Imaging CAR T Cell Trafficking with eDHFR as a PET Reporter Gene

Cell-based therapeutics have considerable promise across diverse medical specialties; however, reliable human imaging of the distribution and trafficking of genetically engineered cells remains a challenge. We developed positron emission tomography (PET) probes based on the small-molecule antibiotic trimethoprim (TMP) that can be used to image the expression of the Escherichia coli dihydrofolate reductase enzyme (eDHFR) and tested the ability of [¹⁸F]-TMP, a fluorine-18 probe, to image primary human chimeric antigen receptor (CAR) T cells expressing the PET reporter gene eDHFR, yellow fluorescent protein (YFP), and Renilla luciferase (rLuc). Engineered T cells showed an approximately 50-fold increased bioluminescent imaging signal and 10-fold increased [¹⁸F]-TMP uptake compared to controls in vitro. eDHFR-expressing anti-GD2 CAR T cells were then injected into mice bearing control GD2⁻ and GD2⁺ tumors. PET/computed tomography (CT) images acquired on days 7 and 13 demonstrated early residency of CAR T cells in the spleen followed by on-target redistribution to the GD2⁺ tumors. This was corroborated by autoradiography and anti-human CD8 immunohistochemistry. We found a high sensitivity of detection for identifying tumor-infiltrating CD8 CAR T cells, ∼11,000 cells per mm³. These data suggest that the [¹⁸F]-TMP/eDHFR PET pair offers important advantages that could better allow investigators to monitor immune cell trafficking to tumors in patients. The ability to track genetically engineered cells in vivo has been a long-standing goal for molecular imaging. Sellmyer et al. show that eDHFR can be used as a positron emission tomography reporter gene to track chimeric antigen receptor (CAR) T cells longitudinally in a rodent model with high sensitivity.