Production of Soluble ScFvs with C-Terminal-Free Thiol for Site-Specific Conjugation or Stable Dimeric ScFvs on Demand

Huguette Albrecht, Patricia A. Burke, Arutselvan Natarajan, Cheng Yi Xiong, Mark Kalicinsky, Gerald L Denardo, Sally J. DeNardo

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)2) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)2 scaffold to form immunoreactive PEG(scFv) 2 bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)2. ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)2 molecules or used as (scFv-Cys)2 for increased sensitivity in IHC.

Original languageEnglish (US)
Pages (from-to)16-26
Number of pages11
JournalBioconjugate Chemistry
Volume15
Issue number1
DOIs
StatePublished - Jan 2004

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Reducing Agents
Sulfhydryl Compounds
Bacteriophages
Polyethylene glycols
Single-Chain Antibodies
Immunoglobulin Fragments
Escherichia coli Proteins
Reducing agents
Drug Delivery Systems
Molecules
Cysteine
Tumors
Neoplasms
Staining and Labeling
Antigens
Forms (concrete)
Antibodies
Scaffolds
Pharmaceutical Preparations
Dimers

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

Cite this

Production of Soluble ScFvs with C-Terminal-Free Thiol for Site-Specific Conjugation or Stable Dimeric ScFvs on Demand. / Albrecht, Huguette; Burke, Patricia A.; Natarajan, Arutselvan; Xiong, Cheng Yi; Kalicinsky, Mark; Denardo, Gerald L; DeNardo, Sally J.

In: Bioconjugate Chemistry, Vol. 15, No. 1, 01.2004, p. 16-26.

Research output: Contribution to journalArticle

Albrecht, Huguette ; Burke, Patricia A. ; Natarajan, Arutselvan ; Xiong, Cheng Yi ; Kalicinsky, Mark ; Denardo, Gerald L ; DeNardo, Sally J. / Production of Soluble ScFvs with C-Terminal-Free Thiol for Site-Specific Conjugation or Stable Dimeric ScFvs on Demand. In: Bioconjugate Chemistry. 2004 ; Vol. 15, No. 1. pp. 16-26.
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