Describing the Clinical and Public Health Utility of a Novel Immuno-PET Imaging Agent, 89Zr-DFO-CD69 Ab

Glioblastoma (GBM) is an aggressive, highly malignant brain tumor with a very poor prognosis, notwithstanding multiple ‘standard of care’ (SOC) treatments. Immune-checkpoint-inhibitor (ICI) therapy is currently being explored for the treatment of GBM. Magnetic resonance imaging (MRI) is the favored approach for treatment assessment; however, MRI cannot adequately survey immune-related changes in the tumor microenvironment. Thus, there is a need for a non-invasive, molecular neuroimaging technique to assess immunotherapeutic response. T-cells are key mediators of cancer immunotherapy responses and upregulation of CD69 is mainly a marker of T-cell activation. In this study, we aimed to non-invasively quantify T-cell activation through CD69 immuno-PET in vivo, following immunotherapy, and correlate the expression to survival. CD69 was evaluated by flow cytometry and immunofluorescence staining on human and mouse T-cells activated in vitro and on dissociated tumors from GL261 glioma-bearing mice treated with anti-PD1/anti-CTLA4 ICIs. Single-cell RNA sequencing (scRNAseq) datasets from recurrent GBM patients receiving (n=14) or not receiving (n=12) ICI were examined for CD69 expression on tumor-infiltrating lymphocyte (TIL) populations. PET/CT was performed on tumor-bearing mice (n=30) receiving radiolabeled anti-CD69 antibody (89Zr-DFO-anti-CD69) to evaluate response to ICI therapy. Standard uptake values (SUV) were compared between ICI-treated and control groups and in relation to survival. We confirmed early CD69 upregulation upon T-cell activation in vitro. Ex vivo, CD69 expression significantly increased on TILs early after ICI treatment compared to controls (63.46% vs 24.37% CD69 + /TILs, respectively, p=0.017). ScRNAseq demonstrated significantly elevated CD69 expression in almost all TILs populations tested in recurrent GBM patients treated with ICI compared with a control group. Immuno-PET demonstrated significantly higher anti-CD69 tracer uptake in ICI-treated mice compared with controls. Most importantly, we observed a strong positive correlation between survival and immune-PET SUV (r=0.9425, p=0.016) in the ICI-treatment group, but not within the control group. Our study results support the potential utility of CD69 Immuno-PET as an early ICI response assessment imaging tool for GBM patients. Emerging PET/CT technologies are being used in a variety of clinical settings, reflecting their broad applicability, potential to improve health outcomes in an array of human diseases, and thus their public health importance