Enhanced Antitumor Immunity of a Globo H‐Based Vaccine Enabled by the Combination Adjuvants of 3D‐MPL and QS‐21

Globo H, a specific carbohydrate antigen overexpressed on various human malignancies, has attracted considerable interest as an antigenic target for anticancer vaccine development. Despite several Globo H-based carbohydrate vaccines that have been designed, efficient access to Globo H hexasaccharide antigen and development of powerful adjuvants for enhancing antitumor immunity remain challenging. Herein, we reported a streamlined chemoenzymatic approach to prepare this hexasaccharide antigen, relying on chemical synthesis of Gb5 pentasaccharide by a stereoconvergent [2+3] strategy and subsequent enzymatic α-fucosylation to easily install α1,2-fucose residue. Separately, a modular assembly approach to efficiently synthesize 3-O-deacyl-monophosphoryl lipid A (3D-MPL) was developed by the integration of stereocontrolled glycosylation, regioselective acylation, site-specific phosphorylation, and facile global deprotection. After efficient construction of Globo H-CRM197 conjugate, we conducted systematic immunological evaluations of Globo H-CRM197 formulated with various adjuvants and adjuvant combinations, comprising saponin QS-21, synthetic 3D-MPL and α-galactosylceramide derivative S34. The results revealed that Globo H-CRM197 conjugate adjuvanted with QS-21 and 3D-MPL elicited robust IgG2a and IgG3 antibody responses and Th1 cellular immunity in mice. Moreover, antibodies induced by this formulation effectively bound to Globo H-positive MCF-7 cancer cells and exhibited superior complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis, holding promise for further development of effective anticancer vaccines.