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Abstract
The goal of this thesis is to develop cytokine therapies to reduce inflammation, promote tissue repair, and restore lost function in neuroinflammatory disease. To this end, we designed, produced, and characterized fusion proteins that bind transcytotic receptors on brain endothelial cells via an antibody fragment and which also contain cytokines to modulate the neuroimmune phenotype. We tested these potential therapeutics in mouse models of neuroinflammatory disease, characterizing the functional effects and assessing the immunomodulatory impacts. In Chapter 1, I introduce the field of neuroinflammatory diseases and current therapeutic approaches to their treatment. To illustrate the importance and relevance of this work, I discuss the pathophysiology and impact of major neurological diseases, with special attention paid to the role of inflammation and the immune system. I address the blood-brain barrier (BBB) in terms of its healthy function as well as its complicating role in the treatment of neurological disease. I introduce pertinent cytokine and neurotrophic therapeutic candidates for treating neuroinflammation. Finally, I discuss the current standard of care in neuroinflammatory diseases and review emerging therapeutic approaches in the field. In Chapter 2, I present the materials and methods used to carry out this research project. I describe the design and production of our experimental protein therapeutics, the cell lines and experiments used to characterize them, and the animals and disease models used to assess their in vivo function and immunological impacts. In Chapter 3, I present the results of the project. I demonstrate that the proteins bind to the target receptor and signal via the expected cytokine pathway. I show that binding to transferrin receptor (TfR) increases the potency and duration of signaling of the fused cytokine payload. I show that binding to TfR confers substantial delivery of IL-4 to the central nervous system in healthy mice, with corresponding modulation of microglia phenotype. I show that in the context of experimental autoimmune encephalomyelitis, significant direct CNS activity is attainable with TfR-binding IL-4, but that in the experiments we conducted this activity provides little additional benefit to function. I present data indicating that BBB-crossing IL-4 may offer functional benefit under the appropriate treatment conditions, which have yet to be optimized. Finally, I show that the TfR-binding approach is generalizable to other payloads, opening the door to a huge variety of potential future research. In Chapter 4, I discuss the significance of the findings presented in this thesis. I address limitations in our work as well as challenges in the broader field of treating neuroinflammatory disease and offer potential approaches to remedy these shortcomings. I present our conclusions and thoughts on the future directions of the work, touching on potential applications in both neurological and peripheral diseases.