Preparation of scfv antibody fragments

An antibody fragment obtainable by a method comprising:

• a) providing a nucleic acid molecule encoding a first antibody variable region or fragment thereof comprised in a source immunoglobulin, wherein the first antibody variable region or fragment thereof is a heavy chain variable region (VH) or a light chain variable region (VL), or a fragment of either of these;
• b) respectively combining (i) the nucleic acid molecule encoding the first antibody VH or VL region or fragment of either with (ii) a plurality of nucleic acid molecules encoding a diverse population of a second antibody variable region or fragment thereof, wherein the second antibody variable region or fragment thereof is a light chain variable region (VL) or a heavy chain variable region (VH), or a fragment of either of these, whereby a population of combined nucleic acid molecules is obtained;
• c) introducing the 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;
• d) selecting at least one antibody fragment displayed in (c) and comprising the VH and VL regions, or a fragment of either or both of these, which specifically binds to an antigen of interest;
• and
• e) isolating the at least one antibody fragment selected in (d);
• wherein the nucleic acid molecule encoding the first antibody variable region or fragment thereof or the nucleic acid molecule encoding the second antibody variable region or fragment thereof 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;
• and
• wherein the source immunoglobulin specifically binds to an antigen of interest, a corresponding antibody fragment of which source immunoglobulin exhibits insufficient soluble recombinant expression.

An antibody fragment obtainable by a method comprising:

• a) providing a nucleic acid molecule encoding a first antibody variable region or fragment thereof comprised in a source immunoglobulin, wherein the first antibody variable region or fragment thereof is a heavy chain variable region (VH) or a light chain variable region (VL), or a fragment of either of these;
• b) respectively combining (i) the nucleic acid molecule encoding the first antibody VH or VL region or fragment of either with (ii) a plurality of nucleic acid molecules encoding a diverse population of a second antibody variable region or fragment thereof, wherein the second antibody variable region or fragment thereof is a light chain variable region (VL) or a heavy chain variable region (VH), or a fragment of either of these, 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;
• d) selecting at least one first antibody fragment displayed in (c) and comprising the VH and VL regions, or a fragment of either or both of these, which specifically binds to an antigen of interest;
• e) respectively combining (i) the nucleic acid molecule encoding the second antibody variable region or fragment thereof with (ii) a plurality of nucleic acid molecules encoding a diverse population of a third antibody variable region or fragment thereof, wherein the third antibody variable region or fragment thereof is a heavy chain variable region (VH) or a light chain variable region (VL), or a fragment of either of these, whereby a second population of combined nucleic acid molecules is obtained;
• f) introducing the second 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;
• g) selecting at least one second antibody fragment displayed in (f) and comprising the VH and VL regions, or a fragment of either or both of these, which specifically binds to the antigen of interest;
• and
• h) isolating the at least one antibody fragment selected in (g);
• wherein the nucleic acid molecule encoding the first antibody variable region or fragment thereof or the nucleic acid molecule encoding the second antibody variable region or fragment thereof of (b) 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;
• and
• wherein the nucleic acid molecule encoding the second antibody variable region or fragment thereof or the nucleic acid molecule encoding the third antibody variable region or fragment thereof of (e) 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 polypepetide moiety, when translated, is linked to the N-terminal end of the second or third antibody variable region;
• and
• wherein the source immunoglobulin specifically binds to an antigen of interest, a corresponding antibody fragment of which source immunoglobulin exhibits insufficient soluble recombinant expression.