CAR-T design: Elements and their synergistic function

Abstract

Chimeric antigen receptor (CAR) T cells use re-engineered cell surface receptors to specifically bind to and lyse oncogenic cells. Two clinically approved CAR-T-cell therapies have significant clinical efficacy in treating CD19-positive B cell cancers. With widespread interest to deploy this immunotherapy to other cancers, there has been great research activity to design new CAR structures to increase the range of targeted cancers and anti-tumor efficacy. However, several obstacles must be addressed before CAR-T-cell therapies can be more widely deployed. These include limiting the frequency of lethal cytokine storms, enhancing T-cell persistence and signaling, and improving target antigen specificity. We provide a comprehensive review of recent research on CAR design and systematically evaluate design aspects of the four major modules of CAR structure: the ligand-binding, spacer, transmembrane, and cytoplasmic domains, elucidating design strategies and principles to guide future immunotherapeutic discovery.

Keywords: CAR-T; Cancer; Cell engineering; Chimeric antigen receptor; Immunotherapy; Synthetic biology.

Fig. 1

Design parameters of each module of the CAR tested in literature.

Fig. 2

scFv properties such as affinity, avidity, aggregation propensity, and its antigen epitope location are critical parameters that can affect CAR function. (a) scFv affinity and avidity can be modulated to improve selective recognition of target cells bearing higher ligand density, thus reducing on-target off-tumor effects. (b) CAR surface aggregation can cause VH-VL mispairing, which can occur at high expression levels or with sub-optimal linker design that limits stabilizing inter-domain interactions. (c) Location of epitope targeted by scFv dictates synaptic cleft distances, which are important for kinetic segregation of phosphatases like CD45.

Fig. 3

Spacer design can be used to modulate synaptic cleft distances, allow flexibility and dimerization and reduce non-specific innate immune responses. (a) Spacer length can be modulated to control synaptic cleft distances, which can possibly regulate signaling. When targeting membrane distal epitopes, short spacers (left) shorten the synaptic cleft, enabling exclusion of phosphatases such as CD45 and hence enhancing phosphorylation of cytoplasmic ITAMs while long spacers (right) lengthen the synaptic cleft and possibly do not exclude phosphatases. (b) Flexible spacers can enable access to sterically hindered epitopes. (c) Dimerization of spacer domains results in increased signal strength and activation stimulus. (d) FcγR interactions arising from IgG based spacers results in activation of innate immune system and decreased efficiency.

Fig. 4

Transmembrane domain interactions can afford novel CAR designs. (a) Association of CAR transmembrane domain with endogenous receptors/endogenous transmembrane domains. (b) Multichain transmembrane association for split CAR systems. (c) Multichain transmembrane associations to create in trans co-stimulatory domain signaling.

Fig. 5

Number, type and order of co-stimulatory domains as well as ITAM multiplicity can affect CAR-T functionality. (a) Based on the number of co-stimulatory domains used, CARs are classified as first-generation (no co-stimulatory domain), second-generation (one co-stimulatory domain), or third-generation (two co-stimulatory domains). (b) The order of the co-stimulatory domains can possibly dictate structural compatibility with the transmembrane region and influence conformation of the CAR. It can also affect accessibility of membrane proximal kinases that are critical for signaling. (c) The number of ITAMs on CD3ζ can be modulated to alter effector functions. Mutations in signaling residues in co-stimulatory domains can also be used to regulate effector functions.