Ozoralizumab, a trivalent anti-TNFα NANOBODY® compound, is resistant to rheumatoid factor-mediated modulation of FcRn-dependent recycling and immune complex uptake

Rheumatoid factor (RF), an autoantibody targeting IgG, is a key diagnostic marker of autoimmune diseases, especially rheumatoid arthritis (RA). In patients with RA, high RF titers have been reported to be associated with disease severity and may influence the pharmacological behavior and clinical response of certain biologic agents, including tumor necrosis factor (TNF) inhibitors. TNF inhibitors are widely used biologic agents in RA treatment and include two structure types: the fragment crystallizable (Fc)-containing protein (e.g. IgG) and the Fc-free protein. Clinical studies have suggested that in RA patients with high RF titers, sufficient circulating drug concentrations are more likely to be maintained with Fc-free TNF inhibitors than with Fc-containing TNF inhibitors. To clarify the mechanisms underlying this clinical observation, we investigated whether RF differentially alters FcRn-dependent recycling and macrophage uptake of the Fc-containing antibody adalimumab and the Fc-free, human serum albumin (HSA)-binding NANOBODY ® compound ozoralizumab (OZR) using an ELISA-based RF-binding assay, an FcRn-mediated recycling assay in FcRn-expressing cells, and a mouse macrophage phagocytosis assay. In a RF binding assay, OZR showed minimal binding to RF. In the FcRn-mediated recycling assay, the recycling efficiency of OZR was not affected by high RF titers. In contrast, adalimumab, an IgG type of TNF inhibitor, exhibited clear interaction with RF and its recycling efficiency decreased in the presence of high RF titers. Furthermore, in a mouse macrophage phagocytosis assay, immune complexes containing the Fc-containing adalimumab, but not the Fc-free OZR, showed increased macrophage uptake in the presence of RF. In summary, our findings demonstrate that the HSA-binding property of OZR enables preservation of HSA-mediated FcRn recycling and avoids RF-enhanced immune-complex phagocytosis under high-RF conditions. These mechanisms may contribute to the maintenance of stable circulating concentrations and therapeutic efficacy of OZR in RA patients with high RF titers.