SYMMETRY OF FV ARCHITECTURE IS CONDUCIVE TO GRAFTING A 2ND ANTIBODY-BINDING SITE IN THE FV REGION

Molecular modeling studies on antibody Fv regions have been pursued to design a second antigen-binding site (chi-site) in a chimeric single- chain Fv (chi sFv) species of about 30 kDa. This analysis has uncovere d an architectural basis common to many Fv regions that permits grafti ng a chi-site onto the Fv surface that diametrically opposes the norma l combining site. By using molecular graphics analysis, chimeric compl ementarity-determining regions (chi CDRs) were defined that comprised most of the CDRs from an antibody binding site of interest. The chain directionality of chi CDRs was consistent with that of specific bottom loops of the sFv, which allowed for grafting of chi CDRs with an over all geometry approximating CDRs in the parent combining site. Analysis of 10 different Fv crystal structures indicates that the positions fo r inserting chi CDRs are very highly conserved, as are the correspondi ng chi CDR boundaries in the parent binding site. The results of this investigation suggest that it should be possible to generally apply th is approach to the development of chimeric bispecific antibody binding site (chi BABS) proteins.