We believe vaccinia virus is a very useful expression vector because it infects most mammalian cell lines, and gives rapid expression of recombinant genes with full mammalian cell quality control and post translational modifications. Vaccinex has developed proprietary technology for the creation of large libraries containing billions of recombinant virus. We have used this technology to construct large full-length immunoglobulin gene libraries in vaccinia.
By creating a fusion protein comprising the Ig heavy chain and a vaccinia virus membrane protein, we have enabled expression of the antibody in association with full-length light chain on the surface of virus shed from the cell. Concurrently, the antibody is also expressed on the surface of the infected cell. This dual display approach allows for a primary, high-throughput “panning” step using purified recombinant virus similar to what one would do with bacteriophage. Post panning, after having enriched for binders, we immediately and without retooling switch to a cell-sorting approach that allows for tunable selection based on antibody specificity and affinity.
Cells from the sorting step are lysed to release the virus and the virus is amplified. Small diversity mini-libraries are subcloned into standard, non-viral based expression plasmid. Several hundred clones are sequenced and unique heavy and lights are transfected into mammalian cells for downstream testing.