Reduced antibody response to streptavidin through site-directed mutagenesis.
Streptavidin provides an effective receptor for biotinylated tumoricidal molecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this bacterial protein to patients repeatedly, so as to maximize the antitumor effect without eliciting an immune response. Therefore, we attempted to reduce the antigenicity of streptavidin by mutating surface residues capable of forming high energy ionic or hydrophobic interactions. A crystallographic image 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 protein sequence. Mutants were screened for tetramer formation, biotin dissociation, and reduced immunoreactivity with pooled patient sera. Patient antisera recognized one minor continuous epitope with binding locus at residue E101 and one major discontinuous epitope involving amino acid residues E51 and Y83. Mutation of residues E51, Y83, R53, and E116 reduced reactivity with patient sera to <10% that of streptavidin, but these mutations were no less antigenic in rabbits. Mutant 37, with 10 amino acid substitutions, was only 20% as antigenic as streptavidin. Rabbits immunized with either streptavidin or mutant 37 failed to recognize the alternative antigen. Biotin dissociated from mutant 37 four to five times faster than from streptavidin. Residues were identified with previously undescribed impact on biotin binding and protein folding. Thus, substitution of charged, aromatic, or large hydrophobic residues on the surface of streptavidin with smaller neutral residues reduced the molecule's ability to elicit an immune response in rabbits.
Meyer
D L
DL
NeoRx Corporation, 410 West Harrison St., Seattle, WA 98119, USA.
Schultz
J
J
Lin
Y
Y
Henry
A
A
Sanderson
J
J
Jackson
J M
JM
Goshorn
S
S
Rees
A R
AR
Graves
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation / Q000235:genetics / Q000276:immunology*; D000941:Antigens / Q000235:genetics / Q000276:immunology; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D017510:Protein Folding; D011817:Rabbits; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*
D000941:Antigens; D002352:Carrier Proteins; D000939:Epitopes; C017071:biotin-binding proteins; D019809:Streptavidin