277. Small Molecule-Regulated Antigen Recognition System for Inducible T Cell Targeting of Cancer Cells

Redirecting T cells against tumors by introducing chimeric antigen receptors (CAR) has demonstrated promising clinical results in certain cancers. However, the constitutive activity of these receptors limits which antigens can be targeted with this approach and may result in persistent elimination of healthy antigen-expressing cells. Successful CAR-T targeting of CD19+ tumors, for example, produces chronic B cell aplasia and necessitates life-long intravenous immunoglobulin (IVIG) treatment. Daric is an alternative antigen targeting approach that aims to: i) minimize the long-term toxicity of CAR-T treatment; ii) allows targeting of previously inaccessible antigens; and iii) is amenable to multiplex antigen targeting and other advanced targeting designs and strategies. The Daric system separates the antigen recognition and signaling functions of a CAR into two distinct polypeptides that contain the FKPB12 and FRB dimerization domains but lack signaling activity in the absence of a dimerization agent. Addition of the FKBP12-FRB bridging drug rapamycin or a non-immunosuppressive rapamycin analogue AP21967 heterodimerizes the signaling and antigen recognition components and restores signaling competency for antigen-dependent T cell activation. Importantly, the FKB12 and FRB dimerization partners are located extracellularly, minimizing interference with endogenously expressed signaling components and eliminating the requirement for rapamycin/AP21967 cell penetrance. A range of extracellular linkers and transmembrane domains were used to design a variety of CD19-targeting Daric constructs that exhibit minimal basal activity and robust drug-dependent antigen-specific cytolytic activity and cytokine production. The CD19-specific Daric T cells exhibited comparable levels of cytokine release, proliferation and cytolytic activity compared to a CD19-targeting CAR T cells. In vitro, Daric T cells were activated at sub-nanomolar to nanomolar concentrations of rapamycin or AP21967. No differences in cellular phenotype, expansion or functional responsiveness of Daric T cells compared to CAR T cells were observed. As expected, rapamycin was immunosuppressive to CAR T cell functionality, however Daric T cells continued to produce high level of cytokines even in the presence of rapamycin. These results highlight the potential of the Daric system to target highly expressed antigens and minimize the off-tumor on-target toxicity associated with traditional CAR designs.