A radiohapten capture system for CAR T cells that tracks them in vivo and improves efficacy

Background: CAR T cell therapy has become an important therapeutic tool for the treatment of B-cell neoplasms. However, the development of CAR T cell therapy outside of hematologic malignancies has stalled. As such, there is an unmet need to understand why CAR T cell therapy fails, which would be aided through the creation of tools to track them in vivo. Additionally, endowing CAR T cells with potent orthogonal anti-tumor activity has emerged as a strategy to improve upon CAR T cell efficacy. To accomplish both goals, we engineered a class of theranostic “THOR” CAR T cells that express a membrane-bound scFv, huC825, that binds DOTA-radiohaptens with pM affinity; these hapten chelands can be conjugated with radionuclides with therapeutic potential or diagnostic imaging capabilities. Methods: We engineered 19BBZ anti-CD19 CAR T cells to express huC825 (THOR CAR T cells) and confirmed transduction of these cells with flow cytometry. To assess the cytolytic ability and effector function of THOR CAR T cells, we used the Matador co-culture assay and measured the cytokine secretion of these co-cultures. We tested the potential to track THOR CAR T cells in vivo using PET/CT following intravenous DOTA-based radiohapten tracer administration in immunodeficient (NSG) mice bearing subcutaneous CD19+ Raji tumors. We also tested the ability of THOR CAR T cells to synergize with therapeutic radionuclide therapy. We evaluated the anti-tumor efficacy using whole animal bioluminescence imaging, tumor size measurements, and median animal survival. Results: We successfully transduced THOR CAR T cells, which demonstrated similar in vitro anti-tumor effects and cytokine secretion as parent 19BBZ CAR T cells. In vivo tracking experiments with 86Y-DOTA-Bn radiotracer showed that THOR CAR T cells can be observed at day 7 post T cell administration, peak at day 14, and persist until at least day 28. 86Y-DOTA-Bn uptake was specific to THOR CAR T cells, as minimal uptake was observed in mice transplanted with 19BBZ CAR T cells. Importantly, we noted rapid renal clearance of unbound tracer resulting in high contrast images. In efficacy assays in vivo, we demonstrated the synergy between 225Ac-Proteus-DOTA and THOR CAR T cells, as shown by a significant decrease in tumor burden and increase in median survival (THOR CAR treated = 26 days, treated with THOR CAR plus 225Ac-Proteus-DOTA = 32 days, p = .04, n = 6 per group). In contrast, mice receiving 19BBZ CAR T cells with or without 225Ac-Proteus-DOTA showed no improvements in antitumor efficacy. Conclusions: We show the pharmacokinetics of THOR CAR T cells can be probed in vivo following systemic administration of radiotracer using serial PET/CT imaging. Furthermore, we demonstrate a CAR T cell can be potentiated by systemically administered cytotoxic radionuclide that are then delivered selectively to tumors by the THOR cells. Conflict of interest: Ownership: MSK has filed for patent protection on behalf of M.M.D, S.M.C., D.R.V., B.H.S., N.K.C., D.A.S., S.M.L. and S.K. for inventions related to this work. Advisory Board: N.K.C. is an advisor to, or owns equity in Abpro, Alexs Lemonade Stand Foundation, Biotec Pharmacon, Eureka Therapeutics, Keystone Symposia, Partner Therapeutics, St. Jude-VIVA Forum, and Y-mAbs Therapeutics that may work in areas related to this study. Other Substantive Relationships: S.M.C. has licensed IP to Y-mAbs.