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.
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Authors | A Goel, G W Beresford, D Colcher, G Pavlinkova, B J Booth, J Baranowska-Kortylewicz, S K Batra |
Journal | Journal of biochemistry
(J Biochem)
Vol. 127
Issue 5
Pg. 829-36
(May 2000)
ISSN: 0021-924X [Print] England |
PMID | 10788792
(Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)
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Chemical References |
- Antibodies, Monoclonal
- Antibodies, Neoplasm
- Antineoplastic Agents
- B72.3 antibody
- DNA Primers
- Immunoglobulin Fragments
- Recombinant Proteins
- immunoglobulin Fv
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Topics |
- Animals
- Antibodies, Monoclonal
(biosynthesis, genetics, pharmacokinetics)
- Antibodies, Neoplasm
(biosynthesis, genetics, metabolism)
- Antineoplastic Agents
(metabolism, pharmacokinetics)
- Base Sequence
- Carcinoma
(drug therapy)
- Colonic Neoplasms
(drug therapy)
- DNA Primers
- Dimerization
- Female
- Immunoglobulin Fragments
(biosynthesis, genetics, metabolism)
- Mice
- Mice, Nude
- Molecular Sequence Data
- Pichia
(genetics)
- Polymerase Chain Reaction
- Protein Engineering
- Recombinant Proteins
(biosynthesis, pharmacokinetics)
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