Chimeric antibodies with anti-dextran-derived complementarity-determining regions and anti-p-azophenylarsonate-derived framework regions.
The framework regions of antibodies fold into a conserved beta-sheet structure that acts as scaffolding for the antigen-contacting complementarity-determining regions (CDR). To test the structural equivalence of the frameworks between two antibodies with widely different combining sites, we created chimeric H and L chains by grafting the CDR of an alpha(1-->6)dextran specific antibody onto the framework of a p-azophenylarsonate (Ars) specific antibody through oligonucleotide-directed mutagenesis of the anti-Ars variable region genes. Antibodies consisting of various chain combinations of the chimeric, anti-dextran, and anti-Ars derived H and L chains were generated in transfectomas and tested for binding to dextran and Ars. Of the newly created chimeric and/or hybrid antibodies, an antibody with the chimeric H chain and the anti-dextran L chain bound to dextran with the same association constant as the parental anti-dextran antibody, and like the anti-dextran antibody was shown by immunochemical mapping to have a site complementary to six glucose residues. None of the other new variable region combinations, including the all-chimeric combination, showed binding to either dextran or Ars. These results indicate that the H chain but not the L chain anti-dextran and anti-Ars frameworks are functionally equivalent. Attempts to confer dextran binding on the H and L chain chimeric antibody, by mutagenizing selected framework residues, were unsuccessful. This study demonstrates the important role of the frameworks in the precise alignment of the CDR for Ag binding.
Kao
C Y
CY
Department of Biochemistry, Boston University School of Medicine, MA 02118.
Sharon
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003911:Dextrans / Q000276:immunology*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D013329:Structure-Activity Relationship; D014162:Transfection; D010132:p-Azobenzenearsonate / Q000276:immunology*
D000906:Antibodies; D003911:Dextrans; D007145:Immunoglobulin kappa-Chains; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D010132:p-Azobenzenearsonate