mouse anti-human her2 mab  (Bio X Cell)

Journal: Analytical chemistry

Article Title: Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels

doi: 10.1021/acs.analchem.9b04485

Figure Lengend Snippet: Quantification of EV concentrations with the 300-nm CuS-MG in 1× PBS or Serum. Calibration curves in 1×PBS using a) anti-CD63, b) anti-HER2; and in serum using c) anti-CD63 or d) anti-HER2. CL0 = chemiluminescence without EVs. [ABEI] = 0.5 mM, [H2O2] =1 mM, pH=11.

Article Snippet: The biotinylated mouse anti-human ErbB2/HER2 (Recombinant Monoclonal Human IgG 1 Clone Hu5), mouse anti-human CD63 (Clone mem-259), and biotinylated mouse anti-human CD63 (Clone NVG-2) was obtained from R&D systems, Sino Biological, and BioLegend, respectively.

Techniques:

Journal: Analytical chemistry

Article Title: Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels

doi: 10.1021/acs.analchem.9b04485

Figure Lengend Snippet: Detection of cell released EVs using CuS-MG. The cell culture medium was collected from three cell lines: MCF-10A, MDA-MB-231 and SK-BR-3. a) Chemiluminescence resulted from EV detection in the culture media of three cell lines targeting CD63 and anti-HER2; b) PCA plot using the chemiluminescence data shown in a); c) Linear correlation between CD63 quantification results obtained by ELISA and the chemiluminescent signals from CuS-MG in our assay targeting CD63. [ABEI] = 0.5 mM, [H2O2] = 1 mM, pH=11.

Article Snippet: The biotinylated mouse anti-human ErbB2/HER2 (Recombinant Monoclonal Human IgG 1 Clone Hu5), mouse anti-human CD63 (Clone mem-259), and biotinylated mouse anti-human CD63 (Clone NVG-2) was obtained from R&D systems, Sino Biological, and BioLegend, respectively.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Expression, cytotoxic activity, and cytokine secretion of T cells expressing altered version of FcγRI. A, Illustration of the modified FcγRI structure with fused signaling domains: A2G (left), FcγRI composed of α-chain and homodimer of FcRγ chain; AG2G (middle), FcγRI α-chain with fused domain of FcRγ chain and homodimer of FcRγ; AGO2GO (right), FcγRI α-chain with fused FcRγ and OX40 domains and homodimer of FcRγ with fused OX40 domain. B, Flow cytometry analysis of A2G, AG2G, and AGO2GO expression following retroviral transduction of healthy donor T cells. C, Mean numbers of HT29 target cells expressing HER2 calculated by incuCyte software through 48-hour incubation with the three FcγRI-based receptor expressing T cells, with 1:1 E:T ratios with or without 6 μg/mL trastuzumab (data shows one of two experiments repeats, n = 4). Each sample is normalized to its cell number at time zero. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. D, Representative incuCyte images showing HER2-expressing HT29 cells, imaged and counted in live imaging system after 48 hours of incubation with different construct-expressing T cells with 1:1 E:T ratio. Scale bar is 100 μm. E, Human Luminex Discovery Assay measurement of cytokine levels in supernatants from 48 hours of culture of T cells 4:1 E:T ratio with HT29 cells expressing HER2 ( n = 4). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Activity Assay, Modification, Flow Cytometry, Retroviral, Transduction, Software, Incubation, Imaging, Construct, Luminex

