title: Delivery of antibody mimics into mammalian cells via anthrax toxin protective antigen.
abstract: Antibody mimics have significant scientific and therapeutic utility for the disruption of protein-protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr-Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co-immunoprecipitation. We also observed inhibition of Bcr-Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA-mediated delivery of an affibody binder that targets hRaf-1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein-protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue 18-596, Cambridge, MA 02193 (USA).
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Liao
authors.forename: Amy E
authors.initials: AE
authors.lastname: Rabideau
authors.forename: Bradley L
authors.initials: BL
authors.lastname: Pentelute
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D000941:Antigens / Q000737:chemistry / Q000378:metabolism*; D000942:Antigens, Bacterial / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D001672:Biocompatible Materials / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D020014:K562 Cells; D020935:MAP Kinase Signaling System; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D048490:raf Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000906:Antibodies; D000941:Antigens; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D001672:Biocompatible Materials; D057846:Cell-Penetrating Peptides; D011993:Recombinant Fusion Proteins; C030325:anthrax toxin; D016044:Fusion Proteins, bcr-abl; D048490:raf Kinases; D018631:ras Proteins
keywords: anthrax toxin; antibodies; intracellular delivery; protein-protein interactions; sortases