Journal: EBioMedicine

Article Title: Rapid detection of bacterial infection using a novel single-tube, four-colour flow cytometric method: Comparison with PCT and CRP

doi: 10.1016/j.ebiom.2021.103724

Figure Lengend Snippet: A schematic diagram on how to calculate the FCBI-index value of the patient with microbiologically confirmed Streptococcus pneumoniae pneumonia using the novel FCBI-index method. (a) The incubation of the whole blood sample with the receptor-specific antibodies, red blood cell lysis, and flow cytometry run were carried out one after another in the same test tube. (b) During the flow cytometry run, the leukocyte population within gate R1 was first gated out from the side scatter (SSC)/MHCI bivariate histogram, after which gates R2 (red) and R3 (blue) were set around neutrophil and lymphocyte populations, respectively, in the SSC/CD64 bivariate histogram containing leukocytes inside gate R1. The stop count of 1000 monocytes inside the monocyte gate (black/R4) was used, leading to the collection of a total of 5000–20000 leukocytes. (c) For the purpose of data analysis, three additional bivariate histograms, namely SSC/CD35, SSC/CD64, and SSC/CD329 were made, showing mean fluorescence intensities (MFI, which correlate with the number of receptors on the cell surface) of gated neutrophil (red) and lymphocyte (blue) populations. In the SSC/CD35 histogram, B-lymphocytes expressing CD35 were left outside the lymphocyte gate. (d) The actual FCBI-index value was calculated by substituting the obtained receptor-specific MFI data from neutrophil and monocyte populations into the bacterial infection algorithm. A more detailed step-by-step description of the development of the FCBI-index method is presented in the method section and Supplementary Figs. 1–5. 1.54 × gCMF-HBSS: 1.54-fold calcium- and magnesium-free HBSS -buffer supplemented with 1.54 mg/mL gelatin.

Article Snippet: Fluorescence-conjugated receptor-specific monoclonal antibodies (test kit): Phycoerythrin (PE)-conjugated anti-human CD35 (Clone E11) was purchased from Ancell (ANC-184-050), PerCP-Vio700-conjugated anti-human CD64 (Clone 10.1.1) was purchased from Miltenyi Biotec (130-101-422), fluorescein isothiocyanate (FITC)-conjugated anti-human CD329 (Clone 191240) was purchased from R&D systems (FAB1139F) and AF647-conjugated anti-human MHC class I molecule (Clone W6/32) was purchased from R&D systems (FAB7098R). (Supplementary Table 5).

Techniques: Incubation, Red Blood Cell Lysis, Flow Cytometry, Fluorescence, Expressing, Infection

Journal:

Article Title:

doi:

Figure Lengend Snippet: Total lysates from TRAMP prostate and MMTV-PyMT mammary tumors were probed for SPARC using two different antibodies. Top and middle panels, SPARC is present as a ~45 kDa protein in SPARC+/−, but not SPARC−/−, tissues. The lower bands in the top panel for the TRAMP samples are non-specific bands detected by the ab14071 SPARC antibody but not by the AF942 SPARC antibody (middle). Bottom, GAPDH loading controls are shown for all samples.

Article Snippet: Membranes were incubated overnight with chicken anti-SPARC antibody (ab14071; 1:500; Abcam, Cambridge, MA), goat anti-SPARC antibody (AF942; 1:500; R&D Systems, Minneapolis, MN) and/or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:5000; Chemicon, Temecula, CA).

Techniques:

Journal: mAbs

Article Title: Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

doi: 10.1080/19420862.2021.1954136

Figure Lengend Snippet: Biochemical and cell-based binding of dostarlimab to PD-1

Article Snippet: Binding of dostarlimab to FcγRI (Cat#1257-FC-050), FcγRIIa (Cat#9595-CD-050), FcγRIIb (Cat#1875-CD-050), FcγRIIIa (Cat#4325-FC-050), and FcγRIIIb (Cat#1597-FC-050; all from R&D Systems) was assessed by SPR using Biacore T200 and Biacore 4000 instruments in which the appropriate Fcγ receptors were immobilized to a Biacore CM5 sensor chip via amine coupling.

