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Site-specific conjugation to interleukin 4 containing mutated cysteine residues produces interleukin 4-toxin conjugates with improved binding and activity.

Fusion of a ligand to another protein frequently impairs the binding of the ligand. Recombinant toxins composed of mutants of Pseudomonas exotoxin (PE) fused to the C-terminus of human interleukin 4 (IL4) are cytotoxic to IL4 receptor- (IL4R-) bearing tumor cells but bind to the IL4R with only 1% the affinity of IL4. We have developed a method to connect a toxin to a ligand which allows the junction to be moved to a location on the ligand which would minimize the binding impairment. We designed mutants of IL4 in which residue 28, 38, 68, 70, 97, or 105 was substituted with cysteine. All purified mutants bound to the IL4R with 60-100% the affinity of IL4, indicating that the IL4 structure was essentially unchanged. The IL4 mutants were then each conjugated through a disulfide bond to PE35, a truncated form of PE which contains a single cysteine. IL4 conjugated to PE35 at residue 28, 38, or 105 of IL4 bound with 10-fold improved affinity and was 10-fold more cytotoxic than the recombinant IL4-toxin in which PE is fused to position 129 at the C-terminus of IL4. IL4 containing PE35 conjugated at position 68, 70, or 97 had lower binding affinity and cytotoxic activity. These results indicate that the location of the ligand-protein junction can be selectively moved to enhance conjugate effectiveness, and implications could be made regarding which regions of IL4 are important for binding.