Objectives: Cancer immunotherapy is becoming an increasingly important treatment strategy across a broad spectrum of tumor types. Non-invasive imaging methods for in vivo tracking of T-cells can be used for longitudinal monitoring of tumor infiltrating T-cells to assess the effect of immune checkpoint therapy or cell-based immunotherapies. The objective of the current work was to develop and optimize Fab-based tracers directed at CD8 for PET imaging of T cells. The imaging performance of radiolabeled Fab fragments was compared to Fab2’ fragments in terms of kinetics and image contrast. Furthermore, Zirconium-89 (89Zr) labeled T cells were tracked in a murine transplantation model.

Methods: Anti-mouse CD8α antibody was cleaved into Fab2’ by enzymatic cleavage followed by reduction of disulfide bridge into Fab fragments. Fab2’ was conjugated to NCS-DFO for 89Zr labeling, while both Fab2’ and Fab were conjugated to SCN-NOTA for Cupper-64 (Cu-64) labeling.

Radiochemical yield, radiochemical purity (RCP) and plasma/buffer stability was assessed by high-performance liquid chromatography. Furthermore, the immunoreactivity and binding affinity was evaluated for the radiolabeled tracers.

In vivo comparison of radiolabeled Fab2’ and Fab fragments was performed by longitudinal PET/CT imaging at various time-points after injection in CT26 (murine colon carcinoma) tumor bearing mice. CD3+ T-cells were directly labeled with 89Zr for tracking experiments. The labeling efficiency and viability of the cells was evaluated ex vivo, and in vivo cell tracking was performed by longitudinal PET/CT imaging after injection of labeled T-cells in naive immune-competent mice.

Results: The cleavage of anti-CD8 into Fab2’ and subsequent purification by HPLC was optimized to achieve a product with high yield and purity. The optimal Fab2’ reduction conditions were determined followed by conjugation to SCN-NOTA An optimized protocol for 89Zr/64Cu-CD8-Fab2’ and 64Cu-CD8-Fab was developed with high radiochemical yield and RCP of >99%. PET/CT imaging with all tracers was performed to compare tumor, spleen and lymph node uptake. The highest uptake was found 24h post injection, but a clear delineation of lymphoid organs and tumors was evident already after 6h for the single Fab allowing same day imaging. T-cells labeled with 89Zr and cell tracking PET/CT imaging showed homing primarily to the spleen.

Conclusions: Different tracers directed at T-cells were successfully developed and evaluated in in vivo PET imaging of T cells. The reduced circulation time of Fab vs. Fab2’ and the use of 64Cu instead of 89Zr allows repeated imaging every 3 days due to the shorter physical half-life (12.7 h) of 64Cu. The study furthermore demonstrates the use of direct radiolabeling of T-cells as an attractive tool for investigating the distribution of cell-based therapies.

Citation Format: Camilla Christensen, Lotte K. Kristensen, Carsten H. Nielsen, Andreas Kjær. ImmunoPET and T cell labeling for in vivo monitoring of immunotherapy: Development of CD8 Fab2’ and Fab tracers for immunoPET and direct labeling of T cells for in vivo tracking [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1133.