Multivalent interactions between streptavidin-based pretargeting fusion proteins and cell receptors impede efficient internalization of biotinylated nanoparticles

Pretargeting represents a promising strategy to enhance delivery of nanoparticles. The strategy involves first introducing bispecific antibodies or fusion proteins (BFP) that can bind specific epitopes on target cells with one arm, and use the other arm to capture subsequently administered effector molecules, such as radionuclides or drug-loaded nanoparticles. Nevertheless, it remains unclear whether BFP that bind slowly- or non-internalizing epitopes on target cells can facilitate efficient intracellular delivery. Here, we investigated the cellular uptake of biotin-functionalized nanoparticles with streptavidin-scFv against TAG-72, a membrane protein on Jurkat T-cell leukemia cells. Unlike conventional active-targeted nanoparticles, we found that pretargeting resulted in preferential retention of ∼100nm nanoparticles at the plasma membrane rather than internalization into cells. We found no improvement in nanoparticle internalization by simply reducing nanoparticle concentration or surface biotin density. Interestingly, by adding both the BFP and a monoclonal antibody against TAG-72, we observed a twofold improvement in internalization of pretargeted nanoparticles. Our work illustrates that the cellular fate of pretargeted nanoparticles can be controlled by carefully tuning the interactions between pretargeting molecules and nanoparticles on the cell surface. Statement of significance Pretargeting is a multi-step strategy that utilizes bispecific proteins that recognize both cellular epitopes and subsequently administered therapeutic molecules. This approach has been extensively studied for radiotherapy of blood cancers; however, pretargeting remains largely underexplored for nanoparticle targeting, including whether pretargeting can facilitate efficient intracellular delivery. Here, we found that high density of targeting proteins on the cell surface can effectively limit internalization of pretargeted nanoparticles. Our work underscores the need to carefully assess specific cell-pretargeting molecule pairs for applications requiring intracellular delivery, and the key design requirements for such bispecific pretargeting molecules.