title: The role of higher-order protein structure in supporting binding by heteroclitic monoclonal antibodies: the monoclonal antibody KIM185 to CD18 also binds C4-binding protein.
abstract: Heteroclitic monoclonal antibodies are characterized by the ability to bind multiple epitopes with little or no similarity. Such antibodies have been reported earlier, but insight into to the molecular basis of this propensity is limited. Here we report that the KIM185 antibody to human CD18 reacts with the plasma protein C4b-binding protein (C4BP). This was revealed during affinity purification procedures where human serum was incubated with surfaces coated with monoclonal antibodies to CD18. Other monoclonal antibodies to CD18 (KIM127 and TS1/18) showed no such interaction with C4BP. We constructed a sandwich-type time-resolved immunofluorometric assay using KIM185 both as capture and developing antibody. By use of proteolytic fragments of KIM185 and recombinant deletion mutants of C4BP the interaction sites were mapped to the variable region of KIM185 and the oligomerization domain of C4BP, respectively. C4BP is a large oligomeric plasma protein that binds activated complement factor C4b and other endogenous ligands as well as microorganisms. By use of the recent crystallographic data on the structure of CD11c/CD18 and prediction of the secondary structure of the C4BP oligomerization domain, we show that epitopes bound by KIM185 in these proteins are unlikely to share any major structural similarity. However, both antigens may form oligomers that would enable avid binding by the antibody. Our report points to the astonishing ability of heteroclitic antibodies to accommodate the binding of multiple proteins with no or little structural similarity within the confined space of the variable regions.
authors.affiliation: Biophysical Immunology Laboratory, Aarhus University Denmark, Denmark.
authors.forename: Louise Carstensen
authors.initials: LC
authors.lastname: Gjelstrup
authors.forename: Stig Henrik
authors.initials: SH
authors.lastname: Andersen
authors.forename: Steen Vang
authors.initials: SV
authors.lastname: Petersen
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.forename: Anna M
authors.initials: AM
authors.lastname: Blom
authors.forename: Thomas
authors.initials: T
authors.lastname: Vorup-Jensen
authors.forename: Steffen
authors.initials: S
authors.lastname: Thiel
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D018821:CD18 Antigens / Q000737:chemistry* / Q000276:immunology*; D050716:Complement C4b-Binding Protein; D003429:Cross Reactions; D006649:Histocompatibility Antigens / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D040681:Structural Homology, Protein
chemical_list: D000911:Antibodies, Monoclonal; C055609:C4BPA protein, human; D018821:CD18 Antigens; D050716:Complement C4b-Binding Protein; D006649:Histocompatibility Antigens