Long-lived Activation of Parenchymal Border Macrophages Using Immunocytokines to Address Aging- and Alzheimer’s Disease-associated Deficits in Cerebrospinal Fluid Dynamics

Cerebrospinal fluid (CSF) perfusion of the brain parenchyma mediates “glymphatic clearance” of brain metabolites and extracellular debris. We (Kipnis) recently identified that leptomeningeal and perivascular macrophages – collectively referred to as parenchymal border macrophages (PBMs) – regulate CSF flow dynamics. Moreover, we found that intrathecal (IT) delivery of macrophage colony-stimulating factor (M-CSF) improves CSF dynamics and decreases the deposition of extracellular matrix proteins in the aged mouse brain, which has significant potential for treating neurodegenerative diseases such as Alzheimer’s disease (AD) and cerebral amyloid angiopathy. However, there has been little clinical translation of these important findings due to the short half-life of M-CSF function in the CSF (hours) and the infeasibility of frequent repeat IT dosing. The overall objective of this proof-of-concept study is to develop immunocytokines – monoclonal antibodies (mAbs) or bispecific antibodies (bAbs) fused to M-CSF – for selective, sustained, and localized delivery of M- CSF depots near PBMs using either IT or intravenous (IV) delivery. For IT delivery, we hypothesize that fusing M-CSF to mAbs targeting proteins near PBMs will result in long-lived cytokine depots that locally activate PBMs. For IV delivery, we hypothesize that fusing M-CSF to bAbs targeting both proteins in the perivascular space – which are largely exclusive to the brain – and blood-brain barrier (BBB) proteins to mediate transport across the brain endothelium will result in M-CSF crossing the BBB and forming depots near PBMs for selective and long- lived PBM activation. Related to IV delivery, we (Tessier and Greineder) recently reported bAbs that target CD98hc, the heavy chain of the large neutral amino acid transporter (LAT1), and mediate the delivery of IgGs and other biologics to the brain parenchyma after IV administration. Notably, we observe superior brain retention of IgGs shuttled via CD98hc relative to similar shuttles targeting transferrin receptor. Moreover, we have validated that cytokines retain their function when fused to CD98hc bAbs. Finally, we have demonstrated CNS delivery and sustained retention of cytokines using our CD98hc bAbs. Therefore, in Aim 1, we will evaluate the optimal perivascular target protein and IT dose of mAb-MCSF immunocytokines for selectively activating PBMs and improving CSF dynamics in the brains of 5xFAD and aged wild-type mice. Next, in Aim 2, we will evaluate the optimal M-CSF potency and IV dose of bAb (AQP4/CD98hc)-MCSF immunocytokines for selectively activating PBMs and improving CSF dynamics in the brains of 5xFAD and aged wild-type mice. A key expected outcome is the development of immunocytokines for long-lived delivery of M-CSF to selectively activate PBMs, which has significant potential for restoring deficits in CSF dynamics and treating AD and related diseases. More generally, these immunocytokines could be used to deliver several additional cytokines to the CNS for treating AD and related neurodegenerative diseases.