Preclinical evaluation of a humanized NR-LU-10 antibody-streptavidin fusion protein for pretargeted cancer therapy.
A humanized single chain Fv antibody fragment specific to the EGP40 antigen was genetically engineered as a streptavidin fusion (scFvSA) for use in pretargeted radioimmunotherapy. The scFvSA construct was expressed as a soluble, tetrameric species in the Escherichia coli periplasm at 110-140 mg/liter. The fusion protein was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of 50-60%. Characterization of the purified protein by SDS-PAGE, light scattering, and size exclusion chromatography demonstrated that the fusion protein was tetrameric with a molecular weight of approximately 172,000. Competitive immunoreactivity assays showed a two-fold greater binding to the antigen than the comparable whole antibody. The purified protein had a biotin disassociation rate identical to recombinant streptavidin and bound an average of three of four possible biotins per molecule. The radiolabeled fusion protein showed a faster blood clearance rate in normal mice than the corresponding whole antibody-streptavidin chemical conjugate. Tumor-specific targeting of a subsequently administered radionuclidechelate/biotin molecule was demonstrated in nude mice bearing SW1222 human colon carcinoma xenografts. A single dose of 800 microCi of 90Y-DOTA-biotin produced cures in mice with established subcutaneous human small cell lung or colon cancer xenografts.
Goshorn
S
S
NeoRx Corporation, 410 W. Harrison St., Seattle, WA 98119, USA.
Sanderson
J
J
Axworthy
D
D
Lin
Y
Y
Hylarides
M
M
Schultz
J
J
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology*; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004591:Electrophoresis, Polyacrylamide Gel; D000071858:Epithelial Cell Adhesion Molecule; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000627:therapeutic use*; D007457:Iodine Radioisotopes / Q000008:administration & dosage; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D016499:Radioimmunotherapy; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics / Q000627:therapeutic use*; D019809:Streptavidin / Q000235:genetics / Q000276:immunology*; D014018:Tissue Distribution; D023041:Xenograft Model Antitumor Assays
D000951:Antigens, Neoplasm; D017252:CD3 Complex; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D007128:Immunoglobulin Fragments; D007457:Iodine Radioisotopes; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D019809:Streptavidin