A Common Small-molecule Blockade Targeting Multiple Siglecs Associated with Inhibitory Immune Checkpoint Suppression of Cytotoxicity and Phagocytosis

Immune checkpoints, such as the PD-1/PD-L1 and CTLA-4 pathways, are key regulators of the immune system that cancer cells exploit to evade immune surveillance. While these pathways have been successfully targeted with antibody therapies, other inhibitory receptors like sialic acidbinding immunoglobulin-type lectins (Siglecs) on immune cells which recognize sialosides on target cells remain underexplored in the clinic setting. For example, Siglec-9 and Siglec-10 are expressed on macrophages within the tumor microenvironment as key inhibitory immune checkpoints to suppress macrophage-mediated phagocytosis and cytotoxicity, while Siglec-7 and Siglec-9 are expressed on NK cells as primary immune checkpoints inhibiting NK cell-mediated cytotoxicity. To develop immunotherapies targeting these Siglecs and improving antibody efficacy, we sought to identify a common small-molecule inhibitor to target these Siglecs. We first synthesized more than 60 glycans, including single-chain and multi-antennary glycans with terminal N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), and assembled them as a glycan array to screen for high-affinity binders to Siglec-10. We identified several strong binders with nanomolar dissociation constant (Kd) in glycan array assays, among them a truncated glycolyl sialoside (R3) was found to strongly interact with Siglec-10 and interfere with the interaction of Siglec-10 with CD24 as well as MCF-7 cells and enhanced macrophagemediated phagocytosis and cytotoxicity in cell-based assays. Notably, this unnatural glycan interact significantly with inhibitory immune checkpoints Siglecs-5, 7, 9, 10, 11, 15 which are present on macrophages, while Siglec-7, Siglec-9 and to a less extent Siglec-10 are also found on NK cells, suggesting its potential to be developed as a common immune checkpoint blockade to enhance phagocytosis and cytotoxicity.