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Single-Chain Nanobody Inhibition of Notch and Avidity Enhancement Utilizing the β-Pore-Forming Toxin Aerolysin
Andrew C D Lemmex1, Jeremy Allred1, Jason Ostergard1
1University of Minnesota, Twin Cities, Minneapolis, Minnesota 55409, United States.
ACS Chemical Biology
|March 13, 2025
Summary
Researchers developed a novel nanobody inhibitor targeting Notch signaling. This engineered antibody, fused with aerolysin, shows enhanced potency for inhibiting T-ALL cell proliferation, offering a new strategy for modulating Notch pathway activity.
Area of Science:
- Molecular Biology
- Immunology
- Biotechnology
Background:
- Notch signaling is crucial in development and disease.
- Modulating Notch activity is a therapeutic goal.
- Existing inhibitors have limitations.
Purpose of the Study:
- To develop a novel nanobody inhibitor of Notch signaling.
- To enhance the potency of a low-affinity nanobody.
- To demonstrate a generalizable strategy for antibody engineering.
Main Methods:
- Utilized phage display to generate a Notch NRR-targeting nanobody.
- Assessed nanobody inhibition in luciferase reporter and cell differentiation assays.
- Engineered a nanobody-aerolysin fusion protein to improve affinity and efficacy.
Main Results:
- Developed a nanobody inhibitor with an IC50 of ~5 μM.
- Fusion with aerolysin significantly improved Notch inhibition potency.
- The nanobody-aerolysin fusion effectively inhibited T-ALL cell proliferation.
Conclusions:
- Successfully developed a potent Notch inhibitory antibody.
- Demonstrated a viable strategy for enhancing low-affinity antibody binders.
- The engineered nanobody-aerolysin fusion shows therapeutic potential for T-ALL.
