title: Helix-stabilized Fv (hsFv) antibody fragments: substituting the constant domains of a Fab fragment for a heterodimeric coiled-coil domain.
abstract: Antibody Fv fragments would in principle be useful for a variety of biotechnological applications because of their small size and the possibility to produce them in relatively large amounts in recombinant form; however, their limited stability is a drawback. To solve this problem, both domains are usually fused via a peptide linker to form a single-chain Fv (scFv) fragment, but in some cases this leads to a dimerization. We present an alternative format for stabilizing antibody Fv fragments. The C(H)1 and C(L) domain of the Fab fragment were replaced with a heterodimeric coiled coil (WinZip-A2B1), which had previously been selected using a protein-fragment complementation assay in Escherichia coli. This new antibody format was termed helix-stabilized Fv fragment (hsFv), and was compared to the corresponding Fv, Fab and single-chain Fv format. Bacterial growth and expression of the hsFv was significantly improved compared to the Fab fragment. The hsFv fragment formed a heterodimer of heavy and light chain with the expected molecular mass, also under conditions where the scFv fragment was predominantly dimeric. The hsFv fragment was significantly more stable than the Fv fragment, and nearly as stable as the scFv fragment under the conditions used (80 nM protein concentration). Thus, the format of a helix-stabilized Fv (hsFv) fragment can be a useful alternative to existing recombinant antibody formats, especially in cases where poor expression of Fab fragments or multimerization of scFv fragments is a problem.
authors.affiliation: Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland.
authors.forename: K M
authors.initials: KM
authors.lastname: Arndt
authors.forename: K M
authors.initials: KM
authors.lastname: Müller
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins