Monoclonal Expression of Anti-Gelsolin Antibody Libraries in Mammalian Suspension Cells for High-Throughput Screening on Color-Coded Beads

Abstract

Phage display technology has become a key player in the selection of affinity reagents from antibody libraries. To obtain the best binders from phage combinatorial libraries, affinity screening has been traditionally done using immunoassay on microtiter plate or filters array. The assay is limited for multiplexing, thus contributes to the bottleneck in screening step. In this study, multiplexed bead-based assay was demonstrated for high-throughput screening of the anti-gelsolin antibody libraries. In addition to the multiplexing, we tackled the problem in producing monoclonal antibody libraries in mammalian cells by testing two approaches, rolling circle amplification (RCA)-transfection and CRISPR/Cas9 knock-in. The monoclonal antibody libraries were produced as fusion protein with SpyCatcher3 to achieve directional coupling onto the SpyTag3-beads. CRISPR/Cas9 knock-in and RCA-transfection were tested using Expi293F cells. In the CRISPR system, Cas9/gRNA vector and destination vector were co-transfected to allow the integration of one copy of multicistronic gene cassette encoding the antibody sequences into target locus. The RCA-transfection approach was done by transfection of direct RCA products from bacteria colony. Furin recognition and 2A self-cleaving peptide sequences were incorporated in both gene cassette and single plasmid vector used for CRISPR and RCA-transfection testing, respectively. SpyTag3-beads were labeled with unique ratio of different fluorophores to allow the multiplexing and simultaneous analysis in flow cytometry. CRISPR experiment was terminated due to cell viability problems. The viability drastically dropped within 3 days after antibiotics addition and could not recover by subculturing or centrifugation separation. Initial test of RCA-transfection was confirmed to produce the parental antibody, with 2.5 µl volume input resulted in the highest expression level (S/B: 1.4), verified by immunoassay. The monoclonal antibody libraries were successfully produced using the RCA-transfection approach, confirmed by the immunoassay on 96-well plate and 12-plex color-coded beads (CCB), albeit the overall low and varying expression level between different clones (Eu signals: 18 133-112 514 counts, APC-A: <20 000 counts). To conclude, RCA-transfection approach is capable to produce monoclonal antibody libraries in mammalian cells, which can be multiplexed using CCB for high-throughput screening. The low expression level from RCA-transfection, however, still requires improvement to use for affinity study.