Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the β-pore forming toxin Aerolysin

Lemmex, A.; Allred, J.; Ostergard, J.; Trask, J.; Bui, H. N.; Anderson, M.; Kopp, B.; Streeter, O.; Smiley, A.; Babilonia-Diaz, N. S.; Blazar, B. R.; Higgins, L.; Gordon, P. M.; Muretta, J. M.; Gordon, W. R.

doi:10.1101/2024.10.22.617501

bioRxiv · 10.1101/2024.10.22.617501

Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the β-pore forming toxin Aerolysin

Lemmex, A.·Allred, J.·Ostergard, J.·Trask, J.·Bui, H. N.·Anderson, M.·Kopp, B.·Streeter, O.·Smiley, A.·Babilonia-Diaz, N. S.·Blazar, B. R.·Higgins, L.·Gordon, P. M.·Muretta, J. M.·Gordon, W. R.

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Abstract

Notch plays critical roles in developmental processes and disease pathogenesis, which has led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling, derived from a synthetic phage-display library targeting the notch Negative Regulatory Region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay and in Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19uM affinity for Notch. We addressed the low affinity by fusion to a membrane-associating domain derived from the {beta}-Pore forming toxin Aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with similar efficacy to other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody, and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

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Lemmex, A., Allred, J., Ostergard, J., Trask, J., Bui, H. N., Anderson, M., Kopp, B., Streeter, O., Smiley, A., Babilonia-Diaz, N. S., Blazar, B. R., Higgins, L., Gordon, P. M., Muretta, J. M., Gordon, W. R.. 2024-10-25. Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the β-pore forming toxin Aerolysin. https://doi.org/10.1101/2024.10.22.617501

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