Abstract
Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.
Highlights
Phage display, first described in 1985, is a practical tool for displaying proteins or peptides of interest in bacteriophage through fusion with viral envelope proteins [1,2,3]
This implied that the phospholipid-magnetic beads complexlabeled cells can be applied to magnetic beads-based bio-panning, as well as to the automated magnetic particle processor for semi-automated cell panning (Figure 1A)
To efficiently enrich scFv binders, a semi-automated cell panning process was combined with conventional bio-panning
Summary
First described in 1985, is a practical tool for displaying proteins or peptides of interest in bacteriophage through fusion with viral envelope proteins [1,2,3]. Phage libraries are used to select and isolate binding molecules with high affinity for the target antigen with applications in monoclonal antibody (mAb) discovery, affinity maturation, and humanization [4,5]. Bio-panning for affinity selection has been used to isolate target protein-binding molecules from phage libraries [1,6,7]. WCP was applied to isolated phage binders using cells intact with cell membrane proteins such as G-protein-coupled receptors, ligand-gate ion channels, receptor tyrosine kinases, and immunoglobulin-like receptors [3]. WCP may select non-specific phage binders to off-target cell surface protein, it could potentially enrich the phage binders recognizing the naturally exposed epitope on the cell surface [12,14,15]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
