Journal: Cancer cell

Article Title: Unbiased Combinatorial Screening Identifies a Bispecific IgG1 that Potently Inhibits HER3 Signaling via HER2-Guided Ligand Blockade.

doi: 10.1016/j.ccell.2018.04.003

Figure Lengend Snippet: Figure 3. Selectivity of PB4188 for HER2/HER3 Signaling (A–D) PathHunter assays to measure ligand-induced dimerization of EGFR/HER2 (A), HER2/HER4 (B), HER2/HER3 (C), and EGFR/HER3 (D). Cells were stim- ulated with an EC80 concentration of EGF (A) or HRG (B–D) and titrations of the indicated antibodies. (E) Human stem cell-derived cardiomyocytes were incubated with the indicated antibodies in combination with doxorubicin. Cell viability was determined by measuring cellular ATP. (F) SKBR-3 cells cultured in serum-free medium were supplemented with 12.5 nM HRG and the indicated antibodies. Twenty-four hours later, protein lysates were analyzed with PathScan array. (G) Phosphorylated (p prefixed) and total levels of signaling proteins after HRG stimulation (12.5 nM) in N87 cells analyzed by western blot. T, trastuzumab; P*, pertuzumab*; T + P*, equimolar combination of trastuzumab plus pertuzumab*. Data in (A–D) are represented as means ± SEM. Boxes in (E) show the middle quartile (25%–75%); horizontal bars represent the median; whiskers show the maximum and minimum values in the dataset.

Article Snippet: AM2016-4759 Chemicals, Peptides, and Recombinant Proteins Human NRG1-beta 1/HRG1-beta 1 EGFlike domain R&D Systems Cat#396-HB Human NRG1-beta 1/HRG1-beta 1 EGFlike domain Prospec Bio Cat#Cyt-733 Human EGF DiscoverX Cat#92-1113 Human HER2 Fc chimera R&D Systems Cat#1129-ER Human HER3 Fc chimera R&D Systems Cat348-RB Human HER4 Fc chimera R&D Systems Cat#1131-ER Human EGFR Fc chimera R&D Systems Cat#344-ER Human HER2 ectodomain Bender MedSystems Cat#362 Critical Commercial Assays Alamar BlueTM Invitrogen Cat#DAL1100 Click-iTTM EdU AlexaFluorTM 488 Imaging Kit Life Technologies Cat#C10337 PathScan RTK Signaling Antibody Array Kit Cell Signaling Cat#7949 PathScan Intracellular Signaling Array Kit Cell Signaling Cat#7323 PathHunter U2OS Dimerization Cell Line DiscoverX N/A Human IgG ELISA ZeptoMetrix Cat# 0801182 VeraTag Assay Monogram Biosciences N/A Cell Signaling Multiplex Assays Millipore Cat# 48-618MAG, 46-645MAG, 46-645M-1K ADCC Reporter Bioassay Promega Cat#G7011, G9790, G7941 Epitope mapping Integral Molecular N/A Deposited Data HER2-ECD:MF3958 structure This paper PDB: 5O4G HER3-ECD:MF3178 structure This paper PDB: 5O4O, 5O7P Experimental Models: Cell Lines Human: JIMT-1 DSMZ Cat#ACC-589; RRID:CVCL_2077 Human: SKBR-3 ATCC Cat#HTB-30; RRID:CVCL_0033 Human: BT-474 DMSZ Cat#ACC-64; RRID:CVCL_0179 Human: MCF-7 DMSZ Cat#ACC-115; RRID:CVCL_0031 Human: MDA-MB-468 Cell Line Service N/A (Continued on next page) e2 Cancer Cell 33, 922–936.e1–e10, May 14, 2018

Techniques: Concentration Assay, Derivative Assay, Incubation, Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Cellular Effects of HER3-Specific Affibody Molecules

doi: 10.1371/journal.pone.0040023

Figure Lengend Snippet: Binding of Alexa Fluor® 488-labelled Affibody molecules (150 nM) to: A. MCF-7, B. SKBR-3 and the HER3-negative cell line SKOV-3. “Bl” = blocking with 15 μM of the corresponding, non-labelled Affibody. MCF-7 cells were stained in a separate experiment, whereas SKBR-3 and SKOV-3 were stained simultaneously.

Article Snippet: As a positive control, a polyclonal goat anti-human HER3 antibody (AF234, R&D systems) was used.

