Notch1 and Notch4 core binding domain peptibodies exhibit distinct ligand-binding and anti-angiogenic properties

Design and characterization of Notch peptibodies

The extracellular domain of the four Notch proteins is composed of a varying number of EGF-like repeats. For Notch1, structure–function analysis coupled with high-resolution crystal structures have identified EGF-like repeats 11–12 as critical for receptor–ligand interaction [21,22,23,24]. In contrast to the Notch1 receptor, the core ligand-binding domain of Notch4, an endothelial-specific Notch gene [25], has not been fully characterized. While Notch4 is the most divergent of the four mammalian Notch receptors, EGFs 11–12 are highly homologous between Notch1 and Notch4. Of the four human Notch proteins, Notch1, Notch2, and Notch4 have the highest levels of conservation between EGF-like repeats 10–14, while a notable divergence appears in Notch3, where EGFs 7–10 of the Notch3 receptor best corresponds to EGFs 11–13 of Notch1 (Fig. S1A) [26]. Further, protein alignment of the human and murine Notch receptors across EGFs 10–14 demonstrates high identity and similarity among these two mammalian species (Fig. S1B). Here, we generated recombinant fragments of the human Notch1 and Notch4 extracellular domains containing the coding sequences of Notch1 and Notch4 EGF-like repeats 10–14 fused to human IgG Fc, referred as N110-14Fc and N410-14Fc in our data. The sequence alignment of these two Notch decoys demonstrated regions of high conservation across EGFs 10–14, including EGFs 11 and 12 (Fig. 1A, B).

Fig. 1figure 1

Construction and expression of Notch decoys. A Notch decoys are composed of specific EGF-like repeats (10–14) of the Notch1 or Notch4 extracellular domain fused to the human Fc domain. B Amino acid alignment of EGFs 10–14 of the Notch1 and Notch4 receptor, respectively. C Coomassie stained TGX-Gel analysis. Purified proteins were loaded under reducing conditions. D Western blot analysis. 100 ng of purified protein were resolved on TGX gels under non-reducing or reducing conditions and immunoblotted with Fc-specific antibody

N110-14Fc and N410-14Fc proteins were produced in HEK293F cells upon transfection with the corresponding expression construct. The secreted proteins were subsequently column purified and resolved in 4–20% SDS-PAGE gels stained with Coomassie, showing approximate molecular weights (MW) of N110-14Fc and N410-14Fc with an expected band at ~ 50 kDa in reducing conditions (Fig. 1C). To confirm the identity of the proteins, western blotting was performed using antibodies specific for human IgG Fc in both non-reducing and reducing conditions, showing specific bands at ~ 100 kDa in non-reducing and ~ 50 kDa in reducing conditions (Fig. 1D). No evidence of cleavage between the Notch EGF-like repeats and Fc domain or other forms of degradation were apparent. As expected, detection of native protein under non-reducing conditions was at double the predicted molecular weight, indicating dimerization of the IgG Fc. To accurately assess their oligomeric states, mass photometry was utilized to evaluate N110-14Fc and N410-14Fc proteins. Both Notch decoys displayed an oligomeric mixture dominated by dimers at 100 kDa (Fig. S2).

Both N110–14Fc and N410–14Fc bind to DLL4 and JAG1

We asked if the homologous ligand-binding domain of Notch4 is sufficient to bind to Notch ligands DLL4 and JAG1. It has been well documented that for Notch1, EGF-like repeats 11 and 12 correspond to the core binding domain and are alone sufficient for ligand interaction [21,22,23,24]. To date, there has been little evidence that Notch4 binds to ligands DLL4 and JAG1. To confirm binding specificity, N110-14Fc, N410-14Fc, full-length FLAG-tagged JAG1, and full-length Myc-tagged DLL4 were co-expressed in 293 T cells and co-immunoprecipitation was performed with Notch decoys acting as the “bait” protein. After pulldown, N110-14Fc and N410-14Fc co-immunoprecipitated with both DLL4 and JAG1, validating pan-ligand binding (Fig. 2A, B). We utilized surface plasmon resonance (SPR) spectroscopy to characterize and quantify the interactions between these proteins. Recombinant Fc-tagged hDLL4 and hJAG1 were immobilized on the sensor chip using amine coupling and multi-cycle kinetic experiments were performed using increasing concentrations of either N110-14Fc or N410-14Fc. As a control, recombinant IgG Fc was immobilized on the sensor chip. Recombinant hDLL4 interacted with N110-14Fc and N410-14Fc with a dissociation constant (KD) of 6.05 × 10–7 M and 9.625 × 10–7 M, respectively (Fig. 3A). Based on previous literature, we had predicted N110–14Fc would bind to DLL4 and JAG1. We report here for the first time a conserved binding domain within Notch4 that promotes interaction with DLL4 and JAG1. A notable feature of the sensorgrams between the two decoys shows N110–14Fc binding to hDLL4 at a fast association and disassociation rate compared to N410-14Fc (Fig. 3B), indicating different binding mechanics. Although no interaction was measured between hJAG1 and N110-14Fc or N410-14Fc (Fig. S3) in SPR-based binding assays, this result aligns with prior published reports [22]. It has been theorized that the interaction of the Notch1 extracellular domain and that of JAG1 is weak and requires a pulling force to stabilize JAG1 into confirmations needed for interaction. We conclude that Notch decoys can readily bind to members of Delta-like or Jagged/Serrate-class Notch ligands.

