%0 Journal Article %A Amita Datta-Mannan %A Bruce A. Molitoris %A Yiqing Feng %A Michelle M. Martinez %A Ruben M. Sandoval %A Robin M Brown %A Daniel Merkel %A Johnny E. Croy %A Kenneth W. Dunn %T Intravital microscopy reveals unforeseen biodistribution within the liver and kidney mechanistically connected to the clearance of a bifunctional antibody %D 2022 %R 10.1124/dmd.122.001049 %J Drug Metabolism and Disposition %P DMD-AR-2022-001049 %X Bifunctional antibody therapeutics offer the potential for novel functionalities beyond those of the individual mono-specific entities. However, combining these entities into a single molecule can have unpredictable effects, including changes in pharmacokinetics that limit the compound's therapeutic profile. A better understanding of how molecular modifications impact in vivo tissue interactions could help inform bifunctional antibody design. The present studies were predicated on the observation that a bifunctional antibody (BfAb) designed to have minimal off-target interactions cleared from the circulation twice as fast as the monoclonal antibody (mAb) from which it was derived. The present study leverages the spatial and temporal resolution of intravital microscopy (IVM) to identify cellular interactions that may explain the different pharmacokinetics of the two compounds. Disposition studies of mice demonstrated that radiolabeled compounds distributed similarly over the first 24 hours, except that BfAb accumulated ~2-3-fold more than mAb in the liver. IVM studies of mice demonstrated that both distributed to endosomes of liver endothelia, but with different kinetics; whereas mAb accumulated rapidly within the first hour of administration, BfAb accumulated only modestly during the first hour, but continued to accumulate over 24 hours, ultimately reaching levels similar to those of the mAb. Although neither compound was freely filtered by the mouse or rat kidney, BfAb (but not mAb) was found to accumulate over 24 hours in endosomes of proximal tubule cells. These studies demonstrate how IVM can be used as a tool in drug design, revealing unpredicted cellular interactions that are undetectable by conventional analyses. Significance Statement Bifunctional antibodies offer novel therapeutic functionalities beyond those of the individual mono-specific entities. However, combining these entities into a single molecule can have unpredictable effects, including undesirable changes in pharmacokinetics. Studies of the dynamic distribution of a bifunctional antibody and its parent monoclonal antibody presented here demonstrate how intravital microscopy can expand our understanding of the in vivo disposition of therapeutics, detecting off-target interactions that could not be detected by conventional pharmacokinetics approaches nor predicted by conventional physicochemical analyses. %U https://dmd.aspetjournals.org/content/dmd/early/2022/12/02/dmd.122.001049.full.pdf