CD4<sup>+</sup> and CD8a<sup>+</sup> PET imaging predicts response to novel PD-1 checkpoint inhibitor: studies of Sym021 in syngeneic mouse cancer models
Authors: 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
Journal: Theranostics (2019)

Predicting the outcome of immunotherapy is essential for efficient treatment.The recent clinical success of immunotherapy is increasingly changing the paradigm of cancer treatment.Accordingly, the development of immune-based agents is accelerating and the number of agents in the global immuno-oncology pipeline has grown 60-70% over the past year.However, despite remarkable clinical efficacy in some patients, only few achieve a lasting clinical response.Treatment failure can be attributed to poorly immunogenic tumors that do not attract tumor infiltrating lymphocytes (TILs).Therefore, we developed positron emission tomography (PET) radiotracers for non-invasive detection of CD4 + and CD8a + TILs in syngeneic mouse tumor models for preclinical studies.Methods: Seven syngeneic mouse tumor models (B16F10, P815, CT26, MC38, Renca, 4T1, Sa1N) were quantified for CD4 + and CD8a + TILs using flow cytometry and immunohistochemistry (IHC), as well as for tumor growth response to Sym021, a humanized PD-1 antibody cross-reactive with mouse PD-1.Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator and radiolabeled with Zirconium-89 ( 89 Zr-DFO-CD4/ 89 Zr-DFO-CD8a).Tracers were optimized for in vivo PET/CT imaging in CT26 tumor-bearing mice and specificity was evaluated by depletion studies and isotype control imaging. 89Zr-DFO-CD4 and 89 Zr-DFO-CD8a PET/CT imaging was conducted in the panel of syngeneic mouse models prior to immunotherapy with Sym021.Results: Syngeneic tumor models were characterized as "hot" or "cold" according to number of TILs determined by flow cytometry and IHC. 89Zr-DFO-CD4 and 89 Zr-DFO-CD8a were successfully generated with a radiochemical purity >99% and immunoreactivity >85%.The optimal imaging time-point was 24 hours post-injection of ~1 MBq tracer with 30 µg non-labeled co-dose.Reduced tumor and spleen uptake of 89 Zr-DFO-CD8a was observed in CD8a + depleted mice and the uptake was comparable with that of isotype control ( 89 Zr-DFO-IgG2b) confirming specificity.PET imaging in syngeneic tumor models revealed a varying maximum tumor-to-heart ratio of 89 Zr-DFO-CD4 and 89 Zr-DFO-CD8a across tumor types and in-between subjects that correlated with individual response to Sym021 at day 10 relative to start of therapy (p=0.0002 and p=0.0354, respectively).The maximum 89 Zr-DFO-CD4 tumor-to-heart ratio could be used to stratify mice according to Sym021 therapy response and overall survival was improved in mice with a 89 Zr-DFO-CD4 ratio >9 (p=0.0018). Conclusion:We developed 89 Zr-DFO-CD4 and 89 Zr-DFO-CD8a PET radiotracers for specific detection and whole-body assessment of CD4 + and CD8a + status.These radiotracers can be used to phenotype preclinical syngeneic mouse tumor models and to predict response to an immune checkpoint inhibitor.We foresee development of such non-invasive in vivo biomarkers for prediction and evaluation of clinical efficacy of immunotherapeutic agents, such as Sym021.