title: A V kappa-J kappa junctional change in an antidigoxin recombinant antibody destroys digoxin-binding activity.
abstract: A set of high affinity antidigoxin antibodies were previously identified with high homologous V kappa 1A L chain sequences but were associated with two entirely different VH regions and two dramatically different specificities for digoxin analogs. Antibodies 40-20, 40-60, 40-90, and 40-100 displayed similar binding specificities but differed from that of antibody 26-10. In a previous study using somatic cell fusion for Ig chain recombination we demonstrated that a recombinant antibody consisting of the H chain of antibody 26-10 and the L chain of antibody 40-20 retained digoxin binding and the 26-10 Id, but displayed a binding specificity pattern dominated by the 26-10 H chain donor. In the present study we produced three additional chain recombinant antibodies that contain the 26-10 H chain recombined with each of the L chains of antibodies 40-60, 40-90, and 40-100. All four recombinants expressed the 26-10 Id indistinguishably from the 26-10 antibody. Two of the recombinants (using the 40-60 and 40-90 L chains) bind digoxin; however, the recombinant using the 40-100 L chain failed to bind digoxin. Complete sequence analyses of the 40-20, 40-60, 40-90, and 40-100 VH and VL regions were performed. Antibodies 40-90 and 40-100 have identical VH region sequences but differed only in their L chains at position 96 (proline/leucine). This single difference at the VK-JK junction abolished digoxin binding in the context of one H chain (26-10), but does not cause a significant change in binding in association with the "normal" parental chains 40-90 and 40-100. Thus, structurally closely related VL regions can recombine with different VH regions to form digoxin binding sites of different specificity; in one binding site the identity of a L chain junctional residue is critical whereas in the second binding site that residue is unimportant. Molecular modeling studies revealed major differences between calculated binding site structures for 26-10 when leucine is substituted for proline at position 96 in the 26-10 VL region.
authors.affiliation: Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46202.
authors.forename: N W
authors.initials: NW
authors.lastname: Hudson
authors.forename: R E
authors.initials: RE
authors.lastname: Bruccoleri
authors.forename: L K
authors.initials: LK
authors.lastname: Steinrauf
authors.forename: J A
authors.initials: JA
authors.lastname: Hamilton
authors.forename: M
authors.initials: M
authors.lastname: Mudgett-Hunter
authors.forename: M N
authors.initials: MN
authors.lastname: Margolies
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002459:Cell Fusion; D003196:Computer Graphics; D004077:Digoxin / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology; D007130:Immunoglobulin Idiotypes / Q000737:chemistry / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry* / Q000276:immunology; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data
chemical_list: D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D046911:Macromolecular Substances; D004077:Digoxin