RT Journal Article SR Electronic T1 Using antibody synergy to engineer a high potency biologic cocktail against C. difficile JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.12.21.473715 DO 10.1101/2021.12.21.473715 A1 Hui Zhao A1 Michael Tasch A1 Michael Dodds A1 Mesfin Gewe A1 Anissa Martinez A1 Melanie Hutton A1 Kristie Keeney A1 Alex Pollock A1 Ben Jester A1 Nhi Khuong A1 Mia Zhang A1 Colin Brady A1 Mark Heinnickel A1 Hannah Tabakh A1 Nathan Sanjaya A1 Kendra Cruickshank A1 Troy Paddock A1 Stacey Ertel A1 Sarah Struyvenberg A1 Jason Dang A1 Chelsea Shanitta A1 David Fletcher A1 Lauren Goetsch A1 Caitlin Gamble A1 Steven J. Mileto A1 Ryan Heselpoth A1 Dena Lyras A1 Craig A. Behnke A1 Vincent Fischetti A1 Brian Finrow A1 James M. Roberts YR 2021 UL http://biorxiv.org/content/early/2021/12/22/2021.12.21.473715.abstract AB We applied a mathematical framework originally used to model the effects of multiple inhibitors on enzyme activity to guide the development a therapeutic antibody cocktail, LMN-201, to prevent and treat C. difficile infection (CDI). CDI causes hundreds of thousands of cases of severe, often recurrent diarrhea and colitis in the United States annually and is associated with significant morbidity and mortality worldwide. Current therapies for preventing recurrent CDI are only partially successful, and there are no options available to prevent initial bouts of CDI in at-risk populations. Almost all antibody therapies have been developed and administered as monotherapies. Antibody cocktails are relatively rare even though they have the potential to greatly increase efficacy. One reason for this is our limited understanding of how antibody interactions can enhance potency, which makes it difficult to identify and develop antibodies that can be assembled into optimally effective cocktails. In contrast to the view that antibody synergies depend on unusual instances of cooperativity or allostery, we show that synergistic efficacy requires nothing more than that the antibodies bind independently to distinct epitopes on a common target. Therefore, synergy may be achieved much more readily than is generally appreciated. Due to synergy the LMN-201 antibody cocktail, which targets the C. difficile exotoxin B (TcdB), is 300- to 3000-fold more potent at neutralizing the most clinically prevalent TcdB toxin types than bezlotoxumab, the only monoclonal antibody currently approved for treatment or prevention of CDI. The efficacy of LMN-201 is further enhanced by inclusion of a phage-derived endolysin that destroys the C. difficile bacterium, and which therefore has a complementary mechanism of action to the antibody cocktail. These observations may serve as a paradigm for the development of high potency biologic cocktails against targets that have proven challenging for single-agent therapies.Competing Interest StatementJ. R. and B. F. are the founders and current employee of Lumen Bioscience, Inc. and each owns stock and stock options in Lumen. H. Z., M. T., M. D., M. G., A. M., K. K., A. P., B. J., N. K., M. Z., C. B., M. H., H. T., N. S., K. C., T. P., S. E., S. S., J. D., C. S., D. F., L. G., C. G., and C. B. are, or were, employees of Lumen; all current and former employees own stock or stock options in Lumen. Lumen has issued U.S. patents including U.S. patent nos. 10,131,870, 10,415,012, 10,336,982, 10,415,013, and pending U.S. patent applications including Nos. 16/570,520, 17/056,306, and 17/264,837 relating to its spirulina transformation, expression, delivery and protein engineering platform and certain research described in this article.