title: Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris: expression, purification, and characterization.
abstract: Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.
authors.forename: A
authors.initials: A
authors.lastname: Goel
authors.forename: G W
authors.initials: GW
authors.lastname: Beresford
authors.forename: D
authors.initials: D
authors.lastname: Colcher
authors.forename: G
authors.initials: G
authors.lastname: Pavlinkova
authors.forename: B J
authors.initials: BJ
authors.lastname: Booth
authors.forename: J
authors.initials: J
authors.lastname: Baranowska-Kortylewicz
authors.forename: S K
authors.initials: SK
authors.lastname: Batra
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000493:pharmacokinetics*; D000912:Antibodies, Neoplasm / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000378:metabolism / Q000493:pharmacokinetics*; D001483:Base Sequence; D002277:Carcinoma / Q000188:drug therapy; D003110:Colonic Neoplasms / Q000188:drug therapy; D017931:DNA Primers; D019281:Dimerization; D005260:Female; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000493:pharmacokinetics*
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000970:Antineoplastic Agents; C091202:B72.3 antibody; D017931:DNA Primers; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv