Predicting Tumor Response to Immunotherapy using Quantitative CD3 PET Imaging

29 Objectives Rapid advances in the field of tumor immunology have led to new therapies aimed at increasing the tumoral immune response against highly malignant cancers. Response rates to these therapies are significantly higher than to standard chemotherapy and those that respond typically have more durable responses than with standard chemotherapy. However, the majority of patients receiving immunotherapy will not respond, yet remain at risk for severe side-effects. Furthermore, evaluation of response to immunotherapies using standard RECIST imaging techniques remains challenging due to immune infiltrates that can masquerade as tumor growth. Determining the appropriate predictive paradigm for tumor immunotherapy could not only detect those patients who are more likely to respond to therapy, but also mitigate unnecessary treatment. Significant evidence has accumulated to suggest that an increased presence of tumor infiltrating lymphocytes, including CD3 positive lymphocytes, is predictive of prognosis and response to immunomodulatory therapy. CD3 is a part of the T-cell receptor complex that serves as a global T lymphocyte marker, and it was therefore hypothesized to be an appropriate marker of T cell infiltration. Methods An anti-murine CD3 antibody was conjugated with DFO and radiolabeled with the PET radiometal 89Zr. The radiolabeled antibody was assessed for purity, specific activity and retained target binding in vitro using cell-based competition and total binding assays. Following confirmation of CD3+ cell binding, the 89Zr-DFO-CD3 antibody was used to monitor the CD3+ T cell infiltration in mice following three treatments at days 4, 7 and 10 post-inoculation with anti-CTLA-4 therapy. Parallel studies examining protein expression in the tumors of CT26 xenograft bearing treated mice by Western blot and immunohistochemistry were undertaken to analyze the variability of CD3+ T cell infiltration. Injection of the radiotracer occurred at day 11 post-inoculation, with subsequent PET imaging occurring on day 14. Mice were grouped based on quantitative imaging and tumor growth measured to assess therapeutic efficacy. Results The DFO-CD3 antibody was labeled with high specific activity (50±7 MBq/mg) and radiochemical purity and demonstrated high affinity (IC50 = 2.8±2.3 nm) binding to CD3+ TK-1 cells in vitro, and the specificity was confirmed by comparing CD3+ TK-1 with CD3- CT26 cell binding which resulted in a significant difference in total binding (47,719±24473 CPM vs. 4,547±2671 CPM P = 0.008). The antibody was used to assess CD3+ T cell infiltration in CT26 tumor bearing mice previously analyzed by ex vivo analysis to confirm differential CD3+ T cell infiltration by Western blot analysis. Quantitative PET imaging resulted in two distinct groups (P Conclusions An 89Zr-DFO-CD3 antibody was generated with high specific activity and affinity. The radiolabeled antibody was used to assess the predictive value of CD3+ T cell infiltration in tumor bearing mice given immunotherapy. The results revealed that high tumor CD3 PET uptake was predictive of response to anti-CTLA-4 therapy prior to divergent changes in tumor volume that separated responders from non-responders, representing a novel approach to predicting immune therapy response. This paradigm permits real-time assessment of immune-mediated T cell infiltration, which has the potential for widespread use across multiple cancer types and therapeutic strategies. $$graphic_C5BE0743-BDF1-41A8-B41A-2F9B7BB196B6$$