A Common Small-Molecule Inhibitor Targeting Multiple Siglecs Associated with Immune Checkpoint Suppression of Cytotoxicity and Phagocytosis.

Immune checkpoints are key regulators of the immune system that cancer cells exploit to evade immune surveillance. While these pathways have been targeted with antibody therapies such as PD-1/PD-L1 inhibitors, other inhibitory receptors, like sialic acid-binding immunoglobulin-type lectins (Siglecs), remain clinically underexplored. Among the many Siglecs on immune cells that interact with sialosides on cancer cells, Siglec-9 and Siglec-10 are prominently expressed on macrophages within the tumor microenvironment, functioning as key inhibitory immune checkpoints that suppress macrophage-mediated phagocytosis and cytotoxicity. Similarly, Siglec-7 and Siglec-9 are expressed on NK cells, acting as primary immune checkpoints that inhibit cellular cytotoxicity. Since all Siglecs recognize sialic acids, we sought to identify a common small-molecule inhibitor to target these receptors for immunotherapy. We first chemoenzymatically synthesized more than 60 glycans, including multiantennary glycans with terminal sialic acids, and assembled them onto a glycan microarray to screen for high-affinity binders to these Siglecs. We identified several strong binders in these array assays; among them, a truncated glycolyl sialoside (R3) was found to strongly inhibit the interaction of Siglec-10 with HER2-low MCF-7 cells, substantially enhancing macrophage-mediated phagocytosis and cytotoxicity. Notably, R3 also interacts significantly with other inhibitory immune checkpoint receptors, including Siglec-5, -7, -9, -11, and -15, thereby relieving Siglec-mediated suppression and enhancing NK cell-mediated cytotoxicity in cell-based assays. Structural studies are underway to investigate the binding mode of R3 with these Siglecs to guide further optimization toward a common small-molecule inhibitor targeting multiple inhibitory glyco-immune checkpoints for cancer immunotherapy.