Development of a Cysteine-Conjugatable Disulfide FRET Probe: Influence of Charge on Linker Cleavage and Payload Trafficking for an Anti-HER2 Antibody Conjugate

Abstract

Disulfide-linked bioconjugates allow the delivery of pharmacologically active or other cargo to specific tissues in a redox-sensitive fashion. However, an understanding of the kinetics, subcellular distribution, and mechanism of disulfide cleavage in such bioconjugates is generally lacking. Here, we report a modular disulfide-linked TAMRA-BODIPY based FRET probe that can be readily synthesized, modified, and conjugated to a cysteine-containing biomolecule to enable real-time monitoring of disulfide cleavage during receptor-mediated endocytosis in cells. We demonstrate the utility of this probe to study disulfide reduction during HER2 receptor-mediated uptake of a Cys-engineered anti-HER2 THIOMAB antibody. We found that introduction of positive, but not negative, charges in the probe improved retention of the BODIPY catabolite. This permitted the observation of significant disulfide cleavage in endosomes or lysosomes on par with proteolytic cleavage of a similarly charged valine–citrulline peptide-based probe. In general, the FRET probe we describe should enable real-time cellular monitoring of disulfide cleavage in other targeted delivery systems for mechanistic or diagnostic applications. Furthermore, modifications to the released BODIPY moiety permit evaluation of physicochemical properties that govern lysosomal egress or retention, which may have implications for the development of next-generation antibody–drug conjugates.

Reduction-Activatable Fluorogenic Nanobody for Targeted and Low-Background Bioimaging

“…Upon endocytic trafficking, the D-body was activated in the lysosome and generated bright red fluorescence that merged well with the green fluorescence of Lyso-Tracker Green (Figure h). Of note, the lysosomal compartments are reductive due to the presence of redox enzymes such as gamma-interferon-inducible lysosomal thiol reductase and reducing species such as cysteine, leading to efficient disulfide cleavage. ,− In this case, the disulfide in the D-body was efficiently cleaved in lysosomes within 1 h, and the released NBO dye was retained in lysosomes after 6 h (Figure S12).…”

“…15 Combined with the stimulus sensitivity of SMFPs, the exquisite specificity of antibodies renders improved selectivity and biocompatibility. In this scheme, a few fluorogenic antibodies have been developed via (1) direct labeling of the antibody with welldesigned SMFPs, 16 (2) conjugation of the fluorophore and quencher FRET pairs to nearby sites of the antibody, 17 and (3) design quench bodies in which the fluorescence is quenched via the PET interaction of the fluorophore with nearby tryptophan residues. 18 However, except for the challenges that lie in complicated fluorophore manipulation and tedious antibody bioconjugation, the cell internalization, fluorogenicity, and biodistribution of these probes are often compromised when coupled to the bulk structure of the antibody.…”

Cathepsin B as a target in cancer therapy and imaging

“…At present, in the research of theranostic ADC molecules, the dual-isotope labeling method and the use of fluorescent probes with FRET reporter groups to both the linker and payload are known to be typical design approaches. 104,105 However, both these methods have many disadvantages, including biological damage caused by radiation elements and the FRET design significantly alters the physical and chemical properties of the ADC, and so may not reflect the actual release and metabolism of the payload in the body.…”

Stimulus-Responsive Nanomedicines for Disease Diagnosis and Treatment

“…Philip et al Proposed the concept of self-immolative structures in 1981 that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [ 157 ]. Self-immolative linkers have subsequently been widely used in prodrug design [ 158 , 159 ], sensors [ 160 , 161 ], drug delivery [ 162 , 163 ] and other applications.…”