A Bidirectional EF1 Promoter System for Armoring CD19 CAR-T Cells with Secreted Anti-PD1 Antibodies

Chimeric antigen receptor (CAR) T cell therapy for B cell malignancies is often limited by T cell exhaustion, which is frequently driven by the PD-1/PD-L1 immune checkpoint axis. To overcome this, we developed an "armored" CAR-T cell strategy using a novel bidirectional promoter system. We engineered a single vector to co-express a CD19-specific CAR alongside a secreted anti-PD1 molecule, in either a full-length antibody or a single-chain variable fragment (scFv) format, using the Sleeping Beauty (SB) transposon system. The sequences for the anti-PD1 modules were derived from the clinical antibody nivolumab. Both armored constructs demonstrated robust CAR expression, comparable to or higher than conventional CAR-T cells, and proliferated significantly more than untransfected controls. The engineered cells successfully secreted their anti-PD1 payloads, with the full-length antibody showing more sustained secretion than the scFv. This autocrine blockade resulted in significantly reduced surface PD1 expression on the armored CAR-T cells. Functionally, the anti-PD1-secreting cells exhibited superior cytotoxicity against PD-L1-positive Raji target cells, particularly at low effector-to-target ratios. Critically, in a serial rechallenge assay designed to simulate chronic antigen exposure, both armored CAR-T cell groups showed markedly enhanced proliferation and persistence compared to conventional CAR-T cells, which failed to expand after repeated stimulation. Our findings validate the bidirectional EF1 promoter as an efficient system for generating multi-functional T cells and demonstrate that armoring CAR-T cells with secreted anti-PD1 antibodies is a potent strategy to enhance their persistence and anti-tumor efficacy.