Noninvasive longitudinal PET/CT imaging of CAR T cells using PSMA reporter gene

Abstract

Purpose: Chimeric antigen receptor (CAR) T cell therapy has achieved great success in treating hematologic malignancies. However, it is yet to prove effective in the treatment of solid tumors. Thus, it is necessary to develop appropriate methodology for the long-term, accurate, and quantitative evaluation of the distribution and activities of CAR T cells in solid tumors. In the present study, we engineered TfR ΔPSMA CAR (CAR-ΔPSMA) T cells, which targeted the transferrin receptor (TfR) expressed by tumor cells and could be tracked in vivo via a reporter gene encoding the truncated prostate specific membrane antigen (ΔPSMA). We then quantitatively monitored these CAR T cells in vitro and in vivo using [⁶⁸Ga]Ga-PSMA-617 positron emission tomography (PET)/computed tomography (CT).

Methods: The CAR-ΔPSMA T cells were genetically engineered by transducing T cells with a lentiviral vector encoding TfR41BBζ-T2A-ΔPSMA. Firstly, the target expression, activation, and cytotoxicity of CAR-ΔPSMA T cells were validated in vitro. Secondly, the minimum thresholds of CAR-ΔPSMA T cells detection for [⁶⁸Ga]Ga-PSMA-617 PET/CT were also determined in vitro and in vivo respectively. Lastly, the feasibility of monitoring the biodistribution and infiltration of CAR-ΔPSMA T cells after systematic administration was evaluated in the breast cancer subcutaneous xenograft model.

Results: The CAR-ΔPSMA T cells retained activation and tumor killing capacity after transduction of the ΔPSMA-encoding reporter gene. Next, the CAR-ΔPSMA T cells could be reliably tracked by [⁶⁸Ga]Ga-PSMA-617 PET/CT, the detection sensitivity of which was 250 cells/mm³ in vitro and 100 cells/mm³ in vivo. Next, the sequential imaging assays revealed that [⁶⁸Ga]Ga-PSMA-617 PET/CT could be used to specifically visualize ΔPSMA⁺ CAR T cells at the tumor site. The increase in the [⁶⁸Ga]Ga-PSMA-617 signal intensity over time allowed us to effectively detect CAR T cells in vivo.

Conclusion: Our findings preliminarily confirmed that [⁶⁸Ga]Ga-PSMA-617 PET/CT could reliably detect CAR-ΔPSMA T cells in vitro and in vivo in solid tumors, laying the foundation for the monitoring CAR T cell therapy in the future.

Keywords: CAR T cells; PET/CT; PSMA; Reporter gene; Solid tumors.