Mechanistic model of chimeric antigen receptor T cell activation

Chimeric antigen receptors (CARs) are engineered receptors that mediate T cell activation. To do so, CARs are comprised of a variety of different activating and co- stimulatory domains derived from endogenous T cells. These intracellular domains initiate signaling required for T cell activation, including ERK activation through the MAPK pathway. The mechanisms by which co-stimulatory domains on CARs influence this signaling are not clear. To better understand how CAR signaling differs from T cell receptor (TCR) signaling and how the individual CAR signaling domains CD3ζ and CD28 influence the downstream activation, I have used a bottom-up modeling approach to build a framework that will allow for the exploration of the specific mechanisms of CAR T cell activation. In chapter one, I develop a computational model to better understand how the main T cell activating kinase, LCK, is autophosphoryated at its inhibitory and activating tyrosine sites. This model, trained using in vitro experimental data of LCK autophosphorylation and phosphorylation by the kinase CSK, identifies key mechanisms required for T cell regulation. In chapter two, I focused on the interaction between LCK and the CD3ζ and CD28 intracellular signaling domains of the CAR. I first collect quantitative phospho-proteomic mass spectrometry measurements to measure the phosphorylation kinetics of different CAR structures. I fit this data using a computational model of CD3ζ phosphorylation by LCK. Testing how different model structures compare to the data, I am able to confirm that these sites are phosphorylated randomly with different rates. The model also predicts that the CD3ζ sites are phosphorylated independently, which I confirm experimentally. When CD28 is added upstream of CD3ζ, the experimental data shows that the phosphorylation rates of all CD3ζ ITAM sites are increased, and the order of the site phosphorylation is altered. In light of this, I use this computational model to explain a mechanism for the appearance of differentially phosphorylated forms of CD3ζ in resting T cells and determine that protein binding to singly phosphorylated ITAM sites plays an important role in influencing the overall phosphorylation of these sites. Finally, in chapter three, I combine the LCK autoregulation and CD3ζ/CD28 phosphorylation models with downstream mechanisms from several other models in the literature to predict how CARs with or without the CD28 co-stimulatory domain affect downstream ERK activation. I use an ensemble modeling approach to explore the specific mechanisms of CD28 that contribute to ERK activation in anti-CD19 CD28-CD3ζ CAR- bearing Jurkat T cells. The model produces several hypotheses for how ERK response time could be affected by various binding events and kinetic changes initiated by CD28 co- stimulation. I then perform experiments to validate the main CD28 mechanism of activation in CAR T cells. Thus, the model provides new mechanistic insights into the functions of T cell co-stimulatory domains. Altogether, my model provides a framework with which to study CAR engineered T cell activation. In the future, the model can be further expanded to study other co- stimulatory domains, like 41-BB, and inhibitory domains, like PD-1. I envision this model as a tool to help understand and optimize CAR T cell activation to improve future CAR therapies.