Date Approved

2013

Document Type

Thesis

Degree Name

Master's in Biomedical Science

Department

Graduate School of Biomedical Sciences

College

School of Osteopathic Medicine

First Advisor

Grant Gallagher, PhD

Committee Member 1

Raymond Yu, PhD

Committee Member 2

Kathryn Iacono, PhD

Committee Member 3

Venkataswar Venkataraman, PhD

Subject(s)

Th17 Cells, Autoimmune Diseases, Interleukin-23, Peptides, Gene Expression Regulation

Disciplines

Cell Biology | Genetic Processes | Immune System Diseases | Laboratory and Basic Science Research | Medical Cell Biology | Medicine and Health Sciences | Molecular Biology | Therapeutics

Abstract

Pathogenic Th17 cells drive progression of many autoimmune diseases. Th17 cells develop from naïve T cells in the immune system after antigen-driven stimulation in a specific cytokine environment. Normally, T cells act to fight off infection, but when not properly controlled, they can cause disease. The cytokine interleukin-23 (IL-23) plays an essential role in the expansion of pathogenic Th17 cells. IL-23 is a heterodimeric protein, composed of a p19 alpha chain and a p40 beta chain. The p40 is also part of IL-12 and binds to the IL-12 receptor beta 1 (IL-12Rβ1) subunit. Thus, it follows that the IL-23 receptor is comprised of the IL-12Rβ1 and IL-23 receptor alpha (IL23Rα) subunits. New research in therapeutics for autoimmune disease is attempting to inhibit IL-23 from binding to its receptor. Our laboratory previously screened a peptide library through display technologies for peptides that bound to the IL-23Rα. Peptides #7 and #2 were identified as having the strongest inhibitory activity on the IL-23 signaling pathway. However, peptides are small, leading them to be cleared from a biological system in a manner of minutes; therefore, the present project created fusion proteins of these peptides with mouse IgG2a-Fc. They were then expressed in mammalian cells, secreted and purified from medium. Peptide #2-Fc and its unmodified counterpart, Peptide #2, were tested and compared using cell-free and cell-based systems to ensure minimal loss of inhibitory activity upon fusion. This work generated a Peptide #2-Fc fusion protein with the capability to bind the IL23Rα chain and thus block IL-23 binding and inhibit subsequent signaling, as well an increased serum half-life over the original Peptide #2.

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