Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 1. Unique fragments of the CDRs within the heavy and light chains of the new mouse monoclonal anti-HER2 antibody. The amino acid sequence corresponds to the characteristic nucleotide sequence of (A) anti-human HER2/70.27.58 mAb and (B) anti-human HER2/70.21.73.67 mAb.

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Sequencing

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 2. Anti-HER2 monoclonal antibodies production and characterization. (A) Morphology of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 hybridoma cells photographed at 20 × and 40 × magnification. (B) FPLC chromatograms were recorded during the purification of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies using affinity chromatography on the Protein A resin. (C) SDS-PAGE analysis of the purified anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded at the amount of 1 µg/well on the 12% polyacrylamide gel under reducing conditions. (D) WB analysis of HER2 in whole cell lysates of the HER2 low expressing (MDA-MB-231) and HER2 high expressing (SK-BR-3, SK-OV-3) cells, probed with the home-made anti-human HER2/70.27.58 monoclonal antibody and detected with the secondary anti-mouse IgG-HRP (upper panel). The recombinant HER2 ECD protein was used as a reference. The loading control was performed with membrane probed with antibody binding β-actin (lower panel). (E) The formaldehyde-fixed SK-OV-3 cells were photographed in the bright field (BF) at the 40 × magnification. Immunofluorescence analysis was performed on cells stained with the commercial anti- HER2 ECD antibody followed by anti-mouse IgG-AlexaFluor594 (AF594) (red channel) and co-stained with the anti-HER2/70.27.58 or anti-HER2/70.21.73.67 antibodies detected with the AlexaFluor488-labeled (AF488) secondary antibody (green channel). Nuclei were stained with DAPI (blue channel) (F) Quantitative ELISA with the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded in a range of 0–5 µg/ ml on the plate coated with the recombinant chimera of the HER2 ECD-Fc protein. The signal generated from secondary antibody anti-mouse IgG-HRP was quantified by measuring absorbance at 450 nm and expressed after background subtraction (A450-A0).

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Bioprocessing, Purification, Affinity Chromatography, SDS Page, Expressing, Recombinant, Control, Membrane, Binding Assay, Immunofluorescence, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Generated

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 3. Parameters of the sandwich ELISA for HER2 detection. (A) HER2 binding kinetics in the standard curve concentration range of 0.156–10 000 ng/well (1.56–100 ng/ml). Results are expressed as absorbance at 450 nm after background subtraction (A450-A0). (B) Assay accuracy was tested by comparison of HER2 level measured by ELISA in the samples of the known antigen concentration (mock samples). Data were collected for 2, 5, 10, 30, and 50 ng/ml of HER2 (given concentration; x-axis), covering both physiological and increased concentrations. Experimentally measured concentration [ng/ml] is shown on the y-axis. Error bars indicate SD.

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Sandwich ELISA, Binding Assay, Concentration Assay, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 4. HER2 expression in tumors from mice with xenografted human cancer cells. (A) Immunohistochemistry staining using anti-HER2/70.27.58 mAb of the mouse tumors induced with the human ovarian cancer cells (SK-OV-3) overexpressing HER2 and (B) human epithelial breast cancer cells (MDA-MB-231) with low expression of HER.

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 1 Schematic representation of the EpiView-D4 mobile pathology platform for breast cancer. The device has a smart- phone base which uses detachable modules for brightfield (top) and fluorescence (bottom) imaging using the cell phone camera. Tumor is first sampled by FNA, and then aspirates are processed for cytology and biomarker evaluation. For cytology, aspirates are smeared on a glass slide and processed with rapid (DiffQuikTM) staining kits and then imaged with the brightfield imaging attachment (top). For biomarker evaluation, aspirates are mixed with lysis buffer and then applied to a D4 immunoassay chip, which quantify an analyte of interest (in this case, HER2). D4 chips are read using the fluorescence imaging attachment. The fluorescence intensity of the cAb spots on the D4 chip correlates with the biomarker expression level.

Article Snippet: To determine analytical sensitivity, analyte binding curves were generated using recombinant HER2 protein (R&D systems, Inc.) spiked into RIPA buffer.

