Journal: Nature chemical biology

Article Title: Inhibition of Delta-induced Notch signaling using fucose analogs

doi: 10.1038/nchembio.2520

Figure Lengend Snippet: NIH3T3 cells expressing mNotch1 (a–c) or mNotch2 (d–f) were co-cultured with cells expressing Dll1 (a, d), Dll4 (b, e), or Jag1 (c, f) in the presence of peracetylated fucose analogs (compounds 9, 10, 11 ). Cells transfected with empty vector (EV) were used as a negative control and cells grown in DMSO were a positive control for Notch signaling. All experiments were performed three independent times, and data represents nine biological replicates (n=9). All plots represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, Tukey post-hoc test, adjusted p values.

Article Snippet: Dll1-Fc (R&D Systems, 3970-DL-050), Dll4-Fc (Sino Biological, 10171-H02H-50) or Jag1-Fc (R&D Systems, 599-JG-100) (each at 0.5 μg/mL) were pre-clustered with fluorescent secondary antibody PE-goat anti-mouse IgG (1:100; Invitrogen, P-852) or PE-anti-human IgG (1:100; Jackson Immuno Research, 109155-098) for 30 min at 4 °C.

Techniques: Expressing, Cell Culture, Transfection, Plasmid Preparation, Negative Control, Positive Control

Journal: Nature chemical biology

Article Title: Inhibition of Delta-induced Notch signaling using fucose analogs

doi: 10.1038/nchembio.2520

Figure Lengend Snippet: Notch ligand binding experiments were performed to measure the effect of fucose analogs on the ability of mNotch1 (a–c) or mNotch2 (d–f) transfectants to bind soluble Dll1-Fc (a, d), Dll4-Fc (b, e), or Jag1-Fc (c, f) using cell-based flow cytometry assays. EV transfected cells were used as a negative control and cells cultured in the presence of DMSO served as a positive control. Six independent experiments were performed for all samples (n=6). All plots represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, Tukey post-hoc test, adjusted p values.

Article Snippet: Dll1-Fc (R&D Systems, 3970-DL-050), Dll4-Fc (Sino Biological, 10171-H02H-50) or Jag1-Fc (R&D Systems, 599-JG-100) (each at 0.5 μg/mL) were pre-clustered with fluorescent secondary antibody PE-goat anti-mouse IgG (1:100; Invitrogen, P-852) or PE-anti-human IgG (1:100; Jackson Immuno Research, 109155-098) for 30 min at 4 °C.

Techniques: Ligand Binding Assay, Flow Cytometry, Transfection, Negative Control, Cell Culture, Positive Control

Journal: Nature chemical biology

Article Title: Inhibition of Delta-induced Notch signaling using fucose analogs

doi: 10.1038/nchembio.2520

Figure Lengend Snippet: (a) Notch1 activation assays using Dll1-Fc (left) or Dll4-Fc (right) as activating ligands coated on plates showing the effect of mutations at EGF8, 12 or both on Dll1 and Dll4-induced Notch1 signaling. Relative Luciferase Unit (RLU) normalized to wild type (WT) Notch1. (b) Plate coating Notch1 activation assays using Dll1-Fc as activating ligand in the presence of DMSO (control) or 100 nM of the indicated compound. RLU for all constructs (WT and mutants) in DMSO was normalized to 1. (c) Plate coating assays for Dll4-induced signaling using 50 μM fucose analogs. The EGF8/12 double mutant was also tested. RLU for all constructs was normalized to the DMSO control. Box and whisker plots represent six biological replicates (n=6). *p<0.05, **p<0.01, ***p<0.001, Tukey post-hoc test, adjusted p values. (d) Model figure for fucose analog mediated inhibition of Notch1 activation. Elimination of the O -fucose site on EGF8, but not EGF12, partially rescues fucose analog inhibition of Notch1 activation by Dll1 suggesting that binding sites corresponding to O -fucose on EGF12 are tolerant of the additional C6 carbon in compounds 10 and 11 . In contrast, the binding site on Dll1 is not tolerant of the additional carbon at EGF8, creating a steric clash, resulting in the observed decreased activation of Notch1. Additional sites contain O -fucose consensus sequences and are modified with O -fucose (red ovals), but contribute little to Notch1-ligand interactions compared to EGF8 and EGF12 .

