anti his gpd mouse antibody  (Bio-Rad)

Journal: Cancer cell

Article Title: Eradication of triple negative breast cancer cells by targeting glycosylated PD-L1

doi: 10.1016/j.ccell.2018.01.009

Figure Lengend Snippet: (A) PD-1/PD-L1 blockade by glycosylated PD-L1 antibodies. Kinetic graph showing quantitative binding of PD-1/Fc protein on BT549 cells expressing PD-L1 at hourly time points after treatment with glycosylated PD-L1 antibodies. (B) Blockade of PD-L1 and PD-1 interaction by the glycosylated PD-L1 antibodies STM004 and STM108. (C) Schematic diagram of various PD-L1 NQ mutants used in this study. The numbers indicate amino acid positions of the PD-L1 protein. (D) Western blot analysis of wild-type and mutant PD-L1 using STM004 or STM108 antibody. (E) Epitope mapping of glycosylated PD-L1–binding antibodies by High-Mass MALDI mass spectrometry (CovalX service). (F) Interaction of human PD-1 (hPD-1) or mouse PD-1 (mPD-1) protein with human PD-L1 (hPD-L1) on BT549 cells or mouse PD-L1 (mPD-L1) or hPD-L1 on 4T1 cells, with or without STM108 antibody. (G) Tumor growth of 4T1 cells expressing human PD-L1 (4T1-hPD-L1) in BALB/c mice treated with STM004 or STM108 antibody. Tumors were measured at the indicated time points and dissected at the endpoint. n = 7 mice per group. (H) Intracellular cytokine stain of IFNγ in CD8+ CD3+ T cell populations. n = 7 mice per group. (I) Immunofluorescence staining of the protein expression pattern of PD-L1, CD8, and granzyme B (GB) in a 4T1-hPD-L1 tumor mass. Scale bar, 100 μm (20 μm in magnified sections). (J) Quantitative binding affinity of gPD-L1 antibody (STM108) to glycan 1 and 2. Glycan array 100 was probed with biotin-labeled gPD-L1 antibody. gPD-L1 antibody bound to two glycans (1 and 2), and the bindings were compromised by a mixture of B3GNT3 substrate or product, mixture of DiLacNAc and GlcNAcβ1,3-Gal. (K) Western blot analysis of glycosylation of PD-L1 protein in BT549 cells by STM108 (gPD-L1). BT549 control (CTRL) or B3GNT3−/− cells were treated with 25 ng/ml EGF or gefitinib overnight. *p < 0.05, statistically significant by Student’s t-test. Error bars, mean ± S.D. of three independent experiments. See also Figure S5, Table S3, and Table S4

Article Snippet: According to histologic scoring, the intensity of staining was ranked into one of four groups: high (score 3), medium (score 2), low (score 1), and negative (score 0). . Epitope mapping by mass spectrometry Epitope mapping for the mouse monoclonal anti-gPD-L1 antibodies STM004 and STM108 was performed by CovalX AG (Switzerland).

Techniques: Binding Assay, Expressing, Western Blot, Mutagenesis, Mass Spectrometry, Staining, Immunofluorescence, Glycoproteomics, Labeling, Control

Journal: Cancer cell

Article Title: Eradication of triple negative breast cancer cells by targeting glycosylated PD-L1

doi: 10.1016/j.ccell.2018.01.009

Figure Lengend Snippet: (A) Internalization of glycosylated PD-L1 antibodies. Internalized antibodies (Ab) in BT549 cells expressing PD-L1 are shown at each time point. Representative phase and red fluorescent merged images at 12 hours are shown. Scale bar, 100 μm. (B) Trafficking of an individual STM108 (top) or STM004 (bottom) antibody in BT549 cells was visualized by the live cell three-dimensional single-molecule tracking system (TSUNAMI). A representative trajectory of antibody moving into the cytoplasm from the cell membrane. (C) The path length of trajectories acquired from IgG, STM004, and STM108 from (B). n.s., not significant. (D) Internalization of STM108 and its co-localization in lysosome. The antibodies were labeled with pHrodoTM Red and then add to PD-L1 WT expressing BT549 cells with LysoTracker® Green. Arrow indicates localization of STM108 at lysosome after internalization. (E) Internalization of STM108 in BT549-PD-L1 cells treated with clathrin-mediated endocytosis (CME) or caveolae-dependent endocytosis (CDE) inhibitors. (F) Western blot analysis of wild-type (WT) PD-L1 in STM108 and/or CME or CDE inhibitor-treated BT549-PD-L1 cells. (G) Western blot analysis of PD-L1 expression. BT549 cells control (CTRL) or B3GNT3−/− were treated with STM108 for 2 days. *p < 0.05, statistically significant by Student’s t-test. Error bars, mean ± S.D. of three independent experiments. See also Figure S6, Movie S1 and Movie S2

Article Snippet: According to histologic scoring, the intensity of staining was ranked into one of four groups: high (score 3), medium (score 2), low (score 1), and negative (score 0). . Epitope mapping by mass spectrometry Epitope mapping for the mouse monoclonal anti-gPD-L1 antibodies STM004 and STM108 was performed by CovalX AG (Switzerland).

Techniques: Expressing, Membrane, Labeling, Western Blot, Control