DiPODS: A Reagent for Site-Specific Bioconjugation via the Irreversible Rebridging of Disulfide Linkages

Abstract

Chemoselective reactions with thiols have long held promise for the site-specific bioconjugation of antibodies and antibody fragments. Yet bifunctional probes bearing monovalent maleimideslong the “gold standard” for thiol-based ligationsare hampered by two intrinsic issues: the in vivo instability of the maleimide–thiol bond and the need to permanently disrupt disulfide linkages in order to facilitate bioconjugation. Herein, we present the synthesis, characterization, and validation of DiPODS, a novel bioconjugation reagent containing a pair of oxadiazolyl methyl sulfone moieties capable of irreversibly forming covalent bonds with two thiolate groups while simultaneously rebridging disulfide linkages. The reagent was synthesized from commercially available starting materials in 8 steps, during which rotamers were encountered and investigated both experimentally and computationally. DiPODS is designed to be modular and can thus be conjugated to any payload through a pendant terminal primary amine (DiPODS–PEG4–NH2). Subsequently, the modification of a HER2-targeting Fab with a fluorescein-conjugated variant of DiPODS (DiPODS–PEG4–FITC) reinforced the site-specificity of the reagent, illustrated its ability to rebridge disulfide linkages, and produced an immunoconjugate with in vitro properties superior to those of an analogous construct created using traditional stochastic bioconjugation techniques. Ultimately, we believe that this work has particularly important implications for the synthesis of immunoconjugates, specifically for ensuring that the attachment of cargoes to immunoglobulins is robust, irreversible, and biologically and structurally benign.

Synthesis and Comparative In Vivo Evaluation of Site-Specifically Labeled Radioimmunoconjugates for DLL3-Targeted ImmunoPET

“…In this investigation, we have harnessed an emergent, thiol-reactive bioconjugation reagent based on a p henyl o xa d iazolyl methyl s ulfone (PODS) core to create a site-specifically modified 89 Zr-radioimmunoconjugate as a companion diagnostic for a DLL3-targeted antibody–drug conjugate (ADC). PODS-based reagents react quickly, cleanly, and (unlike maleimides) irreversibly with thiols (Figure A). − Even more importantly, we have previously demonstrated that the site-selective modification of wild-type antibodies with PODS-bearing chelators produces 177 Lu- and 89 Zr-labeled radioimmunoconjugates with high stability and excellent in vivo performance (Figure B) …”

The Chemistry Behind ADCs

“…They reported a superior stability compared to cysteine maleimide conjugates in human plasma [ 118 , 119 ]. Inspired by this report, Zeglis designed the reagent DiPODS for site-specific, irreversible bioconjugation on native antibodies fragment Fab [ 120 , 121 , 122 ]. This reagent contains two oxadiazolyl methyl sulfone moieties connected by a phenyl group.…”

All-in-one disulfide bridging enables the generation of antibody conjugates with modular cargo loading

“…29–31 These linkers contain two cysteine-reactive groups that may undergo reaction with reduced interchain disulfides in an IgG molecule to effect covalent rebridging of the antibody chains in a site-selective fashion. Next-generation maleimides (NGMs), 32,33 pyridazinediones, 34 bissulfones, 35 divinylpyrimidine (DVP) 36–39 and a variety of other reagents 40–44 have been used to modify antibodies in this way. However, the utility of this approach is currently hampered by the formation of fragmented “half-antibody” species during bioconjugation, which is the result of non-native intrachain cross-linking of the cysteine residues in the hinge region of the antibody (Fig.…”