Abstract 1723: CD4+ and CD8a+ PET imaging in syngeneic mouse models for prediction of immuno-therapeutic response
Authors: Kristensen Lotte K. , Fröhlich Camilla , Christensen Camilla , Melander Maria C. , Poulsen Thomas T. , Galler Gunther R. , Lantto Johan , Horak Ivan D. , Kragh Michael , Nielsen Carsten H. , Kjaer Andreas 
Published: August 2018

Abstract
Introduction: The clinical success of immune-checkpoint inhibitors in oncology has stimulated development of immune-based agents and revolutionized treatment for many types of cancer. With only a subpopulation of patients benefiting from immuno-therapeutic agents, the development of a predictive in vivo biomarker of response is critical to enhance the therapeutic efficacy of such agents. The level of immune infiltration in tumors seems to reflect the outcome of immuno-therapy, and treatment failure is usually attributed to the so-called “cold” tumors that do not attract T-cell infiltration. Thus, we sought to develop specific PET radiotracers for non-invasive in vivo detection, tracking and quantification of CD4+ and CD8a+ tumor-infiltrating lymphocytes (TILs).
Experimental procedures: A panel of syngeneic mouse tumor models was analyzed for the level of TILs using flow cytometry and immunohistochemistry (IHC), and for response to Sym021, a humanized anti-PD1 antibody cross-reactive with mouse PD1. Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Deferoxamine (p-SCN-Bn-DFO) chelator and radiolabeled with 89Zr. Immuno-reactivity of 89Zr-DFO-CD4-F(ab)'2 and 89Zr-DFO-CD8a-F(ab)'2 was assessed with murine T-cells. Tracers were assessed in vivo in a panel of syngeneic mouse models following optimization of dose and imaging time-points.
Results: Based on flow cytometry and IHC analysis, the syngeneic tumor models were characterized as “hot” or “cold” by the level of TILs. The “hot” tumor phenotype clearly correlated with response to Sym021 treatment. 89Zr-labeled CD4- and CD8a-targeting radiotracers were successfully generated with a radiochemical purity &gt;99% and an immuno-reactivity &gt;85%. The optimal imaging parameters were 24 hours post-injection of 1 MBq of tracer. Co-injection with 30 μg unlabeled CD4/CD8a-F(ab)'2 significantly decreased spleen and lymph node uptake, whereas tumor uptake was increased. PET imaging in syngeneic mouse models revealed differential uptake of 89Zr-DFO-CD4-F(ab)'2 and 89Zr-DFO-CD8a-F(ab)'2, with an increased uptake primarily seen in “hot” tumors. In addition, an increase in CD8a+ TILs after fractionated external radiation therapy (XRT) was successfully detected by 89Zr-DFO-CD8a-F(ab)'2 PET imaging.
Conclusions: We have developed 89Zr-DFO-CD4-F(ab)'2 and 89Zr-DFO-CD8a-F(ab)'2 PET imaging radiotracers for whole-body detection and assessment of CD4+ and CD8a+ status. These radiotracers can be used as tools to predict efficacy of immuno-therapeutic treatment in preclinical drug development. In addition, they may be used to develop non-invasive in vivo biomarkers for identifying patients responding to immuno-therapeutic agents, such as Sym021.
Citation Format: Lotte K. Kristensen, Camilla Fröhlich, Camilla Christensen, Maria C. Melander, Thomas T. Poulsen, Gunther R. Galler, Johan Lantto, Ivan D. Horak, Michael Kragh, Carsten H. Nielsen, Andreas Kjaer. CD4+ and CD8a+ PET imaging in syngeneic mouse models for prediction of immuno-therapeutic response [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 1723.