title: Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand.
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.affiliation: Radiodiagnosis and Therapy, Molecular Cancer Institute, University of California Davis Medical Center, Sacramento, California 95816, USA.
authors.forename: Huguette
authors.initials: H
authors.lastname: Albrecht
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Burke
authors.forename: Arutselvan
authors.initials: A
authors.lastname: Natarajan
authors.forename: Cheng-Yi
authors.initials: CY
authors.lastname: Xiong
authors.forename: Mark
authors.initials: M
authors.lastname: Kalicinsky
authors.forename: Gerald L
authors.initials: GL
authors.lastname: DeNardo
authors.forename: Sally J
authors.initials: SJ
authors.lastname: DeNardo
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005285:Fermentation; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry*; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D016296:Mutagenesis; D019151:Peptide Library; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry*; D019163:Reducing Agents / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013439:Sulfhydryl Reagents
chemical_list: D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D007202:Indicators and Reagents; D019151:Peptide Library; D011994:Recombinant Proteins; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D011092:Polyethylene Glycols; D003545:Cysteine