chimeric her2 fc  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: ErbB2 (HER2)-CAR-NK-92 cells for enhanced immunotherapy of metastatic fusion-driven alveolar rhabdomyosarcoma

doi: 10.3389/fimmu.2023.1228894

Figure Lengend Snippet: Lysis of ErbB2-positive, 2D patient-derived tumor organoid aRMS cells by parental NK-92 or NK-92/5.28.z cells. (A) ErbB2 surface expression on RMS102, RMS127 and RMS335 cells was confirmed by flow cytometry. Antigen density per cell quantified by flow cytometry is shown. (B) Cytotoxicity data of NK-92 or NK-92/5.28.z cells during short-term (3 hour) coculture with 2D patient-derived tumor organoid aRMS cells from europium release assays are given. Data of three independent experiments are shown as mean ± SD and differences were analyzed with a two-tailed Student´s t-test and were considered significant for p< 0.05 (*), p< 0.01 (**), p< 0.001 (****), p<0.0001 or ns.

Article Snippet: CAR expression of NK-92/5.28.z cells was analyzed using a chimeric ErbB2-Fc protein (Sino Biological) after nonspecific Fc receptor blocking (Human TruStain FcX, BioLegend) and subsequent staining with an anti-IgG-Fc secondary antibody conjugated with allophycocyanin (APC) (BioLegend, 410712).

Techniques: Lysis, Derivative Assay, Expressing, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: ErbB2 (HER2)-CAR-NK-92 cells for enhanced immunotherapy of metastatic fusion-driven alveolar rhabdomyosarcoma

doi: 10.3389/fimmu.2023.1228894

Figure Lengend Snippet: Cytotoxic capacity against ErbB2-positive 3D tumor spheroids. (A) ErbB2 surface expression on RH30 cells was confirmed by flow cytometry. (B) Expression of the anti-ErbB2-targeted CAR on NK-92/5.28.z cells was confirmed by flow cytometry. (C) Exemplary coculture images of NK-92 or NK-92/5.28.z cells added to established 3D RH30GFP/luc+ tumor spheroids on day 4 are shown. Imaging was performed on days 4, 5, 6, 8 and 10. (D) The areas with green fluorescent signals were quantified using Fiji. Data of three independent experiments are shown as mean ± SD. Differences were analyzed with a one-way ANOVA using the Bonferroni method and were considered significant for p< 0.05 (*), p< 0.01 (**) or ns.

Article Snippet: CAR expression of NK-92/5.28.z cells was analyzed using a chimeric ErbB2-Fc protein (Sino Biological) after nonspecific Fc receptor blocking (Human TruStain FcX, BioLegend) and subsequent staining with an anti-IgG-Fc secondary antibody conjugated with allophycocyanin (APC) (BioLegend, 410712).

Techniques: Expressing, Flow Cytometry, Imaging

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: A flat bottom 96-well plate (MaxiSorp, Nunc, Thermo Fisher Scientific, Waltham, MA, USA) was coated with the Human HER2 Protein Fc Tag chimeric protein (AcroBIOSYSTEMS, Newark, DE, USA) diluted in 0.1 M NaHCO3, pH 9.6 buffer at 0.1 μg/well and incubated overnight at 4 °C.

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: A flat bottom 96-well plate (MaxiSorp, Nunc, Thermo Fisher Scientific, Waltham, MA, USA) was coated with the Human HER2 Protein Fc Tag chimeric protein (AcroBIOSYSTEMS, Newark, DE, USA) diluted in 0.1 M NaHCO3, pH 9.6 buffer at 0.1 μg/well and incubated overnight at 4 °C.

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: A flat bottom 96-well plate (MaxiSorp, Nunc, Thermo Fisher Scientific, Waltham, MA, USA) was coated with the Human HER2 Protein Fc Tag chimeric protein (AcroBIOSYSTEMS, Newark, DE, USA) diluted in 0.1 M NaHCO3, pH 9.6 buffer at 0.1 μg/well and incubated overnight at 4 °C.

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: A flat bottom 96-well plate (MaxiSorp, Nunc, Thermo Fisher Scientific, Waltham, MA, USA) was coated with the Human HER2 Protein Fc Tag chimeric protein (AcroBIOSYSTEMS, Newark, DE, USA) diluted in 0.1 M NaHCO3, pH 9.6 buffer at 0.1 μg/well and incubated overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Staining