Design, construction, and characterization of a large synthetic human antibody phage display library

Abstract

Advances in proteomic research allow the identification of several hundred protein components in complex biological specimens. Structural information is typically lost during proteomic investigations. For this reason, the rapid isolation of monoclonal antibodies specific to proteins of interest would allow the study of structurally intact biological specimens, thus providing complementary proteomic information. Here, we describe the design, construction, characterization, and use of a large synthetic human antibody phage display library (ETH-2-Gold) containing three billion individual antibody clones. A large repertoire of antibodies with similar biochemical properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto three antibody germline segments (DP47, DPK22, and DPL16), which are frequently found in human antibodies. The ETH-2-Gold library exhibits efficient display of antibody fragments on filamentous phage, as assessed by immunoblot. Furthermore, the library is highly functional, since >90% of clones express soluble antibodies in bacteria and since good quality monoclonal antibodies have been isolated against 16 different antigens. The usefulness of the library as a tool for generating monoclonal antibodies for biomedical applications was tested using the C-domain of tenascin-C (a marker of angiogenesis) as antigen and showing that specific antibodies to this target were able to stain vascular structures in tumor sections.

Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

“…Selections of antibodies were carried out on Immunotubes (Nunc, Roskilde, Denmark) coated with recombinant CA IX at 50 mg ml -1 in PBS (20 mM NaH 2 PO 4 , 30 mM Na 2 HPO 4 , 100 mM NaCl, pH 7.4), as described previously by our group (Silacci et al, 2005).…”

“…Recombinant antibody fragments, which were positive in ELISA, in the scFv format were expressed in E.coli TG-1 and purified from culture supernatant by affinity chromatography using Protein A Sepharose Fast Flow resin (GE Healthcare), as described previously (Silacci et al, 2005).…”

“…Mutations at positions 31, 32, 33 of VH and 31, 31a, 32 of VL were introduced by PCR using the same partially degenerate primers (Eurofins MWG Operon, Ebersberg, Germany) as described previously (Villa et al, 2008). A single round of selection on biotinylated antigen (final concentration 10 À7 M) was carried out eluting bound phage with 100 mM triethylamine, as described previously (Silacci et al, 2005). ELISA and Biacore (Otelfingen, Switzerland) screening of dissociation profiles of selected clones on a high-density coated chip yielded the antibody A3.…”

“…SIP(A3) and SIP(CC7), used as primary binding reagents at 1 mg ml -1 , were detected with rabbit-anti-human IgE antibody (Dako), followed by goat anti-rabbit IgG Alexa Fluor 594 antibody (Invitrogen). Primary rabbit polyclonal anti-CA IX antiserum (sc-25599; Santa Cruz Biotechnology, Heidelberg, Germany) was detected with goat anti-rabbit IgG Alexa Fluor 594 antibody (Silacci et al, 2005) was cloned into pHEN1 vector with VH and VL segments of scFvs flanked by the pelB secretion sequence, and an myc-tag followed by an amber codon and the geneencoding pIII. (B, E) Schematic representation of the different antibody formats used in this study.…”

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

“…2 Numerous antibody fragment libraries for phage display have been created, and the creation of new libraries continues as knowledge and technology expands the possibilities. [3][4][5][6][7][8][9][10] Improvements concern stability and functionality of the various antibody fragments themselves, regulation of the expression, the fusion partner utilized in the display system and not least the peptide tag sequences applied (Fig. 1).…”