All-Hydrocarbon Macrocycle Enables Highly Potent Th1-Biased NKT Cell Agonists for Immunotherapy

Abstract Immunotherapy has markedly advanced cancer treatment, with Th1-biased natural killer T (NKT) cell agonists emerging as promising candidates owing to their ability to induce potent antitumor immunity. However, α-galactosylceramide (αGalCer), the prototypical NKT agonist, induces mixed Th1/Th2 responses with antagonistic cytokine effects, limiting therapeutic selectivity. The development of potent immunostimulatory agents promote Th1 polarization remains a major challenge. Herein, we convert the linear aliphatic chain of αGalCer into a conformationally constrained all-hydrocarbon macrocyclic scaffold, establishing the first report of a purely hydrocarbon macrocycle pharmacophore. Guided by the structural architecture of the CD1d antigen-binding groove, this design preorganizes the aliphatic chain to optimize A′ pocket occupancy, reduce entropic penalties, and enhance binding stability. αGalCer macrocyclized (GCC) analogs GCC-1 and GCC-2 elicited potent Th1-biased responses, with an approximately 12-fold increase in in vivo IFN-γ production relative to αGalCer, establishing them as highly potent Th1-biased NKT cell agonists. They exhibited potent immunological activity as effective antitumor immunotherapeutic agents and antiviral SARS-CoV-2 vaccine adjuvants, while retaining robust responsiveness in human NKT cells. Collectively, these findings establish the all-hydrocarbon macrocycle as a rational conformational constraint strategy for selectively biasing NKT cell responses, with potential applications in cancer immunotherapy and vaccine development.