Chimeric antigen receptor- (CAR-) engineered T cells have emerged as a promising treatment for B cell lymphoma, but their success has not transferred to other cancer types. This is, in part, due to a fundamental lack of understanding of the mechanistic signaling events initiated by the CAR intracellular domains. Traditional CARs consist of intracellular signaling domains derived from CD3ζ, the main activating domain in the endogenous T cell receptor, and a co-stimulatory domain, such as CD28. The immuno-tyrosine activating motifs (ITAMs) on CD3ζ are able to induce T cell activation on their own, while CD28 and other co-stimulatory signaling domains augment particular aspects of the response. In this study, we use computational modeling combined with experimental studies to investigate the site-specific kinetics of CD3ζ and CD28 phosphorylation. We apply the model to develop new hypotheses for how to modulate the phosphorylation sites to enable optimal CAR-mediated T cell activation.<br/> <br/>We constructed a model that predicts T cell signaling mediated by CARs containing CD3ζ with and without the CD28 co-stimulatory domain. To account for the many species that emerge from phosphorylation of the six tyrosine sites on CD3ζ and four on CD28, we constructed the model in BioNetGen, a rule-based formalism that allows us to account for the many species that arise due to the complexity of the multiple phosphorylation sites on the CARs. The model is implemented as a set of ordinary differential equations in MATLAB, and the kinetic parameters are estimated by fitting the model to experimental data. We used phospho-proteomic mass spectrometry to measure the level of phosphorylation at each individual tyrosine residue over time.<br/> <br/>Our model provides new insight into how the structure of the CAR intracellular signaling domains influences the rate of phosphorylation and how to control the overall level of CD3ζ phosphorylation in the system. The model predicts that the ability of a CD3ζ ITAM to propagate signal downstream is not controlled by how quickly the two sites get phosphorylated, but by the difference between the catalytic efficiency of LCK at each of the sites. We are currently testing these hypotheses experimentally. These insights can be used to engineer the CD3ζ chain to control the extent of T cell activation and improve CAR therapies. Thus, our model provides a quantitative framework for designing optimal CAR activation of T cells for immunotherapy.

Kinetic analysis of phosphorylation of chimeric antigen receptors: Insight for T cell immunotherapy

255th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.