Building Better Chimeric Antigen Receptors for Adoptive T Cell Therapy

Abstract

The last few years have seen the transfer of two decades of research into Chimeric Antigen Receptors (CARs) into clinical trials. Despite this extensive research, there is still a great deal of debate into the optimal design strategy for these, primarily, anti-cancer entities. The archetypal CAR consists of a single-chain antibody fragment, specific to a tumour-associated antigen, fused to a component of the T-cell receptor complex (typically CD3zeta) which on antigen binding primes the engrafted T-cell for anti-tumour activity. The modular nature of these artificial receptors has enabled researchers to modify aspects of their structure, including the extracellular spacer, transmembrane and cytoplasmic domain, to achieve laboratory defined optimal activity. Despite this there is no consensus on the optimal structure, a problem exacerbated by conflicting results using identical receptors. In this review, we provide a structural overview of CAR development and highlight areas that require further refinement. We also attempt to identify possible reasons for conflicting results in the hope that this information will inspire future rational design strategies for optimal tumour targeting using CARs.

Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies

“…To generate switch molecules specific for the CD19 antigen, the PNE was introduced at defined sites in the anti-CD19 antibody FMC63 (22). This antibody clone is used in the most widely studied conventional CART-19 construct in clinical trials and thus provides a direct comparison for the sCAR-T-cell system (23). By varying the stoichiometry and site of PNE engraftment in FMC63, we could systematically vary the valency and orientation of the 50 of cytotoxicity.…”

CARs on the Highway: Chimeric Antigen Receptor Modified T Cells for the Adoptive Cell Therapy of Malignant Diseases

“…Targeting of some antigens requires a spacer region between the antigen recognition and signaling domains implying some impact of the antigen itself. The spacer is typically derived from the IgG1 or IgG4 constant domain; other spacers such as CD4 or CD8 are also used [2,12]. The spacer length can be varied by using different moieties, for example, IgG1 CH1-CH2-CH3 vs CH2-CH3 vs CH3; for each antigen, there seems to be an optimal CAR design providing the best suitable distance between the interacting CAR T cell and target cell [13].…”

“…CARs typically have a membrane spanning region consisting of 20-23 hydrophobic amino acids, rich in leucines, isoleucines, and valines; a variety of membrane spanning receptor domains have been used in CAR design, including those of CD3ζ, CD4, CD8, CD28, or OX40 [2]. The choice for a transmembrane region is so far empiric, and some evidences imply that CARs with CD3ζ transmembrane domain incorporate into the endogenous TCR/CD3 complex and may be more robust in expression and signaling than others [15].…”

Ligation of the CD2 co-stimulatory receptor enhances IL-2 production from first-generation chimeric antigen receptor T cells

“…8,26 Lentivirus was produced by transient transfection of 293T cells in 150 mm 3 dishes by calcium chloride precipitation. 27 Vectors were added in the ratio 20 mg pSicoR, 13 mg pCMVDR8.91, 7 mg pMD2.g.…”

“…2 In their simplest form, CARs consist of an scFv (single-chain variable fragment) linked to a T-cell-signalling molecule such as CD3z. 3 Upon antigen ligation, these first-generation CARs drive 'signal one'-type signalling resulting in interferon gamma (IFNg) production and target-cell cytotoxicity. However, signal one alone is thought to be sub-optimal as further co-stimulatory signals are required to drive full T-cell activation, survival and proliferation.…”