Directed evolution of a soluble human DR3 receptor for the inhibition of TL1A induced cytokine secretion

Abstract

TNF-like 1A (TL1A) is a cytokine belonging to the TNF superfamily that promotes inflammation in autoimmune diseases. Inhibiting the interaction of TL1A with the endogenous death-domain receptor 3 (DR3) offers a therapeutic approach for treating TL1A-induced autoimmune diseases. Here, we generated improved DR3 variants showing increased TL1A binding affinity and stability using a directed evolution approach. Given the high cysteine content and post-translational modification of DR3, we employed yeast surface display and expression in mammalian cell lines for screening, expression and characterization of improved DR3 variants. A cell-based assay performed with the human TF-1 cell line and CD4+ T cells showed that two improved DR3 mutants efficiently inhibited TL1A-induced cell death and secretion of IFN-γ, respectively. These DR3 mutants can be used as drug candidates for the treatment of inflammatory bowel diseases and for other autoimmune diseases, including rheumatic arthritis and asthma.

Increased Potency of a Bi-specific TL1A-ADAM17 (TACE) Inhibitor by Cell Surface Targeting

“…For the generation of bi-specific DR3-pTACE inhibitor, we utilized an engineered DR3 variant, termed H3, which exhibits improved TL1A binding affinity and higher stability relative to the WT soluble DR3 receptor. In addition, the H3 variant showed higher inhibition of TL1A induced IFN-γ secretion and apoptosis in CD4 + and TF-1 cells, respectively, relative to the WT receptor (Levin et al, 2017 ). For TACE inhibition, we used a previously described pTACE domain containing mutations at the natural furin cleavage site to maintain the integrity of our bi-specific inhibitor (Wong et al, 2015 ).…”

“…The E. coli E. Cloni strain (Lucigen) was used for cloning and plasmid extraction. The A1 and A2 inhibitors (Figure 1 ) were constructed by PCR amplification from ProTACE3mut (Wong et al, 2015 ) and pFUSE-DR3 (H3 variant) (Levin et al, 2017 ). The resulting fragments were purified and assembled by a secondary PCR to form H3-linker-6xHis-proTACE (A2) and proTACE-linker-6xHis-H3 (A1).…”

“… Schematic representation of the bi-specific soluble DR3 and pTACE inhibitor targeting TL1A and TACE. H3 is an engineered variant of soluble DR3 exhibiting high affinity and stability relative to the WT (Levin et al, 2017 ). …”

“…The binding of TL1A to DR3 was shown to boost the secretion of IFN-γ from T cells by acting in synergy with IL-12 and IL-18 and thus direct the immune response toward a T H 1 like response (Papadakis et al, 2004 ; Cassatella et al, 2007 ). To inhibit TL1A, we have recently utilized directed engineered to generate an improved soluble DR3 “trap” receptor (Levin et al, 2017 ). These DR3 extracellular domain (ECD) variants, consisting of 171 residues, exhibit high stability, TL1A binding affinity and potent inhibition of TL1A induced cytokine secretion from T cells.…”

Hydrogen/deuterium exchange mass spectrometry and computational modeling reveal a discontinuous epitope of an antibody/TL1A Interaction

“…5 In the presence of DcR3, TL1A/DR3-mediated cellular responses could be completely abrogated, indicating that the local level of soluble decoy receptor can affect the overall outcome of TL1A effects. 6 Recently, several DR3 mutants have been found to efficiently inhibit TL1A-induced cell death and secretion of IFN-g. 7 Novel therapies that block TL1A/DR3 interaction may thus be efficacious for diverse inflammatory and immune diseases.…”