title: Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin.
abstract: Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to 87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the molecular basis for improved cross-reactivity, we determined the X-ray co-crystal structures of wild-type and cross-reactive antibodies complexed to BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding. The results show how an antibody can be engineered to bind two different antigens despite structural differences in the antigen-antibody interface and may provide a general strategy for tuning antibody specificity and cross-reactivity.
authors.affiliation: Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco Rm. 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, California 94110, USA.
authors.forename: Consuelo
authors.initials: C
authors.lastname: Garcia-Rodriguez
authors.forename: Raphael
authors.initials: R
authors.lastname: Levy
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Arndt
authors.forename: Charles M
authors.initials: CM
authors.lastname: Forsyth
authors.forename: Ali
authors.initials: A
authors.lastname: Razai
authors.forename: Jianlong
authors.initials: J
authors.lastname: Lou
authors.forename: Isin
authors.initials: I
authors.lastname: Geren
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
authors.forename: James D
authors.initials: JD
authors.lastname: Marks
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000276:immunology / Q000378:metabolism*; D015195:Drug Design; D018604:Epitope Mapping / Q000379:methods*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D019274:Botulinum Toxins, Type A