Techniques: Imaging, Biomarker Discovery, Staining, Lysis, Expressing

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 2 D4 sandwich immunoassay for quantification of HER2 expression level and its in vitro assessment. a–c, Schematic and operation of D4 immunoassay. a Spots of immobile cAb and an excess of “soluble” fluorescently labeled dAb are printed directly onto POEGMA-coated glass. b Dispensing sample fluid onto chip surface leads to dissolution of soluble dAb spots, followed by diffusion-driven mixing and antibody “sandwich” formation if HER2 is present. HER2 binding is detected by fluorescence imaging. c Representative fluorescence image of D4 assay after exposure to HER2-spiked RIPA buffer. Scale bar, 1 mm. d Representative dose-response curve generated from D4 chips for RIPA buffer spiked with recombinant HER2. Error bars: mean ± s.d. of duplicate assays. e, Assessment of cultured breast cancer cell lines (BT474, BT20, MDA-MB-231, MDA-MB-468) by D4 assay and comparison with western blotting. Inset: Western blot against HER2 for each cell line, with representative D4 cAb spots underneath. Main: D4 signal intensity (mean ± 95% CI) of ≥6 replicates. Significant difference by one-way ANOVA (F(4, 31) = 179.4, p < 0.0001). Bars with different letters indicate different groups (Tukey post hoc test, p ≤0.05). f Concordance analysis of 8 different patient-derived tumor cell lines in culture for HER2 expression by D4 assay vs. ELISA performed by clinical lab; specimens indexed “a” through “h”. D4 results are mean ± s.d. of duplicate assays. Pearson’s r = 0.975 (p < 0.0001, 95% CI: 0.845–0.996).

Article Snippet: To determine analytical sensitivity, analyte binding curves were generated using recombinant HER2 protein (R&D systems, Inc.) spiked into RIPA buffer.

Techniques: Expressing, In Vitro, Labeling, Dissolution, Diffusion-based Assay, Binding Assay, Imaging, Generated, Recombinant, Cell Culture, Comparison, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 3 EpiView mobile microscope for brightfield and fluorescence imaging. a–d Optomechanical design of EpiView. Labeled 3D schematic and photographs of the EpiView scope with brightfield (a, b) and epifluorescence (c, d) assemblies. e, f Raw resolution of brightfield imaging from unprocessed images of USAF 1951 test target. Brightfield images of test target (green channel extracted) are shown in e, with red and blue dashed lines in image corresponding to vertical (red) and horizontal (blue) intensity profiles of test target features in f. Comparison of brightfield imaging of FNA cytology from human breast cancer specimen using standard benchtop microscopy (g) and EpiView device (h). Left panels: standard microscope image obtained by a 10× objective lens (top) and native view on EpiView (bottom). Scale bar, 0.2 mm. Right panels: standard microscope image obtained by a 40× objective (top), showing the same ROI outlined with red dashed line in the 10× image, and similar view obtained by digital zoom on EpiView (bottom). Scale bar, 30 µm. i, j Resolution testing with USAF 1951 in a manner similar to e and f but in epifluorescence mode based on extracting green channel image. k Left panel: Widefield view of 200 nm fluorescence beads using EpiView in epifluorescence mode. Scale bar, 0.1 mm. Right panel: Comparison of EpiView versus and benchtop microscope (40× objective, NA = 0.6) of the 200 nm beads outlined by the red and blue ROIs in the widefield image. Images were extracted from green channel. Scale bars, 40 µm. l Dose-response curve of HER2 with D4-EpiView and a conventional glass slide scanner. Limit of detection for this experiment using conventional glass scanner (LODG) is 21 pM, while that of the D4-EpiView (LODE) is 77 pM. Data represent log signal intensity ± s.d for duplicate assays for both EpiView-D4 and the slide scanner.

Article Snippet: To determine analytical sensitivity, analyte binding curves were generated using recombinant HER2 protein (R&D systems, Inc.) spiked into RIPA buffer.

Techniques: Microscopy, Imaging, Labeling, Comparison

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 4 Evaluation of solid tumor xenografts from nude mice orthotopically engrafted with human breast cancer cell lines with EpiView- D4. a Schematic of workflow. Tumor aspirates are divided into two aliquots; one aliquot from an aspirate is processed as a cytology specimen on a glass slide using a Diff-QuikTM rapid staining kit and then imaged using the brightfield imaging attachment (top row), The second aliquot is lysed in RIPA buffer and applied to a D4 chip and its HER2 level is quantified using the fluorescence attachment on the EpiView-D4. b Representative western blots against HER2 for each human breast cancer cell line used for xenografting (BT20, BT474, and MDA-MB-453). Vinculin used as loading control. c Results of HER2 credentialing for 16 different solid tumor specimens from BT20, BT474, and MDA-MB-453 xenografts (N = 5, 8, 3, respectively). Each data point represents the D4 fluorescence intensity (average of duplicates) measured by the EpiView-D4 for individual mouse tumors, categorized by xenograft line. Also shown is the mean ± 95% CI fluorescence intensity for each category, which mirrors the western blots shown in panel b. There was a statistically significant difference between groups as determined by one-way ANOVA (F (2, 13) = 76.10, p < 0.0001). Multiple comparison testing showed significant differences between each group (Tukey post hoc test, p ≤0.05). d D4 fluorescence intensity by EpiView-D4 for each mouse tumor from f plotted against corresponding HER2 ELISA. Pearson r = 0.968, p < 0.0001, 95% CI: 0.910–0.989). e–g Representative LPF (main panel) and HPF (inset) images of FNA preparations for each xenograft line confirm malignant cytology (see Supplementary Fig. 4 for representative images with standard microscope). Scale bars for LPF and HPF images are 0.2 mm and 30 µm, respectively.

