Background/Objectives: This study evaluated a novel PET tracer, ⁶⁸Ga-NOTA-CYT-200, which targets human granzyme B (GZB) as a biomarker for cytotoxic T-cell activation in a clinically relevant model of melanoma-bearing mice with a humanized immune system treated with immune checkpoint inhibitor (ICI) therapy. Methods: The binding affinity of the tracer was determined using an enzymatic colorimetric assay. Tumor-bearing humanized NSG mice underwent PET imaging before and during ICI monotherapy or combination therapy to assess ⁶⁸Ga-NOTA-CYT-200 uptake within tumors and other organs. The tumor growth was carefully monitored. The treatment response was evaluated based on the percentage change in tumor size at days 5 and 15 after the treatment started. A tracer biodistribution study and immunohistochemical staining of the tumors and organs were also performed. Results: The inhibition constant (Ki) of ⁶⁸Ga-NOTA-CYT-200 was estimated at 4.2 nM. PET imaging showed a significantly higher ⁶⁸Ga-NOTA-CYT-200 uptake in mice receiving the combination therapy compared to those receiving monotherapy or a vehicle (p < 0.0001 or p = 0.0005, respectively), which correlated with the greatest reduction in tumor size in the combination ICI group. Regardless of treatment, the responders presented with a significantly higher ⁶⁸Ga-NOTA-CYT-200 uptake at days 4 or 7 after the treatment began (p = 0.0002 and p = 0.0109, respectively). An increased uptake of ⁶⁸Ga-NOTA-CYT-200, especially in the intestines and liver within the combination ICI group, suggested immune-related adverse events (IrAEs). Conclusions: Our study demonstrates that ⁶⁸Ga-NOTA-CYT-200 PET imaging can predict the early treatment response in melanoma models treated with ICI and may also help in detecting IrAEs.

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.