Abstract 3031: Positron emission tomography imaging of activated T cells by targeting OX40 reveals spatiotemporal immune dynamics and predicts response to in situ tumor vaccination
Authors: Mayer Aaron T. , Alam Israt S. , Sagiv-Barfi Idit , Wang Kezheng , Vermesh Ophir , Czerwinski Debra K. , Johnson Emily M. , James Michelle L. , Levy Ronald , Gambhir Sanjiv S. 
Published: August 2018

Abstract
Clinical success of cancer immunotherapies has renewed interest in imaging the behavior of immune cells. Due to the spatiotemporally varying signatures of immune response, it has been difficult to monitor and predict patient outcomes using traditional clinical tests. ImmunoPET, defined herein as positron emission tomography utilizing radiolabeled antibodies, has the potential to enable noninvasive, sensitive and longitudinal interrogation of immune cell subset and state. Cell states including activation, anergy, and exhaustion may be more prognostic of disease outcome than the presence of tumor-infiltrating immune cells alone. In particular, T cell activation is thought to be critical to treatment success across many classes of cancer immunotherapy. In this work, we present the first radionuclide imaging of OX40, a novel and specific biomarker of activated antigen-specific T cells. Activation dependent and T cell restricted expression of OX40 was validated in vitro via flow cytometric analysis. Cell uptake studies with radiolabeled 64Cu-DOTA-AbOX40 demonstrated ~11 fold [p &lt; .0001] higher uptake in dyna-bead activated T cells compared to resting. The tracer showed negligible nonspecific uptake in OX40 blocked or OX40-/- T cells and low background levels across a panel of 5 cancer cell lines tested. In vivo, ImmunoPET imaging revealed new insights into response following in situ tumor vaccination with CpG, an adjuvant immunotherapy currently in clinical trials. Balb-C mice bearing dual A20 lymphoma tumors were administered low dose CPG directly in the left tumor (n=7-10), while vehicle control mice received PBS (n=7-10). Early after vaccination, imaging revealed increased OX40 radiotracer uptake in the CPG treated tumor (TT) [~37%; p&lt;0.05] confirmed by immunofluorescent staining. ViSNE, a visualization technique for high-dimensional cytometry data, classified OX40+ single cells in a cluster associated with a nonregulatory, activated CD4 T cell phenotype. CPG treatment led to local expansion of this unique OX40 cell population [~63%; p&lt;0.05]. By late time points, a full systemic response emerged as evidenced by increased Luminex cytokine measurements in the plasma of CPG-treated mice. Unsupervised hierarchical clustering based on radiotracer or cytokine measurements correctly assigned mice into CPG-treated or vehicle cohorts, with few exceptions. More remarkably, a generalized linear regression model indicated early PET signal (mean %ID/g) in the local tumor environment to be highly predictive of response outcomes at late timepoints [r2=0.746]. OX40 ImmunoPET provides a readily translatable approach for monitoring activated T cells with high sensitivity and specificity. In this instance, integration of molecular imaging and computational immunology enabled systems-level interrogation of vaccine response.
Citation Format: Aaron T. Mayer, Israt S. Alam, Idit Sagiv-Barfi, Kezheng Wang, Ophir Vermesh, Debra K. Czerwinski, Emily M. Johnson, Michelle L. James, Ronald Levy, Sanjiv S. Gambhir. Positron emission tomography imaging of activated T cells by targeting OX40 reveals spatiotemporal immune dynamics and predicts response to in situ tumor vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3031.