Article Snippet: To determine analytical sensitivity, analyte binding curves were generated using recombinant HER2 protein (R&D systems, Inc.) spiked into RIPA buffer.

Techniques: Staining, Imaging, Western Blot, Control, Comparison, Enzyme-linked Immunosorbent Assay, Microscopy

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Generation of multispecific DB-VHH constructs. Trastuzumab IgG1-K409R mAb (anti-HER2) C-termini are fused to VHHs directed against EGFR, IL6R or NKG2D via a GS-linker. CS06 IgG1-F405L mAb (anti-c-MET) is fused to the same VHH molecules in the same manner. After recombinant production and purification, parental IgG-VHHs are recombined pairwise by reduction and reoxidation. The matching K409R and F405L mutations drive the generation of heterodimeric multispecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Construct, Recombinant, Purification

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Binding affinities measured for each paratope using recombinant antigens

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Construct

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Biolayer interferometry analysis of simultaneous antigen binding of tri- and tetraspecific DB-VHHs. (a), (b) and (c) show exemplary sensorgrams for trispecific molecules and (d) a tetraspecific DB-VHH. The first association step represents binding of the DB-VHH (200 nM) via its CS06 paratope to biotinylated c-MET immobilized to streptavidin biosensors. Second (and third for (D)) association step is performed using an IL6R, EGFR or NKG2D recombinant protein (200 nM). The last association step is performed using HER2 (200 nM). Kinetic buffer (KB) controls were applied as negative controls for each association step.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Specific cell clustering due to simultaneous binding of the DB-VHHs to three different cancer cell lines. Flow cytometry cytograms represent the fluorescence signals of the different cell populations. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2 +++ ) cells stained with DeepRed, lower left gate = MDA-MB-468 (EGFR +++ ) cells stained with CMRA, lower right gate = EBC-1 (c-MET ++ ) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (cell triplets) were marked in red. Cells were incubated in the presence of 1 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Simultaneous interaction of the DB-VHHs with HCC-1954 and EBC-1 target cells and binding of recombinant IL6R. Flow cytometry cytograms represent the fluorescence signals of the two cell populations and bound recombinant IL6R, detected with an anti-His6 detection antibody. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2+++) cells stained with DeepRed, lower right gate = EBC-1 (c-MET++) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (HCC-1954 + EBC-1 + bound recombinant IL6R-His Tag) were marked in blue. Cells were incubated in the presence of 10 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: DB-VHHs elicit potent and specific NK cell-mediated target cell killing. Tumor cells were incubated with primary effector cells (NK cells) at a 1:5 ratio in the presence of antibody constructs in different concentrations. Error bars represent standard deviation of two biological replicates. Wildtype CS06 and trastuzumab with active Fc effector functioning were used as an ADCC reference (green). (a) and (b): c-MET-positive EBC-1 target cells, (c) and (d) HER2-overexpressing SK-BR-3 target cells. (a) and (c): NK cell cytotoxicity triggered by parental antibodies. (b) and (d): DB-VHHs serve as NK cell engager and mediate tumor cell killing.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Incubation, Construct, Standard Deviation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet:

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Variant Assay, Hydrophobic Interaction Chromatography, Mutagenesis, Molecular Weight, High Performance Liquid Chromatography

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: FASN phosphorylation by HRG in SKBR3 cells . (A) SKBR3 cells were incubated in serum-free medium overnight and then treated with 50 ng/mL heregulin (HRG) for the indicated times. FASN was immunoprecipitated and immunoblotted by the use of a monoclonal phosphotyrosine (PT66) and anti-FASN antibodies. (B) Immunoprecipitated FASN substrate was incubated with HER2 kinase and radiolabeled with (γ-P 32 )-ATP for 30 minutes at 30ºC, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualized by autoradiography. Recombinant HER2 kinase was used as a positive control. Western blotting for FASN and HER2 was performed to confirm that equal amounts of FASN or HER2 were used in each kinase assay.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography, Recombinant, Positive Control, Western Blot, Kinase Assay

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: Colocalization of FASN and HER2 in breast cancer cells . Treatment of SKBR3 cells with HRG caused HER2 (red) and FASN (green) to colocalize at the cell membrane (yellow on merged images).

