ABSTRACT
Antibodies that target the blood-brain barrier (BBB) in vivo are of particular interest for the treatment of neurological diseases. Here, we screened a phage display single-chain antibody (scFv) library by brain perfusion in an attempt to isolate scFv that target the rat BBB. After four rounds of screening, the resulting antibody pool remained highly complex and discrete clonal sampling did not identify any scFvs capable of binding to the rat BBB. Thus, the heavy chain CDR3 in the resulting pools was subjected to NGS, and the resulting data was used to identify 12 scFv clones that were of high abundance and/or enriched from round 3 to 4, signifying potential hits. Of these, two scFv, denoted scFv 4 and scFv 40, were identified that bound the rat BBB. Neither of these scFvs was identified by discrete sampling, motivating NGS as a tool to identify lead antibodies from complex in vivo screens.
ABSTRACT
The blood-brain barrier (BBB) represents an obstacle in targeting and delivering therapeutics to the central nervous system. In order to discover new BBB-targeting molecules, we panned a phage-displayed nonimmune human single-chain antibody fragment (scFv) library against a representative BBB model comprised of hydrocortisone-treated primary rat brain endothelial cells. Parallel screens were performed with or without pre-subtraction against primary rat heart and lung endothelial cells in an effort to identify antibodies that may have binding selectivity toward brain endothelial cells. After three rounds of screening, three unique scFvs, scFv15, scFv38, and scFv29, were identified that maintained binding to primary rat brain endothelial cells, both in phage and soluble scFv format. While scFv29 and to a lesser extent, scFv15, exhibited some brain endothelial cell specificity in tissue culture, scFv29 did not appear to bind a BBB antigen in vivo. In contrast, both scFv15 and scFv38 were capable of immunolabeling rat brain vessels in vivo and displayed brain vascular selectivity with respect to all peripheral organs tested other than heart. Taken together, scFv15 and scFv38 represent two new antibodies that are capable of binding antigens that are expressed at the BBB in vivo.