Fig. 2figure 2

Binding of Notch ligands and Notch decoys examined by co-immunoprecipitation. Co-immunoprecipitation assays show interaction of Notch ligands DLL4 and JAG1 with Notch peptibodies. A N110-14Fc and full-length DLL4-MYC or JAG1-FLAG were transiently co-transfected into HEK-293Ts. Protein A/G beads were used to immunoprecipitate N110-14Fc acting as the “bait” protein from whole cell lysates. Binding of N110-14Fc and Notch ligands were determined by immunoblot using anti-Fc, anti-FLAG, and anti-MYC antibodies. B N410-14Fc was evaluated similarly to panel A

Fig. 3figure 3

Binding of Notch ligands and Notch decoys examined by SPR. SPR measurements show binding affinity of N110-14Fc and N410-14Fc to DLL4. A Recombinant Fc-tagged hDLL4 was immobilized on the sensor chip using amine coupling and multi-cycle kinetic experiments were performed using increasing concentrations of either N110-14Fc or N410-14Fc. B Normalization of the N410-14Fc sensorgram to N110-14Fc. The resulting sensorgrams were normalized using Biacore sensorgram fitting algorithms and similarity scores

N110–14Fc suppresses endothelial Notch signaling

Interaction of endothelial Notch with Notch ligands, such as DLL4, causes the cleavage of the intracellular domain of Notch1 and subsequent translocation to the nucleus. Notch decoys that inhibit DLL4–Notch interactions would be expected to inhibit cleavage of endogenous Notch1 expressed on the surface of endothelial cells. To evaluate whether Notch decoys can block DLL4-mediated Notch1 activation, we seeded HUVECs onto DLL4-coated plates and subsequently dosed with increasing concentrations of N110-14Fc. At the highest doses tested, N110-14Fc reduced the DLL4-induced cleavage of endogenous Notch1 expressed in HUVECs (Fig. 4A).

Fig. 4figure 4

N110–14Fc suppresses endothelial Notch signaling. A N110-14Fc inhibits DLL4-induced cleavage of the Notch1 receptor. HUVECs were plated onto 1 μg/mL of recombinant hDLL4 in the presence of N110-14Fc or IgG Fc isotype control for 24 h and quantitated for cleaved Notch1 by Western blot. (B, C) RT-qPCR analysis of Notch decoy-induced gene changes in HUVECs. Cells were treated with either Notch peptibody or IgG Fc at indicated concentrations for 24 h. B Expression of targets of canonical Notch signaling in HUVECs treated with N110-14Fc. C Expression of targets of canonical Notch signaling in HUVECs treated with N410-14Fc. For all figures, error bars represent standard error of mean (SEM) and ***P value < 0.001, **P value < 0.01, *P value < 0.05

Once the NICD enters the nucleus, it interacts with the transcription factor RBPJ/CSL to regulate expression of canonical Notch target proteins. To directly test whether Notch decoys affect the canonical Notch signaling pathway, a crucial regulator in endothelial cells, we examined the inhibitory effects of N110-14Fc and N410-14Fc on the Notch pathway in HUVECs. Increasing concentrations (0, 0.5, 1, 5, and 10 ug/ml) of either IgG Fc, N110-14Fc, or N410-14Fc were used to treat HUVECs for 24 h. Differences in mRNA expression of Notch target genes were then examined by RT-qPCR. Compared to the control group, N110-14Fc significantly downregulated Notch target genes NRARP, HEY2, RND1, DLL4, and Notch1 at multiple concentrations (Fig. 4B). N410-14Fc decoy downregulated Notch target genes to a lesser degree in HUVECs (Fig. 4C).

N110–14Fc inhibits angiogenesis in vitro

Angiogenesis is a tightly controlled, multi-step process in endothelial cells that involves proliferation, cell migration, and tube formation. To assess the effects of our Notch decoys on endothelial sprout formation, a three-dimensional in vitro assay was used. Cytodex beads were coated with HUVECs and subsequently embedded into a fibrinogen matrix. To support HUVEC growth, fibroblast cells were cultured on top of the matrix to provide growth factors. In this assay, endothelial sprouts grow out from the bead, mimicking the nascent stages of angiogenesis. We evaluated the number of outgrowths and their corresponding length in HUVECs treated with increasing concentrations of either IgG Fc, N110-14Fc, or N410-14Fc. After treatment with N110-14Fc, the number of angiogenic sprouts and the length of the newly formed sprouts were reduced at concentrations of 5 and 10 ug/ml of the Notch decoy (Fig. 5A–C). No significant reduction was seen in either sprout length or sprout number after treatment with N410-14Fc (Fig. 5A–C).

