Cell-based therapies have emerged as a promising treatment modality for diseases such as cancer and autoimmunity. T cells engineered with synthetic receptors known as chimeric antigen receptors (CARs) have proven effective in eliminating chemotherapy-resistant forms of B cell cancers. Such CAR T cells recognize antigens on the surface of tumor cells and eliminate them. However, CAR T cells also have adverse effects, including life-threatening inflammatory side effects associated with their potent immune activity. Risks for severe toxicity present a key challenge to the effective administration of such cell-based therapies on a routine basis.

Concerns about the potential for severe toxicity of cellular therapeutics primarily stem from a lack of precise control over the activity of the therapeutic cells once they are infused into patients. Exogenously imposed specific regulation over the location, duration, and intensity of the therapeutic activities of engineered cells would therefore be desirable. One way to achieve the intended control is to use small molecules to gate cellular functions. Small molecules with desired pharmacologic properties could be systemically or locally administered at varying dosages to achieve refined temporal and spatial control over engineered therapeutic cells.

We developed an ON-switch CAR that enables small molecule–dependent, titratable, and reversible control over CAR T cell activity. ON-switch CAR T cells required not only a cognate antigen but also a priming small molecule to activate their therapeutic functions. Depending on the amount of small molecule present, ON-switch CAR T cells exhibited titratable therapeutic activity, from undetectable to as strong as that of conventional CAR T cells. The ON-switch CAR was constructed by splitting key signaling and recognition modules into distinct polypeptides appended to small molecule–dependent heterodimerizing domains. The ON-switch CAR design is modular; different antigen recognition domains and small-molecule dimerizing modules can be swapped in.

The ON-switch CAR exemplifies a simple and effective strategy to integrate cell-autonomous decision-making (e.g., detection of disease signals) with exogenous, reversible user control. The rearrangement and splitting of key modular components provides a simple strategy for achieving integrated multi-input regulation. This work also highlights the importance of developing optimized bio-inert, orthogonal control agents such as small molecules and light, together with their cellular cognate response components, in order to advance precision-controlled cellular therapeutics.

A conventional CAR design activates T cells upon target cell engagement but can yield severe toxicity due to excessive immune response. The ON-switch CAR design, which has a split architecture, requires a priming small molecule, in addition to the cognate antigen, to trigger therapeutic functions. The magnitude of responses such as target cell killing can be titrated by varying the dosage of small molecule to mitigate toxicity. scFv, single-chain variable fragment; ITAM, immunoreceptor tyrosine-based activation motif.