Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris: Expression, purification, and characterization

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 SDSPAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10⁷, 2.75 x 10⁷, and 1.14 x 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 (scFv)₂-His₆ 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.