Non-invasive and long-lived central nervous system delivery of anti-inflammatory cytokines - multiple sclerosis (MS) is an inflammatory demyelinating disease that involves immune cell infiltration…

Non-invasive and long-lived central nervous system delivery of anti-inflammatory cytokines - multiple sclerosis (MS) is an inflammatory demyelinating disease that involves immune cell infiltration into the central nervous system (CNS), breakdown of the myelin sheath, axonal loss, and gliosis. The immune system plays a major role in MS progression and associated neuroinflammation, primarily mediated by autoreactive T cells and microglia. Regulatory T cells (TREGS) have been shown to suppress the activity of these immune cells, as well as prevent neurotoxic astrogliosis and promote myelin regeneration, which overall reduces disease severity. However, MS patients possess reduced treg numbers and function, limiting their neuroprotective capacity. Cytokines can selectively expand TREGS, including wild-type (WT) IL-2 and mutants thereof (referred to herein as muteins) that increase IL-2RΑ dependence. To improve clinical translation, methods for delivering treg-expanding cytokines to the CNS are needed with four key attributes: I) intravenous (IV) administration; II) efficient crossing of the intact blood-brain (BBB) and blood-spinal cord (BSCB) barriers; III) long-lived CNS retention to reduce dosing frequency; and IV) strong expansion of TREGS in the CNS without expansion of effector T cells (TEFFS). We propose an immunocytokine technology that possesses all of these desired attributes for preventing and/or reversing neurodegeneration in an MS mouse model. The feasibility of this proposal is supported by our three key discoveries. First, we have developed bispecific antibodies (BABS) that target a) CD98HC - the heavy chain of the large neutral amino acid transporter (LAT1) - for mediating CNS delivery of biologics with distinct advantages over similar BABS that target transferrin receptor, and B) myelin oligodendrocyte glycoprotein (MOG) - a CNS-exclusive antigen on the myelin sheath of oligodendrocytes - for localization and retention at the site of injury in MS. Second, we have fused WT IL-2 to these BABS and confirmed both their delivery to the CNS of mice at functionally relevant concentrations and the expansion of TREGS in the CNS. Third, we have developed a panel of IL-2 muteins that more selectively activate treg-specific VS. Teff- specific signaling relative to WT IL-2 and confirmed that their activity is maintained when fused to BABS. Our central hypothesis is that CNS delivery of IV-administered IL-2 immunocytokines will mediate strong and long- lived expansion of TREGS in the CNS, reduce neuroinflammation and pathology, and improve clinical scores in a mouse model of MS [experimental autoimmune encephalomyelitis (EAE)]. To test this hypothesis, in aim 1, we propose to evaluate the ability of our first-generation WT IL-2 immunocytokine to reduce neuroinflammation and pathology and improve clinical scores in EAE mice. Next, in aim 2, we will evaluate second-generation IL-2 mutein immunocytokines for their ability to further increase the extent, duration, and selectivity of CNS treg expansion in order to reduce the required dosing frequency. Our innovation is achieving long-lived, functionally relevant concentrations of treg-expanding cytokines in the CNS while only using IV administration, leading to strong CNS treg expansion while minimizing teff expansion and dosing frequency.