Neuroinflammation is linked to diverse acute and chronic disorders. Regulatory T cells (Tregs) have long been recognized to suppress inflammation and effector T cell (Teff) activation in the periphery, but their role in the CNS has only been fully realized in the last few years. Notably, selective expression of IL-2 in the CNS is effective at differentiating CNS-resident T cells into Tregs and preventing neuropathology in multiple models of neuroinflammation, including traumatic brain injury (TBI), stroke, and multiple sclerosis (MS). However, there has been little clinical translation of these findings due to the lack of methods for CNS delivery of IL-2 or other Treg-inducing cytokines with a combination of four attributes: i) non-invasive administration (intravenous, IV); ii) ability to efficiently cross the intact blood-brain and blood-spinal cord barriers; iii) localization to and retention at the surface of CNS cells (e.g., oligodendrocytes) to form long-lived cytokine depots; and iv) ability to selectively induce Tregs in the CNS relative to in the periphery. The overall objective of this proof-of-concept study is to develop bispecific antibodies (bAbs) fused to IL-2, referred to as IL-2 immunocytokines, that possess all four of these attributes. We have developed a bAb shuttle technology that targets CD98hc, the heavy chain of the large neutral amino acid transporter (LAT1), and mediates the delivery of IgGs to the brain parenchyma. Notably, we observe superior brain retention of IgGs shuttled via CD98hc as compared to similar shuttles targeting transferrin receptor. We have also identified myelin oligodendrocyte glycoprotein (MOG)-specific antibodies and demonstrated that MOG/CD98hc bAbs specifically target oligodendrocytes and are retained in the CNS for at least two weeks following IV administration. Finally, we have developed a panel of IL-2 muteins that more selectively activate Treg-specific vs. Teff-specific signaling relative to wild-type (WT) IL-2 and confirmed that their activity is maintained when conjugated to bAbs. Therefore, in Aim 1, we will evaluate the optimal format and dosing of WT IL-2 immunocytokines to maximize Treg induction in the CNS while minimizing Treg induction in the periphery. We posit that MOG/CD98hc bAb-IL2 immunocytokines will induce Tregs more efficiently in the CNS than in the periphery due to their short half-life in circulation relative to their long CNS half-life. Next, in Aim 2, we will evaluate the effectiveness of a panel of designed IL-2 mutein immunocytokines to selectively induce Tregs relative to Teffs in the CNS. We expect that the IL-2 muteins will mediate Treg induction in the CNS over a wide range of doses while minimizing Teff induction. Finally, in Aim 3, we will evaluate the efficacy of WT IL-2 and/or IL-2 mutein immunocytokine CNS depots for protecting against neuroinflammation pathology in mouse models of TBI and MS. A key expected outcome is the development of IL-2 immunocytokine shuttles for non-invasive and long-lived delivery of Treg-inducing cytokines to the CNS, which has significant potential for enabling future fundamental and translational studies.

The goal of this proposal is to improve the delivery of agents that combat neuroinflammation – which are named cytokines – in a sustained and long-lived manner in the brain and spinal cord. This research will involve conjugating anti-inflammatory cytokines (IL-2) to binding proteins (antibodies) that penetrate the blood-brain barrier, target cell-surface proteins on brain cells (oligodendrocytes), and present their conjugated cytokines to locally induce proliferation of regulatory T cells and thereby reduce neuroinflammation. Successful completion of this research would enable a broad range of fundamental and therapeutic applications, including treating neuroinflammatory disorders such as multiple sclerosis and traumatic brain injury.