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

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Simultaneous binding to VEGF and Dll4 by HD105 bispecific antibody leads to effective blockade of VEGF/VEGFR2 and Dll4/Notch1 interactions. The HD105 bispecific antibody was constructed of the C-terminal of the anti-VEGF (bevacizumab-similar) IgG backbone linked with a single-chain Fv targeting Dll4 (A). The binding affinity of the HD105 bispecific antibody against human VEGF or human Dll4 was determined by Biacore assays (B) and ELISAs (C, D). The KD values of each antibody against VEGF or Dll4 are summarized in Table (B). The HD105 bispecific antibody (closed circle) dose-dependently bound to human VEGF (C) or Dll4 (D). In addition, the HD105 bispecific antibody simultaneously bound to each antigen, human VEGF and human Dll4, in dual-antigen capture ELISAs (E). The anti-Dll4 antibody (open circle in C) or the anti-VEGF (bevacizumab-similar) antibody (open circle in D, E) was used as negative control. Competitive ELISAs demonstrated that the HD105 bispecific antibody inhibited the interaction between VEGF/VEGFR2 (F) or Dll4/Notch1 (G) in a dose-dependent manner. The EC50 (half maximal effective concentration) values of the anti-VEGF (bevacizumab-similar) antibody (open circle) and HD105 bispecific antibody (closed circle) for VEGF/VEGFR2 inhibition were 2.98 ± 0.5 nM and 2.84 ± 0.41 nM, respectively (F). The EC50 values of the anti-Dll4 antibody (open circle) and HD105 bispecific antibody (closed circle) were 0.65 ± 0.06 nM and 1.14 ± 0.06 nM, respectively (G).

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Binding Assay, Construct, Negative Control, Concentration Assay, Inhibition

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Blockade of both VEGF/VEGFR2 and Dll4/Notch1 signaling pathways by HD105 bispecific antibody leads to inhibition of each signaling-induced cellular response. The HD105 bispecific antibody inhibited both the VEGF/VEGFR2 and the Dll4/Notch1 signaling pathways in HUVECs (A). The VEGF/VEGFR2 signaling pathway was monitored by the activation of VEGFR2 and ERK (phosphorylation). The Dll4/Notch1 signaling pathway was monitored by the generation of NICD (Notch-induced intracellular domain). HUVEC sprouting assays were performed in a fibrin gel in the presence of PBS (B), anti-VEGF (bevacizumab-similar) antibody (C), anti-Dll4 antibody (D), or HD105 bispecific antibody (E). Representative images show sprouting tip cells of HUVECs from the beads under basal media (B, arrowheads) and more sprouting under anti-Dll4 antibody treatment (D, arrows) but much less sprouting under anti-VEGF antibody (C) or HD105 bispecific antibody treatment (E). Scale bar (B-E), 150 μm. The bar graph (F) shows the measurement of sprouting HUVECs at 225 μm from beads (n = 20 beads/group, mean ± SE). *, P < 0.05 versus PBS. †, P < 0.05vs. anti-Dll4 antibody. The HD105 bispecific antibody inhibited VEGF-dependent HUVEC proliferation (G) and Dll4-induced Notch-1-dependent activation of luciferase in SKOV-3-RBP-J Κ luciferase cells (H) in a dose-dependent manner. The IC50 values of the anti-VEGF (bevacizumab-similar) antibody (open circle) and HD105 bispecific antibody (closed circle) on HUVEC proliferation were 1.49 ± 0.04 nM and 1.58 ± 0.08 nM, respectively (G). The IC50 values of the HD105 bispecific antibody (closed circle) and the anti-Dll4 antibody (open circle) on luciferase activation were determined to be 0.62 ± 0.23 nM and 0.58 ± 0.03 nM, respectively (H).

