Production of nanobodies targeting il-1R1

IL-1R1, a member of the IL-1 receptor family, is essential for innate immunity and regulation of inflammation. IL-1α and IL-1ꞵ are the ligands of IL-1R1 and have various roles in the inflammatory response, such as induction of fever, stimulation of T and B cell proliferation, and pro-inflammatory cytokine secretion. Dysregulation in IL-1 signaling leads to inflammatory diseases which are generally treated with Canakinumab, Anakinra or Rilonacept. Despite their effectiveness in causing remission of disease symptoms, these drugs still have limitations. Nanobodies, derived from variable fragments of heavy-chain-only antibodies, offer promising alternatives due to their small size, high affinity, stability, and solubility. Regarding advantageous properties of nanobodies in therapeutic applications, this study aimed to develop anti-IL-1R1 nanobodies as potential anti-inflammatory agents. Two strategies were employed: synthesis of a synthetic nanobody gene against IL-1R1 and production of anti-IL-1R1 nanobodies derived from immunized llamas. Although the nanobody gene synthesis was successful, its expression in bacteria was not possible. On the other hand, purification and characterization of the nanobodies from the immune library identified over ten candidates with high affinity to IL-1R1. Among them, 3WEB23 and 2TED12 effectively inhibited human IL-1R1 in cell culture. These two nanobodies are promising therapeutic candidates against inflammatory diseases but their in vivo functionality needs further investigation. To enhance inhibitory activities, developing a multivalent nanobody can be considered. Epitope binning experiments confirmed that the binding capability of these two candidate nanobodies were unaffected by any other high affinity nanobodies, supporting their potential use in multivalent nanobody production. Overall, this study identified two anti-IL-1R1 nanobodies as alternative therapeutic candidates for inflammatory diseases.