Imaging activated immune response following therapeutic vaccination in an orthotopic glioma model with 89Zr-DFO-OX40 mAb PET (2020)

2 Objectives: Glioblastoma (GBM) is one of the most difficult malignancies to eradicate. Recently, vaccines targeting GBM have shown potential clinical promises (1,2). In the tumor microenvironment, CD4+ T cells play an important role in tumor clearance not only by augmenting cytotoxic CD8+ T cells, but also by directly killing cancer cells (3,4). Based on the accumulating evidence of vaccine efficacy, it is important to establish an imaging tool which specifically delineates activated immune cells involved in therapeutic responses. Here, we targeted OX40 as an imaging biomarker, which is a co-stimulatory transmembrane receptor in the Tumor Necrosis Factor superfamily mainly expressed on activated effector CD4+ T cells. OX40 promotes proliferation and extends survival of effector and memory T cells, and represents a potential surrogate biomarker to monitor T cell activation and immunotherapy responses. The purpose of this study was to test the ability of our novel monoclonal antibody (mAb) PET tracer, 89Zr-DFO-OX40 mAb (89Zr-OX40 mAb), to visualize T cells activation following therapeutic vaccination in an orthotopic glioma model.
 Methods: An orthotopic mouse glioma model was prepared for this study (Fig. 1). Albino C57BL/6 mice were inoculated in the right brain hemisphere with GL26 cells expressing luciferase. Tumor growth was confirmed after 10 to 14 days by bioluminescence imaging (BLI). Vaccinated mice (n = 9) received a subcutaneous injection cocktail of CpG, GL26 tumor lysate, and OX40 mAb in their left shoulders. A control group of mice (n = 7) received PBS injections at the same site. 89Zr-OX40 mAb PET was used for subsequent evaluation of systemic immune response, beginning on 6 days post vaccination. 89Zr-OX40 mAb was administered once via tail vein injection and PET/CT scans were acquired on 1,2, and 5 days post tracer injection. An isotype control tracer, 89Zr-DFO-rat IgG, was generated and administered to determine the specificity of 89Zr-OX40 mAb binding in both vaccinated and control groups (n = 5, each). Immediately following final PET/CT scans, animal organs were harvested for ex vivo biodistribution (BioD) study. Cells isolated from the left axillary lymph nodes (LN) and the spleen were also investigated for CD4 and OX40 expression by flow cytometry. The treatment response was evaluated by quantifying the OX40 tracer uptake (%ID/g) and the tumor BLI signals.
 Results: The vaccinated group showed a significant decrease in tumor BLI signal compared to the control (p = 0.018). 89Zr-OX40 mAb PET exhibited 6.2 ± 1.0-fold increase in uptake in vaccinated group in the left axillary LN (p < 0.0001) and 1.9 ± 0.4 fold increase in the spleen (p = 0.020), relative to the control group (Fig. 2) or in mice injected with the control 89Zr-DFO-rat IgG. BioD results demonstrated a significant linear correlation with PET signals (p < 0.0001). Flow cytometry confirmed OX40 expression to be highly restricted to CD4+ T cells and revealed that the OX40+ cell ratio was significantly higher in the left axillary LN (p = 0.017), and the spleen (p = 0.036) in the vaccinated group, compared to the control group.
 Conclusions: 89Zr-OX40 mAb PET successfully visualized the activation of systemic immune responses that are triggered upon therapeutic vaccination. The increase in 89Zr-OX40 mAb PET signals correlated with treatment efficacy in the glioma model. Our results highlight the ability of 89Zr-OX40 mAb PET to monitor T cells activation in vaccine strategies targeting GBM.