In vivo monitoring of CAR T cells using [¹⁸F]fluoropropyl-trimethoprim

Objectives: Cell based therapeutics have immense promise across a variety of fields, from metabolic engineering to cancer therapies. Perhaps the most prominent example of this approach is the recent FDA approval of chimeric antigen receptor (CAR) T cells for targeting relapsed/refractory B cell acute lymphoblastic leukemia (ALL). However, imaging genetically engineered cells in vivo remains a challenge and new technologies are needed. We recently developed a positron emission tomography (PET) probe based on the small molecule antibiotic trimethoprim (TMP) for imaging genetically engineered cells that express bacterial dihydrofolate reductase (dhfr) in small animals.(1) Here, we tested the ability of this PET probe and reporter gene pair to image engineered primary human T cells, with and without CARs targeted to solid tumor xenografts. We monitored the in vivo trafficking of CAR T cells to solid tumors in mice using a triple imaging reporter construct that encoded yellow fluorescent protein (YFP), the PET reporter gene dhfr, and Renilla luciferase. Methods [¹⁸F]fluoropropyl-trimethoprim, [¹⁸F]FPTMP, was synthesized as previously reported.(2) Lentiviral vectors were generated with the EF-1α promoter followed by the dhfr-YFP fusion protein, a 2A ribosomal skip site, and Renilla luciferase (DYR) and with CAR recognizing the GD2 disialoganglioside with mCherry (CAR). Primary human T cells were activated with CD3/CD28-coated beads and lentivirally transduced with DYR alone or DYR and CAR vector. Positive transductants were sorted with two color FACS. HCT116 (human colon carcinoma; GD2-) and 143b (human osteosarcoma; GD2+) subcutaneous xenografts were grown for 10 days in immunodeficient (NSG) mice on contralateral shoulders; once tumors were palpable, mice were injected with approximately 1 million DYR-CAR T cells via the tail vein. T cell trafficking to tumors was monitored with bioluminescence imaging (BLI) using coelantrazine IV and [¹⁸F]FPTMP PET/CT on days 2 and 4 post T cell injection. [¹⁸F]Fluorodeoxyglucose (FDG) measured the metabolic activity in the tumors on day 3. Mice were sacrificed on day 4 and tumor T cell IHC is pending. Results Primary DYR T cells showed over 50-fold increased BLI signal compared to untransduced primary T cell controls in vitro and showed approximately 10-fold increased uptake after incubation with [¹⁸F]FPTMP for 2h as compared to control T cells. In vivo imaging of tumor-bearing NSG mice via BLI and [¹⁸F]FPTMP PET/CT on days 2 and 4 following administration of DYR-CAR T cells demonstrated increased signal in 143b tumors as compared to HCT116 tumors, suggesting greater trafficking of CAR T cells to GD2-positive tumors at both time points. Furthermore, the BLI signal increased from 7 to 70-fold from day 2 to day 4, likely also due to preferential targeting of GD2-positive tumors mediated by epitope recognition. In a similar fashion, PET/CT showed increased uptake of [¹⁸F]FPTMP within 143b tumors as compared to HCT116 tumors and expansion of this signal from day 2 to day 4; notably this uptake was localized in the center of tumors, suggesting successful infiltration of the tumor by CAR T cells. On day 4 there was 2.4-fold increased [¹⁸F]FPTMP uptake in 143b tumors compared to HCT116 tumors, which correlated with the optical imaging results. FDG PET confirmed the metabolic activity of the tumors. Conclusion Imaging will likely play a central role in understanding immune cell trafficking to tumors, evaluating sites of potential toxicity, and determining the efficacy of cell-based therapies. We show in vivo BLI and PET/CT monitoring of CAR T cells trafficking to solid tumors. This triple reporter imaging approach allows facile monitoring of cells in preclinical studies (via fluorescence and/or BLI), and we anticipate that the [¹⁸F]FPTMP/dhfr PET probe and reporter gene pair will be useful for tracking CAR T cells in the clinical setting. Future studies will test whether trafficking of CAR T cells correlates with therapeutic response.