Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus

HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respira tory syncytial virus (RSV) and neutralizes the virus, both lit vitro and ir t vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equ ally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfaci ng requires that surface residues not normally found in a human Fv be mutat ed to the expected human amino acid, thereby eliminating potentially immuno genic sites. We describe the construction and characterization of two human ized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both mo lecules show unaltered binding affinities to the RSV antigen (purified F pr otein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (K-a approximate to 1x10(9) M-1). A competition ELISA usi ng captured whole virus confirmed that the binding affinities of the parent al scFv and also of hu7 and hu10 scFvs were identical. However, when compar ed with the original scFv, hu10 scFv was shown to have significantly decrea sed antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient fo r neutralization. We speculate that neutralization may involve the inhibiti on or induction of conformational changes in the bound antigen, thereby int erfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection.