Divalent Forms of CC49 Single-Chain Antibody Constructs in Pichia pastoris. Expression, Purification, and Characterization.

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概要

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• 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) CC 49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDSPAGE, HPLC, and competitive ELISA. The binding affinity constant (KA), determined by surface plasmon resonance analysis using BlAcore, was 4.28×107, 2.75×107, and 1.14×108 M-1 for (scFv)2-His6, sc (Fv)2 His6, and CC 49 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-174 T human colon carcinoma xenografts showed equivalent tumor-targeting of CC 49 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 scFv s suggest possible therapeutic applications.

• 社団法人 日本生化学会の論文

著者

• Batra Surinder

  Department Of Biochemistry And Molecular Biology University Of Nebraska Medical Center

• Booth Barbara

  Department Of Pathology And Microbiology University Of Nebraska Medical Center

• Baranowska-kortylewicz Janina

  Department Of Radiation Oncology University Of Nebraska Medical Center

• COLCHER David

  Coulter Pharmaceutical Inc.

• Goel Apollina

  Department Of Biochemistry And Molecular Biology University Of Nebraska Medical Center

• Pavlinkova Gabriela

  Department Of Pathology And Microbiology University Of Nebraska Medical Center

• Beresford Guy

  Department of Microbiology and Immunology, Emory University

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