title: The effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody.
abstract: In recent years a variety of recombinant methods have been developed for efficient production of bispecific antibodies (BsAb) in various formats. Bispecific diabody (bDAb), a 55-60 kDa molecule comprising two non-covalently associated cross-over single chain Fv (scFv) polypeptides, represents one of the most promising as well the most straightforward approaches to BsAb production. Here we constructed a bDAb, using two human scFv, 11F8 and A12, directed against the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR), respectively, as the building blocks. A total of 8 scFv and diabody constructs were prepared comprising the same two variable heavy (V(H)) and variable light (V(L)) chain domains but arranged in different orientations. V(H)/V(L) orientation, i.e., V(H)-linker-V(L) or V(L)-linker-V(H), showed significant effects on the expression and antigen-binding activity of scFv and monospecific diabody of both 11F8 and A12. Further, only 2 out of the 4 possible V(H)/V(L) orientations/arrangements in bDAb construction yielded active products that retain binding activity to both EGFR and IGFR. Both active bDAb preparations retained their original antigen-binding activity after incubation at 37 degrees C in mouse serum for up to 7 days, indicating excellent stability of the constructs. Taken together, our results underscore the importance of identifying/selecting optimal V(H)/V(L) orientation/arrangement for efficient production of active bDAb.
authors.affiliation: Department of Antibody Technology, ImClone Systems Incorporated, New York, NY 10014, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Lu
authors.forename: Xenia
authors.initials: X
authors.lastname: Jimenez
authors.forename: Larry
authors.initials: L
authors.lastname: Witte
authors.forename: Zhenping
authors.initials: Z
authors.lastname: Zhu
mesh_terms: D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D004355:Drug Stability; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007700:Kinetics; D015202:Protein Engineering / Q000379:methods; D017451:Receptors, Somatomedin / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D018033:Antibodies, Bispecific; D007135:Immunoglobulin Variable Region; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins; D066246:ErbB Receptors