Techniques: Binding Assay, Blocking Assay, Staining

Journal: PLoS ONE

Article Title: Cellular Effects of HER3-Specific Affibody Molecules

doi: 10.1371/journal.pone.0040023

Figure Lengend Snippet: Images showing AU565, SKBR-3, MCF-7 and SKOV-3 cells stained with HER3-specific Affibody molecules Z05416 and Z05417, respectively. The polyclonal anti-HER3 antibody A234 and ZTaq were used as positive and negative staining controls, respectively. Affibody molecules and antibodies binding to cells are shown in green while nuclear staining by DAPI is given in blue. AU565 and SKOV-3 images were acquired on the same day using the same detection gain and laser power, enabling comparison between staining intensities. Cell staining of MCF-7 and SKBR-3 was analysed on different days, using different detection gains for optimal image acquisition. Additionally, MCF-7 images were acquired using increased laser power.

Article Snippet: As a positive control, a polyclonal goat anti-human HER3 antibody (AF234, R&D systems) was used.

Techniques: Staining, Negative Staining, Binding Assay, Comparison

Journal: PLoS ONE

Article Title: Cellular Effects of HER3-Specific Affibody Molecules

doi: 10.1371/journal.pone.0040023

Figure Lengend Snippet: Histograms showing ELISA absorbance results for detection of: A. Phospho-HER2 in MCF-7 cells, B. Phospho-HER3 in MCF-7 cells, C. Phospho-HER2 in SKBR-3 cells and D. Phospho-HER3 in SKBR-3 cells. Cells were incubated without HRG (grey bars) or with 0.05 nM HRG (black bars), in combination with the Affibody molecules (100 nM) before being lysed and analysed in an ELISA.

Article Snippet: As a positive control, a polyclonal goat anti-human HER3 antibody (AF234, R&D systems) was used.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation

Journal: Journal of Biological Chemistry

Article Title: Direct Binding of the EGF-like Domain of Neuregulin-1 to Integrins (αvβ3 and α6β4) Is Involved in Neuregulin-1/ErbB Signaling

doi: 10.1074/jbc.m110.113878

Figure Lengend Snippet: FIGURE 2. Docking simulation of v3-NRG1 interaction. a, a model of NRG1-integrin v3 interaction predicted by docking simulation by using AutoDock3 is shown. The headpiece of integrin v3 (PDB code 1LG5) was used as a target. The model predicts that the EGF-like domain of NRG1 (PDB code 1HAF, blue) binds to the RGD-binding site of the integrin v3 head- piece (green and red). b, the Lys residues at positions 180, 184, and 186 of NRG1 are located at the interface between NRG1 and v3 and were selected for mutagenesis studies. c, superposition of TGF and NRG1 is shown. d, the Lys residues at positions 180, 184, and 186 of NRG1 are not located in the binding site for EGFR. We replaced TGF in the TGF-EGFR complex (PDB code 1MOX) with NRG1 (PDB code 1HAF) by superposing. ErbB3 or ErbB4 is homologous to EGFR.

Article Snippet: We obtained recombinant human NRG1 EGF-like domain peptide (residues Ser-177— Lys-241, synthesized in Escherichia coli, 97% purity), recombinant human NRG1 isoform SMDF (296 amino acids, Spodoptera frugiperda Sf21(baculovirus)-derived), and recombinant human ErbB3 Fc chimera from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Direct Binding of the EGF-like Domain of Neuregulin-1 to Integrins (αvβ3 and α6β4) Is Involved in Neuregulin-1/ErbB Signaling

doi: 10.1074/jbc.m110.113878

Figure Lengend Snippet: FIGURE 4. The 3KE mutant of NRG1 binds to ErbB3. a, binding of the 3KE mutant of NRG1 to recombinant ErbB3 is shown. Recombinant soluble ErbB3 Fc fusionprotein(R&Dsystem)wascoatedontowellsofa96-wellmicrotiterplate(1 g/ml). NRG1 WT or 3KE mutant was added to the wells and incubated for 1 h at room temperature. GST was used as a control. After washing the wells, bound GST NRG1 was determined by using anti-GST antibody HRP conjugate. The resultssuggestthatthe3KEmutantofNRG1bindstoErbB3atlevelsnearlycom- parable with WT NRG1. The data are shown as the means S.E. of triplicate ex- periments. b, shown is a competitive binding assay. Recombinant soluble ErbB3 Fc fusion protein was coated onto wells of 96-well microtiter plate (1 g/ml). Bindingofbiotin-labeledNRG1WT(20nM)inthepresenceofincreasingconcen- trationsofNRG1WT,3KE,orGSTisshown.Afterwashingthewells,boundbiotin- labeledNRG1WTwasdeterminedbyusingstreptavidinHRPconjugate.Thedata are shown as the means S.E. of triplicate experiments. The results suggest that the 3KE mutant of NRG1 binds to ErbB3 at levels comparable with WT NRG1. c, binding of soluble ErbB3-Fc to immobilized NRG1 is shown. We immobilized NRG1 WT and 3KE proteins to the wells of the 96-well microtiter plate at the indicatedcoatingconcentrationsandincubatedwithsolubleErbB3-Fc(1g/ml) as described above. Bound ErbB3 was detected using HRP-conjugated anti-His6 tag antibodies and peroxidase substrate. ErbB3-Fc has a His6 tag.

