Abstract NG01: Effects of altering receptor structure in CAR T cells: Predictions from an experimentally-validated systems biology model

Abstract

Abstract Background and motivation. As T cell immunotherapy applications have expanded, it has become increasingly important to better understand the signaling events that lead to T cell activation. We are particularly interested in chimeric antigen receptor- (CAR-) engineered T cells (CAR-T cells). CAR-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, which prevents us from being able to engineer an optimal CAR. Traditional CARs consist of intracellular signaling domains derived from CD3ζ, the main activating domain in the endogenous T cell receptor (TCR), and a co-stimulatory domain, such as CD28. The immuno-tyrosine activating motifs (ITAMs) on CD3ζ are able to induce T cell cytotoxicity on their own, while CD28 and other co-stimulatory signaling domains augment particular aspects of the response. It is known that phosphorylation of the six tyrosine sites on CD3ζ and four on CD28 by lymphocyte-specific protein tyrosine kinase (LCK) results in downstream signaling that leads to T cell activation. However, the specific mechanisms that control LCK's binding and catalytic activity are not well defined. These ten phosphorylation sites on the CAR contribute to different signaling events in the T cells, so understanding the mechanisms through which these sites are activated can inform the best strategies for engineering the next generations of CARs. To date, development of CAR constructs is largely achieved using a trial-and-error experimental approach, which has several disadvantages: it does not uncover why a particular CAR construct works; it does not necessarily identify the optimal design, just a particular design that works; and it does not generate insight that can be generalized to different cancer types. Additionally, this trial-and-error approach does not consider the intracellular signaling induced by the CAR, which ultimately governs T cell function and promotes cell killing. Our proposed research uses computational modeling to address these limitations. In this study, I describe our work to develop a predictive mechanistic computational model that describes the signaling events that occur upon CAR activation. This work builds on our published model of LCK autoregulation [1]. Here, we use quantitative phospho-proteomic mass spectroscopy and computational modeling to quantify the site-specific kinetics of CD3ζ and CD28 phosphorylation. We apply the model to improve our understanding of how CAR structure influences activation and to develop new hypotheses for the optimal design of CAR-engineered T cell systems. Citation Format: Jennifer A. Rohrs, Dongqing Zheng, Nicholas A. Graham, Pin Wang, Stacey D. Finley. Effects of altering receptor structure in CAR T cells: Predictions from an experimentally-validated systems biology model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr NG01.