Single-Chain Nanobody Inhibition of Notch and Avidity Enhancement Utilizing the β-Pore-Forming Toxin Aerolysin

Abstract

Notch plays critical roles in developmental processes and disease pathogenesis, which have 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 with an IC₅₀ of about 5 μM and in a Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19 μM affinity for the Notch NRR. We addressed the low affinity by fusion to a mutant varient of the β-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 efficacy similar to that of 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.

Figure 1. Identification of a Notch1 NRR binding Nanobody.

- (A) Cartoon schematic of the biopanning strategy employed. (B) Anti-NRR ELISA of 12 candidates at 3 conditioned media dilutions. (C) Additional ELISA testing conditioned media dilutions of S7 at 4 different concentrations as well as testing of Strain 7 conditioned media on plates coated with an irrelevant protein M04–6xHis, BSA, or NRR to identify possible non-specific binding. (D) Results of two biological replicates in a Microscale Thermophoresis Assay using the Strain 7 nanobody and Notch1 NRR.

Figure 2. S7 inhibits Notch activation in cell-based assay.

- (A) Schematic of signaling assay. Cells are reverse transfected with N1-Gal4 constructs and reporter plasmids and are plated in plates precoated with Notch ligand. The Receptor binds the Notch ligand (1), leading to cleavage of the intracellular Gal4 domain (2) and translocation to the nuclease where it drives transcription of luciferase (3). Luciferase signal is normalized against the constitutive expression of Renilla luciferase. (B) Effect of serial dilution of S7 on Notch1-Gal4 signaling. Each point represents 3 technical replicates (C) 10 point serial dilution of S7 against Notch1-Gal4. Each point represents 3 technical replicates.

Figure 3. S7 inhibits differentiation of Hemogenic Endothelial cells into Definitive Hematopoietic cells.

– (A) Schematic representation of experimental workflow. iPSC cells are differentiated into Hemogenic Endothelial Cells, after which they are cultured in Notch stimulating conditions and treated with various treatments, then read out via flow cytometry. (B) Representative flow plots from select treatments. Cells are first gated on CD43−, then definitive HPCs are gated by CD235−CD34+. 10uM S7 showed only 2.89% of gated cells differentiating to definitive HPCs compared to 15.2% for the buffer only control. (C) Quantification of data from the experiment described in panel B splitting out absolute number of CD43+ HSPCs, the absolute number of primitive and definitive HSPCs, and the ratio of primitive to definitive. There was a statistically significant decrease of the number of definitive HSPCs at the 10μM S7 treatment, as well as statistically significant increases in the primitive:definitive ratio.

Figure 4. S7 fused to Aerolysin improves efficacy of S7 inhibitory effect.

- (A) Cartoon representation of S7 fused to the N-terminus of Aerolysin and the localization of S7 to the surface of the cell via Aerolysin. (B) Cartoon representation of the assay in C, in which plates are coated with a titration of DLL4 ligand, cells with the Notch reporter are plated and then treated with a fixed concentration of S7 or S7-aerolysin to determine if the treatments shift the response to DLL4.. (D) The response of U2OS cells in the signaling assay plated on different concentrations of DLL4 ligand while treating with either 10uM S7 or 250nM S7-Aerolysin.

Figure 5. Notch binders conjugated to Aerolysin alters cell surface staining populations of Notch1

– (A) Cartoon schematic depicting the doxycycline induced expression of FLAG-human-Notch1-GFP in U2OS with anti-FLAG-APC binding the extracellular FLAG tag and GFP fused to the C-terminal intracellular side. (B) Representative flow plots of S7-Aerolysin and control treatments or α-Notch1-Fab-Aerolysin and α-Notch1-Fab treatments. Treatments in which a Notch binder couple to Aerolysin showed a measurable decrease in APC signal compared to the respective controls, while levels of GFP remained unchanged. (C) Levels of GFP and APC fluorescence normalized to the respective controls of each treatment showed statistically significant decreases of APC signal while changes in GFP signal were not statistically significant (n = 3 with each point being a biological replicate). (D) Cartoon schematic of the hypothesized effect of Aerolysin when conjugated to a Notch binder. GPI-AP and Notch receptors are normally trafficked with pools of the various populations being recycled to the membrane and some portion being trafficked for degradation. When Aerolysin is introduced with a Notch binder, it introduces cross-talk between the normal trafficking processes governing Notch and GPI-AP, and leads to a measurable decrease in the surface levels of Notch without necessarily leading to an increase in overall Notch degradation.

Figure 6. S7-Aerolysin inhibits the proliferation of T-ALL cell line sensitive to Notch-inhibition but does not alter proliferation of Notch-insensitive cell line.

– (A) DNA staining and cell cycle analysis with Jurkat cells (Notch insensitive) vs HPB-ALL cells (Notch sensitive) with the indicated treatments. (B) Quantification of the percentage of cells in G1-phase of the corresponding treatments.