Article Snippet: We obtained recombinant human NRG1 EGF-like domain peptide (residues Ser-177— Lys-241, synthesized in Escherichia coli, 97% purity), recombinant human NRG1 isoform SMDF (296 amino acids, Spodoptera frugiperda Sf21(baculovirus)-derived), and recombinant human ErbB3 Fc chimera from R&D Systems (Minneapolis, MN).

Techniques: Mutagenesis, Binding Assay, Recombinant, Incubation, Control, Competitive Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Direct Binding of the EGF-like Domain of Neuregulin-1 to Integrins (αvβ3 and α6β4) Is Involved in Neuregulin-1/ErbB Signaling

doi: 10.1074/jbc.m110.113878

Figure Lengend Snippet: FIGURE 5. Effect of the 3KE mutation on NRG1 signaling. a and b, the 3KE mutant of NRG1 is defective in inducing ErbB3 phosphorylation, AKT activation, and ERK1/2 activation. MCF-7 cells were serum-starved over- night and stimulated with 10 nM WT and the 3KE mutant of NRG1 for 5 min (a) or 30 min (b). Cell lysates were analyzed by Western blotting. Data are representative of three independent experiments. c–e, levels of phos- phorylation were quantified using a luminescence analyzer from triplicate experiments. Data were normalized using WT NRG1 as 1. f, recruitment of p85 of PI3K is shown. Cells were stimulated with WT NRG1 or 3KE, ErbB3 was immuno-purified (IP) from lysates using anti-ErbB3, and immuno-purified materials were analyzed by Western blotting. The p85 subunit of PI3K was detected in lysates of cells stimulated with WT NRG1. Much lower levels of p85 were detected in cells stimulated with 3KE. Data shown are representative of three inde- pendent experiments. g, shown is the effect of WT and 3KE NRG1 on the proliferation of MCF-7 cells. Human MCF-7 breast cancer cells were serum-starved overnight and cultured for 48 h with WT or 3KE mutant NRG1. GST was used as a control. Cell number was measured by MTS assays (OD490). The data are shown as the means S.E. (n 3). p 0.05 by 2-way ANOVA in each case. Data are representative of three independent experiments performed.

Article Snippet: We obtained recombinant human NRG1 EGF-like domain peptide (residues Ser-177— Lys-241, synthesized in Escherichia coli, 97% purity), recombinant human NRG1 isoform SMDF (296 amino acids, Spodoptera frugiperda Sf21(baculovirus)-derived), and recombinant human ErbB3 Fc chimera from R&D Systems (Minneapolis, MN).

Techniques: Mutagenesis, Phospho-proteomics, Activation Assay, Western Blot, Purification, Cell Culture, Control

Journal: Journal of Biological Chemistry

Article Title: Direct Binding of the EGF-like Domain of Neuregulin-1 to Integrins (αvβ3 and α6β4) Is Involved in Neuregulin-1/ErbB Signaling

doi: 10.1074/jbc.m110.113878

Figure Lengend Snippet: FIGURE 6. WT NRG1 induced co-precipitation of integrin 3 and 4 with ErbB3, whereas 3KE is defective in this function. a, MCF-7 cells were for serum-starved 24 h and stimulated with 10 nM NRG1 WT or 3KE for 5 min. We used 0.7 mg of protein of cell lysate for immunoprecipitation with anti-ErbB3 antibody. Immunoprecipitated materials were analyzed by Western blotting (IB). The levels of ErbB3 phosphorylation were less with the 3KE mutant. Inte- grin 4 was co-immunoprecipitated with the ErbB3 upon stimulation with WT NRG1, whereas the 3KE mutant was defective in this function. Integrin 3 was not detected under the conditions used. The three bands in co-precipi- tated 4 in MCF-7 are considered to be (from the top) 64 heterodimer, intact 4, and a fragment of 4 based on size. Data are representative of three independent experiments. b, we detected co-precipitation (IP) of 3 with ErbB3 when we used 5 more MCF-7 lysate for co-precipitation experiments using WT NRG1. Data are representative of three independent experiments.

Article Snippet: We obtained recombinant human NRG1 EGF-like domain peptide (residues Ser-177— Lys-241, synthesized in Escherichia coli, 97% purity), recombinant human NRG1 isoform SMDF (296 amino acids, Spodoptera frugiperda Sf21(baculovirus)-derived), and recombinant human ErbB3 Fc chimera from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Mutagenesis