Co-localized pixels were identified from the overlap of the masked images of each fluorophore. (525K) GUID:?5491578A-79EB-491F-B905-41F1359EB5AB 03: Figure S3 F(ab)2-ARG1 internalization and (22R)-Budesonide endosomal localization is similar to full length ARG1. MNA cells were co-transfected with rabies G and WT-GFP-Rab5a (A) or YFP-Rab9a (B) and incubated with F(ab)2-ARG1 (red) for given time points in complete medium. Data are representative of two separate experiments. Arrows indicate regions of co-localization. Bar, 5 m. NIHMS331569-supplement-03.tif (6.5M) GUID:?E0FB95C5-42CC-41A9-9E48-8EFC86AFEC09 04: Figure S4 Ligand and antibody-mediated internalization of the TfR localizes with clathrin. (A, B) HEK cells were transfected with 1 g clathrin-GFP followed by incubation with either transferrin (Tf) or antibody (CD71). Graphs show the amount of Tf/clathrin (A) or CD71/clathrin (B) co-localized pixels from addition to cells (T = 0 or 400) to 20 min. following addition (T = 1200). Images for analysis were taken using TIRF microscopy. Data are representative of three separate experiments. NIHMS331569-supplement-04.tif (828K) (22R)-Budesonide GUID:?86185F6F-0354-407B-8EE8-23460E6D9216 05: Figure S5 Ligand-mediated transferrin receptor internalization localizes with early and recycling endosomes, but not late endosomes. HEK cells were transfected with 1 g GFP-Rab4a (A) GFP-Rab5a (B), GFP-Rab11a (C) or YFP-Rab9a (D) and incubated with Tf (red) for given time points in complete medium. Arrows indicate regions of co-localization. Data are representative of three separate experiments. Bar, 5 m. NIHMS331569-supplement-05.tif (12M) GUID:?78DCD791-53E4-4470-93EB-6CF3EE292B2D Abstract Antibody-mediated intracellular delivery of therapeutic agents has been considered for treatment of a variety of diseases. These approaches involve targeting cell-surface receptor proteins expressed by tumors or viral proteins expressed on infected cells. We examined the intracellular trafficking of a viral cell-surface-expressed protein, rabies G, with or without binding a specific antibody, ARG1. We found that antibody binding shifts the native intracellular trafficking pathway of rabies G in an Fc-independent manner. Kinetic studies indicate that the ARG1/rabies G complex progressively co-localized with clathrin, early endosomes, late endosomes, and lysosomes after addition to cells. This pathway was different from that taken by rabies G without addition of antibody, which localized with recycling endosomes. Findings were recapitulated using a cellular receptor with a well-defined endogenous recycling pathway. We conclude that antibody binding to cell-surface proteins induces redirection of intracellular trafficking of unbound or ligand bound receptors to a specific degradation pathway. These findings have broad implications for future developments of antibody-based therapeutics. Keywords: Endosomes, degradation, antibody, trafficking, internalization Introduction1 The use of antibodies to target specific cells or cell types has become an increasingly desirable method of treatment for a variety of infections and diseases. Several publications have indicated the benefit to using monoclonal antibodies to specifically deliver drugs, including the targeted delivery of cytokines and siRNAs to tumor cells (Kamizuru et al., 2001; Kaspar et al., 2007; Marecos et al., 1998; Peer et al., 2007; Polson et al., 2007; Trachsel et al., 2007). It has also been shown that glycoprotein-specific antibodies can mediate internalization of viral glycoproteins (Favoreel et al., 1999; Favoreel et al., 2004; Sarmiento et al., 2007; Van de Walle et al., 2001). Rabies virus infections occur in over 100 countries and territories and are fatal once symptoms develop (WHO, 2007). To prevent development of the disease, treatment of exposed individuals includes administration of the rabies vaccine and human rabies immune globulin, which helps to neutralize the virus. There are several ways that rabies G-specific antibodies have been Mouse monoclonal to ERBB3 shown to mediate inhibition of the virus. Neutralizing antibodies can bind to the virion-expressed glycoprotein to either block infection of target cells or to inhibit escape of the virus from endosomal compartments following entry (Dietzschold et al., 1987), an important step in (22R)-Budesonide (22R)-Budesonide viral uncoating. Antibodies can also bind to rabies G expressed on the surface.