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Characterization of AG2G-expressing T cells, activation, and cytotoxicity. A, ELISA measurement of cytokine levels of AG2G-expressing T cells after 48-hour incubation with HT29 cells expressing HER2. B, IFNγ and TNFα levels measured by ELISA in supernatants from 48-hour incubation of AG2G-expressing T cells and HT29 cells expressing HER2 in different E:T ratios. C, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with 0, 2.5, 5, 10 mg/mL IVIG, with or without 30 μg/mL trastuzumab ( n = 4). D, ELISA measurement of IFNγ levels of AG2G-expressing cells cocultured with HT29 cells expressing HER2 described in B . E, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with different concentrations of trastuzumab over 96 hours ( n = 2). F, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with different E:T ratios of AG2G-expressing T cells that were isolated for their CD4 (left) or CD8 (middle) population or with no isolation (right). G, ELISA measurement of cytokine levels from supernatants of 48-hour culture of 4:1 E:T ratios of isolated CD4, isolated CD8, or AG2G-expressing T cells with HT29 cells expressing HER2 and 30 μg/mL trastuzumab. Graphs show mean ±SD. Statistical significance was calculated using Student t test for a, two-way ANOVA with Sidak correction for multiple comparisons for B and C , two-way ANOVA with Tukey's correction for multiple comparisons for E and F . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Software, Isolation

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: AG2G-expressing T cells differentiate between cells expressing high and low antigen levels, are more specific, and less exhausted, compared with classic CAR T cells. A, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with transduced T cells in combination with antibodies (60 μg/mL each, n = 4). B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-transduced T cells incubated with HER2-expressing HT29 target cells, and with tumor-binding trastuzumab, or irrelevant antibodies cetuximab and rituximab (60 μg/mL). C, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. D, Normalized confluence of kidney epithelial cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell confluence at time zero. E, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D ( n = 4). F, ELISA measurement of IFNγ, granzyme B and TNFα levels in 24-hour supernatants of 10 5 AG2G-expressing T cells or CAR T cells incubated on immobilized trastuzumab or rHER2, in rising concentrations (mmol/mL) respectively. G, Confocal microscope imaging of internalization kinetics of PE-labeled IgG by AG2G-expressing cells (top), or PE-labeled HER2 by CAR T cells (bottom). H, PD-1, LAG-3, and TIM3 flow cytometry analysis of AG2G-expressing cells or CAR T cells after 48-hour incubation with or without 12 mmol/mL immobilized trastuzumab or HER2, respectively. Graphs show mean ± SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for A , C , D , and H . Two-way ANOVA with Sidak correction for multiple comparisons for E and F . One-way ANOVA with Dunnett correction for multiple comparisons for B . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Software, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay, Microscopy, Imaging, Labeling, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Human T cells expressing AG2G exert specific tumor cytotoxicity while sparing normal cells. A, Flow cytometry expression analysis of HER2 on different tumor cell lines and primary human normal cells. B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-expressing T cells with different tumor cells or primary normal cells in 4:1 E:T ratio and 60 μg/mL trastuzumab (results shown are from 3 different donors combined, n = 12). C, Quantification of HER2 receptor numbers using DAKO QIFIKIT (Agilent) beads by flow cytometer. Individual populations of the calibration are gated, a linear regression is calculated from the MFI and the ABC values of the five calibration bead populations using MS Excel. Using the linear regression, the ABCs of the beads coated with αHER2 is calculated from their MFI values. SDs are also retrieved from gated populations and transformed using linear regression. D, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. E, Normalized confluence of kidney epithelial (epithel) cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab. Each sample is normalized to its cell confluence at time zero. Graphs are identical and described in . and but with the comparison to ACTR707 instead of CAR T. F, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D and E ( n = 4). G, ELISA measurement of IFNγ levels in 24 hours supernatants of AG2G-expressing T cells or ACTR707 incubated with medium containing different concentration of IVIG ( n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. ****, P < 0.0001. Error bars represent standard error. Endothel, endothelial cells.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Transformation Assay, Software, Incubation, Comparison, Concentration Assay

