title: A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy.
abstract: Single-chain Fv antibody fragments from the CD20-specific murine monoclonal antibody B9E9 were genetically engineered as streptavidin fusions [single-chain Fv-streptavidin (scFvSA) fusion protein] for use in pretargeted radioimmunotherapy. The scFvSA constructs were expressed as soluble, tetrameric species in the periplasm of Escherichia coli. Expression levels were affected by the order of the variable regions and the length and composition of the single-chain Fv linker. The best expressor was obtained with the variable regions in the heavy chain-light chain configuration separated by a 25-mer Gly4Ser linker. This construct produced 250-300 mg of soluble, tetrameric fusion protein per liter of fermentor culture. The fusion protein (Mr 173,600) was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of about 50% and was analyzed for functionality both in vitro and in vivo. Immunoreactivity of the scFvSA fusion protein and its nanomolar affinity to CD20-positive Ramos cells were comparable with the B9E9 monoclonal antibody. The fusion protein had a biotin dissociation rate identical to recombinant streptavidin and bound an average of 3.6 biotins/molecule of a possible 4 biotins/molecule. Labeled fusion protein cleared from the blood of BALB/c mice with a P half-life of about 16 h. In nude mice bearing Ramos xenografts, the fusion protein demonstrated sufficient tumor localization of functional streptavidin to enable efficient, tumor-specific targeting of a subsequently administered radionuclide-chelate/biotin molecule. These results suggest that large quantities of functional scFvSA can be produced for clinical testing as a therapy for non-Hodgkin's lymphoma.
authors: NeoRx Corporation, Molecular Biology Research, Seattle, Washington 98119-4007, USA.
authors: J
authors: J
authors: Schultz
authors: Y
authors: Y
authors: Lin
authors: J
authors: J
authors: Sanderson
authors: Y
authors: Y
authors: Zuo
authors: D
authors: D
authors: Stone
authors: R
authors: R
authors: Mallett
authors: S
authors: S
authors: Wilbert
authors: D
authors: D
authors: Axworthy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018951:Antigens, CD20 / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007457:Iodine Radioisotopes / Q000008:administration & dosage; D008228:Lymphoma, Non-Hodgkin / Q000532:radiotherapy / Q000628:therapy; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D015202:Protein Engineering; D016499:Radioimmunotherapy; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019809:Streptavidin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014018:Tissue Distribution; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D007128:Immunoglobulin Fragments; D007457:Iodine Radioisotopes; D011993:Recombinant Fusion Proteins; D019809:Streptavidin