title: A fully human scFv phage display library for rapid antibody fragment reformatting.
abstract: Phage display libraries of human single-chain variable fragments (scFvs) are a reliable source of fully human antibodies for scientific and clinical applications. Frequently, scFvs form the basis of larger, bivalent formats to increase valency and avidity. A small and versatile bivalent antibody fragment is the diabody, a cross-paired scFv dimer (∼55 kDa). However, generation of diabodies from selected scFvs requires decreasing the length of the interdomain scFv linker, typically by overlap PCR. To simplify this process, we designed two scFv linkers with integrated restriction sites for easy linker length reduction (17-residue to 7-residue or 18-residue to 5-residue, respectively) and generated two fully human scFv phage display libraries. The larger library (9 × 10(9) functional members) was employed for selection against a model antigen, human N-cadherin, yielding novel scFv clones with low nanomolar monovalent affinities. ScFv clones from both libraries were reformatted into diabodies by restriction enzyme digestion and re-ligation. Size-exclusion chromatography analysis confirmed the proper dimerization of most of the diabodies. In conclusion, these specially designed scFv phage display libraries allow us to rapidly reformat the selected scFvs into diabodies, which can greatly accelerate early stage antibody development when bivalent fragments are needed for candidate screening.
authors.affiliation: Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 570 Westwood Plaza, Box 951770, Los Angeles, CA 90095, USA.
authors.forename: Keyu
authors.initials: K
authors.lastname: Li
authors.affiliation: Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 570 Westwood Plaza, Box 951770, Los Angeles, CA 90095, USA.
authors.forename: Kirstin A
authors.initials: KA
authors.lastname: Zettlitz
authors.affiliation: Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 570 Westwood Plaza, Box 951770, Los Angeles, CA 90095, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Lipianskaya
authors.affiliation: Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, 1001 Potrero Ave, Rm 3C-38, San Francisco, CA 94110, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, 1001 Potrero Ave, Rm 3C-38, San Francisco, CA 94110, USA.
authors.forename: James D
authors.initials: JD
authors.lastname: Marks
authors.affiliation: Canary Center for Cancer Early Detection, Stanford University, Palo Alto, CA 94304, USA.
authors.forename: Parag
authors.initials: P
authors.lastname: Mallick
authors.affiliation: Department of Urology, UCLA, Los Angeles, CA 90095, USA Molecular Biology Institute at UCLA, Los Angeles, CA 90095, USA Jonsson Comprehensive Cancer Center at UCLA, Los Angeles, CA 90095, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Reiter
authors.affiliation: Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 570 Westwood Plaza, Box 951770, Los Angeles, CA 90095, USA Jonsson Comprehensive Cancer Center at UCLA, Los Angeles, CA 90095, USA awu@mednet.ucla.edu.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D019151:Peptide Library; D057127:Single-Chain Antibodies
keywords: Antibody fragment; N-cadherin; diabody; phage display; scFv