Identification of blood-brain barrier targeting antibodies for proteomic and drug delivery applications

Vertebrate brain endothelium is impermeable to most materials and has therefore been called the blood-brain barrier (BBB). The barrier properties of the BBB are conferred by its unique complement of membrane proteins, including tight junction proteins, molecular transporters and immunoregulatory proteins. However, our current knowledge of the BBB membrane proteome is very limited, and this has important consequences since BBB dysfunction is related to many central nervous system (CNS) diseases. Although BBB impermeability is required for normal brain function, the BBB also provides a bottleneck for the treatment of neurological disease. A promising approach for overcoming the BBB employs monoclonal antibodies targeted against endogenous transporter proteins at the BBB. These antibodies can act as molecular Trojan horses allowing noninvasive delivery of brain pharmaceuticals from the blood into the brain. Therefore, there exists a significant need for the identification of novel transporters and their cognate targeting antibodies.; To identify BBB-binding antibodies for BBB membrane proteomic research or brain drug delivery applications, we have combined antibody yeast surface display technology with BBB endothelial cells in a novel screening strategy. This combinatorial strategy allowed discovery of novel BBB-binding antibodies via yeast-endothelial cell conjugates without a priori knowledge of the antibody or endothelial cell membrane proteins. We first developed the screening procedure known as yeast biopanning by employing a model antibody-hapten system, and critical parameters governing the screening methodology were investigated. The fast and efficient screening strategy was then successfully applied to the mining of a nonimmune human antibody library displayed on yeast surface for antibodies that bind to the surface of brain endothelial cells. Discovery of many unique BBB-binding antibodies using yeast biopanning has for the first time proven the feasibility of using yeast surface display for cell-based selections. Several antibodies that could be internalized by BBB endothelial cells were discovered and shown to target a specific BBB molecular transport system, offering promise as noninvasive drug delivery reagents. Finally, using a novel technique that employs antibody-displaying yeast as immunoprecipitation particles, the corresponding antigens targeted by these antibodies were investigated as an important initial step taken towards expanding the BBB membrane protein profile.