Figure 2
From: Identification of lamprey variable lymphocyte receptors that target the brain vasculature
BBBVLR library screening and characterization workflow. (i) Immunized lamprey BBBVLR libraries are expressed on the yeast surface by fusion to the C-terminus of the Aga2p protein. Each yeast cell displays thousands of copies of a single VLR clone on its surface. (ii) and (iii) The immune, unsorted library is enriched for binders to BBB antigens expressed in vivo via MACS and FACS with detergent-solubilized BMPMs. The percentage of antigen-binding yeast clones before (ii) and after (iii) sorting are reported. (iv) The FACS-enriched BMPM-binding pool is next screened for VLRs that bind extracellular domains of BMPM epitopes via biopanning against an MBEC cell line while also employing negative selection against ECM in each round. (v) Unique MBEC binding clones are identified via a monoclonal biopanning and sequencing workflow. (vi) Unique VLRs are reformatted as rabbit IgG-Fc fusion proteins, secreted from HEK293 cells, and used to immunolabel brain sections to verify binding to relevant BBB antigens in the mouse brain. (vii) VLR capability for BBB transport is assessed in vitro with an internalization assay using cultured MBECs. (viii) Lead VLR-Fcs coming out of this pipeline are further characterized in various in vitro and in vivo assays.
