Inhibitory CARs fail to protect from immediate T cell cytotoxicity.

Chimeric antigen receptors (CARs) equipped with an inhibitory signaling domain (iCARs) have been proposed as strategy to increase on-tumor specificity of CAR-T cell therapies. iCARs inhibit T cell activation upon antigen recognition and thereby program a Boolean NOT gate within the CAR-T cell. If cancer cells do not express the iCAR target antigen while it is highly expressed on healthy tissue, CAR/iCAR coexpressing T cells are supposed to kill cancer cells but not healthy cells expressing the CAR antigen. In this study, we employed a well-established reporter cell system to demonstrate high potency of iCAR constructs harboring BTLA-derived signaling domains. We then created CAR/iCAR combinations for the clinically relevant antigen pairs B7-H3/CD45 and CD123/CD19 and show potent reporter cell suppression by iCARs targeting CD45 or CD19. In primary human T cells αCD19-iCARs were capable of suppressing T cell proliferation and cytokine production. Surprisingly, the iCAR failed to veto immediate CAR-mediated cytotoxicity. Likewise, T cells overexpressing PD-1 or BTLA did not show impaired cytotoxicity toward ligand-expressing target cells, indicating that inhibitory signaling by these receptors does not mediate protection against cytotoxicity by CAR-T cells. Future approaches employing iCAR-equipped CAR-T cells for cancer therapy should therefore monitor off-tumor reactivity and potential CAR/iCAR-T cell dysfunction.

Interrogating ligand-receptor interactions using highly sensitive cellular biosensors.

Interactions of membrane-resident proteins are important targets for therapeutic interventions but most methods to study them are either costly, laborious or fail to reflect the physiologic interaction of membrane resident proteins in trans. Here we describe highly sensitive cellular biosensors as a tool to study receptor-ligand pairs. They consist of fluorescent reporter cells that express chimeric receptors harboring ectodomains of cell surface molecules and intracellular signaling domains. We show that a broad range of molecules can be integrated into this platform and we demonstrate its applicability to highly relevant research areas, including the characterization of immune checkpoints and the probing of cells for the presence of receptors or ligands. The platform is suitable to evaluate the interactions of viral proteins with host receptors and to test for neutralization capability of drugs or biological samples. Our results indicate that cellular biosensors have broad utility as a tool to study protein-interactions.