Synthesis and characterization of membrane-active GALA-OKT9 conjugates

Cellular processing of immunotoxins is inefficient, limiting the overall ef fectiveness of current immunotoxin therapies. Specifically, translocation o f ribosome-inactivating toxins across intracellular membranes is agonizingl y slow. In one strategy to improve immunotoxin efficacy, membrane-active pe ptides are attached to immunotoxins to facilitate transfer of the toxic moi ety across a cellular membrane to the cytosol. pH-sensitive peptides are of particular interest, as the membrane activity can be localized to the endo somal/lysosomal pathway, reducing nonspecific interactions at the cell surf ace. In this study, GALA, a pH-sensitive peptide that forms multimeric pore s in membranes, was chemically attached to OKT9, an anti-transferrin recept or mAb. Conjugates were tested by measuring release of encapsulated dyes fr om liposomes to determine the extent to which the membrane-lytic properties of GALA were retained. The most significant feature affecting the lytic pr operties of GALA-OKT9 conjugates was the number of attached GALA per OKT9. Conjugates with a single GALA per OKT9 caused almost no leakage while conju gates with two or three GALA per OKT9 caused significant leakage in a conce ntration-dependent manner. Invariably, GALA-OKT9 conjugates were significan tly less active than unconjugated GALA, attributable to a decrease both in partitioning and in surface aggregation. No improvement in membrane-lytic a ctivity was achieved by using a longer, more flexible poly(ethylene glycol) cross-linker. Attachment of GALA via C- versus N-terminal linkage had no e ffect on membrane-lytic properties. Size-selective release of high molecula r weight dextrans was almost identical for conjugated and unconjugated GALA , suggesting that GALA forms the same pore structure regardless of conjugat ion state.