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Systemic administration of AG2G-expressing T cells in combination with trastuzumab eradicates HER2-expressing tumor cells in vivo . A, NCI-N87 tumor volume measured by caliper over 42 days following treatment initiation. NSG mice were injected with 2.5×10 6 NCI-N87 tumor cells, after tumor reached 70 mm 3 mice were randomized and treated once a week for 3 times (treatments are marked with arrows) with either intraperitoneal injection of saline or 250-μg trastuzumab and intravenous injection of saline or 5×10 6 AG2G-expressing T cells ( n = 6). B, Endpoint analysis of tumor volume measured by caliper, 42 days following treatment initiation. C, Endpoint analysis of tumor weight measured using scales, 42 days following treatment initiation. D, Histology images taken by x4 objective light microscopy, showing whole sections of tumors stained with H&E ( n = 6; N.D., not detected - 2 mice from AG2G+trastuzumab group). Scale bar is 5 mm. E, Representative image taken by x4 and x40 objectives using light microscopy, of one tumor from AG2G+trastuzumab group stained with anti-human CD3 antibody by IHC. F, Tumor volume measured by caliper in NSG mice treated with a single injection of T cells (10 7 ) 13 days after tumor inoculation (2.5×10 6 NCI-N87). Antibodies were injected once a week for a total of 3 times (250 μg/mouse), n = 5. G, NSG mice were injected with 1.7×10 6 NCI-N87 tumor cells, after tumor reached 100 to 200 mm 3 mice were randomized and treated once with intravenous injection of 5×10 6 AG2G-expressing T cells and with intraperitoneal injection of saline or 250-μg trastuzumab. On days 1, 7, 14, 28 following treatment, 3 mice from each group were sacrificed and RNA was extracted from blood and tumors for real-time PCR analysis of AG2G-expressing cells ( n = 12). Graphs show mean ± SD. Graphs A – C show one of two independent experiments performed. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for A and B , one-way ANOVA with Holm-Sidak correction for multiple comparisons for C . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, In Vivo, Injection, Saline, Light Microscopy, Staining, Real-time Polymerase Chain Reaction

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Retroviral transduction of γδ-T cells with AG2G endows them with antitumor ADCC. A, Flow cytometry analysis of sham or AG2G-transduced αβ-T cells (two left panels, activated with IL2 and anti-CD3); sham or AG2G-transduced γδ-T cells (two right panels, activated with IL2 and zoledronic acid). B, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with AG2G-expressing γδ-T cells or AG2G-expressing αβ-T cells, with or without trastuzumab ( n = 3). C, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with sham untransduced or AG2G-expressing γδ-T cells with or without trastuzumab, in E:T ratios of 1:1, 2:1 or 4:1 ( n = 4). D, ELISA measurement of IFNγ levels in supernatants of 48-hour sham or AG2G-expressing γδ-T cells cocultured with HT29-HER2 expressing cells, with or without trastuzumab (E:T ratio 4:1, n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for B and C . Two-way ANOVA with Sidak correction for multiple comparisons for D . *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Retroviral, Transduction, Flow Cytometry, Expressing, Software, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

doi: 10.1371/journal.pone.0049829

Figure Lengend Snippet: A. Immunostaining showing regional diversity of HER2 expression in high-grade papillary serous ovarian adenocarcinoma. HER2 expression levels were graded in a 0–3 scale (score 0 = undetectable, score 3 = strong staining). Original magnification was 200×. B. Heatmap illustration of HER2 expression level in 50 primary and metastatic ovarian carcinoma cases, as scored by IHC staining. C. Frequency distribution of sites expressing HER2 at levels ranging from score 0 to 3. The mean number of evaluated sites for cases with undetectable or detectable HER2 expression was similar (4.2 vs. 3.7 respectively; P = 0.19). D. Frequency distribution of either primary or metastatic sites expressing HER2 at levels ranging from score 0 to 3. A greater frequency of primary tumor sites expressed HER2 (36%; 30/84) compared to metastatic sites (24%; 27/111) and had a higher mean HER2 expression score (0.37 vs. 0.21, P = 0.04). No statistically significant difference was observed comparing the expression levels among the primary and metastatic sites that expressed HER2 at any level (1.03 vs. 0.86, P = 0.26). P values were calculated using unpaired student’s t-test analysis.

Article Snippet: Protein was transferred from gel to Immobilon-P transfer membrane for 30 minutes, blocked overnight with 5% milk/PBST and blotted using 1 ug/ml of mouse anti-human HER2 mAb (clone 3B5, BD biosciences).

