Journal: EBioMedicine

Article Title: A novel HER2-targeting antibody 5G9 identified by large-scale trastuzumab-based screening exhibits potent synergistic antitumor activity

doi: 10.1016/j.ebiom.2020.102996

Figure Lengend Snippet: Characterization of trastuzumab-optimal-synergistic-epitope (TOSE) -binding monoclonal antibodies. (a) Schematic workflow of trastuzumab-based synergistic functional screening of TOSE-binding antibodies (4H2, 4C9, 4G6, 5F12 and 5G9). 1st screening: ELISA binding and competitive ELISA with trastuzumab or pertuzumab; 2nd screening: binding to HER2-positive cells; 3rd screening: trastuzumab-based cell proliferation inhibition assay. (b) Five monoclonal antibodies (4H2, 4C9, 4G6, 5F12 and 5G9) showed trastuzumab-synergistic bioactivity higher than pertuzumab in the BT-474 cell proliferation inhibition assay. TRA: trastuzumab; PER: pertuzumab. (c) The antibody epitope grouping assay was performed by competitive ELISA. Five monoclonal antibodies (4H2, 4C9, 4G6, 5F12 and 5G9) bound to a unique epitope-TOSE, which was different from both the trastuzumab-binding epitope and pertuzumab-binding epitope. (d) The binding specificity and species cross-reactivity were determined by ELISA. 5G9 bound to HER2 protein, but not to other EGFR family members, including EGFR, HER3 and HER4. (e) The cross-species binding reactivity was determined by ELISA. 5G9 was able to bind cynomolgus HER2 protein but not murine HER2 protein. The BT-474 cell proliferation inhibition assay, antibody epitope grouping assay and the cross-reactivity assay were repeated three times and the results were shown as the mean ± SD ( n = 3). Two-way ANOVA, *** P< 0.001.

Article Snippet: The recombinant proteins huEGFR (Cat# 10001-H02H), huHER2 (Cat# 10004-H02H), huHER3 (Cat# 10201-H02H), huHER4 (Cat# 10363-H02H), rhesus HER2 (Cat# 90020-K02H) and mouse HER2 (Cat# 50714-M02H) were purchased from Sino Biological Inc.

Techniques: Binding Assay, Functional Assay, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Inhibition

Journal: EBioMedicine

Article Title: A novel HER2-targeting antibody 5G9 identified by large-scale trastuzumab-based screening exhibits potent synergistic antitumor activity

doi: 10.1016/j.ebiom.2020.102996

Figure Lengend Snippet: 5G9 and trastuzumab synergistically inhibit HER2-positive cancer cell growth. (a) Inhibition of BT-474 cell viability was examined by Cell Titer Glo after 6 days of antibody treatment. The initial antibody concentration was 100 nM for both the monoclonal antibody and the antibody combination (1:1 ratio). Growth inhibition was calculated as the fraction of viable cells in the antibody treated group compared with untreated control cells. The cell proliferation inhibition assay was repeated three times and the results were shown as the mean ± SD ( n = 3). Two-way ANOVA, ** P< 0.01. TRA: trastuzumab; PER: pertuzumab. (b) Dose-effect plots and combination index (CI.) plots of the two combinations were calculated by using the method of Chou and Talalay with the commercial software Calcusyn. C.I. values for effective doses at which 50%, 75%, and 90% (ED 50 , ED 75 , and ED 90 , respectively) of cells were killed. Drug synergy was defined by C.I. values less than 1, the lower C.I. value, the more synergy. (c) Representative images from the EdU assay of BT-474 cells. The viable cells were stained blue by DAPI (blue) and the proliferation cells were stained red by EdU (red).The EdU assay was repeated three times and the representative images were shown. (d) Human breast cancer cell lines (SK-BR-3, AU-565, MCF-7) and human gastric cell lines (NCI-N87) were treated with 100 nM antibodies for 3 or 6 days and cell viability was determined by Cell Titer Glo. The experiments were repeated three times and the cell viability is shown as the mean ± SD ( n = 3). The relative HER2 expression of cell lines was detected and analyzed by flow cytometry and FlowJo software.

Article Snippet: The recombinant proteins huEGFR (Cat# 10001-H02H), huHER2 (Cat# 10004-H02H), huHER3 (Cat# 10201-H02H), huHER4 (Cat# 10363-H02H), rhesus HER2 (Cat# 90020-K02H) and mouse HER2 (Cat# 50714-M02H) were purchased from Sino Biological Inc.

