Antibody-drug conjugates (ADCs) showed strong anticancer efficacy in the clinic. However, the current conventional
technologies generate conjugates with undefined attachment sites and heterogeneous profiles containing different sub-populations,
leading to potential off-target toxicity. In order to reduce the variability and heterogeneity associated with the ADCs generated using
conventional technologies, several site-specific antibody-drug conjugation strategies were developed for the next generation of ADCs.
These strategies include cysteine-targeted conjugation by engineering a free cysteine into the antibody or by placing a thiol bridge on
cysteines in hinge disulfides. Glutamine-targeted conjugation was also demonstrated by coupling the drug-linker to glutamine residues
through an engineered glutamine tag or a native glutamine, as well as an additionally introduced glutamine residue in aglycosylated
antibody mutant using microbial transglutaminase. The site-specific conjugation of drug-linker to antibody carbohydrates was developed
either through metabolic engineering or a chemo-enzymatic approach. Other amino acids, such as unnatural amino acids or amino acid
derivatives introduced through protein engineering, have also been shown to be efficient targets for site-specific conjugation. The sitespecific
ADCs with homogeneous profiles and well-defined conjugation sites were obtained using these second generation ADC methods
and showed potent in vitro cytotoxicity and strong in vivo antitumor activity. These results suggest that newly developed site-specific
conjugation technologies can potentially be applied in producing the next generation ADC for cancer treatment in the clinic with high
Keywords: Antibody-drug conjugate; conjugation through unnatural amino acids or amino acid derivatives, glutamine conjugation,
glycoconjugation, lysine conjugation, site-specific conjugation, cysteine conjugation.
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