Fig. 5figure 5

N110–14Fc affects endothelial viability and modulates angiogenesis. A Representative images of Fibrin bead assays (FiBA). HUVEC-coated beads were embedded in fibrin gel with increasing doses of human IgG Fc, N110-14Fc, or N410-14Fc for 12 days. After 12 days, N110-14Fc significantly reduced both sprout number and sprout length at dosages of 5 and 10ug/ml. No significant effect was seen with N410-14Fc at any dosage. B Quantification of mean sprout number per bead for treated HUVECs. Box-and-whisker plots show median, minimum, and maximum values. C Quantification of mean sprout length for treated HUVECs. D Cell viability assay of HUVECs treated with increasing dose of IgG Fc, N110-14Fc, or N410-14Fc for 72 h. IgG Fc-treated control group was set at 100% and was compared with that of peptibody-treated groups. E Identical experiments conducted with mLMVEC. **P value < 0.01, *P value < 0.05

To assess if Notch decoys have cytotoxic effects on human or mouse endothelial cells, we treated cells with increasing concentrations of either IgG Fc, N110-14Fc, or N410-14Fc for 72 h. The viability of HUVECs was dose-dependently inhibited by N110-14Fc but not by N410-14Fc (Fig. 5D). The viability of mouse lung microvascular endothelial cells (mLMVEC) was dose-dependently inhibited by both peptibodies. We asked if the anti-angiogenic effect observed in vitro is due in part to a migration defect or solely viability. Previous literature has demonstrated that inhibition of DLL4–Notch signaling has been shown to induce the migration of endothelial cells. To understand the role of Notch decoys on endothelial cell migration, we utilized a scratch wound-healing assay in which the extent of migration of cells into the scratched areas was examined. HUVECs or mLMVECs treated with either IgG Fc, N110-14Fc, or N410-14Fc were assessed and no migration alterations were detected (Fig. S4). These results indicate that N110-14Fc, but not N410-14Fc, modulates human endothelial viability and both peptibodies reduce mouse endothelial viability.

To assess whether Notch peptibodies affect other cell types, we examined T cell acute lymphoblastic leukemia (T-ALL) cells which require Notch signaling for survival [8,9,10,11]. N110-14Fc and N410-14Fc showed no significant effects on survival of human T-ALL KopTK cells or mouse T6E cells (Fig. S5).

N110-14Fc inhibits murine retinal angiogenesis

We assessed N110-14Fc treatment during postnatal murine angiogenesis to better understand how peptibody-based Notch inhibitors effect angiogenesis in vivo. To deliver the recombinant proteins, either human IgG Fc or N110-14Fc were injected intragastrically into murine neonates. Compared to the Fc-treated group, N110-14Fc showed a reduction in both the vascular area of the angiogenic front and radial vascular outgrowth (Fig. 6A). Further, filopodia-extending endothelial sprouts, termed Tip Cells, were observed to be no more abundant in mice treated with N110-14Fc than Fc. While not the focus of this investigation, we observed that approximately half the mice treated with N110-14Fc exhibited unusual enlargement of retinal veins (Fig. S6), which warrants future investigation.

Fig. 6figure 6

N110–14Fc inhibits retinal angiogenesis in murine neonates. C57BL/6 mice were injected intragastrically with 12.5 mg/kg of recombinant N110-14Fc peptibody or IgG Fc for three days postnatally (P1-P3). A Representative images and quantification of postnatal day 5 (P5) retinal vasculature stained with Isolectin B4 (red). Radial outgrowth and percent vascular coverage near the angiogenic front were reduced in N110–14Fc-treated mice (N = 7–8), while tip cell density and percent vascular coverage of the mature plexus were not statistically altered. Scale bars: 1000 μm. B Representative images and quantification of postnatal day 5 (P5) retinal vasculature stained with Isolectin B4 (endothelium, red) and α-SMA (vascular smooth muscle cells, green). No difference was observed in the percentage of smooth muscle coverage in control and N110-14Fc-treated mice (N = 4). Scale bars: 106 μm. Box-and-whisker plots show median, minimum, and maximum values. **P value < 0.01, *P value < 0.05. (Color figure online)

In some vascular development settings, Notch ligands JAG1 and DLL4 play a crucial role in the recruitment of vascular smooth muscle cells to nascent arteries during the maturation process of angiogenesis [5, 17, 27,28,29]. Vascular smooth muscle cell coverage of mice treated with N110-14Fc remained unchanged compared to the control group (Fig. 6B), indicating that N110-14Fc inhibited angiogenesis with no effect on vascular remodeling at this time point. These results suggest that N110-14Fc can cause inhibition of angiogenic vessels in vivo.

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