Site-specific antibody-drug conjugation through glycoengineering

Area of Science:

• Bioconjugation Chemistry
• Antibody Engineering
• Cancer Therapeutics

Background:

• Classical antibody-drug conjugate (ADC) preparation methods targeting amines or disulfides yield heterogeneous products with unstable linkages.
• Limitations in current ADC conjugation chemistries hinder reproducible drug loading and product profiles.

Purpose of the Study:

• To develop a novel, site-specific conjugation method for antibody-drug conjugates (ADCs) by utilizing native glycosylation sites.
• To address limitations of heterogeneity and linkage instability in conventional ADC preparation.

Main Methods:

• Enzymatic remodeling of native glycans at Asn-297 using galactosyl and sialyltransferases to introduce terminal sialic acids.
• Periodate oxidation of sialic acids to create aldehyde groups for subsequent oxime ligation with aminooxy-functionalized cytotoxic agents.
• Demonstration with trastuzumab and two cytotoxic agents, followed by analysis using hydrophobic interaction chromatography and LC-MS.

Main Results:

• Successful glyco-conjugation of approximately 1.6 cytotoxic agents per antibody molecule, correlating with sialic acid residues.
• Glyco-conjugated ADCs displayed target-dependent antiproliferative activity against antigen-positive tumor cells.
• Significant greater antitumor efficacy observed in a Her2-positive tumor xenograft model compared to naked antibodies.

Conclusions:

• Enzymatic glycan remodeling combined with oxime ligation offers an attractive strategy for generating ADCs with well-defined product profiles.
• This site-specific conjugation approach provides a viable alternative to methods requiring antibody sequence re-engineering or complex chemical processes.
• The developed method ensures reproducible drug loading and improved therapeutic efficacy for antibody-drug conjugates.