Abstract / Summary
Adoptive cell therapy often shows limited efficacy against solid tumors as PD-1-expressing anti-tumor T-cells are suppressed by PD-L1 in the tumor microenvironment. To overcome this, T-cells have been engineered with PD-1 receptor-based CARs to convert PD-1/PD-L1 engagement into an activating signal. As PD-L1 is widely expressed on healthy cells, this strategy risks on-target, off-tumor T-cell activation. Monoclonal antibodies (mAbs) that block PD-1/PD-L1 interactions are widely used in clinical practice. Here, we developed a mutated PD-1 receptor-based CAR (PD-1 K78A ) that binds anti-PD-1 mAbs but not PD-L1, enabling controlled, antigen-independent activation. In co-culture assays, PD-1 K78A CARs showed no binding to PD-L1 + target cells, compared to wild-type PD-1 (PD-1 WT ) CAR model T-cells. mRNA-based CARs, with expression levels determined by their molecular design (with/without hinge), induced model T-cell activation upon soluble anti-PD-1 mAb administration. Lentivirally transduced CARs failed to trigger activation due to lower CAR-expression levels, likely limiting CAR cross-linking. In primary T-cells, both PD-1 mRNA CAR variants demonstrated enhanced cytotoxic responses when combined with tumor-targeting T cell receptors. These findings show that anti-PD-1 mAbs can drive antigen-independent activation of CAR T-cells when expressing high levels of a PD-1 K78A CAR alongside a tumor-targeting element, providing a foundation for a novel therapeutic approach.