Poster X18 - Identification of Antibodies for Specific Targeting of the Brain Following Ischemic Stroke

Introduction: Stroke currently holds the second highest rate of death and disability globally. There is a lack of treatment for chronic stroke with the gold standard in clinics being tissue plasminogen activator (tPA) and mechanical thrombectomy, both of which are dangerous and can cause more damage to the brain following stroke (Powers et al 2019). Improved outcomes following ischemic stroke and other central nervous system (CNS) diseases is impaired by the highly protective blood brain barrier (BBB) that conducts tight regulatory control of molecules, preventing entry of therapeutics into the brain parenchyma. Improved means of delivering therapeutics to the brain in CNS diseases. Variable lymphocyte receptors (VLRs) offer a novel means of targeting the BBB following ischemic stroke. The large evolutionary gap that exists between jawless vertebrates, who produce VLRs in their adaptive immune system, and humans can be exploited to identify novel targets. Identifying VLRs that can bind to receptors at the BBB can offer an alternate pathway for the delivery of therapeutic cargo to treat CNS diseases.

Materials and

Methods: A library of VLRs was biopanned against induced pluripotent stem cell derived brain microvascular endothelial-like cells (BMECs). VLRs were expressed in yeast surface display (YSD). BMECs exposed to hypoxia for six hours and allowed to recover for 18 hours before incubation with yeast. Successive biopanning experiments enriched for targeting VLR antibodies that express a higher affinity to the ischemic brain. Lead candidate VLR proteins have been further validated in vitro.

Results, Conclusions, and Discussions: An existing VLR antibody library was used to identify binding VLRs to ischemic BMECs. Our systematic study discovered lead VLR antibody candidates that bind specifically to ischemic BMECs. YSD and biopanning were completed in successive rounds to enrich for binding VLR antibodies to ischemic BMECs. Enrichment of binding VLRs was observed comparing the first and third round of biopanning on streaked plates. Following three rounds of enrichment, individual clones were sequenced to identify specific VLR protein sequences of those high binding antibodies. Each individual clone was then transformed into fresh yeast for purification for further experiments to validate unique individual VLR proteins binding to ischemic BMECs. Future experiments include construction of a soluble VLR-Fc fusion protein for in vivo testing of VLR BBB transcytosis and brain uptake in mice models and human post mortem tissues.

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