Helix-stabilized Fv (hsFv) antibody fragments: Substituting the constant domains of a Fab fragment for a heterodimeric coiled-coil domain

Antibody Fv fragments would in principle be useful for a variety of biotech nological applications because of their small size and the possibility to p roduce them in relatively large amounts in recombinant form; however, their limited stability is a drawback. To solve this problem, both domains are u sually 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 CL domain of t he Fab fragment were replaced with a heterodimeric coiled coil (WinZip-A2B1 ), which had previously been selected using a protein-fragment complementat ion assay in Escherichia coli. This new antibody format was termed helix-st abilized 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 for med 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 n early as stable as the scFv fragment under the conditions used (80 nM prote in concentration). Thus, the format of a helix-stabilized Fv (hsFv) fragmen t can be a useful alternative to existing recombinant antibody formats, esp ecially in cases where poor expression of Fab fragments or multimerization of scFv fragments is a problem. (C) 2001 Academic Press.