Background Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is a novel treatment option for patients with B-cell lineage derived cancers. Other cancer entities are less successfully targeted due to the lack of antigens that are expressed on cancer cells but not healthy tissue cells. One way to address this issue is the concept of inhibitory chimeric antigen receptors (iCARs) that deliver an inhibitory signal upon antigen encounter on off-target cells. The activating effect of CARs depends on a multitude of factors including expression levels, affinity, different signaling domains and steric effects. It has to be expected that co-expression of an iCAR would result in even more complexity. Consequently, there is a demand for a robust high-throughput cellular system to evaluate iCAR-formats.

Materials and Methods Our approach is based on a previously published Jurkat based triple parameter reporter cell (TPR) system. This setup allows for molecular monitoring of the T cell activation state by measuring fluorescent reporter gene expression via flow cytometry. Inhibitory effects of receptors are determined as the ratio of cellular geometric mean fluorescent intensity (gMFI) in the presence of the inhibitory ligand versus without inhibitory ligand.

Results To test if inhibitory receptors would measurably reduce reporter activation, PD-1 as a well-characterized inhibitory receptor was expressed on Jurkat TPRs. When PD-L1 was present during stimulation reporter activation was reduced by 26–34% proving feasibility of our approach. Intracellular domains of other inhibitory receptors including BTLA, ILT-2 and KIR2DL1 were evaluated. All three domains outperformed PD-1 in a series of experiments with a mean reduction of gMFI by 48–57%, 50–53% and 38–41% respectively. To assess if the reporter platform could be used to study downstream signaling pathways of inhibitory constructs we created a SHP-2 knock-out reporter cell line using the CRISPR/Cas9 gene editing technique. To simulate different degrees of activating signal strength, peptide-MHC complex recognizing activating receptors were created. The reporter activation correlated with the concentrations of peptide in the stimulation cultures. Co-expression of iCAR and CAR could be achieved using selection for two separate antibiotic resistance genes introduced into the respective vector. Preliminary experiments showed greatly reduced inhibitory efficacy of iCAR molecules due to an adhesion effect resulting from the high affinity extracellular domain of the iCARs that lead to tighter cell-cell contact and stronger stimulation through the CAR.

Conclusions We present a highly flexible and controllable Jurkat-based reporter cell platform for the thorough study of inhibitory signaling mechanisms. This project was supported by the Austrian Science Fund, FWF; Project P32411

Disclosure Information M.A. Funk: None. A. De Sousa Linhares: None. C. Battin: None. K. Radakovics: None. J. Leitner: None. P. Steinberger: None.