Antibodies have been explored for decades for the delivery of small molecule cytotoxins directly to diseased cells. In antibody-directed enzyme prodrug therapy (ADEPT), antibodies are armed with enzymes that activate nontoxic prodrugs at tumor sites. However, this strategy failed clinically due to off-target toxicity associated with the enzyme prematurely activating prodrug systemically. We describe here the design of an antibody-fragment split enzyme platform that regains activity after binding to HER2, allowing for site-specific activation of a small molecule prodrug. We evaluated a library of fusion constructs for efficient targeting and complementation to identify the most promising split enzyme pair. The optimal pair was screened for substrate specificity among chromogenic, fluorogenic, and prodrug substrates. Evaluation of this system on HER2-positive cells revealed 7-fold higher toxicity of the activated prodrug over prodrug treatment alone. Demonstrating the potential of this strategy against a known clinical target provides the basis for a unique therapeutic platform in oncology.

Reconstruction of Moving Morphology (XROMM), which uses videofluoroscopy and computed tomography to reconstruct 3D skeletal shape and movement 20,21. Here, we used XROMM to measure the flexion of the vertebral column and the 3D rotations of ribs relative to their corresponding vertebrae (i.e. at the costovertebral joints) during slow treadmill locomotion in savannah

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT ® , Promega ® ) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production. K E Y W O R D Saffinity chromatography, enzyme complementation, molecular tag, protein production, split-enzyme, split-luciferase

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