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: Association of FASN and HER2 in breast cancer cells . SKBR3 (A) and BT474 (B) cells were treated for one hour with: 1) no agent (control); 2) 50 ng/mL HRG; 3) 1 μM lapatinib or 4) 50 ng/mL HRG plus 1 μM lapatinib. The immunoprecipitated FASN complexes were subjected to Western blotting for FASN, HER2 and PT66. (C) Whole-cell lysates from SKBR3 (left panel) and BT474 cells (right panel) were subjected to Western blotting for pHER2 (Y1248), pHER3 (Y1289), pAkt (S473), pErk1/2, HER2, HER3, Akt, Erk1/2, FASN and β-actin.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Immunoprecipitation, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: FASN phosphorylation in breast cancer cells treated with C75 . SKBR3 (A) and BT474 (B) cells were pretreated with 10 μM C75 for five hours and then treated for one additional hour with 1) no agent (control); 2) 50 ng/mL HRG; 3) 10 μM C75; or 4) 50 ng/mL HRG plus 10 μM C75. The immunoprecipitated FASN complexes were assessed for FASN, HER2 and PT66 by Western blotting. (C) Whole-cell lysates from SKBR3 and BT474 cells were subjected to Western blotting for pHER2 (Y1248), pHER3 (Y1289), pAkt (S473), pErk1/2, HER2, HER3, Akt, Erk1/2, FASN and β-actin.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Immunoprecipitation, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: HRG-induced invasion in HER2-positive BT474 breast cancer cells treated with lapatinib or siRNA targeting FASN . (A) BT474 cells were treated with 50 ng/mL HRG or 50 ng/mL HRG plus 0.2 μM lapatinib for 36 hours. The invaded BT474 cells were stained with crystal violet and counted under a microscope. Representative microscopic images are shown. The changes in cell invasion compared with HRG-induced cell invasion are shown in a bar graph. All experiments were done three times. A simple t -test was used to assess differences in the number of invaded cells between any two experimental conditions. *, statistically significant compared with cells treated with HRG; P < 0.05 was considered statistically significant. (B) BT474 cells transfected with scrambled siRNA or siRNA targeting FASN (si-FASN) were treated with 50 ng/mL HRG for 36 hours. The efficiency of si-FASN knockdown was confirmed by Western blotting for FASN and β-actin (lower left). The changes in cell invasion compared with the invaded scrambled siRNA-transfected untreated cells are shown as a percentage in the bar graph (lower right). All experiments were done three times. A simple t -test was used to assess differences in the number of invaded cells between any two experimental conditions. *, statistically significant compared with scrambled si-RNA-transfected cells treated with HRG; P < 0.05 was considered statistically significant.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Staining, Microscopy, Transfection, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: Cell invasion and activity of MMP9 in HER2-positive BT474 breast cancer cells with suppressed FASN . (A) BT474 cells were treated with 50 ng/mL HRG or 50 ng/mL HRG plus 10 μM C75 for 36 hours. Cells shown in the microscopic image represent invasive cells. The changes in cell invasion compared with HRG-induced cell invasion are shown as a percentage in the bar graph. All experiments were done three times. A simple t -test was used to assess differences in the number of invaded cells between any two experimental conditions. *, statistically significant compared with cells treated with HRG; P < 0.05 was considered statistically significant. (B) BT474 cells were treated as described in Figures 7A, B and 8A, and the medium was harvested for zymogram assay as described in Materials and methods. MMP-9 activity is shown as the digested clear band in Coomassie blue-stained gel containing 0.2% gelatin.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Activity Assay, Staining

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: Regulation of cell invasion through functional interaction between FASN and HER2 in HER2-overexpressing breast cancer cells . When HRG binds to HER3, HER2 interacts with HER3 and induces tyrosine phosphorylation of HER3. The activated HER2 not only stimulates the PI3K/Akt and MAPK signaling pathways but also induces tyrosine phosphorylation of FASN and upregulation of FASN activity, leading to increased cell invasion. Suppression of FASN indirectly (for example, with lapatinib) or directly (for example, with C75 or si-FASN) inhibits invasion of HER2-overexpressing breast cancer cells.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Functional Assay, Activity Assay

