title: Insect cell-based expression and characterization of a single-chain variable antibody fragment directed against blood coagulation factor VIII.
abstract: A recombinant single-chain variable antibody fragment (scFv) KM33 was previously described as a ligand that can inhibit the function of blood coagulation factor VIII (FVIII). This scFv was previously derived from an individual with anti-FVIII antibodies manifested in FVIII functional deficiency (Hemophilia A) and expressed in bacteria. In the present work, we describe an alternative approach for fast and easy production of KM33 in insect cells (Spodoptera frugiperda). The KM33 gene was codon-optimized and expressed in secreted form using a baculovirus system. The protein was isolated using metal-affinity and size-exclusion chromatography to purity of about 96% and yield of 0.4-1.2 mg per 120 mL of culture, based on several independent expression experiments. In a binding assay using surface plasmon resonance, the insect cell-derived KM33 (iKM33) was qualified as a high-affinity ligand for FVIII. Epitope specificity of iKM33 on FVIII (C1 domain) was confirmed by testing the binding with a relevant mutant of FVIII. In several FVIII functional tests (factor Xa generation, APTT clotting, thrombin generation and video microscopy clot growth assays), iKM33 strongly inhibited FVIII activity in accordance with the clinical effect of the parental antibody. Therefore, the expressed protein was concluded to be fully functional and applicable in various assays with FVIII.
authors.affiliation: Division of Hematology, Office of Blood Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD 20852, USA.
authors.forename: James H
authors.initials: JH
authors.lastname: Kurasawa
authors.forename: Svetlana A
authors.initials: SA
authors.lastname: Shestopal
authors.forename: Naveen K
authors.initials: NK
authors.lastname: Jha
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Ovanesov
authors.forename: Timothy K
authors.initials: TK
authors.lastname: Lee
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Sarafanov
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001777:Blood Coagulation; D002460:Cell Line; D005169:Factor VIII / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D015870:Gene Expression; D007313:Insecta / Q000166:cytology / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D013917:Thrombin / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D005169:Factor VIII; D013917:Thrombin; D015951:Factor Xa