Figure 4.

Synthetic biology approach to creating chimeric antigen receptor (CAR) “AND” gates. (A) CARs are composed of distinct combinatorial units, specifically: individual antigen-binding motifs [scFv specific for anaplastic lymphoma kinase (ALK) or GFRA3], linking domains (CD8 linker and TM) and intracellular signaling domains (illustrated are components of a second-generation CAR derived from CD137 and CD3-zeta, and an incomplete intracellular signaling domain composed of only CD28 transmembrane and signaling sequences). In addition to scFv, ligands for overexpressed receptors on the tumor surface such as Artemin, the ligand for GFRA3, can be used in CAR construction. (B) Two antigens on a tumor cell could be targeted by expressing two complete and independent CARs on the T cell surface, creating a logical “OR” gate for antitumor targeting. Either construct can fully activate the CAR-expressing lymphocyte (CAR-T) expressing it. (C) CAR components can also be used to split activation signals and thus require two binding interactions to fully activate the CAR-expressing lymphocyte, creating a logical “AND” gate. Here, the scFv specific for ALK expresses only one intracellular signaling domain. Complete activation of the T cell requires the binding of a second CAR. The second portion of the “AND” gate may be contributed by either an scFv specific for GFRA3 or the natural ligand of the tumor-expressed receptor, artemin (ARTN).