Article Snippet: Dll1-Fc (R&D Systems, 3970-DL-050), Dll4-Fc (Sino Biological, 10171-H02H-50) or Jag1-Fc (R&D Systems, 599-JG-100) (each at 0.5 μg/mL) were pre-clustered with fluorescent secondary antibody PE-goat anti-mouse IgG (1:100; Invitrogen, P-852) or PE-anti-human IgG (1:100; Jackson Immuno Research, 109155-098) for 30 min at 4 °C.

Techniques: Activation Assay, Luciferase, Construct, Mutagenesis, Whisker Assay, Inhibition, Binding Assay, Modification

Journal: Nature chemical biology

Article Title: Inhibition of Delta-induced Notch signaling using fucose analogs

doi: 10.1038/nchembio.2520

Figure Lengend Snippet: Representative flow cytometric profiles of cells produced from bone marrow LSK cells co-cultured with OP9-GFP, OP9-Dll1 or OP9-Dll4 stromal cells in the presence of DMSO, compound 9 or compound 10 for 8 days. (a) Production of CD25 + T-cell progenitors was evaluated by the expression of CD44 and CD25. (c) Percentage of CD25 + T-cells from mice with a profile typical of panel a (n=3). Mean ± SEM, each symbol represents average data from duplicate wells of LSK cells from one mouse. Data shown are representative of three experiments performed in duplicate. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, unpaired two-tailed Student’s t-test.

Article Snippet: Dll1-Fc (R&D Systems, 3970-DL-050), Dll4-Fc (Sino Biological, 10171-H02H-50) or Jag1-Fc (R&D Systems, 599-JG-100) (each at 0.5 μg/mL) were pre-clustered with fluorescent secondary antibody PE-goat anti-mouse IgG (1:100; Invitrogen, P-852) or PE-anti-human IgG (1:100; Jackson Immuno Research, 109155-098) for 30 min at 4 °C.

Techniques: Produced, Cell Culture, Expressing, Two Tailed Test

Journal: Nanoscale

Article Title: Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature

doi: 10.1039/D0NR02962A

Figure Lengend Snippet: Fig. 1 Binding specificity and selectivity of VNAR E4-Fc. Binding ELISAs of (A) VNAR E4-Fc to human and murine DLL4 and the closely related ligand DLL1 and (B) binding of the VNAR E4-Fc and YW26.82 mAb to a truncated version of DLL4 displaying only DSL and EGF repeats 1–3.

Article Snippet: A carboxymethylated dextran CM5 sensor chip (GE healthcare) was initially activated with 0.4 M EDC and 0.1 M NHS enabling chip functionalisation with 20 μg ml−1 recombinant human DLL4 Fc chimera protein (Sino Biological) via carbodiimide chemistry.

Techniques: Binding Assay

Journal: Nanoscale

Article Title: Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature

doi: 10.1039/D0NR02962A

Figure Lengend Snippet: Fig. 2 DLL4 blockade by VNAR E4-Fc results in an alternative sprouting phenotype. (A) HUVEC coated beads in a fibrin matrix were cultured for 7 days in media only (no treatment) or supplemented with 250 nM VNAR E4-Fc or mAb YW26.82. The branch points of each sprout are indicated by a star. Representative images under phase contrast and confocal microscopy post F-actin staining are shown. (B) The number of branch points per bead were counted and a total of 10 beads were analysed per treatment (n = 3).

Article Snippet: A carboxymethylated dextran CM5 sensor chip (GE healthcare) was initially activated with 0.4 M EDC and 0.1 M NHS enabling chip functionalisation with 20 μg ml−1 recombinant human DLL4 Fc chimera protein (Sino Biological) via carbodiimide chemistry.

