Receptor Crosslinking: A General Method to Trigger Internalization and Lysosomal Targeting of Therapeutic Receptor:Ligand Complexes

Abstract

A major unmet clinical need is a universal method for subcellular targeting of bioactive molecules to lysosomes. Delivery to this organelle enables either degradation of oncogenic receptors that are overexpressed in cancers, or release of prodrugs from antibody–drug conjugates. Here, we describe a general method that uses receptor crosslinking to trigger endocytosis and subsequently redirect trafficking of receptor:cargo complexes from their expected route, to lysosomes. By incubation of plasma membrane receptors with biotinylated cargo and subsequent addition of streptavidin to crosslink receptor:cargo–biotin complexes, we achieved rapid and selective lysosomal targeting of transferrin, an anti-MHC class I antibody, and the clinically approved anti-Her2 antibody trastuzumab. These three protein ligands each target a receptor with a distinct cellular function and intracellular trafficking profile. Importantly, we confirmed that crosslinking of trastuzumab increased lysosomal degradation of its cognate oncogenic receptor Her2 in breast cancer cell lines SKBR3 and BT474. These data suggest that crosslinking could be exploited for a wide range of target receptors, for navigating therapeutics through the endolysosomal pathway, for significant therapeutic benefit.

Characteristic rotational behaviors of rod-shaped cargo revealed by automated five-dimensional single particle tracking

“…The siRNA oligonucleotide sequence 5′-GAGCAUGUGCACGCUGGCCA-3′ targeting μ2-2 subunit of AP-2 is highly effective to achieve nearly complete depletion of AP-2 with two cycles of transfection, as reported by Motley et al 44 and confirmed by others 45, 46 . The siRNA treatment was performed following the established protocol.…”

Revisiting the value of competition assays in folate receptor-mediated drug delivery

“…It should be noted that Moody et al demonstrated that the recycling rate of transferrin and Her2 receptors was altered and that the cargo was redirected to the lysosome when the receptors were crosslinked with biotinylated cargo and the addition of streptavidin. (44) Therefore, it seems possible that multivalent particles, such as the folate decorated micelleplexes described here, which can bind and occupy multiple receptors on the cell surface, can alter the receptor's natural endocytic pathway to enhance the delivery of therapeutic agents to the lysosome. This agrees with other multivalent FRα targeting studies suggesting that multivalent particles follow a lysosomal pathway to the cytosol.…”

Characteristic Rotational Behaviors of Rod-Shaped Cargo Revealed by Automated Five-Dimensional Single Particle Tracking

“…Furthermore, specific mean to inhibit clathrin-mediated endocytosis was carried out by siRNA knockdown of AP-2 µ2 subunit in A549 cells. The siRNA oligonucleotide sequence 5′-GAGCAUGUGCACGCUGGCCA-3′ targeting μ2-2 subunit of AP-2 is highly effective to achieve nearly complete depletion of AP-2 with two cycles of transfection, as reported by Motley et al 44 and confirmed by others 45,46 . The siRNA treatment was performed following the established protocol.…”