Computational analysis of 4-1BB-induced NFκB signaling suggests improvements to CAR cell design.

Background: Chimeric antigen receptor (CAR)-expressing cells are a powerful modality of adoptive cell therapy against cancer. The potency of signaling events initiated upon antigen binding depends on the costimulatory domain within the structure of the CAR. One such costimulatory domain is 4-1BB, which affects cellular response via the NFκB pathway. However, the quantitative aspects of 4-1BB-induced NFκB signaling are not fully understood.

Methods: We developed an ordinary differential equation-based mathematical model representing canonical NFκB signaling activated by CD19scFv-4-1BB. After a global sensitivity analysis on model parameters, we ran Monte Carlo simulations of cell population-wide variability in NFκB signaling and quantified the mutual information between the extracellular signal and different levels of the NFκB signal transduction pathway.

Results: In response to a wide range of antigen concentrations, the magnitude of the transient peak in NFκB nuclear concentration varies significantly, while the timing of this peak is relatively consistent. Global sensitivity analysis showed that the model is robust to variations in parameters, and thus, its quantitative predictions would remain applicable to a broad range of parameter values. The model predicts that overexpressing NEMO and disabling IKKβ deactivation can increase the mutual information between antigen levels and NFκB activation.

Conclusions: Our modeling predictions provide actionable insights to guide CAR development. Particularly, we propose specific manipulations to the NFκB signal transduction pathway that can fine-tune the response of CD19scFv-4-1BB cells to the antigen concentrations they are likely to encounter. Video Abstract.