A Genetically Encoded Photocaged Cysteine for Facile Site-Specific Introduction of Conjugation-Ready Thiol Residues in Antibodies

Abstract

Antibody−drug conjugates (ADCs) have emerged as a powerful class of anticancer therapeutics that enable the selective delivery of toxic payloads into target cells. There is increasing appreciation for the importance of synthesizing such ADCs in a defined manner where the payload is attached at specific permissive sites on the antibody with a defined drug to antibody ratio. Additionally, the ability to systematically alter the site of attachment is important to fine-tune the therapeutic properties of the ADC. Engineered cysteine residues have been used to achieve such site-specific programmable attachment of drug molecules onto antibodies. However, engineered cysteine residues on antibodies often get "disulfide-capped" during secretion and require reductive regeneration prior to conjugation. This reductive step also reduces structurally important disulfide bonds in the antibody itself, which must be regenerated through oxidation. This multistep, cumbersome process reduces the efficiency of conjugation and presents logistical challenges. Additionally, certain engineered cysteine sites are resistant to reductive regeneration, limiting their utility and the overall scope of this conjugation strategy. In this work, we utilize a genetically encoded photocaged cysteine residue that can be site-specifically installed into the antibody. This photocaged amino acid can be efficiently decaged using light, revealing a free cysteine residue available for conjugation without disrupting the antibody structure. We show that this ncAA can be incorporated at several positions within fulllength recombinant trastuzumab and decaged efficiently. We further used this method to generate a functional ADC site-specifically modified with monomethyl auristatin F (MMAF).

Late-Stage Desulfurization Enables Rapid and Efficient Solid-Phase Synthesis of Cathepsin-Cleavable Linkers for Antibody–Drug Conjugates

“…Antibody–drug conjugates (ADCs) have rapidly emerged as a leading class of drugs in oncology, capitalizing on the precision of monoclonal antibodies (mAbs) and the potency of cytotoxic drugs to effectively target and eliminate cancer cells. − ADCs are composed of three key elements: (i) a mAb that selectively binds to an antigen on the tumor cell surface, (ii) an anticancer cytotoxic payload that is generally too toxic to be administered systemically, and (iii) a linker that provides a bridge between the payload and mAb and defines the payload release rate, both on- and off-target (Figure ). − …”