P156 Investigating the novel mechanism of action for NI-0501, a human interferon gamma monoclonal antibody

Abstract

The aim of this project was to investigate the underlying mechanism of action of NI-0501, a fully human monoclonal antibody (mAb) directed against interferon gamma (IFNγ). NI-0501 is being developed for the treatment of inflammatory and autoimmune conditions. Biacore analysis was performed to assess the binding kinetics and affinity of NI-0501 for IFNγ. Furthermore, in conjunction with flow cytometry, the interaction of NI-0501/IFNγ complexes with IFNγ-Receptor-1 (IFNγR1) was investigated. The potency of NI-0501 was established using STAT-1 Reporter HeLa cells. Confocal microscopy was used to evaluate the interaction and internalization of IFNγ/IFNγR1 complexes. NI-0501 demonstrated a binding kinetics and affinity (KD) in the pM range for IFNγ and a potent ability to neutralize the biological activity of IFNγ. In both Biacore and flow cytometry experiments, NI-0501 was observed to bind to both free and IFNγR1-bound IFNγ. Using an in situ proximity ligation assay, NI-0501 was observed to impair IFNγR1 and IFNγ-Receptor-2 (IFNγR2) interaction induced by IFNγ at the cell surface. Finally, using confocal microscopy, we observed that IFNγ-mediated IFNγR1 internalization into Lysosomal-Associated Membrane-Protein-1 (LAMP-1) positive lysosomes was not affected by NI-0501. In this study, we demonstrate a mechanism of action by which NI-0501 neutralizes IFNγ. Thus, our data show that NI-0501 is a non competitive inhibitor of IFNγ binding to its receptor and that NI-0501 binding to IFNγ prevents IFNγR2 recruitment but allows for IFNγR1 endocytosis and internalization into lysosomes.