Reduced antibody response to streptavidin through site-directed mutagenesis

Streptavidin provides an effective receptor for biotinylated tumoricidal mo lecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this b acterial protein to patients repeatedly, so as to maximize the antitumor ef fect without eliciting an immune response. Therefore, we attempted to reduc e the antigenicity of streptavidin by mutating surface residues capable of forming high energy ionic or hydrophobic interactions. A crystallographic i mage of streptavidin was examined to identify residues with solvent-exposed side chains and residues critical to streptavidin's structure or function, and to define loops. Mutations were incorporated cumulatively into the pro tein sequence. Mutants were screened for tetramer formation, biotin dissoci ation, and reduced immunoreactivity with pooled patient sera. Patient antis era recognized one minor continuous epitope with binding locus at residue E 101 and one major discontinuous epitope involving amino acid residues E51 a nd Y83. Mutation of residues E51, Y83, R53, and E116 reduced reactivity wit h patient sera to <10% that of streptavidin, but these mutations were no le ss antigenic in rabbits. Mutant 37, with 10 amino acid substitutions, was o nly 20% as antigenic as streptavidin. Rabbits immunized with either strepta vidin or mutant 37 failed to recognize the alternative antigen. Biotin diss ociated from mutant 37 four to five times faster than from streptavidin. Re sidues were identified with previously undescribed impact on biotin binding and protein folding. Thus, substitution of charged, aromatic, or large hyd rophobic residues on the surface of streptavidin with smaller neutral resid ues reduced the molecule's ability to elicit an immune response in rabbits.