A Drug-Regulated CAR Platform (DARIC) Induces Effective and Reversible Tumor Control In Vivo Using Non-Immunosuppressive Rapamycin Dosing

Redirecting T cells by introducing anti-CD19 antigen-specific chimeric antigen receptors (CARs) has shown promising clinical results as a potential treatment strategy for B cell malignancies. However, traditional CARs are constitutively active, resulting in two potential liabilities - first, long term-toxicity due to loss of all target cells including normal tissues/cells that express the target antigen (off-tumor, on-target activity) and second, acute T cell activation associated with cytokine release syndrome (CRS). Furthermore, the potential for persistent T cell activation caused by constant exposure to normal-tissue antigens may drive T cells to a state of immunological exhaustion. While "off switches" based on suicide cassettes or other cell depleting approaches are being explored, such systems by definition result in the elimination of the therapeutic cells. Here we have developed a novel drug-regulated CAR platform termed Dimerizing Agent Regulated Immune-receptor Complex (DARIC) that aims to: i) provide control over any long-term toxicity of CAR T treatment; ii) lessen persistent T cell activation and subsequent exhaustion; and iii) regulate CAR T cell activity to potentially control CRS. The DARIC platform separates the antigen recognition and signaling functions of a CAR into two distinct polypeptides that are further engineered to contain the human-derived FKPB12 and FRB protein small-molecule regulated dimerization domains. In the absence of the dimerizing drug (e.g. rapamycin or the non-immunosuppressive rapalog AP21967) the DARIC system lacks signaling activity. However, the addition of dimerizing agent drives the interaction of the two DARIC subunits, fully restoring CAR function. Using CD19-expressing cell lines in vitro, we demonstrated that treatment of anti-CD19 DARIC+ T cells with rapamycin or AP21967 results in equivalent cytotoxicity, cytokine production and proliferation compared to a standard anti-CD19-targeting CAR. Importantly, anti-CD19 DARIC T cells were activated by low (10pM) levels of rapamycin and exhibited a higher antigen sensitivity than standard anti-CD19-CAR T cells in vitro . In an aggressive CD19+Nalm-6 xenograft tumor mouse model, anti-CD19 DARIC T cells did not exhibit anti-tumor activity in the absence of dimerizing agent. However, anti-CD19 DARIC treated mice that received either rapamycin or AP21967 showed robust tumor control equivalent or better to animals treated with the standard anti-CD19-CAR T cells. Importantly, full anti-tumor function of anti-CD19 DARIC T cells in vivo were observed using a non-immunosuppressive daily dose of rapamycin (0.01 mg/kg) resulting in trough levels of rapamycin near the lower level of assay quantitation (1.5 ng/ml). To model future applications designed to spare antigen-expressing normal tissues, we discontinued rapamycin injections in a subset of treated animals. Cessation of drug treatment resulted in the loss of anti-CD19 DARIC T cell activity and the expansion of Nalm-6 tumors cells in the anti-CD19 DARIC T cell treated mice, consistent with the ability to switch off CD19-DARIC T cells in vivo by withdrawing drug. Taken together, these results highlight the potential of the DARIC platform to facilitate the regulation of CAR T cell function in vivo .