HOMEPRODUCTSCOMPANYCONTACTFAQResearchDictionaryPharmaSign Up FREE or Login

Divalent forms of CC49 single-chain antibody constructs in Pichia 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)-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.
AuthorsA Goel, G W Beresford, D Colcher, G Pavlinkova, B J Booth, J Baranowska-Kortylewicz, S K Batra
JournalJournal of biochemistry (J Biochem) Vol. 127 Issue 5 Pg. 829-36 (May 2000) ISSN: 0021-924X [Print] England
PMID10788792 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)
Chemical References
  • Antibodies, Monoclonal
  • Antibodies, Neoplasm
  • Antineoplastic Agents
  • B72.3 antibody
  • DNA Primers
  • Immunoglobulin Fragments
  • Recombinant Proteins
  • immunoglobulin Fv
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)

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.
Realize the full power of the drug-disease research graph!


Choose Username:
Email:
Password:
Verify Password:
Enter Code Shown: