Development of a Potent QS‐21‐Derived Adjuvant Photoaffinity Probe: Identification of Cathepsin D as the Molecular Receptor

The saponin natural product QS‐21 is a potent immunological adjuvant used in human vaccines to enhance the immune response but suffers from scarcity, instability, and dose‐limiting toxicity, precluding its advancement as a stand‐alone adjuvant. Further, putative cellular receptor(s) and its molecular mechanisms of action are unknown, which has prevented the rational design of saponin adjuvants, emphasizing the need for discovering these receptors and elucidating mechanisms. Herein, we have developed novel saponin photoaffinity probes for molecular target identification, merging synthetic chemistry, photoaffinity labeling, and chemical proteomics. One of these potent adjuvants generated superior antibody responses to QS‐21 with less toxicity and enabled identification of Cathepsin D (CatD) as the selective receptor driving saponin adjuvant effects. Binding to surface‐exposed CatD, lipid raft interactions, and lysosomal acidification were shown to be key for CatD‐mediated saponin‐induced immune activation, whereas neither photocrosslinking nor apparent binding was observed in comparative experiments with other lysosomal cathepsins, confirming the specificity of the interaction. These studies highlight the promise of this saponin probe as a potent vaccine adjuvant, unveiling CatD as the molecular target. This breakthrough sets the stage for elucidating the precise mechanisms of saponin immunopotentiation, facilitating rational adjuvant design and development of optimized adjuvant–antigen combinations for future vaccines.