Modification of Cysteine-Substituted Antibodies Using Enzymatic Oxidative Coupling Reactions

Area of Science:

• Bioconjugation Chemistry
• Immunotherapy
• Enzymatic Synthesis

Background:

• Cysteines are crucial for site-specific antibody conjugation in targeted immunotherapy.
• Current methods like maleimide-thiol Michael additions face challenges in scalability and linkage stability.
• Existing linkages can be reversible under physiological conditions, limiting therapeutic efficacy.

Purpose of the Study:

• To introduce a novel enzymatic approach for antibody conjugation using tyrosinase.
• To develop more stable and reliable antibody-drug conjugates (ADCs) for improved targeted immunotherapy.
• To explore the versatility of this method for attaching various payloads to antibodies.

Main Methods:

• Utilized the enzyme tyrosinase to oxidize phenols into reactive ortho-quinone intermediates.
• Engineered cysteines on antibody surfaces for site-specific modification.
• Investigated the stability of the resulting thiol-ortho-quinone linkages under physiological conditions.
• Constructed antibody conjugates with cytotoxic payloads, fluorophores, and oligonucleotides.

Main Results:

• Demonstrated successful attachment of phenolic cargo to antibody cysteines via tyrosinase-mediated oxidation.
• Showcased that the thiol-ortho-quinone linkages exhibit enhanced resistance to reversion compared to maleimide linkages.
• Successfully synthesized antibody conjugates with cytotoxic payloads that effectively kill targeted cells.
• Validated the method for conjugating diverse cargo, including fluorophores and oligonucleotides, to antibodies.

Conclusions:

• The tyrosinase-based method offers a robust and scalable alternative for antibody conjugation.
• This enzymatic approach yields more stable antibody conjugates, improving their potential for targeted immunotherapy.
• The demonstrated versatility supports its application in developing advanced antibody-based therapeutics and diagnostics.