Abstract LB379: A novel saponin-based adjuvant induces antigen cross-presentation in inflammatory type 2 conventional dendritic cells and promotes cytotoxic T lymphocyte activation through autophagy inhibition

Abstract Saponin-based adjuvants (SBA), such as QS-21, show profound abilities to enhance antibody production as well as to activate cellular immunity. They are being used in more than 70 cancer vaccines clinical trials, particularly due to the nature of activated cellular immune response; however, only a few evidence was given SBA could modulate antigen cross-presentation in dendritic cells to activate CD8+ T-cell response. It is still elusive how SBAs could promote T-cell cross-priming in vivo comprehensively. Our study has shown that SBA rendered a robust anti-tumor effect by boosting antigen-specific cytotoxic T lymphocyte (CTL) and Th1-inclined immunity. Vaccinated mice showed an increased proportion of CD26+CD64+CD172a+MAR-1+ inflammatory cDC2 (inf-cDC2) subset in the draining lymph node. In both interferon receptor signaling-deficiency KO mice (AGB6) models, losing the maturation of inf-cDC2 completely abolished the antigen specificity in CD8+ T cell activation, but there is no significant change of the antigen-specific CD8+ T cell while cDC1 is depleted in Batf3-/- mice. Collectively, SBA-recruited inf-cDC2 but not cDC1 is mainly dependent on promoting T cell cross-priming in vivo. At 24 h post-vaccination, SBA-induced inf-cDC2 was superior in priming to OT-1 CD8 T cells compared to sorted inf-cDC2 from antigen alone. It showed no difference from cross-priming in both cDC1 subsets, indicating SBA specifically raised the efficacy of cross-presentation in inf-cDC2. Newcoming RNA-Seq (RNA sequencing) analysis unraveled the potentials between cross-presentation induction and the hidden mechanisms associated with protein ubiquitination and autophagy pathway. The expression of LC3-II/LC3-I was significantly elevated upon SBA exposure, which a characteristic response is indicative of autophagy inhibition in immunoblotting. Our late-breaking results illustrated that inhibiting autophagy enhanced the efficacy of cross-priming under treatment of both early and late-stage autophagy inhibitors, such as 3-MA, Wortmannin, Bafilomycin A1, and Chloroquine. In summary, SBA-induced cross-presentation on murine inf-cDC2 was in a fashion of autophagy inhibition, leading to impairing antigen lysosomal degradation and antigen long-term cross-presentation to drive T cell cross-priming. To date, hundreds of clinical trials have been conducted using chloroquine and hydroxychloroquine as therapeutic strategies for autophagy inhibition; however, the induced immunomodulatory effects from trial results significantly differ between high and low doses. Dose-limiting hindrances qualify the efficacy in different cancer types, making it a massive challenge to achieve curative outcomes due to its off-targeting drawback. Therefore, we propose that prioritizing autophagy inhibition, specifically in cross-presenting cells, could effectively overcome recent clinical challenges. This study highlights a novel therapeutic strategy to efficiently harness bona fide cross-presenting DCs, bridging the gap between preclinical and clinical investigations in cancer vaccines. Citation Format: Yu-Chi Huang, Chun-Kai Chang, Mridula Prakash Menon, Pei-Chuan Chiang, Ching-Hui Tsai, Yeh Fong Tan, Chen-Yun Yeh, Pi-Hui Liang, Yungling Lee. A novel saponin-based adjuvant induces antigen cross-presentation in inflammatory type 2 conventional dendritic cells and promotes cytotoxic T lymphocyte activation through autophagy inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB379.