Homogeneous Antibody-Drug Conjugates via Glycoengineering.

Conventional antibody-drug conjugates (ADCs) randomly assemble small-molecule drugs onto Lys or Cys residues of a tumor-targeting antibody, featured with heterogeneity in payload numbers and conjugation positions. Glycosite-specific ADCs (gsADCs) link payload drugs onto IgG Fc N-glycans with high homogeneity that facilitates structural optimization and quality control for ADC drug development. In this protocol, we report two strategies for preparation of homogeneous ADCs via chemoenzymatic glycoengineering. First, an azido-tagged unnatural N-glycan substrate is transferred onto Fc glycosites of a therapeutic antibody through Endo-S-catalyzed glycoremodeling, followed by click reaction with an alkyne-tagged payload drug to give a well-defined gsADC. In an alternative way, glycoengineering of antibody with a natural sialylated N-glycan and successive selective oxidation of sialic acid moieties using sodium periodate provided an aldehyde handle on the glycans for conjugation with an aminooxy-assembled payload. These two strategies both enable gsADCs with high homogeneity in their conjugation sites, payload numbers, and glycoforms, which are characterized of a single mass under mass-spectral detection.