Quantitative dynamic granzyme B PET imaging to characterize novel combination immunotherapy response in preclinical glioblastoma models

Rationale: Immune-targeted positron emission tomography (PET) allows for the non-invasive monitoring of immune populations with potential to provide early markers of response to novel immunotherapies in glioblastoma (GBM). Dynamic PET acquisitions can inform on tracer kinetics via dynamic modeling to enhance the GBM tumor microenvironment characterization, while its impact has yet to be understood. The objective of this study is to evaluate the quantitative description of dynamic granzyme B (GZP)-PET imaging and its ability to inform on combination immunotherapy response in orthotopic syngeneic GBM models. Methods: Orthotopic GBM murine models (GSC005-luc) were imaged with dynamic [⁶⁴Cu]-NOTA-GZP PET (0-80 min) and T₂ -weighted MRI one week post-treatment with saline or combination M002 virotherapy and anti-PD1 immunotherapy. One-, two-, and three-tissue compartment models were evaluated for suitability to tracer kinetics via Akaike information criterion. Biological validation consisted of ex vivo brain PET imaging, autoradiography, H&E and immunofluorescence for granzyme B. A subset of mice was longitudinally monitored via dynamic [⁶⁴Cu]-NOTA-GZP PET at days 4 and 7. Changes in viable cell bioluminescence and radiological tumor volume (MRI) were used to determine response. Imaging-derived metrics including k₁ , k₂ , k₃, k₄ and SUV were evaluated across treatment and response groups via unpaired two-tailed T test. Results: Intratumoral [⁶⁴Cu]-NOTA-GZP PET tracer kinetics showed improved fitting based on a two-tissue compartment model (p < 0.05). Intratumoral effector cell function (SUV_mean TBR) and tracer binding rate (k₃ ) were positively correlated with histological granzyme B density (p < 0.01). Increases in tumor tracer influx (k₁ ) were observed in responders relative to non-responders (p < 0.01). Conclusions: Mathematical description of tracer kinetics via dynamic [⁶⁴Cu]-NOTA-GZP PET offers complementary quantitative metrics for the characterization of immunotherapy response in GBM. Integration of dynamic protocols to immune-target PET approaches can provide clinically translatable metrics to differentiate immunotherapy-induced effects from tumor progression in GBM.