Techniques: Cell Culture, Confocal Microscopy, Staining

Journal: Nanoscale

Article Title: Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature

doi: 10.1039/D0NR02962A

Figure Lengend Snippet: Fig. 4 E4 (anti-DLL4) conjugated NPs bind specifically to recombinant DLL4 (A) size and PDI of E4 and 2V (isotype) conjugated PLGA-PEG-Mal NPs and amount of conjugated VNAR as measured by DLS and mBCA respectively. (B) FLISA assessment of DLL4 binding by nude, E4- and 2V-conjugated NPs. Assay performed in triplicate and data presented as mean of three independent experiments. Statistical significance was established by two- way ANOVA and Tukey’s post-hoc test (****p ≤0.0001). (C) PLGA-PEG-Mal polymeric NPs were incubated with anti-DLL4 clone E4 VNAR. Post- incubation, the ability of the NPs to bind DLL4 was determined by SPR. Data presented as representative SPR binding sensorgram and (D), corres- ponding details for relative binding response.

Article Snippet: A carboxymethylated dextran CM5 sensor chip (GE healthcare) was initially activated with 0.4 M EDC and 0.1 M NHS enabling chip functionalisation with 20 μg ml−1 recombinant human DLL4 Fc chimera protein (Sino Biological) via carbodiimide chemistry.

Techniques: Recombinant, Fluorophore-linked Immunoabsorbent Assay, Binding Assay, Incubation

Journal: Nanoscale

Article Title: Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature

doi: 10.1039/D0NR02962A

Figure Lengend Snippet: Fig. 5 Anti-DLL4 VNAR conjugation is maleimide dependent. Rhodamine 6G loaded PLGA-PEG-Mal and PLGA-PEG-NHS polymeric NPs were incu- bated with anti-DLL4 clone E4 VNAR. Post-incubation, NPs were assessed (A) in terms of the success of the conjugation reaction, as determined by mBCA. Rhodamine 6G loaded PLGA-PEG-NHS-E4 and PLGA-PEG-Mal-E4 NPs binding assessment via (B) FLISA method. Data expressed as mean ± standard deviation (SD). Representative of three independent experiments. Statistical significance was established by two-way ANOVA and Tukey’s post-hoc test (****p ≤0.0001). (C) Binding assessment of PLGA-PEG-Mal-E4 and PLGA-PEG-NHS-E4 NPs by SPR. Data presented as relative binding response with (D), corresponding SPR binding sensorgram details. Representative of three independent experiments. (E) Rhodamine 6G-loaded PLGA-PEG-Mal polymeric NPs possessing varying proportions of PLGA-PEG-Mal copolymer, were conjugated with anti-DLL4 clone E4 VNAR and binding assessed by FLISA. Data expressed as mean ± standard deviation (SD). Representative of two independent experiments. Statistical signifi- cance was established by two-way ANOVA and Tukey’s post-hoc test (**p ≤0.01; ****p ≤0.0001).

Article Snippet: A carboxymethylated dextran CM5 sensor chip (GE healthcare) was initially activated with 0.4 M EDC and 0.1 M NHS enabling chip functionalisation with 20 μg ml−1 recombinant human DLL4 Fc chimera protein (Sino Biological) via carbodiimide chemistry.

Techniques: Conjugation Assay, Incubation, Binding Assay, Fluorophore-linked Immunoabsorbent Assay, Standard Deviation

Journal: Nanoscale

Article Title: Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature

doi: 10.1039/D0NR02962A

Figure Lengend Snippet: Fig. 6 Basal expression of DLL4 in PDAC cell lines correlates with enhanced uptake of anti-DLL4 VNAR conjugated PLGA-PEG-Mal NPs (A) western blot of lysates of MIA PaCa-2 and PANC-1 cells (N = 4). (B–C) Representative histograms of cell fluorescence intensity for MIA PaCa-2 and PANC-1 cells either unstained or stained with YW26.82 and an anti-human PE-conjugated secondary antibody (D) mean fluorescence intensity measure- ments (relative to unstained control). Significance assessed by unpaired t-test (ns p > 0.05) (N = 3) (E–F) uptake of fluorescent NPs into MIA PaCa-2 and PANC-1 cells respectively. Significance was assessed by two-way ANOVA and Tukey’s post-hoc test (****p < 0.0001).

Article Snippet: A carboxymethylated dextran CM5 sensor chip (GE healthcare) was initially activated with 0.4 M EDC and 0.1 M NHS enabling chip functionalisation with 20 μg ml−1 recombinant human DLL4 Fc chimera protein (Sino Biological) via carbodiimide chemistry.

