Molecular Approaches to Safe and Controlled Engineered T-cell Therapy

Abstract

Chimeric antigen receptor-modified T-cell therapy (CAR-T therapy) is one of the fastest developing areas of immuno-oncology. Over the past decade, it has revolutionized the cell therapy modality and expedited its pace of development, from optimization of the structure of chimeric antigen receptors and animal model experiments to successful clinical application. The initial designs of the CAR configuration focused on increasing T-cell activation, cytotoxicity, and persistence. However, the first attempts to treat patients with CAR T cells have demonstrated the need for increased safety and controlled activation of genetically modified T cells. Herein, we summarize the different molecular approaches to engineering chimeric antigen receptors for reducing the potential clinical risks of T-cell therapy.

Keywords: T-cells; cancer cells; cell therapy; chimeric antigen receptors.

Figure

The methods used to regulate CAR T cells. A – the general structure of CAR. B – elimination of CAR by exogenous molecules. In the right-hand side of the Figure, HSV-TK phosphorylates ganciclovir to ganciclovir monophosphate, which is further sequentially converted to the di- and triphosphate forms by cellular kinases. Ganciclovir triphosphate is incorporated into DNA at the elongation and replication stages, resulting in cell death. In the left-hand side of the Figure, the truncated variant of caspase-9 and the FK506 fragment are dimerized in the presence of rimiducid (AP1903) and induce the apoptotic cascade. The antigen targeting monoclonal antibodies, which is capable of eliminating CAR T cells, is added to the surface of CAR T cells or to the linker domain of CAR. D – iCAR interacts with the antigen present on healthy cells and inhibits the CAR function via the intracellular domain of PD-1 or CTLA-4. This inhibition is reversible, which allows T cells to function when they subsequently encounter a tumor cell. E – after the additional receptor (synNotch) interacts with one tumor antigen, transcription factors (TFs) induce expression of CAR, which recognizes the second antigen and induces cytotoxicity. F – CAR T cells are sufficiently activated only when two CARs interact with two different tumor antigens. G – modular CARs. The left-hand side of the Figure shows that the activation ability of CARs is restored only upon dimerization of the protein binding FK 506 (FKBP) with the T2089L mutant of FKBP-rapamycin (FRB*) via the exogenously inserted rapamycin analogue (AP21967). In the right-hand side of the Figure: CAR is activated only through an exogenous “mediator molecule.” H – modification of the extracellular domain of CAR by a masking peptide, which is cleaved in the tumor microenvironment, thus allowing CAR to bind to its antigen.