title: Synthesis and characterization of membrane-active GALA-OKT9 conjugates.
abstract: Cellular processing of immunotoxins is inefficient, limiting the overall effectiveness of current immunotoxin therapies. Specifically, translocation of ribosome-inactivating toxins across intracellular membranes is agonizingly slow. In one strategy to improve immunotoxin efficacy, membrane-active peptides are attached to immunotoxins to facilitate transfer of the toxic moiety across a cellular membrane to the cytosol. pH-sensitive peptides are of particular interest, as the membrane activity can be localized to the endosomal/lysosomal pathway, reducing nonspecific interactions at the cell surface. In this study, GALA, a pH-sensitive peptide that forms multimeric pores in membranes, was chemically attached to OKT9, an anti-transferrin receptor mAb. Conjugates were tested by measuring release of encapsulated dyes from liposomes to determine the extent to which the membrane-lytic properties of GALA were retained. The most significant feature affecting the lytic properties of GALA-OKT9 conjugates was the number of attached GALA per OKT9. Conjugates with a single GALA per OKT9 caused almost no leakage while conjugates with two or three GALA per OKT9 caused significant leakage in a concentration-dependent manner. Invariably, GALA-OKT9 conjugates were significantly less active than unconjugated GALA, attributable to a decrease both in partitioning and in surface aggregation. No improvement in membrane-lytic activity was achieved by using a longer, more flexible poly(ethylene glycol) cross-linker. Attachment of GALA via C- versus N-terminal linkage had no effect on membrane-lytic properties. Size-selective release of high molecular weight dextrans was almost identical for conjugated and unconjugated GALA, suggesting that GALA forms the same pore structure regardless of conjugation state.
authors.affiliation: Department of Chemical Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, Wisconsin 53706-1607, USA.
authors.forename: J
authors.initials: J
authors.lastname: Kuehne
authors.forename: R M
authors.initials: RM
authors.lastname: Murphy
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000493:pharmacokinetics*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004337:Drug Carriers / Q000138:chemical synthesis / Q000493:pharmacokinetics; D016503:Drug Delivery Systems; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D000922:Immunotoxins / Q000493:pharmacokinetics; D008081:Liposomes; D051379:Mice; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics*; D014168:Transferrin / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D004337:Drug Carriers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000922:Immunotoxins; D008081:Liposomes; D010455:Peptides; D014168:Transferrin; C066951:GALA peptide; C007740:fluorexon