Journal: Cancer Research

Article Title: Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

doi: 10.1158/0008-5472.can-07-6051

Figure Lengend Snippet: Figure 1. Expression of the scFv and scFv-CD40 CRs in murine DC. A, schematic representation of the scFv and scFv-CD40 CRs. Each construct consisted of the ErbB2-specific scFv, a c-myc tag, the hinge region of murine CD8a, and the transmembrane (TM) and/or cytoplasmic regions of murine CD40 signaling chain. B, after 5 d of DC differentiation, surface expression of MHC and costimulatory molecules in immature DC was assessed by immunofluorescent staining with antibodies specific for CD40, CD86, MHC class I Kd, and MHC class II I-Ad along with anti-CD11c antibody. DC treated with 1 Ag/mL LPS for 24 h were included as a positive control. C, analysis of expression of either GFP or CR expression in DC after adenoviral infection at 100 MOI. Top, GFP expression in uninfected DC or GFP-DC; bottom, CR expression in GFP-DC, scFv-DC, or scFv-CD40-DC. D, lysates of GFP-DC (lanes 1 and 4), scFv-DC (lanes 2 and 5), and scFv-CD40-DC (lanes 3 and 6) were separated by SDS-PAGE under reducing (R) or nonreducing (NR) conditions. Immunoblot analysis was performed with an anti–c-myc or anti–a-tubulin mAb followed by chemiluminescent detection. The position of molecular weigh standards (kd) is indicated.

Article Snippet: For nuclear Rel-B blotting, infected DC were stimulated in 24-well plates (106 cells per well) by various concentrations of immobilized bovine serum albumin (BSA), recombinant ErbB2 protein (R&D Systems), or 1 Ag/mL LPS (SigmaAldrich) for 24 h. Nuclear lysates were prepared using the Transfactor Extraction kit (Clontech Laboratories) and probed with mouse Rel-B– and Sp1-specific mAb (Santa Cruz Biotechnology).

Techniques: Expressing, Construct, Staining, Positive Control, Infection, SDS Page, Western Blot

Journal: Cancer Research

Article Title: Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

doi: 10.1158/0008-5472.can-07-6051

Figure Lengend Snippet: Figure 2. The scFv-CD40 CR induced NF-nB–dependent DC activation and effector function after ErbB2 stimulation. A, NF-nB mRNA levels from GFP-DC, scFv-DC, or scFv-CD40-DC stimulated by 1 Ag/mL BSA or ErbB2 protein for 24 h were detected by real-time PCR. Results are normalized to glyceraldehyde-3-phosphate dehydrogenase and expressed as fold increase relative to uninfected DC. B, nuclear lysates from infected DC stimulated by titrated levels of ErbB2 protein (1, 100, and 1000 ng/mL) were prepared for RelB/Sp1-specific Western blots. Infected DC stimulated by 1 Ag/mL LPS was included as a positive control. C, l, infected DC were cultured with the irradiated D2F2 or D2F2/E2 tumor cells at a DC to tumor ratio of 5:1 at 37jC. After 24 h, DC were analyzed for the expression of costimulatory and MHC molecules by flow cytometry. Results are normalized to uninfected-DC MFI. II, infected DC were treated as described in I before supernatants were collected and cytokine levels were determined by ELISA. D, l, infected DC were incubated with the D2F2 or D2F2/E2 tumor cells at a DC to tumor ratio of 5:1 at 37jC for 24 h and cytotoxicity was evaluated by MTT assay. Wells with medium alone were used as background control (BG) and wells for total viability/spontaneous death of untreated cells (TS) contained only medium and tumor cells. Experimental (Ex) wells contained tumor cells and infected DC. Percentage of cytotoxicity was calculated using the following formula: % cytotoxicity = [(TS BG) (Ex BG)]/(TSBG) 100%. II, D2F2 or D2F2/E2 tumor cells were stained with 4 Ag/mL Hoechst 33342 for 30 min. Cells were washed and maintained in culture for additional 3 h before washing again and adding infected DC at a DC to tumor ratio of 5:1 at 37jC or 4jC. After 24 h, the cells were harvested and stained with anti-mouse CD11c mAb and PI before flow cytometry analysis. Viable DC (CD11c+PI) were gated for analysis, and MFI of Hoechst 33342 staining is reported. All results are representative of three independent experiments. *, P < 0.05; **, P < 0.01; NF-nB mRNA, costimulatory, and MHC molecules expression, cytokine levels, or tumoricidal activity significantly increased in scFv-CD40-DC after ErbB2 ligation compared with the controls (GFP-DC or scFv-DC).

