(4aa) Modeling the Human Blood-Brain Barrier: Leveraging in Vitro Models for the Identification of Novel Brain Targeting Antibodies

The blood-brain barrier (BBB) performs the critical role of protecting the brain from circulating blood species and strictly maintaining brain homeostasis, allowing for the free and active transport of nutrients into the brain and restricting the transport of all other species. This tight control of transport inhibits the ability of therapeutics to enter the brain. The BBB is implicated in many central nervous system diseases, not in just the difficulty it causes in delivery of therapeutics, but the breakdown of the BBB contributes to disease progression and neuronal breakdown. Modeling and understanding the role of the BBB in health and disease is critical to the understanding of neurodegenerative disease progression and the development of novel therapeutic delivery strategies that are able to penetrate the brain parenchyma at physiologically relevant doses. To this aim my research has focused on the development of in vitro models of the human BBB using stem cell derived cells to model the 3D geometry and transport at the human BBB during health and disease, as well as the identification and development of novel cerebrovascular targeting and penetrating antibodies for improved delivery.

Graduate Research: During my PhD my focus was on the development of human in vitro BBB models, investigating the role of brain microvascular endothelial cells (BMECs) in the formation of the barrier and regulation of transport. To do this BMEC-like-cells (iBMECs) were derived from induced pluripotent stem cells (iPSCs) that display the critical restricted transport characteristics of the BBB [1]. Subsequently, iPSCs sourced from individuals with genetic mutations encoding for neurodegenerative diseases were used to differentiate iBMECs, the transport properties of the resultant iBMECs were investigated to determine the potential role of non-cell autonomous cerebrovascular dysfunction and its potential role in disease progression. It was determined that many of the neurodegenerative lines resulted in iBMECs with reduced barrier function and dysfunctional transport resulting in an ultimately leakier barrier, particularly dysfunction of efflux transporters [2]. While these results are preliminary, when taken together with similar results seen by others they suggest that BMEC dysfunction likely contributes in a non-cell autonomous mechanism to CNS disease progression. In addition to simple transport modeling, the iBMECs were used to develop a 3D model of the BBB, where cell phenotype and phenomenon can be elucidated in real time via live cell microscopy [3]. The model has been further validated and developed to visualize additional BMEC phenomenon [4]. These tools provide a valuable model to elucidate barrier formation and dysfunctional transport at the human BBB.

Postdoctoral Research: My postdoctoral research has centered on identifying and developing novel antibodies for BBB targeting and delivery utilizing murine model systems. The antibodies used in this research are variable lymphocyte receptors (VLRs), the adaptive immune response of the lamprey; VLRs offer an expanded potential pool of target ligands due to their unique binding geometry, enhanced glycan binding, and the large evolutionary distance from humans. Lamprey were immunized against murine brain microvessel fragments [5], the resultant VLRs were then bio-panned in yeast surface display format over BMECs to enrich and identify BBB binding clones. These clones were subsequently sequenced and produced as VLR-Fc fusion proteins and their binding and internalization was validated both in vitro, in murine and human brain tissue sections, as well as following injection in a murine model. The murine screen has generated three candidate antibodies that are able to bind to cerebrovasculature and penetrate the parenchyma.

Together with the in vitro model I developed in my graduate studies, this will provide a valuable platform to study perturbations in the human BBB and screen potential therapeutic delivery systems.

Teaching Interests:

As both a graduate student and a postdoctoral fellow I have made teaching and mentorship a central focus. I have had the opportunity to serve as a teaching assistant for the undergraduate materials engineering thermodynamics course, where I was able to lead a few of the lectures and assist students understanding the material in office hours. I also was a teaching assistant for the biomaterials lab capstone course where I assisted the students in selecting and designing their final lab project. I have also mentored a number of undergraduate and graduate students from a wide variety of backgrounds. This has given me an opportunity to employ a number of different teaching strategies individually suited for each student as I helped them learn new techniques, proper data handling and analysis methods, experimental design, and helped them on their way to independence. Watching my students make the transition from taking direct instruction on experimental design, to designing their experiments that will be able to answer their own relevant scientific questions is the ultimate reward. I look forward to teaching graduate and undergraduate level chemical engineering courses particularly focused on biomaterials and transport as well as develop protein engineering or tissue engineering courses.

References:

1. Katt, M.E., Xu, Z.S., Gerecht, S., and Searson, P.C. (2016). Human brain microvascular endothelial cells derived from the BC1 iPS cell line exhibit a blood-brain barrier phenotype. PloS one 11.
2. Katt, M.E., Mayo, L.N., Ellis, S.E., Mahairaki, V., Rothstein, J.D., Cheng, L., and Searson, P.C. (2019). The role of mutations associated with familial neurodegenerative disorders on blood–brain barrier function in an iPSC model. Fluids and Barriers of the CNS 16, 20.
3. Katt, M.E., Linville, R.M., Mayo, L.N., Xu, Z.S., and Searson, P.C. (2018). Functional brain-specific microvessels from iPSC-derived human brain microvascular endothelial cells: the role of matrix composition on monolayer formation. Fluids and Barriers of the CNS 15, 7.
4. Linville, R.M., DeStefano, J.G., Sklar, M.B., Xu, Z., Farrell, A.M., Bogorad, M.I., Chu, C., Walczak, P., Cheng, L., and Mahairaki, V. (2019). Human iPSC-derived blood-brain barrier microvessels: Validation of barrier function and endothelial cell behavior. Biomaterials 190, 24-37.
5. Lajoie, J.M., Katt, M.E., Waters, E.A., Herrin, B.R., and Shusta, E.V. (Under Revision). Identification of lamprey variable lymphocyte receptors that target the brain vasculature.