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

Apollina Goel, Guy W. Beresford, David Colcher, Gabriela Pavlinkova, Barbara J M Booth, Janina Baranowska-Kortylewicz, Surinder Kumar Batra

Research output: Contribution to journalArticle

33 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)829-836
Number of pages8
JournalJournal of Biochemistry
Volume127
Issue number5
DOIs
StatePublished - Jan 1 2000

Fingerprint

Single-Chain Antibodies
Pichia
Purification
Tumors
Bearings (structural)
Neoplasms
Pharmacokinetics
Surface Plasmon Resonance
Surface plasmon resonance
Heterografts
Nude Mice
Dimers
Yeast
Escherichia coli
Bacteria
Colon
Blood
Immunoglobulin G
Yeasts
Enzyme-Linked Immunosorbent Assay

Keywords

  • Colon carcinoma xenografts
  • Pharmacokinetics
  • Single-chain Fv
  • Valency
  • Yeast

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris : Expression, purification, and characterization. / Goel, Apollina; Beresford, Guy W.; Colcher, David; Pavlinkova, Gabriela; Booth, Barbara J M; Baranowska-Kortylewicz, Janina; Batra, Surinder Kumar.

In: Journal of Biochemistry, Vol. 127, No. 5, 01.01.2000, p. 829-836.

Research output: Contribution to journalArticle

Goel, Apollina ; Beresford, Guy W. ; Colcher, David ; Pavlinkova, Gabriela ; Booth, Barbara J M ; Baranowska-Kortylewicz, Janina ; Batra, Surinder Kumar. / Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris : Expression, purification, and characterization. In: Journal of Biochemistry. 2000 ; Vol. 127, No. 5. pp. 829-836.
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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 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.",
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AU - Pavlinkova, Gabriela

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AU - Baranowska-Kortylewicz, Janina

AU - Batra, Surinder Kumar

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