title: Genetically engineered tetravalent single-chain Fv of the pancarcinoma monoclonal antibody CC49: improved biodistribution and potential for therapeutic application.
abstract: Failure of radiolabeled monoclonal antibodies (MAbs) in the treatment of solid tumors, for the most part, is a result of undesirable pharmacokinetics that lead to significant radiation exposure of normal tissues and an inadequate delivery of radiation doses to tumors. Using genetic engineering, antitumor MAbs can be optimized for desirable clinical applications. In the present study, we report the generation of a tetravalent single-chain Fv [[sc(Fv)2]2] of the murine MAb CC49 that recognizes the tumor-associated glycoprotein, TAG-72. [Sc(Fv)2]2 was expressed as a secreted soluble protein in Pichia pastoris under the regulation of alcohol oxidase 1 promoter. The in vitro binding properties of the tetravalent construct were analyzed by solid-phase RIA and surface plasmon resonance studies using BIAcore. The binding affinity constant (K(A)) for the [sc(Fv)2]2 and CC49 IgG were similar, i.e., 1.02 x 10(8) M(-1) and 1.14 x 10(8) M(-1), respectively, and were 4-fold higher than its divalent scFv [sc(Fv)2; 2.75 x 10(7) M(-1)]. At 6 h postadministration, the percentage of injected dose accumulated/g of LS-174T colon carcinoma xenografts was 21.3+/-1.3, 9.8+/-1.3, and 17.3+/-1.1 for radioiodinated [sc(Fv)2]2, sc(Fv)2, and IgG, respectively. Pharmacokinetic analysis of blood clearance studies showed the elimination half-life for [sc(Fv)2]2, sc(Fv)2, and IgG as 170, 80, and 330 min, respectively. The gain in avidity resulting from multivalency along with an improved biological half-life makes the tetravalent construct an important reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.
authors.affiliation: Department of Biochemistry, University of Nebraska Medical Center, Omaha 68198-4525, USA.
authors.forename: A
authors.initials: A
authors.lastname: Goel
authors.forename: D
authors.initials: D
authors.lastname: Colcher
authors.forename: J
authors.initials: J
authors.lastname: Baranowska-Kortylewicz
authors.forename: S
authors.initials: S
authors.lastname: Augustine
authors.forename: B J
authors.initials: BJ
authors.lastname: Booth
authors.forename: G
authors.initials: G
authors.lastname: Pavlinkova
authors.forename: S K
authors.initials: SK
authors.lastname: Batra
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000627:therapeutic use*; D000970:Antineoplastic Agents / Q000493:pharmacokinetics* / Q000627:therapeutic use*; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005818:Genetic Engineering; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D008819:Mice, Nude; D008954:Models, Biological; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D010843:Pichia / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011863:Radioimmunoassay; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014018:Tissue Distribution
chemical_list: D000912:Antibodies, Neoplasm; D000970:Antineoplastic Agents; C091202:B72.3 antibody; D007074:Immunoglobulin G; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase