Structural studies of the interaction between Death Receptor 3 and anti- DR3 antibody

Abstract

The interaction between tumor necrosis factor (TNF) and its receptor (TNFR), expressed by cells, leads to various signaling events that regulate cell survival and apoptosis, maintaining cellular homeostasis. Specifically, DR3, one of the TNF receptor superfamily (TNFRSF), is highly expressed in T lymphocytes, playing a crucial role in maintaining the homeostasis of these cells. T lymphocytes can differentiate into various subclasses, each performing diverse functions after differentiation. While most T lymphocyte subclasses contribute to immune enhancement and induction of inflammatory responses, regulatory T (Treg) cells play a role in preventing excessive immune reactions by regulating immune suppression, thereby preventing the onset of autoimmune diseases and allergies. Therefore, abnormalities in the activation and proliferation of Treg cells can lead to immune homeostasis disorders. If the activation and proliferation of Treg cells can be effectively regulated, this could be a promising therapeutic option for immune cell treatments. Therefore, I aimed to structurally elucidate the binding of DR3, which shows high expression in Treg cells, with anti-DR3 antibodies to understand the mechanism by which antibodies regulate cells. Additionally, I sought to enhance the stability of the yet-unrevealed structure of DR3 through antibodies for structural clarification. In an attempt to express and purify DR3 for these purposes, there was a recurring issue of insufficient expression levels for crystallography experiments. To enhance expression, different constructs were transfected and examined, with the full-form DR3 (a.a25-199) exhibiting the highest expression levels, while other constructs showed lower expression. Particularly, constructs with introduced N67D and N106D mutations for crystallography experiments had challenges with either no expression or very limited expression. There was also an issue with insufficient expression levels of the antibody binding to DR3. To address this, cloning was performed into the pSGHP0 and pLEXm vectors, and various vector combinations were tested to compare the expression levels of kappa chain and heavy chain. However, it was not possible to obtain sufficient amounts for crystallography with these various vector combinations. However, DR3 was successfully purified using methods such as Nickel purification, ion-exchange, and size-exclusion chromatography. Additionally, the expressed antibody was confirmed to be purified through a protein G column. The binding between the purified antibody and DR3 was also verified through gel filtration.