Novel biologic therapeutics targeting vacuolar ATPase localized on plasma membrane

The long-term goal of this project is to validate plasma membrane vacuolar ATPase (pmVATPase) as a therapeutic target and to develop novel antibodies targeting pmVATPase as anti-cancer therapy. Vacuolar ATPase (V-ATPase) is a complex molecular machine that transports proton ions across organelle membranes or the plasma membrane. An increase in the abundance of pmVATPase are associated with KRAS-driven, challenging cancers, and it promotes tumor cell survival through multiple mechanisms. Currently available inhibitors against V-ATPase are not selective to pmVATPase because they are cell permeable and hence inhibit V-ATPase at intracellular membranes as well as pmVATPase. This lack of subcellular selectivity compromises V-ATPase functions that are crucial for normal cell functions such as endocytosis, lysosomal degradation and autophagy, leading to high toxicity. We propose to utilize antibodies, which are inherently cell impermeable, to achieve higher selectivity toward pmVATPase and to minimize toxicity. Early three-dimensional structures showed that pmVATPase has minimal exposure on the extracellular side of the plasma membrane, suggesting that pmVATPase is an extremely challenging antigen for developing antibodies. However, recent structures of complete mammalian V-ATPases revealed that the ATP6AP1 (also known as Ac45) subunit presents a sizeable globular domain on the extracellular surface. Unlike previously targeted subunits, ATP6AP1 is common in all populations of pmVATPase (harboring different subunit a isotypes) and crucial for localization of V-ATPase on the plasma membrane. Based on these data, we hypothesize that ATP6AP1 presents surfaces (epitopes) that are more accessible by antibodies, and that we can identify anti-ATP6AP1 antibodies that can selectively inhibit pmVATPase. We propose the following Aims. 1. We will develop anti-ATP6AP1 antibodies that recognize pmVATPase on cells. We have already produced a water-soluble form of the ATP6AP1 globular domain and generated a panel of antibodies against it, which has substantially de-risked this project. 2. We will develop a strategy to selectively inhibit or kill cancer cells that have high levels of pmVATPase with anti-ATP6AP1 antibodies. If successful, this project will establish pmVATPase as an actionable therapeutic target and advance development of novel therapeutics against multiple cancer types.