Development of Efficient Chemistry to Generate Site-Specific Disulfide-Linked Protein– and Peptide–Payload Conjugates: Application to THIOMAB Antibody–Drug Conjugates

Conjugation of small molecule payloads to cysteine residues on proteins via a disulfide bond represents an attractive strategy to generate redox-sensitive bioconjugates, which have value as potential diagnostic reagents or therapeutics. Advancement of such “direct-disulfide” bioconjugates to the clinic necessitates chemical methods to form disulfide connections efficiently, without byproducts. The disulfide connection must also be resistant to premature cleavage by thiols prior to arrival at the targeted tissue. We show here that commonly employed methods to generate direct disulfide-linked bioconjugates are inadequate for addressing these challenges. We describe our efforts to optimize direct-disulfide conjugation chemistry, focusing on the generation of conjugates between cytotoxic payloads and cysteine-engineered antibodies (i.e., THIOMAB antibody–drug conjugates, or TDCs). This work culminates in the development of novel, high-yielding conjugation chemistry for creating direct payload disulfide connec...