Divalent Forms of CC49 Single-Chain Antibody Constructs in Pic) 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,-His₆] and covalent [sc(Fv)₂-His₆] 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 × 10⁷, 2.75 × 10⁷, and 1.14 × 10⁸ M⁻¹ for (scFv)₂-His⁶, sc(Fv)₂-His₆, 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 (scFvJj-HiSg and bacteria (scFv)a 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