Disulfide stabilization of antibody Fv: computer predictions and experimental evaluation.
Using molecular modeling technology we have recently identified positions in conserved framework regions of Fvs which can be used to stabilize antibody Fvs by an interchain disulfide bond engineered in between the structurally conserved framework positions of the variable domains of heavy (VH) and light (VL) immunoglobulin chains (disulfide-stabilized Fv; dsFv). The computer model indicated the existence of other potential sites in the framework regions that might be suitable for disulfide bond formation between VH and VL. The possibility of obtaining dsFvs using these positions is evaluated here experimentally by constructing dsFv immunotoxins in which the Fv moiety is fused to a truncated form of Pseudomonas exotoxin. We analyzed the extent of dsFv formation and the activity of the resulting dsFv immunotoxins, and compared various dsFv molecules with the scFv immunotoxin. Our results demonstrate that position H44-L105 is the only one which gives high production yields of active dsFv. All other positions gave either low yields and activity or completely failed to produce active dsFv. With one exception, the formation and activities of the dsFvs corresponded to the C alpha-C alpha distance between the VH and VL positions, with an optimal distance of 5.7 A producing the best dsFv. Distances of 6.0-6.9 A resulted in a low yield of protein that was still capable of binding antigen, whereas distances > 7.0 A resulted in molecules in which dsFv formation was not obtained.
Reiter
Y
Y
Division of Cancer Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Brinkmann
U
U
Jung
S H
SH
Pastan
I
I
Lee
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects; D003198:Computer Simulation; D017931:DNA Primers / Q000737:chemistry; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D000922:Immunotoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011489:Protein Denaturation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D014364:Tryptophan / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
D017931:DNA Primers; D004220:Disulfides; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D014508:Urea