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Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand.
Bioconjug Chem. 2004 Jan-Feb; 15(1):16-26.BC

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.

Authors+Show Affiliations

Radiodiagnosis and Therapy, Molecular Cancer Institute, University of California Davis Medical Center, Sacramento, California 95816, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

14733579

Citation

Albrecht, Huguette, et al. "Production of Soluble ScFvs With C-terminal-free Thiol for Site-specific Conjugation or Stable Dimeric ScFvs On Demand." Bioconjugate Chemistry, vol. 15, no. 1, 2004, pp. 16-26.
Albrecht H, Burke PA, Natarajan A, et al. Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand. Bioconjug Chem. 2004;15(1):16-26.
Albrecht, H., Burke, P. A., Natarajan, A., Xiong, C. Y., Kalicinsky, M., DeNardo, G. L., & DeNardo, S. J. (2004). Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand. Bioconjugate Chemistry, 15(1), 16-26.
Albrecht H, et al. Production of Soluble ScFvs With C-terminal-free Thiol for Site-specific Conjugation or Stable Dimeric ScFvs On Demand. Bioconjug Chem. 2004 Jan-Feb;15(1):16-26. PubMed PMID: 14733579.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand. AU - Albrecht,Huguette, AU - Burke,Patricia A, AU - Natarajan,Arutselvan, AU - Xiong,Cheng-Yi, AU - Kalicinsky,Mark, AU - DeNardo,Gerald L, AU - DeNardo,Sally J, PY - 2004/1/22/pubmed PY - 2004/3/10/medline PY - 2004/1/22/entrez SP - 16 EP - 26 JF - Bioconjugate chemistry JO - Bioconjug. Chem. VL - 15 IS - 1 N2 - 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. SN - 1043-1802 UR - https://www.unboundmedicine.com/medline/citation/14733579/Production_of_soluble_ScFvs_with_C_terminal_free_thiol_for_site_specific_conjugation_or_stable_dimeric_ScFvs_on_demand_ L2 - https://dx.doi.org/10.1021/bc030018+ DB - PRIME DP - Unbound Medicine ER -