Techniques: Expressing, Western Blot, Staining, Control

Journal: Nanoscale

Article Title: Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature

doi: 10.1039/D0NR02962A

Figure Lengend Snippet: Fig. 7 Anti-DLL4 conjugated vNAR NPs enhance payload delivery to DLL4 expressing endothelial cells (A) HUVECs were treated with FGF and/or VEGF for 24 h. Following treatment, cells were lysed and DLL4 expression validated by western blot. α-Tubulin was used as a loading control. (B) Densitometry quantification of western blot DLL4 expression levels. (C) HUVEC cells (pre-stimulated with FGF and VEGF) were treated with either rhodamine 6G loaded PLGA-PEG-Mal or PLGA-PEG-Mal-E4 NPs for 3 h, then washed, fixed and nuclear regions stained with DAPI. Cells were sub- sequently imaged by confocal microscopy to assess formulation uptake. Three representative images per treatment group are shown. Scale bars = 10 μm. (D) HUVEC cells stimulated with VEGF/FGF were treated with CPT-loaded NPs for 1 h at 4 °C and cell viability was assessed 24 h and (E) 48 h post treatment via CellTiter-Glo®. Cells were treated with either 0.1 mg mL−1 or 0.2 mg mL−1 NP treatments, corresponding to 0.22 μg mL−1 or 0.44 μg mL−1 CPT respectively. Representative of two independent experiments. Statistical significance was established by one-way ANOVA and Tukey’s post-hoc test (*p ≤0.05; **p ≤0.01).

Article Snippet: A carboxymethylated dextran CM5 sensor chip (GE healthcare) was initially activated with 0.4 M EDC and 0.1 M NHS enabling chip functionalisation with 20 μg ml−1 recombinant human DLL4 Fc chimera protein (Sino Biological) via carbodiimide chemistry.

Techniques: Expressing, Western Blot, Control, Staining, Confocal Microscopy, Formulation

Journal: Nanoscale

Article Title: Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature

doi: 10.1039/D0NR02962A

Figure Lengend Snippet: Fig. 8 HUVEC tube morphology is altered upon treatment with anti-DLL4 VNAR conjugated NPs (A) Representative DLL4 western blot of lysates derived from HUVECs cultured under varying oxygen conditions (5, 10 or 21% oxygen), β-actin used as loading control (B) quantification of total mesh area from the below phase contrast images using Angiogenesis Analyzer FIJI plugin. Data pooled from 3 independent experiments and nor- malized to untreated control. Statistical significance determined by two-way ANOVA (**p < 0.01, ****p < 0.0001). (C) Representative 10× phase-con- trast images of HUVECs taken 6 hours after seeding with no treatment (untreated), E4 NPs (100 or 500 nM of NP-bound VNAR) or a matched polymer concentration of nude NP. (D) Quantification of total mesh area from the below phase contrast images using Angiogenesis Analyzer FIJI plugin. Data pooled from 3 independent experiments and normalized to untreated control. Statistical significance determined by two-way ANOVA (****p < 0.0001). (E) Representative 10× phase-contrast images of HUVECs taken 6 hours after seeding with no treatment (untreated), E4 NPs (100 nM of NP-bound VNAR) or free E4 (100 nM).

Article Snippet: A carboxymethylated dextran CM5 sensor chip (GE healthcare) was initially activated with 0.4 M EDC and 0.1 M NHS enabling chip functionalisation with 20 μg ml−1 recombinant human DLL4 Fc chimera protein (Sino Biological) via carbodiimide chemistry.

Techniques: Western Blot, Derivative Assay, Cell Culture, Control, Polymer, Concentration Assay

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Generation and characterization of therapeutic anti-NOTCH3 mAbs (A) NOTCH3-dependent report gene assay with NOTCH3-targeted and control mAbs. Data represent mean ± SEM from 3 biological replicates (n = 4 per replicate), ns, non-significant. (B) qRT-PCR of HES1 gene expression in treated cells. Data represent mean ± SD from 2 biological replicates (n = 3 per replicate), ns, non-significant. (C) NOTCH3 immunoblot from mAb-treated cells and xenografts. GAPDH is shown as a loading control. Schematic diagram of NOTCH3 cleavage events and protein fragments. M, mouse number. (D and E) Immunoblot using N- or C-terminal domain antibodies to detect NOTCH3 fragments after DLL4 activation in treated cells. β-actin is shown as a loading control. (F) Epitope mapping of anti-NOTCH3 mAbs using NRR3-NRR1 domain swap chimeric constructs. NRR3 domains are shown in black and NRR1 domains are shown in gray. Representative data represent mean (n = 2). (G) Binding interface of anti-N3(i) Fab on the NOTCH3-NRR domain as determined from the co-crystal structure is shown in red, LNR-A (green), LNR-B (beige), LNR-C (blue), HD1 (pink), and HD2 (magenta).

