CT-167 Using the Sodium Iodide Symporter to Visualize CAR-T Cell Trafficking

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy revolutionized the treatment of refractory B cell malignancies, but relapses occur through a variety of incompletely understood mechanisms. Currently, tracking CAR-T expansion and persistence in patients is limited to peripheral blood sampling, but this approach provides no insights into how and where CAR-T cells traffic beyond the peripheral blood or how cellular localization may impact persistence and efficacy. Furthermore, costimulatory endodomains that are known to influence CAR-T cell phenotype have unclear downstream effects on how CAR-T cells traffic. The sodium iodide symporter (NIS) is a plasma membrane glycoprotein that actively uptakes iodide and its analogs, and it can be transduced into various cell types in preclinical models allowing sensitive and specific PET/SPECT imaging. Coexpression of NIS with CAR on T cells would allow real-time, in vivo imaging of CAR-T cells and aid in understanding CAR-T cell biology and trafficking. Using Golden Gate modular vector construction, multiple anti-CD20 CAR constructs with specificity for both human and non-human primate (NHP) CD20 antigens were generated. Each construct included one of several costimulatory domains (4-1BB, CD28, or CD27), a standard CD3-zeta signaling domain, and either the rhesus-NIS reporter or truncated EGFR following a short ribosomal skip sequence peptide, allowing transduction under control of the same EF1a promoter. CAR was effectively cotransduced with NIS in both human and NHP primary T cells with no effect of NIS on CAR-T cell-mediated cytotoxicity against CD20-expressing Raji cells in vitro compared to control. Cytotoxicity was antigen-specific as no difference was seen against CD20- control cells. Functional iodide and [18F]-TFB, an iodide analog, uptake via NIS was specific to NIS-transduced cells and was appropriately inhibited by coincubation with KClO4. An NSG mouse xenograft model will be used to track the localization, expansion, efficacy, and persistence of CAR-T constructs against Raji lymphoma cells. Different costimulatory domains appear to elicit only slight differences in cytokine production in vitro but will be further studied for differences in efficacy and trafficking in vivo in mice and later in a syngeneic NHP model.