Abstract: Monoclonal antibodies have significantly advanced cancer therapy, with over 570 antibody therapeutics in clinical trials and 79 FDA-approved for clinical use. They offer precise antigen density measurements and specific targeting of tumor cells. Although tumor accumulation can rapidly reach substantial levels within a few hours post-injection,¹ circulating activity persists for days due to their slow blood clearance profile, delaying optimal imaging contrast to 3-7 days post-injection. This prolonged circulation not only adds to background and blood pool signal resulting in low-quality images, but also increases the radiation dose to healthy organs.² In this study, we explored the application of biorthogonal chemistry to tackle the challenges associated with antibody-based imaging using the CD20-targeting antibody rituximab. Our objective is to introduce flexibility into antibody imaging, allowing high-contrast imaging at any chosen time point following antibody administration.

This method, which we term a Tetrazine Knock-Out (TKO), is rooted in transcyclooctene-tetrazine (TCO-Tz) iDDEA chemistry, entails the straightforward modification of antibodies with a TCO linker. This linker is then conjugated to different radioisotopes, including Ga-68 and Zr-89. For the Ga-68 study, I45-CD20⁺ tumors were grown in mice for approximately 2 weeks prior to the administration of [⁶⁸Ga]Ga-NOTA-TCO-rituximab, which was allowed to accumulate in the tumors for 2 h. At this point, tetrazine (50 mg/kg) or vehicle (5% EtOH/PBS) was injected intravenously, followed by a 1 h wait before performing PET/BioD. Ex vivo biodistribution showed a 50-60% decrease in signal in the blood as well as in other background organs such as the heart, spleen, bone, and lymph nodes.

We also used a clinically relevant model of B-cell lymphoma to investigate the TKO approach using Zr-89. Nude mice bearing Raji tumors, grown for approximately 3 weeks, were administered [⁸⁹Zr]Zr-DFO-TCO-rituximab. After 24 h, an intravenous injection of tetrazine or a vehicle was performed, followed by imaging and biodistribution. A substantial reduction (56%) in blood radioactivity and a decline (27%) in liver uptake upon Tz treatment were noticed. A decrease in activity was also seen in lymph nodes (62%), and small and large intestines (45% and 23%, respectively). Importantly, tumor-associated activity remained unaffected, yielding high-contrast visualization of the tumor in the Tz-treated group.

Tz reacted with the radiolabeled antibodies in circulation. During this reaction, small molecules bearing the radioisotope became detached from the antibody, and the liberated radioactivity was rapidly cleared from the bloodstream, accumulating in the bladder within 10 minutes. In both cases, the TKO strategy resulted in a 50-60% decrease in radioactivity in non-target organs following a single dose of Tz injection. Although, in the I45-CD20⁺ tumor model with [⁶⁸Ga]Ga-labeled rituximab, a decrease in tumor uptake was observed in Tz-treated groups, likely due to the slower internalization rate of the radioimmunoconjugates into the engineered mesothelioma cells. However, in Raji xenograft models with [⁸⁹Zr]Zr-labeled rituximab, no such decrease in tumor uptake was noticed, and the tumor-to-background ratio increased by more than 2-fold, a difference easily observed on PET images.

Thus, the TKO approach can improve the imaging contrast of radioimmunoconjugates at different time points. This approach holds significant promise for advancing cancer imaging and diagnostics, as well as radiotherapy delivered by biologic agents.

Acknowledgements: We would like to thank the Eric Blankemeyer (SAIF), Cyclotron and Radiochemistry Facility, the Flow Cytometry and Cell Sorting Facility for making this research possible.

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