Techniques: Immunostaining, Expressing, Staining, Immunohistochemistry

Journal: PLoS ONE

Article Title: Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

doi: 10.1371/journal.pone.0049829

Figure Lengend Snippet: A. ERBB2 mRNA levels in ovarian cancer cell lines by q-PCR. ERBB2 mRNA levels of ovarian cancer cell lines are relative to that of ErbB2-negative CEM cells. All ovarian cancer cell lines express ERBB2 mRNA. B-actin was used as an endogenous gene control. Results depict the mean ± SD of triplicate wells. Mean relative ERBB2 mRNA expression amongst established and short-term OvCa cell lines was not statistically different ( P = 0.45). P value was calculated using unpaired student’s t-test analysis. B. Detection of surface HER2 protein expression (filled histograms) by human ovarian cancer cell lines by flow cytometry; isotype antibody control (open histograms). C. Western blot analysis of HER2 protein expression in representative cell lines expressing differential amounts of HER2. HER2 protein is expressed at variable levels in all the ovarian cell lines tested. B-actin was used as control.

Article Snippet: Protein was transferred from gel to Immobilon-P transfer membrane for 30 minutes, blocked overnight with 5% milk/PBST and blotted using 1 ug/ml of mouse anti-human HER2 mAb (clone 3B5, BD biosciences).

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: PLoS ONE

Article Title: Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

doi: 10.1371/journal.pone.0049829

Figure Lengend Snippet: HER2 surface protein expression in established and primary ovarian cell lines.

Article Snippet: Protein was transferred from gel to Immobilon-P transfer membrane for 30 minutes, blocked overnight with 5% milk/PBST and blotted using 1 ug/ml of mouse anti-human HER2 mAb (clone 3B5, BD biosciences).

Techniques: Expressing, Negative Control

Journal: PLoS ONE

Article Title: Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

doi: 10.1371/journal.pone.0049829

Figure Lengend Snippet: A . ERBB2 mRNA quantitation in CD45-depleted primary ascites cancer cells. SKOV-3 and CEM were used as positive and negative HER2-expressing cell line controls respectively. Bars depict the mean ± SD values of triplicate wells. B . ERBB2 mRNA quantitation in CD45-depleted primary solid tumor cells. No significant difference in mean ERBB2 mRNA level was observed between ascites and solid tumors ( P = 0.46). C. Surface HER2 expression (solid histograms) by representative Ber-Ep4 + CD45 − gated ascites and solid tumor-derived cancer cells monitored by flow cytometry; isotype antibody control (open histograms). No significant difference in HER2 protein level was observed between ascites or solid tumors derived cells ( P = 0.95). D. Western blot analysis of HER2 protein expression in bulk solid tumor lysates. B-actin was used as endogenous gene control. OVCAR-3 and CEM were used as positive and negative controls for HER2 expression. P values were calculated using unpaired student’s t-test analysis.

Article Snippet: Protein was transferred from gel to Immobilon-P transfer membrane for 30 minutes, blocked overnight with 5% milk/PBST and blotted using 1 ug/ml of mouse anti-human HER2 mAb (clone 3B5, BD biosciences).

Techniques: Quantitation Assay, Expressing, Derivative Assay, Flow Cytometry, Western Blot

Journal: PLoS ONE

Article Title: Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

doi: 10.1371/journal.pone.0049829

Figure Lengend Snippet: HER2 surface protein expression in fresh primary ovarian ascites, solid tumor and normal OSE.

