Highlights d MVBs are complex organelles with intraluminal vesicles bound by the limiting membrane d Intraluminal membranes are in a dynamic equilibrium with the limiting membrane d Retrofusion of internal vesicles is controlled by processes used for viral fusion d Exosomes arise from internal MVB vesicles not participating in retrofusion

Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species. The latter approach necessitates the creation of fusions whose behavior may not accurately reflect that of their unmodified counterparts. Here, we report a method for the characterization of protein internalization in real time through sortase-mediated, site-specific labeling of single-domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. Quenched probes of this type have been used to measure enzyme activity in complex environments and for different cell types, but not as a sensor of protein movement into living cells. This approach allows a quantitative assessment of the movement of proteins into protease-containing endosomes in real time in living cells. We demonstrate considerable variation in the rate of endosomal delivery for different cell surface receptors. We were also able to characterize the kinetics of influenza virus delivery to cathepsin-positive compartments, showing highly coordinated arrival in endosomal compartments. This approach should be useful for identifying proteins expressed on cells of interest for targeted endosomal delivery of payloads, such as antibody–drug conjugates or antigens that require processing.

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Reagents that label specific subsets of cells are broadly useful for the treatment of cancer and modulation of cells of the immune system. Labeling is often achieved through the identification and characterization of proteins specifically expressed on selected cells but not others. Knowledge of cell membrane protein routing dynamics can be leveraged to efficiently deliver cytotoxic or immune stimulating payloads. The most common method for monitoring internalization relies on labeling proteins with antibodies modified with a fluorophore or other tag that can also be used to report on whether the protein of interest has been internalized. This approach requires indirect methods, such as multiple rounds of cell staining, to differentiate extracellular protein from protein that has been internalized and recycled to the cell surface. Here we report a method for the characterization of protein internalization in real time through the sortase-mediated, site-specific labeling of single domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. This approach allows quantitative measurement of the movement of proteins into protease-containing endosomes in real time in live cells. This method revealed variation in the rate of internalization for different cell surface receptors and allowed for kinetic characterization of influenza virus internalization. These findings help to explain the utility of certain single domain antibody-antigen conjugates for inducing humoral immune responses. The tools and methods described here should be useful for the identification of proteins expressed on target cells that are ideal for antibody-mediated drug delivery or for promotion of specific types of immune responses. Citation Format: Ross W. Cheloha, Zeyang Li, Djenet Bousbaine, Andrew Woodham, Priscillia Perrin, Jana Volaric, Hidde L. Ploegh. Real-time analysis of cell membrane protein and virus internalization using site-specific conjugation of protease-sensitive probes [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B149.

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