Granzyme B PET Imaging Enables Detection of CAR T-Cell Therapy Response in a Human Melanoma Mouse Model

Background/Objectives: Granzyme B (GZB) PET Imaging is a non-invasive tool that can determine tumoral and systemic effects in immunotherapy. We aim to evaluate ⁶⁸Ga-NOTA-CYT-200 PET Imaging as a molecular imaging approach to determine CAR T-cell therapy response in a human melanoma mouse model. Our goal is to provide a method to monitor CAR T-cell therapy for patients with melanoma and other solid tumors. Methods: A human melanoma mouse model was generated by implanting naïve NSG mice (n = 28) with a human melanoma cell line (A375) subcutaneously (s.c.). After tumor implantation, mice were randomly assigned to receive either the treatment (CAR T) or vehicle solution (controls). After treatment, tumor sizes were measured every other day up to 35 days after cell implantation. ⁶⁸Ga-NOTA-CYT-200 PET Imaging was performed on days 2, 7, and 14 after CAR T-cell administration to assess T-cell activity within the tumors and organs. The PET Imaging results were correlated with IHC and immunofluorescent staining and cytokine assessment of tumor samples. Results: Tracer uptake within tumors of the CAR T group was significantly greater on days 2 (3.1 ± 1.2 vs. 1.1 ± 0.4, p = 0.002) and 7 (2.0 ± 1.1 vs. 1.1 ± 0.1, p = 0.01) after treatment, even before the CAR T group first presented with significantly lower tumor volumes on day 11 after treatment (61.8 mm³ ± 8.7 vs. 287.1 mm³ ± 157.6, p = 0.05). GZB (p = 0.03) and CAR T (p = 0.001) staining were also significantly greater in tumors of CAR T-cell-treated mice. Inflammatory cytokines such as IFN gamma (p = 0.03), CXCL10 (p = 0.004), and CCL5 (p = 0.02) concentrations were also significantly greater in CAR T-cell-treated tumors. Conclusions: CAR-T-treated tumors show significantly elevated ⁶⁸Ga-NOTA-CYT-200 uptake compared with controls, consistent with enhanced effector activity.