Enzyme-Responsive Linkers Enable In Vivo Clearance of Circulating Radioimmunoconjugates

The application of full-length antibodies as radioimmunoconjugates necessitates a prolonged period between administration and imaging to achieve optimal target visualization. Transcyclooctene-tetrazine chemistry, which enables displacement of radioisotopes from immunoconjugates, can enhance in vivo imaging of target signal relative to background through rapid clearance of circulating, nontargeted radioactivity. An alternative approach, which may accelerate clinical translation, repurposes recombinant enzymes that are approved by the Food and Drug Administration for therapeutic use to trigger cleavage of radioimmunoconjugate linkers. Methods: Radioimmunoconjugate linkers cleavable by β-glucuronidase, carboxypeptidase G2, and tissue plasminogen activator were designed, synthesized, and conjugated to the CD20-targeting antibody rituximab using NOTA or desferrioxamine (DFO) to chelate ⁶⁸Ga or ⁸⁹Zr. CD20-positive Raji cells were incubated with radioimmunoconjugate followed by a cleaving enzyme. Radioimmunoconjugate uptake was measured in CD20-positive Raji cells to confirm immunoreactivity and accumulation in target cells. Biodistribution studies were performed in healthy mice after administration of radioimmunoconjugate and either cognate enzyme or saline to quantify the clearance of radioactivity from the circulation. [⁸⁹Zr]DFO-methotrexate-rituximab was administered to CD1 nude mice carrying subcutaneous Raji xenografts, followed by carboxypeptidase G2 or saline, with subsequent PET imaging and biodistribution studies performed to assess enzyme-mediated changes in imaging contrast. Results: Radioimmunoconjugates were successfully prepared with greater than 95% radiochemical purity. In vitro uptake studies in Raji cells confirmed internalization of radioimmunoconjugates after antigen binding. After internalization, radioimmunoconjugates were no longer susceptible to enzymatic cleavage. Intravenous administration of carboxypeptidase G2 and β-glucuronidase cleared approximately 50% and 70% of injected radioactivity, respectively, from the circulation of non-tumor-bearing mice. In immunocompromised mice bearing subcutaneous Raji xenografts, administration of [⁸⁹Zr]DFO-MTX-rituximab followed by carboxypeptidase G2 enhanced the tumor-to-blood imaging signal by greater than 40% compared with saline-treated controls. Conclusion: The use of Food and Drug Administration-approved enzymes to clear radioactivity from circulating radioimmunoconjugates showed significant efficacy in vivo, providing proof of concept for future clinical applications, particularly in hematologic malignancies.