Article Snippet: For nuclear Rel-B blotting, infected DC were stimulated in 24-well plates (106 cells per well) by various concentrations of immobilized bovine serum albumin (BSA), recombinant ErbB2 protein (R&D Systems), or 1 Ag/mL LPS (SigmaAldrich) for 24 h. Nuclear lysates were prepared using the Transfactor Extraction kit (Clontech Laboratories) and probed with mouse Rel-B– and Sp1-specific mAb (Santa Cruz Biotechnology).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Infection, Western Blot, Positive Control, Cell Culture, Irradiation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, MTT Assay, Control, Staining, Activity Assay, Ligation

Journal: Cancer Research

Article Title: Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

doi: 10.1158/0008-5472.can-07-6051

Figure Lengend Snippet: Figure 4. In vivo antitumor effects of the CR-modified DC. A, D2F2/E2 tumors. BALB/c mice were injected s.c. in the left flank on day 0 with 2 105 ErbB2-positive D2F2/E2 mammary tumor cells. On day 8 and 11, 5 105 scFv-DC or scFv-CD40-DC were injected i.v. B, D2F2 tumors. BALB/c mice were injected s.c. on day 0 with 2 105 ErbB2-negative D2F2 mammary tumor cells. On day 8 and 11, 5 105 scFv-DC, scFv-CD40-DC was injected i.v. C, EL4/E2 tumors. C57BL/6 mice were injected s.c. on day 0 with 2 105 ErbB2-positive EL4/E2 thymoma cells. On day 8 and 11, 5 105 scFv-DC or scFv-CD40-DC were injected i.v. D, EL4 tumors. C57BL/6 mice were injected s.c. on day 0 with 2 105 ErbB2-negative EL4 thymoma cells. On day 8 and 11, 5 105 scFv-DC or scFv-CD40-DC was injected i.v. In all tumor models, mice injected with PBS were used as an additional control. Tumor size was measured twice weekly and survival was monitored for 12 wk. *, P < 0.05; **, P < 0.01, tumor growth was significantly inhibited in the scFv-CD40-DC–treated group compared with the scFv-DC or PBS treatment group.

Article Snippet: For nuclear Rel-B blotting, infected DC were stimulated in 24-well plates (106 cells per well) by various concentrations of immobilized bovine serum albumin (BSA), recombinant ErbB2 protein (R&D Systems), or 1 Ag/mL LPS (SigmaAldrich) for 24 h. Nuclear lysates were prepared using the Transfactor Extraction kit (Clontech Laboratories) and probed with mouse Rel-B– and Sp1-specific mAb (Santa Cruz Biotechnology).

Techniques: In Vivo, Modification, Injection, Control

Journal: Cancer Research

Article Title: Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

doi: 10.1158/0008-5472.can-07-6051

Figure Lengend Snippet: Figure 5. Induction of tumor-specific CTLs after i.v. administration of scFv- CD40-DC. Mice were implanted s.c. in the left flank on day 0 with 2 105 ErbB2- positive D2F2/E2 mammary tumor cells. On day 8 and 11, 5 105 scFv-DC, scFv-CD40-DC, or PBS alone was injected i.v. Ten days after treatment, the splenocytes were isolated and restimulated in vitro for 5 d with the irradiated D2F2/E2 cells and assayed for CTL activity against 51Cr-labeled D2F2/E2 (A), D2F2 (B), and NIH3T3 targets (C) or D2F2/E2 targets in the presence of neutralizing or isotype-matched control mAb (10 Ag/mL) as indicated (D). Points, the mean of triplicate cultures, representative of three independent experiments; bars, SE.

Article Snippet: For nuclear Rel-B blotting, infected DC were stimulated in 24-well plates (106 cells per well) by various concentrations of immobilized bovine serum albumin (BSA), recombinant ErbB2 protein (R&D Systems), or 1 Ag/mL LPS (SigmaAldrich) for 24 h. Nuclear lysates were prepared using the Transfactor Extraction kit (Clontech Laboratories) and probed with mouse Rel-B– and Sp1-specific mAb (Santa Cruz Biotechnology).

Techniques: Injection, Isolation, In Vitro, Irradiation, Activity Assay, Labeling, Control