Abstract Cancer therapy has been revolutionized by inhibiting immune checkpoints to harness the power of the immune system in fighting cancer. However, the majority of patients are resistant to the current immuno-oncology drugs. There is a strong need to identify novel mechanisms of immune evasion in cancer and explore novel therapeutic modalities. Recently, glyco-immune checkpoints have been identified as an important modulator in cancer immune escape. Upon ligation of cancer-specific sialylated glycans, glyco-immune checkpoints suppress multiple facets of cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation anticancer T-cell immunity. However, due to the complexity and heterogeneity of cancer sialylated glycans and the profound species differences of the glyco-immune checkpoints between human and mouse, this immunomodulatory pathway remains unexplored for therapeutic interventions. Here we described a novel multifunctional antibody-like therapeutic modality, named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), to target this glyco-immune checkpoint axis. EAGLE overcomes the complexity of cancer glycans and specifically cleaves the immunosuppression-mediating key sugar moieties, the terminal sialic acids of various sialylated glycans on tumor cells. We hypothesized that EAGLE could block the immunosuppressive glyco-immune checkpoints in the tumor microenvironment and potentiate innate and adaptive immune responses to treat cancer. Indeed, systematic delivery of an EAGLE molecule decreased the levels of immunosuppressive sialic acids on tumor cells, and increased T-cell infiltration and activation in syngeneic tumor models. EAGLE treatment led to 50% complete regressions of established tumors as a monotherapy, and 100% cures in combination with anti-PD1 mAb. Furthermore, cured mice from EAGLE treatment completely rejected the rechallenge of tumor cells, suggesting that EAGLE induced antitumor immunologic memory. In summary, we demonstrated that this novel therapeutic modality, EAGLE, can release glyco-immune checkpoint-mediated immunosuppression in the tumor microenvironment and potentiate innate and adaptive antitumor immunity, which offers a novel and promising immunomodulatory strategy to treat cancer. This abstract is also being presented as Poster A91. Citation Format: Li Peng, Adam Petrone, Lizhi Cao, James Broderick. A novel immunomodulatory strategy of targeting glyco-immune checkpoints using EAGLE technology to treat cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR18.