Developing Cell-Specific Antibodies to Endothelial Progenitor Cells Using Avian Immune Phage Display Technology

Abstract

This study aims at generating immune chicken phage display libraries and single-chain antibodies (scFvs) specifically directed against cell surface markers of cultured peripheral blood mononuclear cells (PBMCs) that contain endothelial progenitor cells (EPCs). In contrast to previous approaches that use well-defined recombinant antigens attached to plastic surfaces that may alter the structure of the proteins, the authors describe a method that maintains the cell surface markers on live cells while providing the opportunity to rapidly screen entire libraries for antibodies that bind to unknown cell surface markers of progenitor/stem cells. Chickens immunized with live EPCs, consisting of a heterogeneous population of lymphocytes and monocytes, demonstrated a robust immune response. After three rounds of biopanning, the authors purified and characterized three unique scFvs called UG1-3. Codon-optimized recombinant UG1 (gUG-1) shows binding by flow cytometry to circulating CD14-positive cells in peripheral blood consistent with predominant expression of a target protein on monocyte subsets. The authors describe the successful use of immunization of chickens for the generation of scFvs against a heterogenous population of EPCs displaying unknown cell surface markers and demonstrate the strong potential of phage display technology in the development of reagents for the isolation and characterization of stem/progenitor cells.

Expression of human lambda expands the repertoire of OmniChickens

“…The lack of widespread use of chicken antibodies in clinical research derives from the lack of a fusion partner for chicken B cells, the frequency of disulfide bridges in CDR-H3 [12] and the presence of additional N-glycosylation sites [30]. As a result, chicken monoclonal antibodies have traditionally been derived from phage and other display libraries [31][32][33]. Subsequent humanization is then required to minimize immunogenicity.…”

Reconciling the Structural Attributes of Avian Antibodies

“…The chicken antibody repertoire has been extensively examined at the genomic level and also in terms of both functional isotype content (4,42). Recently, a number of studies have exploited the chicken repertoire to generate high quality antibodies to a wide range of antigens (10,19,22,(43)(44)(45)(46)(47)(48), have extensively examined the characteristics of the repertoire with detailed analysis of amino acid diversity in both naïve and selected repertoires (20), and have presented the crystal structure of an chicken Fv domain (19).…”

A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain

“…Consequently, strategies for obtaining mAbs directed against surface receptors have primarily involved the use of intact cells as antigen. This approach can yield mAbs directed towards native epitopes in a random fashion, which can be useful for some applications [3, 4], but simultaneously yields mAbs against unwanted surface targets. An alternative approach is to use small peptides (conjugated to carrier proteins for immune response) comprising predicted loops from crystal structures of target receptors.…”