Abstract Cancer therapy has been revolutionized by the recent developments of immune-checkpoint inhibitors (ICI) to harness the power of the immune system in fighting cancer. However, most patients fail to have durable responses or become resistant to ICI, highlighting the need to identify new mechanisms of immune evasion in cancer and develop novel therapeutic modalities. Recently, the glyco-immune checkpoint axis (sialoglycan/Siglec pathway) has emerged as a new mechanism of immune regulation involving both innate and adaptive immunity and an important mechanism of cancer immune escape. Upon ligation of sialoglycan to ITIM-containing Siglecs on immune cells, this pathway suppresses multiple facets of anti-cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation of anti-cancer T cells. However, a therapeutic intervention of this axis remains a great challenge due to the overlapping and promiscuous receptor-ligand interactions between 15 Siglecs and dense array of various sialoglycans in humans. To overcome this hurdle and block the glyco-immune checkpoint axis, we describe here a new therapeutic modality named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which is antibody-like, multi-functional, and comprised of a tumor-associated antigen-binding moiety and a sialidase moiety, allowing selectively removing terminal sialic acids, the critical binding carbohydrate of Siglecs, from sialoglycans on tumor cells. We demonstrated that EAGLE decreased sialic acid levels of tumor cells and enhanced anti-tumor immune responses using multiple human system models mimicking immunosuppressive tumor microenvironment and immunocompetent syngeneic mouse tumor models. EAGLE treatment released cancer cell-mediated immunosuppression, restored dendritic cell functions, enhanced CD8+ T-cell proliferation/activation, and induced proinflammatory cytokines IFNγ, IL-17A, IL-2, IL-6, and TNFα in human coculture assays of cancer cells with dendritic cells or PBMC in the presence or absence of primary endothelial cells. Systematic administration of EAGLE increased tumor-infiltrating immune cells and led to significant anti-tumor activities with complete regressions as monotherapy in syngeneic mouse tumor models. Re-challenge experiments in cured mice from the EAGLE treatment resulted in a complete rejection of tumor cells, demonstrating that EAGLE induced anti-tumor immunological memory. We further revealed that the mechanism of action of EAGLE involved both innate and adaptive immunity because depleting macrophages or CD8+ T-cells decreased or abolished its efficacy. Moreover, EAGLE in combination with anti-PD1 mAb treatment achieved ~100% cures in syngeneic EMT6-Her2 models. In summary, EAGLE is a novel and promising immunomodulatory therapeutic modality inhibiting the glyco-immune checkpoints and has the potential to overcome resistance to current immunotherapies. Citation Format: Lizhi Cao, Adam Petrone, Wayne Gatlin, Jenny Che, Abhishek Das, Robert LeBlanc, Zakir Siddiquee, Sujata Nerle, Michal Stanczak, Michele Mayo, Lihui Xu, Karl Normington, Jeff Brown, Wei Yao, Carolyn Bertozzi, James Broderick, Heinz Läubli, Li Peng. A novel therapeutic modality of inhibiting the glyco-immune checkpoint axis to treat cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-109.