Orthogonal Conjugation via Endoglycosidase and Microbial Transglutaminase Enables Efficient Synthesis of Dual-Payload Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are an important class of targeted therapeutics that leverage the specificity of antibodies to deliver highly cytotoxic agents to antigen-expressing cancer cells. Site-specific conjugation strategies are preferred over conventional stochastic methods because they yield homogeneous ADCs with well-defined structures and improved pharmacological properties. Here, we report an orthogonal antibody conjugation strategy that combines microbial transglutaminase (mTG)-catalyzed transglutamination with endoglycosidase S2 (Endo-S2)-catalyzed Fc glycan remodeling, enabling the site-specific installation of two distinct payloads or ligands within the Fc domain. We found that mTG exhibits prominently higher activity toward Endo-S2 deglycosylated antibody substrate bearing a residual Fucα1,6GlcNAc disaccharide at the N297 glycosylation site than the fully deglycosylated antibody generated by PNGase F treatment. This preference allows for efficient and selective introduction of a tag or cytotoxic drug at the Q295 residue of the Fc domain. Conversely, Endo-S2-mediated glycan remodeling at N297 was found to tolerate prior modification at Q295 to introduce a second payload, despite the close spatial proximity of the two conjugation sites. Notably, mTG retained robust transglutamination activity even when an azide-tetrasaccharide was present at N297, indicating a broader substrate tolerance than previously reported. Collectively, these results demonstrate that the combined and orthogonal use of mTG and Endo-S2 enzymes provides a versatile and practical platform for the construction of site-specific dual-payload antibody-drug conjugates.