A Branched Tetraspecific Nanobody Reprograms the Tumor Glyco-Immune Interface through Coupled Protein–Glycan Checkpoint Modulation

Abstract Glycan immune checkpoints cooperate with canonical protein checkpoints to drive immune suppression at the tumor–immune interface. However, current tumor immunotherapies typically target these pathways separately, and coordinated regulation of glycan- and protein-mediated signaling remains challenging because of their spatial and functional interdependence. Here, we developed a branched tetraspecific nanobody for simultaneous protein checkpoint blockade and localized glycocalyx remodeling on both tumor cells and immune cells. Through SpyTag/SpyCatcher-mediated bioorthogonal ligation, a sialidase module, anti-CTLA-4 and anti-PD-L1 nanobodies, and a pharmacokinetic extension domain were chemically integrated into a branched four-arm macromolecular framework. This branched architecture minimizes steric interference between functional modules while maintaining their independent activities and target accessibility at the tumor–immune interface. As a result, the branched tetraspecific nanobody restores the effector functions of T cells, NK cells, and macrophages, thereby enhancing antitumor immunity. In multiple murine tumor models, including large, established tumors, this nanobody induced complete tumor regression in a subset of mice and generated a durable antitumor immune memory. These results establish branched multivalent macromolecular architectures as a strategy for coordinated glycan and protein immune checkpoint modulation and glycoimmunology reprogramming.