Antibody glyco-optimization and site-specific conjugation enhance the immune-stimulating activity of antibody-TLR ligand conjugates.

Immune-stimulating antibody conjugates (ISACs) represent an emerging class of immunotherapeutics that combine tumor-targeted antibodies with innate immune agonists. However, current ISAC platforms often rely on heterogeneous conjugation strategies and poorly defined antibody architectures, making it difficult to disentangle how Fc glycosylation, payload presentation, and linker design collectively govern immune activation. Here, we report a dual-enzymatic antibody-editing strategy that integrates Fc glyco-engineering with site-specific payload conjugation to construct structurally defined immune-stimulating antibody conjugates. Using microbial transglutaminase-mediated conjugation at the Fc Q295 residue together with Endo-S2-mediated Fc glycan remodeling at N297, we generated trastuzumab-based ISACs with defined Fc glycoforms and linker architectures. Systematic evaluation reveals that Fc glycosylation state and payload presentation act as orthogonal and synergistic determinants of immune activation. In particular, an intact and afucosylated Fc glycan is essential for robust tumor-cell killing and cytokine induction, while cleavable linkers further enhance innate immune activation through efficient intracellular release of the TLR7/8 agonist. This work establishes a versatile chemical strategy for controlling antibody architecture and immune activation, providing a broadly applicable platform for the rational design of next-generation antibody therapeutics.