-1BB co-stimulation, and demonstrated superior antitumor function after adoptive transfer in xenograft models of GBM and metastatic osteosarcoma. Thus, a CPR molecule with 4-1BB co-stimulation that curtails PD-1 inhibition and complements CAR signaling to optimize T-cell activation could enhance CART efficacy against solid tumors.