(394e) Engineering Variable Lymphocyte Receptors to Target Blood Brain Barrier Disruption Via Exposed Neural ECM

Introduction: Normally the blood-brain barrier (BBB) prevents interaction between blood components and brain tissue, including neural extracellular matrix (ECM). However, certain neurological diseases, such as traumatic brain injury, stroke, and brain tumors, exhibit pathologic BBB disruption that is localized to the disease region, thus, exposing normally inaccessible neural ECM to circulating blood components. We propose exploiting differential exposure of neural ECM as a method for targeting therapeutics to pathologies that induce BBB disruption. Targeting therapeutics to the site of BBB disruption, as opposed to utilizing differential expression of cellular receptors, could have multiple benefits including targeting both diseased and supporting stromal cells, identifying multiple sites of BBB disruption within a single patient, and have generalizable application to multiple types of neuropathologies that exhibit a disrupted BBB phenotype.

Methods: We developed a semi-high throughput screen to identify Variable Lymphocyte Receptors (VLRs, the antigen recognition system from Lamprey) that demonstrate high specificity for neural ECM. Initial VLR clones underwent further protein engineering for selective neural ECM binding using in vitro murine tissue accumulation and immunostaining assays. Results were confirmed in vivo, using manitol disruption and murine glioblastoma models.

Results: Multiple neural ECM-binding VLRs were identified. The lead candidate identified after protein engineering, named P1C10, demonstrates diffuse binding to parenchymal neural ECM, without detectable binding to ECM from clearance organs. Additionally, P1C10 demonstrates nanomolar affinity for in vitro derived neural ECM. P1C10 accumulated in murine brains exposed to mannitol as well as at the tumor site of intracranial GL261 and U87 murine models of GBM. Finally, doxorubicin-loaded liposomes targeted with P1C10 conferred a significant survival benefit to mice bearing intracranial U87 GBM.

Conclusions: Here we present the rationale and proof-of-concept demonstration for targeting sites of BBB disruption via exposure of neural ECM. Novel protein engineering methods were used to screen and modify ECM-binding VLRs. Neural ECM-binding VLRs selectively target intracranial sites of GBM and functionally deliver therapeutic cargo. Thus, this new physiological-based targeting scheme may be well suited to target sites of BBB disruption in a variety of neural pathologies.

Acknowledgements: This work was supported by National Institutes of Health grants NS091851 (E.V.S and B.R.H.) and NS099158 (E.V.S.), a Falk Medical Research Trust Catalyst Award (E.V.S. and J.S.K.) and a Defense Threat Reduction Agency grant HDTRA1-15-1-0012 (E.V.S.).