© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into

Internalizing antibodies have great potential for the development of targeted therapeutics. Antibodies that internalize via the macropinocytosis pathway are particularly promising since macropinocytosis is capable of mediating rapid, bulk uptake and is selectively upregulated in many cancers. We hereby describe a method for identifying antibodies that internalize via macropinocytosis by screening phage-displayed single-chain antibody selection outputs with an automated fluorescent microscopy-based high-content analysis platform. Furthermore, this method can be similarly applied to other endocytic pathways if other fluorescent, pathway-specific, soluble markers are available.