A bivalent disulfide-stabilized Fv with improved antigen binding to erbB2.
We have used protein engineering to generate a stable bivalent Fv molecule of the anti-erbB2 monoclonal antibody e23. The VH and VL domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a flexible 15 amino acid residue (Gly4-Ser)3 linker. The e23 (dsFv)2 molecule is fused to a truncated form of Pseudomonas exotoxin to generate a bivalent disulfide-stabilized, (dsFv)2, immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro and purified to about 95% purity. Binding studies demonstrated that the (dsFv)2 molecule has a much higher affinity for erbB2 than a monovalent dsFv molecule and a similar binding affinity as the parental antibody e23. The (dsFv)2 immunotoxin was 5 to 20-fold more cytotoxic to two e23 antigen-positive cell lines than the monovalent dsFv immunotoxin. The bivalent dsFv molecule is very stable, retaining 94% of its activity after a 24 hours incubation in human serum at 37 degreesC. Two other molecules with shorter linkers five and ten amino acid residues in length were produced and showed similar activities as the molecule containing a 15 amino acid residue linker. The bivalency, stability and the relative ease of purification makes these e23 (dsFv)2 molecules valuable reagents for cancer immunotherapy and diagnosis.
National Cancer Institute, National Institutes of Health, Building 37, Bethesda, MD, 20892-4255, USA.
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Brinkmann
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Pastan
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000915:Antibody Affinity; D001427:Bacterial Toxins; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry; D005098:Exotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000922:Immunotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D002479:Inclusion Bodies; D008958:Models, Molecular; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012709:Serum Albumin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D012709:Serum Albumin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C055966:immunoglobulin Fv