Targeting the major pro-inflammatory Interleukin-6-type cytokine receptor gp130 by antagonistic single domain antibodies

Silke Pudewell¹Julia Heuser¹Inna Dorogobed²Britta Lipinski²Thi Hong Hue Tran²Pia Metzenmacher¹Richard Kunze¹Felix Geyer²Stefan Zielonka²Doreen Floss¹Jens Moll¹Harald Kolmar²Jürgen Scheller^1*

• ¹Heinrich Heine University of Düsseldorf, Düsseldorf, North Rhine-Westphalia, Germany
• ²Technical University of Darmstadt, Darmstadt, Germany

Although Interleukin (IL)-6-type cytokine signaling is critical for maintaining the body's homeostasis, aberrant signaling has been observed in numerous diseases including autoimmunity and cancer. Currently, all approved biologics that inhibit IL-6-type cytokines specifically target the key proinflammatory mediator IL-6 or its receptor (IL6R). Historically, direct inhibition of glycoprotein 130 (gp130)-the shared transmembrane receptor for IL-6-type cytokines-was avoided due to concerns that broad suppression might cause more harm than benefit. However, this view is being reconsidered in light of the clinical success of Janus kinase (JAK) inhibitors, which broadly disrupt cytokine signaling, including pathways mediated by gp130. Here we developed four single domain antibodies, consisting out of a camelid-derived nanobody and a human Fc-fragment, which directly bind gp130 in the cytokine binding module (CBM) and largely inhibit IL-6-type cytokine signaling by interfering with the high-affinity binding site of IL-6, IL-11, CLCF1, CT1, CNTF, OSM and LIF. The four nanobodies were characterized by direct protein interaction analysis, epitope binding, epitope binning, as well as inhibition of cytokine-induced stimulation and proliferation of appropriate Ba/F3 cell lines. Furthermore, we functionally demonstrate the inhibitory effect of the selected nanobodies in cell-based transmigration assays of the human colorectal cancer cell line HT-29. In summary, our study has identified and characterized four novel inhibitory high-affinity gp130 nanobodies with potential for use in cytokine-dependent autoimmunity or cancer therapy.