Article Snippet: Protein was transferred from gel to Immobilon-P transfer membrane for 30 minutes, blocked overnight with 5% milk/PBST and blotted using 1 ug/ml of mouse anti-human HER2 mAb (clone 3B5, BD biosciences).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

doi: 10.1371/journal.pone.0049829

Figure Lengend Snippet: A. ERBB2 mRNA quantitation in normal OSE cells (398, IOSE-4, IOSE-6 and 1744) by q-PCR. SKOV-3 and CEM were used as positive and negative HER2 expressing cell lines respectively. Bars show the mean ± SD value of triplicate wells. B. Surface HER2 protein expression (solid histograms) by normal ovarian epithelial cells by flow cytometry; isotype antibody control (open histograms). C–D. Comparison of the ERBB2 mRNA and protein levels between ovarian cancer ascites, solid tumor and normal OSE cells. ( C ) Vertical scatter plots of the ERBB2 mRNA in ascites, solid tumor and normal OSE determined by q-PCR. The mean of each group is indicated by the horizontal line. P = 0.0498 when comparing ERBB2 mRNA in ascites vs normal OSE cells; P = 0.0210 when comparing ERBB2 mRNA in solid tumors vs normal OSE cells. ( D ) Vertical scatter plots of the protein levels in ascites, solid tumor and normal OSE determined by flow cytometry. The mean of each group is indicated by the horizontal line P = 0.0616 when comparing HER2 protein in ascites vs normal OSE cells; P = 0.1749 when comparing HER2 protein in solid tumors vs normal OSE cells. P values were calculated using unpaired student’s t-test analysis.

Article Snippet: Protein was transferred from gel to Immobilon-P transfer membrane for 30 minutes, blocked overnight with 5% milk/PBST and blotted using 1 ug/ml of mouse anti-human HER2 mAb (clone 3B5, BD biosciences).

Techniques: Quantitation Assay, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

doi: 10.1371/journal.pone.0049829

Figure Lengend Snippet: A . Schematic representation of Anti-HER2 Chimeric Antigen Receptor (CAR) construct containing the CD3ζ cytosolic domain alone (C6.5-z). C6.5, anti-HER2 scFv; VL, variable light chain; L, Linker; VH, variable heavy chain; TM, transmembrane region. C6.5 scFv CAR expression (gray histograms) was detected on human CD4 + and CD8 + -gated T cells using recombinant human HER2-Fc chimeric protein 10 days after transduction, compared to untransduced T cells (open histograms). Percentage of CAR transduction is indicated. B. Anti-HER2 CAR transduced T cells produce IFN-γ specifically after stimulation with human ovarian cancer cell lines. CAR transduced or untransduced T cells were cultured alone (none) or stimulated overnight with human HER2 + established and short-term ovarian cancer cell lines or HER2 − control lines CEM and MDA468. IFN-γ was quantified from cell-free supernatants by ELISA. C. Anti-HER2 CAR T cells secrete IFN-γ after stimulation by HER2 + primary ascites (left) or solid ovarian (middle) tumor cells. Minimal amounts of IFN-γ were detected after stimulation with the normal ovarian surface epithelial cells 398, IOSE-4, IOSE-6 or 1744 (right). Cytokine concentrations (pg/ml) are reported as the mean ± SEM of triplicate wells.

Article Snippet: Protein was transferred from gel to Immobilon-P transfer membrane for 30 minutes, blocked overnight with 5% milk/PBST and blotted using 1 ug/ml of mouse anti-human HER2 mAb (clone 3B5, BD biosciences).

Techniques: Construct, Expressing, Recombinant, Transduction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

doi: 10.1371/journal.pone.0049829

Figure Lengend Snippet: C6.5 CAR T cells degranulate and express T cell activation markers in response to HER2-specific stimulation. C6.5 CAR T cells were cultured without target cells (none) or with the indicated HER2-negative or -positive established and primary tumor cell targets or normal OSE cells for 5 h while being stained by an anti-CD107a, b antibody conjugated with FITC. After the incubation period, T cells were stained for CD8 and CD69 and analyzed by flow cytometry.

Article Snippet: Protein was transferred from gel to Immobilon-P transfer membrane for 30 minutes, blocked overnight with 5% milk/PBST and blotted using 1 ug/ml of mouse anti-human HER2 mAb (clone 3B5, BD biosciences).

Techniques: Activation Assay, Cell Culture, Staining, Incubation, Flow Cytometry