INTERBIZ 2022: anti-DR3 Poster submission 

<Abstracat>

DR3 (death receptor 3, TNF Receptor Super Family 25, TNFRSF25) is one of tumor necrosis factor receptor superfamily. Although DR3 is constitutively expressed on regulatory T cells at high level, it can also be induced in conventional T cells upon TCR stimulation. DR3 signaling promoted the activation and proliferation of the Tregs under steady state as demonstrated with agonistic anti-DR3 in mice. Indeed, small dose (10 g/head) of anti-DR3 single injection drove maximum Treg expansion systemically including secondary lymphoid organs and intestinal compartments between day 4 and 7, without the expansion of none-targeted immune cell population. It is a major difference from IL-2 complex, which also expands conventional CD4 and CD8 T cells. As the expansion of Tconvs can cause a major drawback in treating diverse inflammatory diseases, a novel method to selectively expand Treg is highly demanded. In this purpose, we developed agonistic anti-human DR3 through human ScFv phage display and screening system in house. In brief, six individual human ScFv phage libraries were subjected to panning against recombinant human DR3, through which a total of eighty-four clones were selected. They were regenerated as monoclonal ScFv phages, and tested for binding against plate-bound DR3 (ELISA) and cell membrane expressing DR3 (flow cytometry). Next, six of the final clones of ScFvs were further subjected for functional analysis. These antibodies had distinct features in the epitope recognition and binding strength. Competitive binding assay showed that three clones of anti-DR3 bound to the TL1A interacting domain of DR3. As expected, these induced stronger NK-B activation on human CD4 T cells, demonstrating the successful development of agonistic anti-DR3. Interestingly, they induced strong activation of AKT, which is a critical signaling molecule for T cell activation. As it was unobservable in TL1A, it is speculated that it was attributed from a stronger affinity with DR3, compared to the TL1A. The selected clone 4-4 also strongly bound to the monkey DR3, and further efficacy tests will be followed.