Engineering CAR-T cells: Design concepts

Abstract

Despite being empirically designed based on a simple understanding of TCR signaling, T cells engineered with chimeric antigen receptors (CARs) have been remarkably successful in treating patients with advanced refractory B cell malignancies. However, many challenges remain in improving the safety and efficacy of this therapy and extending it toward the treatment of epithelial cancers. Other aspects of TCR signaling beyond those directly provided by CD3ζ and CD28 phosphorylation strongly influence a T cell's ability to differentiate and acquire full effector functions. Here, we discuss how the principles of TCR recognition, including spatial constraints, Kon/Koff rates, and synapse formation, along with in-depth analysis of CAR signaling might be applied to develop safer and more effective synthetic tumor targeting receptors.

Figure 1. Signaling of conventional and CAR T cells

Left: Delivery of signals 1 and 2 to conventional T cells is initiated by the TCR interacting with pMHC on an APC. The spatial distance between the T cell and the APC is ~15nm, which physically excludes from the synapse the inhibitory receptor CD45 because of its large ectodomain. Interaction of CD4/CD8 co-receptors with MHC recruits Lck to the TCR complex, where it phosphorylates and activates Zap70, which provides signal 1. Ligation of the co-stimulatory receptor CD28 by CD80/CD86 results in PI3K activation and delivers signal 2 for full T cell activation. Right: Single receptor design showing a second-generation CAR containing CD3z and CD28 endodomains in cis. Activation of these CARs by a single tumor antigen is sufficient to deliver both signals 1 and 2 in cis, resulting in T cell activation. The spatial distance between CAR T cells and target tumor cells is not known, nor is it known whether this distance is small enough to physically exclude the phosphatase CD45 from the synapse. It is also unknown whether CARs interact with endogenous TCR/CD3z or CD4/CD8 co-receptors.

Figure 2. Split receptor designs to enhance tumor selective recognition

A. Enhancing tumor selectivity using chimeric costimulatory receptors. To reduce the potential for CAR-T cells to recognize normal tissues that may express the target antigen, dual CARs have been designed such that two molecules both of which are expressed on tumor cells must be engaged to deliver signals 1 and 2 and fully activate CAR-T cells. Normal cells that express only one of these antigens do not signal T cells sufficiently for full activation. The CAR that delivers signal 1 may need to be attenuated in signaling, such as by mutating one or more ITAMs in CD3ζ or by reducing the affinity of the scFv. B. Enhancing tumor selectivity using inhibitory chimeric receptors. In this scenario, a CAR that delivers a dominant inhibitory signal such as might be mediated via a PD1 endodomain for example, is co-expressed with a CAR capable of full T cell activation. Engagement of both target antigens on normal cells would suppress T cell activation, whereas recognition of only the activating ligand on tumor cells would result in T cell activation and effector function.