Techniques: Inhibition, Concentration Assay, Software, EdU Assay, Staining, Expressing, Flow Cytometry

Journal: EBioMedicine

Article Title: A novel HER2-targeting antibody 5G9 identified by large-scale trastuzumab-based screening exhibits potent synergistic antitumor activity

doi: 10.1016/j.ebiom.2020.102996

Figure Lengend Snippet: 5G9 and trastuzumab enhance cell apoptosis, HER2 internalization and disruption. (a) BT-474 cells were treated with 100 nM antibodies for 3 days. The number of viable cells was detected by Trypan Blue, and annexin-V and PI were used to stain early apoptotic cells and dead cells. TRA: trastuzumab; PER: pertuzumab. (b) BT-474 cells were treated with 100 nM antibodies for 3 days, and cell lysates were obtained and immunoblotted with a poly (ADP-ribose) polymerase (PARP) polyclonal antibody. Cleaved PARP is represented by 89 KD-fragments and full-length PARP (116 KD). (c) HER2 internalization induced by 5G9, trastuzumab or pertuzumab in SK-BR-3 cells. (d) The effect of trastuzumab on the internalization of 5G9 or pertuzumab in SK-BR-3 cells. FITClabeled 5G9 or pertuzumab was detected and analyzed by flow cytometry and FlowJo software. (e) The effect of 5G9 or pertuzumab on the internalization of trastuzumab in SK-BR-3 cells. APC labeled trastuzumab was detected and analyzed by flow cytometry and FlowJo software. (f) Effect of HER2 single agents or combination on receptor phosphorylation and downstream signaling cascade. BT-474 cells were treated with 100 nM antibodies for 3 days, and cell lysates were obtained and immunoblotted with antibodies against EGFR, p-EGFR, HER2, p-HER2, HER3, p-HER3, AKT, p-AKT, ERK, p-ERK, and GAPDH. All experiments were repeated three times and the representative images and data were shown. Two-way ANOVA, * P< 0.05, *** P< 0.001.

Article Snippet: The recombinant proteins huEGFR (Cat# 10001-H02H), huHER2 (Cat# 10004-H02H), huHER3 (Cat# 10201-H02H), huHER4 (Cat# 10363-H02H), rhesus HER2 (Cat# 90020-K02H) and mouse HER2 (Cat# 50714-M02H) were purchased from Sino Biological Inc.

Techniques: Staining, Flow Cytometry, Software, Labeling

Journal: JNCI Journal of the National Cancer Institute

Article Title: Quantitative Assessment of Effect of Preanalytic Cold Ischemic Time on Protein Expression in Breast Cancer Tissues

doi: 10.1093/jnci/djs438

Figure Lengend Snippet: Biomarkers used in the study*

Article Snippet: Vendor Biomarkers for companion diagnostic tests Estrogen receptor-alpha (ERα) Mouse 1D5/IgG1kappa M7047 DAKO Rabbit SP1/IgG RM-9101 Thermo Scientific Progesterone receptor (PgR) Mouse PgR636/IgG1kappa M3569 DAKO Rabbit PgRA/B (C89F7) 3153 Cell Signaling Technology HER2, also known as ERBB2 Mouse CB11/IgG1 CM 076 AA Biocare Medical Rabbit polyclonal A0485 DAKO Ki67 Mouse MIB-1/IgG1kappa M7240 DAKO Rabbit SP6/IgG 9106-S0 Lab Vision Markers of cold ischaemia Beta-actin (ACTB) Rabbit 13E5/IgG 13E5/IgG Cell Signaling Technology Beta-tubulin (TUBB) Rabbit pF3/IgG 2128 Cell Signaling Technology Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Rabbit 14C10/IgG 2118 Cell Signaling Technology Histone 4 (HIST4H4) Mouse L64C1 2935 Cell Signaling Technology Histone 3 (HIST3H3) Mouse 96C10/IgG1, kappa 3680 Cell Signaling Technology Lamin A/C (LMNA) Rabbit polyclonal 2032 Cell Signaling Technology Lactate dehydrogenase A (LDHA) Rabbit IgG, C4B5 3582 Cell Signaling Technology Cytokeratin (KRT X) Mouse AE1/AE3/IgG1 M3515 DAKO Rabbit polyclonal ZO622 DAKO Markers of hypoxia Cyclin D1 (CCND1) Rabbit IgG/SP4 RM-9104 Thermo Fisher Fremont Cyclin B1 (CCNB1) Mouse GNS-11/IgG2 554178 BD Biosciences A kinase (PRKA) anchor protein 13 (AKAP13) Mouse IgG2a/ZX-18 sc-81902 Santa Cruz Biotechnology Cell division cycle 42 (CDC42) Mouse IgG3/B-8 sc-8401 Santa Cruz Biotechnology Cleaved caspase3 (CASP3) Rabbit polyclonal 9661 Cell Signaling Technology Hypoxia inducible factor 1-alpha (HIF1A) Rabbit polyclonal NB 100–449 Novus Biological Hypoxia inducible factor 2-alpha (HIF2A) Mouse ep190b/IgG1 ab8365 abcam Markers of phosphorylated proteins Phosphorylated tyrosine (4G10) Mouse IgG2b 05-1050 Millipore Markers of post-translational modification SMT3 suppressor of mif two 3 homolog 1 (SUMO1) Rabbit Y299/IgG ab32058 abcam Acetylated lysine Rabbit polyclonal, purified 9441 Cell Signaling Technology Neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) Rabbit IgG, 19E3 2754 Cell Signaling Technology Open in a separate window * Antibodies were validated and used for quantitative immunofluorescence assays as described in the Patients and Methods section.

Techniques: Diagnostic Assay, Modification, Purification