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Inhibition, Activation Assay, Luciferase

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Suppression of tumor progression in several cancer xenograft models by HD105 bispecific antibody. Human A549 lung cancer (A) or human SCH gastric cancer (B, C) was subcutaneously implanted into nude mice. After tumors were grown to an average volume of 150–200 mm3, PBS (open triangle), anti-VEGF (bevacizumab-similar) antibody (2.5 mg/kg, open circle), anti-mouse Dll4 antibody (2.5 mg/kg, closed triangle), or mouse HD105 bispecific antibody (3.25 mg/kg, closed circle) was intraperitoneally injected twice (A549) or once (SCH) per week (A, B). Tumor volume was calculated by the formula width2 × length × 0.52. The dose dependency of the mouse HD105 bispecific antibody was evaluated in human SCH gastric cancer xenograft model (C). PBS (open triangle) or mouse HD105 bispecific antibody (0.361 mg/kg, closed triangle; 1.083 mg/kg, open circle; 3.25 mg/kg, closed circle) was intraperitoneally injected once per week. The response to mouse HD105 bispecific antibody (6.5 mg/kg, once per week, closed circle) was also determined using other human gastric cancer xenograft models, including MKN-74 (D), SNU-5 (E), and SNU-16 (F). Tumor progression was not inhibited by the mouse HD105 bispecific antibody in MKN-74 and SNU-5 but was inhibited in SNU-16 similarly to SCH.

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Injection

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Suppression of tumor angiogenesis in cancer xenograft models by HD105 bispecific antibody. Fluorescence micrographs compare the vasculature of A549 human lung cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), or mouse HD105 bispecific antibody (D). Scale bar (A-D), 50 μm. The tumor vasculature was stained for CD31 immunoreactivity (green), and the vascular basement was stained for type IV collagen (red). Tumor vessels were decreased after treatment with anti-VEGF (bevacizumab-similar) antibody or mouse HD105 bispecific antibody, whereas tumor vessels were markedly increased after treatment with anti-mouse Dll4 antibody compared to PBS. Higher-resolution images compare the phenotype changes of tumor vessels in detail after PBS (E), anti-VEGF (bevacizumab-similar) antibody (F), anti-mouse Dll4 antibody (G), or mouse HD105 bispecific antibody treatment (H). Scale bar (E-H), 20 μm. The tumor vasculature was stained for CD31 immunoreactivity (red), and the perivascular pericyte was stained for NG2 (green). The nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole). Tumor vessels after treatment with anti-mouse Dll4 antibody were conspicuously thinner and more branched than the tumor vessels of other groups. Bar graph (I) measuring tumor vessel density of A549 tumor tissues in xenograft mice confirms the conspicuous increase of tumor vessels after anti-mouse Dll4 antibody treatment but decreases after anti-VEGF (bevacizumab-similar) antibody, mouse HD105 bispecific antibody, or combination treatment with anti-mouse Dll4 antibody and anti-VEGF (bevacizumab-similar) antibody. †, P < 0.05 versus PBS. *, P < 0.05vs. anti-Dll4 antibody. However, the functional tumor vessels in SCH gastric cancer tissues assessed by intravenous FITC-labeled Lycopersicon esculentum (Tomato) lectin staining were significantly decreased after treatment with anti-VEGF (bevacizumab-similar) antibody as well as anti-mouse Dll4 antibody (J). †, P < 0.05 versus PBS. ‡, < 0.05vs. anti-VEGF (bevacizumab-similar) antibody. *, P < 0.05 versus anti-Dll4 antibody. Functional tumor vessels were more decreased after treatment with mouse HD105 bispecific antibody compared to the other groups.

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Fluorescence, Staining, Functional Assay, Labeling

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Increase in apoptotic tumor cells in cancer xenograft models treated with HD105 bispecific antibody. Fluorescence micrographs show apoptotic cells stained for activated caspase-3 antibody (red) in SCH human gastric cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), and mouse HD105 bispecific antibody (D and E). Scale bar (A-D), 50 μm; (E), 20 μm. Nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole, blue). The higher-resolution image confirms that activated caspase-3 antibody was stained in the cytoplasm of the apoptotic cells after mouse HD105 bispecific antibody treatment (E). The bar graph (F) measuring the cell density of apoptotic cells in SCH cancer tissues confirms the significant increase in apoptotic cells after mouse HD105 bispecific antibody treatment. *, P < 0.05vs. PBS. ‡, < 0.05 versus anti-VEGF (bevacizumab-similar)) antibody. *, P < 0.05vs. anti-Dll4 antibody.

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Fluorescence, Staining