Modular delivery of co-stimulatory signals through a PD-1-based immunoswitch receptor improves the functionality of...

In chronic hepatitis B virus infection (cHBV), T-cell responses are skewed. This prevents the elimination of HBV-infected hepatocytes, contributes to hepatocellular carcinoma development, and limits the efficacy of T-cell therapies. To counteract the immune checkpoint PD-1/PD-L1-interaction in adoptive T-cell therapy, we developed PD-1-based immunoswitch receptors that convert PD-L1 engagement into co-stimulation. We designed immunoswitch receptors linking a PD-1 ectodomain to intracellular CD28, 4-1BB, or OX40 signaling domains and expressed them in HBV-specific chimeric antigen receptor (CAR)- and T-cell receptor-engineered T cells. T-cell activity was assessed in antigen- and PD-L1-dependent co-cultures, transcription factor reporter systems, and an HBV carrier mouse model. Second-generation S-CAR functionality was not improved by signal stacking of additional co-stimulatory domains, resulting in a dysfunctional activation state rather than an additive benefit. In contrast, modular delivery of co-stimulation through PD-1-based immunoswitch receptors enhanced antigen sensitivity, functionality, and cytotoxicity of engineered T cells. PD-L1 supplied in trans was sufficient to support on-target functionality of engineered T cells by immunoswitch receptor co-stimulation. PD-1_4-1BB proved superior at inducing sustained NF-κB signaling and reducing exhaustion-associated pathways. In vivo, PD-1_4-1BB improved T-cell persistence and decreased TOX upregulation. PD-1-based immunoswitch receptors offer a modular strategy to strengthen engineered T-cell responses against HBV antigen-expressing cells in the liver's tolerizing environment. This ligand-driven approach enables precise tuning of co-stimulation, generating a versatile platform for enhancing adoptive T-cell therapies in chronic infections and cancer.