Monitoring In Vivo Performances of Protein-Drug Conjugates UsingSite-Selective Dual Radiolabeling and Ex Vivo Digital Imaging

In preclinical models, the development and opti-mization of protein-drug conjugates require accurate determinationof the plasma and tissue profiles of both the protein and itsconjugated drug. To this aim, we developed a bioanalytical strategybased on dual radiolabeling and ex vivo digital imaging. Bycombining enzymatic and chemical reactions, we obtainedhomogeneous dual-labeled anti-MMP-14 Fabs (antigen-bindingfragments) conjugated to monomethyl auristatin E where theprotein scaffold was labeled with carbon-14 (14C) and theconjugated drug with tritium (3H). These antibody-drugconjugates with either a noncleavable or a cleavable linker werethen evaluated in vivo. By combining liquid scintillation countingand ex vivo dual-isotope radio-imaging, it was possible not only tomonitor both components simultaneously during their circulation phase but also to quantify accurately their amount accumulatedwithin the different organs.

Automated summary

This article presents a bioanalytical strategy based on dual radiolabeling and ex vivo digital imaging for accurate determination of the plasma and tissue profiles of protein-drug conjugates. The strategy involves enzymatic and chemical reactions to obtain dual-labeled anti-MMP-14 Fabs conjugated to monomethyl auristatin E, allowing for simultaneous monitoring and accurate quantification of the components during circulation phase and accumulation in different organs.