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

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 full-length recombinant trastuzumab and decaged efficiently. We further used this method to generate a functional ADC site-specifically modified with monomethyl auristatin F (MMAF).

An Efficient Opal-Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells.

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual-ncAA incorporation. Using these platforms, we site-specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site-specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.