ENGINEERING BIVALENT SINGLE-DOMAIN ANTIBODIES TARGETING TGF-β2

Abstract

Transforming Growth Factor β (TGF-β) is a signaling protein found in various tissues within the body. It is essential for wound healing, tissue homeostasis, and regulates several cellular processes including proliferation, differentiation, and apoptosis. However, when irregularities occur within the signaling process, it contributes to the pathogenesis of inflammation, fibrogenesis, and other detrimental diseases. One such disease is ocular fibrosis, a condition in the eye due to irregular healing processes that causes irreversible damage. Inflammation, fibroblast activation, and extracellular matrix (ECM) deposition are characteristics of this disease, and are processes regulated by TGF-β. One isoform, TGF-β2, is found to have elevated levels in the occurrence of ocular fibrosis and is the main target molecule of this project. This thesis presents single-domain antibodies (sdAb) constructed that bind to TGF-β2, with a potential to disrupt the signaling of the cytokine. The first research aim investigates whether bivalent sdAbs have better binding affinity when compared to monovalent sdAbs. It also investigates different configurations, by altering the number of GS linkers connecting the sdAbs. The second aim investigates whether hetero-bivalent or homo-bivalent constructs have better affinity to TGF-β2. Finally, the strongest binders found in these aims are compared to each other to determine the construct with the highest binding affinity to TGF-β2.