PET imaging of OX40+ activated T cells predicts therapeutic response in a murine cancer vaccine model (2018)

73 Objectives: The complex and varying spatiotemporal dynamics of both the innate and adaptive immune response in the tumor microenvironment has rendered monitoring and predicting patient response to cancer immunotherapy challenging. Monitoring T cell activation, considered critical to treatment success across many classes of cancer immunotherapy could likely be more prognostic of treatment outcome than the presence of tumor infiltrating T cells alone. We report a novel PET tracer (64Cu-DOTA-mAbOX40) that enables non-invasive and longitudinal imaging of OX40, a cell surface marker expressed on antigen-specific activated T cells [1]. Using a dual tumor mouse model treated with an in situ CpG vaccine, we demonstrated that the radiotracer not only enabled visualization of T cell activation following treatment but that tracer uptake in the tumor at day 2 post-treatment initiation was predictive of tumor responses at day 9 and with higher accuracy than both anatomical and blood based measurements.

 Methods: Radiolabelling and in vitro evaluation: anti-OX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86, BioXcell) was DOTA-conjugated. Immunoreactivity of the conjugate was tested with cell binding studies using activated murine T cells, which showed that binding was comparable to unconjugated antibody. The conjugate was subsequently radiolabeled with 64CuCl2 (final specific activity; 5-10μCi/μg and radiochemical purity; >99%). The tracer was also evaluated in vitro to confirm its specificity for activated versus resting murine T cells (supplementary data). In vivo characterization: female Balb-C mice bearing dual A20 lymphoma tumors on the shoulders were administered either low dose CpG oligonucleotide (50μg in PBS, n=7-10) or PBS alone directly in the left tumor (vehicle control, n=7-10). PET/CT imaging of mice with 64Cu-DOTA-mAbOX40 (80-100 µCi, i.v.) was performed at an early (day 2) and late (day 9) time point post-treatment initiation. Mice were euthanized following PET, and radioactivity in dissected tissues measured using a gamma-counter. Flow cytometry and histological analyses of tumor draining lymph nodes (TDLN), tumors, and spleen from vehicle and CpG-treated mice was also performed at the same treatment time points to determine OX40 protein expression on cells.
RESULTSIn vivo OX40-ImmunoPET, revealed new insights into response following in situ tumor vaccination with CpG, an adjuvant immunotherapy currently in clinical trials [2]. Early time point imaging after CpG administration revealed increased radiotracer uptake in the CpG-treated tumor [CpG 10.3±0.7; Veh. 7.5±0.3 %ID/g; p<0.05] and the associated TDLN [CpG 12.92 ± 1.15; Veh. 10.67 ± 0.94%ID/g; p<0.01] (Fig. 1A and B). Quite powerfully, a generalized linear regression model indicated early PET signal (mean %ID/g) in the local tumor environment to be highly predictive of response at late time points [r2=0.746] (Fig. 1C). An increase in the frequency of OX40+CD3+ expression in the CpG-treated tumor and associated TDLN at the early time point vs. untreated sites (p<0.05) and vehicle cohorts was confirmed by FACS and histology suggesting in situ CpG vaccination triggered induction of a local adaptive immune response. ViSNE, a visualization technique for high-dimensional cytometry data, showed that OX40+ cells were highly restricted to clusters associated CD4 helper T cells (data not shown).
CONCLUSION To our knowledge, this is the first report of an OX40 PET tracer that enables specific imaging of OX40+ effector T cell driven immune responses. OX40-immunoPET provides a readily translatable approach for monitoring activated T cells with high sensitivity and specificity for clinical cancer immunotherapy strategies.