Improvement of an antibody neutralizing the anthrax toxin by simultaneous mutagenesis of its six hypervariable loops.
The enhancement of antibody affinity by mutagenesis targeting only complementarity determining regions has the advantage of respecting the framework regions, which are important for tolerance if clinical use is envisaged. Here, starting from a Fab (antigen-binding fragment; 35PA(83)) capable of neutralizing the lethal toxin of anthrax and having an affinity of 3.4 nM for its antigen, a phage-displayed library of variants where all six complementarity determining regions (73 positions) were targeted for mutagenesis was built. This library contained 5 x 10(8) variants, and each variant carried four mutations on average. The library was first panned with adsorbed antigen and washes of increasing stringency. It was then screened in parallel with either small concentrations of soluble biotinylated antigen or adsorbed antigen and long elution times in the presence of soluble antigen. The stringencies of both selections were pushed as far as possible. Compared with 35PA(83), the best selected clone had an affinity enhanced 19-fold, to 180 pM, and its 50% inhibitory concentration was decreased by 40%. The results of the two selection methods were compared, and the generality of these methods was considered.
Laffly
Emmanuelle
E
Groupe de Biotechnologie des Anticorps, Laboratoire d'Immunobiologie, Centre de Recherches du Service de Santé des Armées, 24 Avenue des Maquis du Grésivaudan, 38702 La Tronche, France.
Pelat
Thibaut
T
Cédrone
Frédéric
F
Blésa
Stéphane
S
Bedouelle
Hugues
H
Thullier
Philippe
P
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009500:Neutralization Tests; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011897:Random Allocation; D015203:Reproducibility of Results
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; C030325:anthrax toxin