Modification of Cysteine-Substituted Antibodies Using Enzymatic Oxidative Coupling Reactions

Abstract

Cysteines are routinely used as site-specific handles to synthesize antibody–drug conjugates for targeted immunotherapy applications. Michael additions between thiols and maleimides are some of the most common methods for modifying cysteines, but these functional groups can be difficult to prepare on scale, and the resulting linkages have been shown to be reversible under some physiological conditions. Here, we show that the enzyme tyrosinase, which oxidizes conveniently accessed phenols to afford reactive ortho-quinone intermediates, can be used to attach phenolic cargo to cysteines engineered on antibody surfaces. The resulting linkages between the thiols and ortho-quinones are shown to be more resistant than maleimides to reversion under physiological conditions. Using this approach, we construct antibody conjugates bearing cytotoxic payloads, which exhibit targeted cell killing, and further demonstrate this method for the attachment of a variety of cargo to antibodies, including fluorophores and oligonucleotides.

Recent Technological and Intellectual Property Trends in Antibody–Drug Conjugate Research

“…Site-specific conjugation methods present a groundbreaking approach in ADC development, aiming to precisely attach the payload at specific locations and overcome challenges associated with traditional coupling methods. For example, the introduction of engineered reactive cysteine residues selectively inserted at specific positions enables precise conjugation at that site, enhancing the homogeneity of ADCs and providing tunable reactivity through the alteration of the modification site [ 32 , 33 , 34 ]. In enzymatic conjugation methods, a variety of enzymes, such as bacterial-derived formyl glycine-generating enzymes, transglutaminases, glycotransferases, and sortases, have been utilized for tag-free antibody modification techniques [ 35 , 36 , 37 , 38 , 39 ].…”

Site-selective radiolabeling using mushroom tyrosinase and the strain-promoted oxidation-controlled 1,2-quinone cycloaddition

“…11 While the synthesis of ADCs using this bioconjugation strategy has previously been reported, this work represents the rst application of this technology to radiopharmaceutical chemistry andmore importantlythe rst in vivo evaluation of any immunoconjugate synthesized in this manner. [12][13][14] Indeed, we contend that this strategy represents a step forward compared to extant chemoenzymatic approaches to radiolabeling because it (a) is faster, (b) involves fewer steps, (c) relies on cheaper and easier-to-make reagents, and (d) employs more widely used and commonly available enzymes.…”