title: Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv.
abstract: Using molecular modeling technology, we have recently identified two positions in conserved framework regions of antibody Fv fragments (Fvs) that are distant from CDRs, and potentially can be used to make recombinant Fv fragments in which the unstable VH and VL heterodimer is stabilized by an interchain disulfide bond inserted between structurally conserved framework positions. A disulfide bond has been introduced at one of these positions, VH44-VL105, and shown to stabilize various Fvs that retain full binding and specificity. Recombinant immunotoxins, e.g. B3(dsFv)-PE38KDEL in which this disulfide-stabilized Fv moiety is connected to a truncated form of Pseudomonas exotoxin (PE; PE38KDEL) which contains the translocation and ADP ribosylation domains, are indistinguishable in binding and specificity from its single-chain immunotoxin counterparts. We have now analyzed the alternative position, (VH111-VL48), predicted by the modeling methodology, for disulfide stabilization of mAb B3(Fv) by producing a recombinant immunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxin was also very active and retained full specificity to B3 antigen-positive cells. However, it was 2- to 3-fold less active than the VH44-VL105 dsFv-molecule. We also tested various biochemical features of VH44-VL105 and VH111-VL48 dsFv immunotoxins and compared them with the corresponding single-chain immunotoxin. We found the dsFv immunotoxins were more stable in human serum and more resistant to thermal and chemical denaturation than the single chain (sc) Fv immunotoxin. Because dsFv immunotoxins and dsFvs have full activity and specificity and improved stability, they may be more useful than scFv immunotoxins as therapeutic and diagnostic agents.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Y
authors.initials: Y
authors.lastname: Reiter
authors.forename: U
authors.initials: U
authors.lastname: Brinkmann
authors.forename: K O
authors.initials: KO
authors.lastname: Webber
authors.forename: S H
authors.initials: SH
authors.lastname: Jung
authors.forename: B
authors.initials: B
authors.lastname: Lee
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry*; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000737:chemistry; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D000922:Immunotoxins / Q000737:chemistry*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A