title: Antibodies capable of releasing diphtheria toxin in response to the low pH found in endosomes.
abstract: Diphtheria toxin (DT) undergoes a rapid conformational change in response to the acidity encountered within endosomes. That transition is integral to the passage of its catalytic domain into the cytosol and thus its lethal action. The importance of this translocation mechanism led us to develop several monoclonal antibodies that bind DT at neutral pH but spontaneously release the toxin when critical epitopes denature or unfold upon lowering the pH to 4.5-5.5. Hybridomas were selected using a microtiter plate assay that measured the pH-dependent detachment of antibody from immobilized toxin. The acid-sensitive epitopes involved were on the catalytic, transmembrane, and receptor binding domains of DT. This pH-induced disruption of the binding of toxin to these monoclonal antibodies was analyzed by sedimentation velocity ultracentrifugation. Antibody combining sites were fully occupied at pH 5.5, partially bound at pH 5.0, and totally empty at pH 4.5. It was estimated that the Ka for antibody-toxin binding was approximately 1000-fold lower at pH 5.0 than at neutral pH. This novel acid-triggered release mechanism provides a basis for delivery of antibody-bound toxin into cells accompanied by its immediate dissociation as the complex enters acidic vesicles.
authors.affiliation: Boston Biomedical Research Institute, Boston, Massachusetts 02114, USA. RASO@bbri.harvard.edu
authors.forename: V
authors.initials: V
authors.lastname: Raso
authors.forename: M
authors.initials: M
authors.lastname: Brown
authors.forename: J
authors.initials: J
authors.lastname: McGrath
authors.forename: S
authors.initials: S
authors.lastname: Liu
authors.forename: W F
authors.initials: WF
authors.lastname: Stafford
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D001666:Binding Sites, Antibody; D004167:Diphtheria Toxin / Q000276:immunology / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; D004167:Diphtheria Toxin; D007457:Iodine Radioisotopes