Responsive Antibody Conjugates Enable Quantitative Determination of Intracellular Bond Degradation Rate.

Degradable crosslinkers that respond to intracellular biological stimuli are a critical component of many drug delivery systems. With numerous stimuli-responsive drug delivery systems in development, it is important to quantitatively study their intracellular processing. Herein we report a framework for quantifying the rate of intracellular bond degradation in the endocytic pathway. Toward this end, we devised and synthesized a reduction-sensitive FRET-based crosslinker that can be readily conjugated to a variety of targeting ligands. This crosslinker was conjugated to trastuzumab, a humanized monoclonal antibody against the HER2 receptor. We developed a model based on mass-action kinetics to describe the intracellular processing of this conjugate. The kinetic model was developed in conjunction with live-cell experiments to extract the rate constant for intracellular disulfide bond degradation. This framework may be applied to other endocytosis pathways, bond types, and cell types to quantify this fundamental degradation rate parameter.