Introduction:Many immunotherapies focus on (re)invigorating CD8+ T cell anti-cancer responses. Different nuclear imaging techniques have been developed to measure CD8+ T cell distributions. Comprehensive comparisons of in vivo and ex vivo T cell labeling methods with respect to tumor and normal tissue targeting and correlation with CD8⁺ T cell presence are lacking, but essential for accurate clinical interpretation. We performed a head-to-head comparison of three CD8+ T cell imaging approaches: 89Zr-labeled Fc-silent anti-CD8 antibody ([89Zr]Zr-anti-CD8-IgG2asilent), ex vivo 89Zr-labeled ovalbumin-specific CD8+ T cells ([89Zr]Zr-OT-I), and 18F-labeled IL2 ([18F]AlF-RESCA-IL2). Methods:B16F10/OVA tumor-bearing C57BL/6 mice (n = 10/group) underwent PET/CT imaging at 72 ([89Zr]Zr-anti-CD8-IgG2asilent), 24 and 48 h ([89Zr]Zr-OT-I), and 10 min ([18F]AlF-RESCA-IL2) pi. Subsequently, biodistribution analysis was performed, followed by flow cytometry to evaluate intratumoral CD8+ T cell numbers. Intratumoral radiolabel distributions were assessed by autoradiography and immunohistochemistry. Results:All approaches showed uptake in CD8-rich tissues, with preferential spleen targeting. Biodistribution analyses showed tumor uptake exceeded blood level for [89Zr]Zr-anti-CD8-IgG2asilent and [89Zr]Zr-OT-I. Furthermore, their tumor uptake correlated to intratumoral CD8+ T cells presence even though intratumoral distribution patterns differed significantly. Conclusion:[89Zr]Zr-anti-CD8-IgG2asilent and [89Zr]Zr-OT-I PET/CT imaging can evaluate intratumoral CD8+ T cell infiltration. [89Zr]Zr-anti-CD8-IgG2asilent might be suited for TME immunophenotyping, while ex vivo labeling visualizes tumor migration and invasion dynamics of tumor-specific T cells. [18F]AlF-RESCA-IL2 uptake did not correlate to the intratumoral CD8+ T cell presence. Here, we provide new insights to guide the selection of imaging strategies for assessing relevant immunotherapy-specific aspects of the TME and support the correct interpretation of clinical CD8 imaging.

One of the main challenges of PET imaging with 89Zr-labeled monoclonal antibodies (mAbs) remains the long blood circulation of the radiolabeled mAbs, leading to high background signals, decreasing image quality. To overcome this limitation, here we report the use of a bioorthogonal linker cleavage approach (click-to-release chemistry) to selectively liberate [89Zr]Zr-DFO from trans-cyclooctene-functionalized trastuzumab (TCO-Tmab) in blood, following the administration of a tetrazine compound (trigger) in BT-474 tumor-bearing mice. Methods: We created a series of TCO-DFO constructs and evaluated their performance in [89Zr]Zr-DFO release from Tmab in vitro using different trigger compounds. The in vivo behavior of the best performing [89Zr]Zr-TCO-Tmab was studied in healthy mice first to determine the optimal dose of the trigger. To find the optimal time for the trigger administration, the rate of [89Zr]Zr-TCO-Tmab internalization was studied in BT-474 cancer cells. Finally, the trigger was administered 6 h or 24 h after [89Zr]Zr-TCO-Tmab- administration in tumor-bearing mice to liberate the [89Zr]Zr-DFO fragment. PET scans were obtained of tumor-bearing mice that received the trigger 6 h post-[89Zr]Zr-TCO-Tmab administration. Results: The [89Zr]Zr-TCO-Tmab and trigger pair with the best in vivo properties exhibited 83% release in 50% mouse plasma. In tumor-bearing mice the tumor-blood ratios were markedly increased from 1.0 ± 0.4 to 2.3 ± 0.6 (p = 0.0057) and from 2.5 ± 0.7 to 6.6 ± 0.9 (p < 0.0001) when the trigger was administered at 6 h and 24 h post-mAb, respectively. Same day PET imaging clearly showed uptake in the tumor combined with a strongly reduced background due to the fast clearance of the released [89Zr]Zr-DFO-containing fragment from the circulation through the kidneys. Conclusions: This is the first demonstration of the use of trans-cyclooctene-tetrazine click-to-release chemistry to release a radioactive chelator from a mAb in mice to increase tumor-to-blood ratios. Our results suggest that click-cleavable radioimmunoimaging may allow for substantially shorter intervals in PET imaging with full mAbs, reducing radiation doses and potentially even enabling same day imaging.