Patent ID: 8247194

Claim:
A method of preparing an antibody fragment of a source immunoglobulin, which source immunoglobulin specifically binds to an antigen of interest, a corresponding antibody fragment of which source immunoglobulin exhibits insufficient soluble recombinant expression, comprising: (a) providing a nucleic acid molecule encoding a first antibody variable region comprised in the source immunoglobulin, wherein the first antibody variable region is a heavy chain variable region (VH) or a light chain variable region (VL); (b) respectively combining (i) the nucleic acid molecule encoding the first antibody VH or VL region with (ii) a plurality of nucleic acid molecules encoding a diverse population of a second antibody variable region, wherein the second antibody variable region is a light chain variable region (VL) or a heavy chain variable region (VH), whereby a first population of combined nucleic acid molecules is obtained; (c) introducing the first population of combined nucleic acid molecules into a display system chosen from a phage display system, a prokaryotic display system, a eukaryotic display system, or an mRNA display system, whereby the nucleic acid molecule encoding the first antibody variable region or the nucleic acid molecule encoding the second antibody variable region is operably linked to a nucleic acid molecule encoding an N-terminal, cis-acting amphipathic polypeptide moiety such that said N-terminal, cis-acting amphipathic polypeptide moiety, when translated, is linked to the N-terminal end of the first or second antibody variable region; (d) selecting at least one first antibody fragment displayed in step (c) and comprising the VH and VL region, which specifically binds to the antigen of interest and is in soluble form; and (e) isolating the at least one first antibody fragment selected in step (d); wherein the amphipathic polypeptide moiety is chosen from the pro regions of any of the following polypeptides: papain, cruzain, thermolysin, cathepsin B, cathepsin L, protease A, protease B, IgA protease and carboxypeptidase Y, or the N2 domain of a filamentous phage.