Article Snippet: To generate the ligand-expressing cells, HEK293 cells were transfected with a vector for expression of human DLL4 in the pCMV6-AC-HA-His backbone (Origene, Rockville, MD) and termed HEK-DLL4.

Techniques: Gene Assay, Quantitative RT-PCR, Expressing, Western Blot, Activation Assay, Construct, Binding Assay

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs transendocytose into DLL4 ligand cells (A and B) TEC of labeled mAbs (green) that were bound to U2OS-hN3 cells and then co-cultured with HEK-DLL4 or HEK-parental cells (red) from live-cell confocal imaging. (A) Single optical sections of a U2OS-hN3 cell and a migrating HEK-DLL4 cell (asterisk) before and after contact (arrows). Scale bar, 10 μm. (B) z stack of maximum intensity projections. Arrows, anti-NOTCH3 mAbs inside HEK-DLL4 cells. (C) Confocal images of maximum intensity projections acquired from indirect immunofluorescence of mAbs bound to U2OS-hN3 cells (magenta) and then co-cultured with HEK-DLL4 (green) from. Dashed white line demarcates anti-NOTCH3 mAbs inside HEK-DLL4 cells. Scale bar, 10 μm.

Article Snippet: To generate the ligand-expressing cells, HEK293 cells were transfected with a vector for expression of human DLL4 in the pCMV6-AC-HA-His backbone (Origene, Rockville, MD) and termed HEK-DLL4.

Techniques: Labeling, Cell Culture, Imaging, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce cytotoxicity in both receptor and ligand cells (A) General structure of NOTCH3-targeted ADCs that were generated with mAbs, a cleavable dipeptide-based linker and the Aur0101 payload (blue). (B) NOTCH3-ADC induction of caspase-3/7 activity. Data represent mean ± SEM of 3 biological replicates (n = 3 per replicate). (C) In vitro cytotoxicity of NOTCH3-ADCs after control ( Control:siRNA ) or siRNA knockdown of NOTCH3 mRNA ( N3:siRNA ). Data represent mean ± SEM of 2 biological replicates (n = 3 per replicate). (D) In vitro cytotoxicity of NOTCH3-ADCs using parental MDA-MB-468 cells under 2D and 3D culture conditions. Data represent mean ± SEM (n = 3). (E and F) TEC of NOTCH3-ADCs induces caspase activity in HEK-DLL4 cells. (E) z stack of maximum intensity projections from live-cell confocal imaging of anti-N3(i) ADC bound to U2OS-hN3 cells and co-cultured with HEK-DLL4 cells labeled with pHrodo Red dextran. Caspase compartments (magenta line), pHrodo Red dextran compartments (blue line) and the merged image. (F) Percentage of the caspase-positive compartments that were calculated after treatment with NOTCH3-ADCs. Data represent mean ± SEM for 3 biological replicates (n = 15 fields imaged per replicate), ns, non-significant.

Article Snippet: To generate the ligand-expressing cells, HEK293 cells were transfected with a vector for expression of human DLL4 in the pCMV6-AC-HA-His backbone (Origene, Rockville, MD) and termed HEK-DLL4.

Techniques: Generated, Activity Assay, In Vitro, Imaging, Cell Culture, Labeling

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet:

Article Snippet: To generate the ligand-expressing cells, HEK293 cells were transfected with a vector for expression of human DLL4 in the pCMV6-AC-HA-His backbone (Origene, Rockville, MD) and termed HEK-DLL4.

Techniques: Plasmid Preparation, Blocking Assay, Electron Microscopy, Recombinant, Antibody Labeling, Sequencing, Software