Techniques: Binding Assay

Journal: mAbs

Article Title: Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

doi: 10.1080/19420862.2021.1954136

Figure Lengend Snippet: Dostarlimab binding to native PD-1 receptor expressed on human and cynomolgus monkey CD3 + T cells. Dostarlimab binds with high affinity to native human and cynomolgus monkey PD-1 expressed on the surface of CD3 + T cells. Occupancy of dostarlimab on cynomolgus monkey and human CD3 + T cells was assessed by preincubating PBMCs from either (a) cynomolgus monkey or (b) human donors with dostarlimab at varying concentrations and then incubating with either a saturating concentration (20 μg/mL) of dostarlimab or IgG4 isotype control before detection with either FITC-anti-CD3 or PE-anti-IgG4 antibodies and analysis by flow cytometry. The number of CD3 + /IgG4 + cells was quantified, and percentage receptor occupancy was calculated as described in Materials and Methods. FITC: fluorescein isothiocyanate; IgG: immunoglobulin G; PBMC: peripheral blood mononuclear cells; PD-1: programmed cell death protein 1; PE: phycoerythrin

Article Snippet: Binding of dostarlimab to FcγRI (Cat#1257-FC-050), FcγRIIa (Cat#9595-CD-050), FcγRIIb (Cat#1875-CD-050), FcγRIIIa (Cat#4325-FC-050), and FcγRIIIb (Cat#1597-FC-050; all from R&D Systems) was assessed by SPR using Biacore T200 and Biacore 4000 instruments in which the appropriate Fcγ receptors were immobilized to a Biacore CM5 sensor chip via amine coupling.

Techniques: Binding Assay, Concentration Assay, Control, Flow Cytometry

Journal: mAbs

Article Title: Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

doi: 10.1080/19420862.2021.1954136

Figure Lengend Snippet: Dostarlimab disrupts binding of PD-1 to its ligands. Dostarlimab potently blocks the interaction of PD-1 with its ligands PD-L1 and PD-L2 and inhibits PD-1/PD-L1-driven signaling. (a) Dostarlimab inhibits the binding of labeled PD-L1-mFc-DyL650 and PD-L2-mFc-DyL650 to CHO-K1 cells stably expressing recombinant human PD-1. (b) Dostarlimab relieves PD-1/PD-L1-mediated repression of an NFAT-luciferase reporter downstream of TCR signaling. For a detailed description of the assay, see Materials and Methods. CHO: Chinese hamster ovary; EC 50 : half-maximal effective concentration; IC 50 : half-maximal inhibitory concentration; NFAT: nuclear factor of activated T cells response element; PD-1: programmed cell death protein 1; PD-L1(2): programmed cell death ligand 1(2); RLU: relative light units; TCR: T-cell receptor

Article Snippet: Binding of dostarlimab to FcγRI (Cat#1257-FC-050), FcγRIIa (Cat#9595-CD-050), FcγRIIb (Cat#1875-CD-050), FcγRIIIa (Cat#4325-FC-050), and FcγRIIIb (Cat#1597-FC-050; all from R&D Systems) was assessed by SPR using Biacore T200 and Biacore 4000 instruments in which the appropriate Fcγ receptors were immobilized to a Biacore CM5 sensor chip via amine coupling.

Techniques: Binding Assay, Labeling, Stable Transfection, Expressing, Recombinant, Luciferase, Concentration Assay

Journal: mAbs

Article Title: Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

doi: 10.1080/19420862.2021.1954136

Figure Lengend Snippet: Functional activity of dostarlimab in primary T-cell–based assays. The functional activity of dostarlimab was evaluated in several in vitro assays, including (a) allogeneic MLR, (b) SEB activation, and (c) flu/PPD/TT activation assays. In the MLR assay, dendritic cells and allogeneic CD4 + T cells were incubated in the presence of dostarlimab or isotype control at the indicated concentrations for 48 hours, and activation of T cells was determined by quantifying the level of IL-2 secretion. Representative data from 4 donor combinations is shown in (a). PBMCs from 5 and 3 donors, respectively, were incubated as described in Materials and Methods with either SEB or flu/PPD/TT in the presence of dostarlimab or isotype control at the indicated concentrations for 72 hours and 5 days, respectively. Activation of T cells was determined by measuring the level of IL-2 or IFN-γ secretion. Results shown are the normalized mean ± SEM values across all donors. IFN: interferon; IL: interleukin; MLR: mixed lymphocyte reaction; PBMC: peripheral blood mononuclear cell; PPD: purified protein derivative from Mycobacterium tuberculosis tuberculin; SEB: staphylococcal enterotoxin B; SEM: standard error of the mean; TT: tetanus toxoid antigen

Article Snippet: Binding of dostarlimab to FcγRI (Cat#1257-FC-050), FcγRIIa (Cat#9595-CD-050), FcγRIIb (Cat#1875-CD-050), FcγRIIIa (Cat#4325-FC-050), and FcγRIIIb (Cat#1597-FC-050; all from R&D Systems) was assessed by SPR using Biacore T200 and Biacore 4000 instruments in which the appropriate Fcγ receptors were immobilized to a Biacore CM5 sensor chip via amine coupling.

Techniques: Functional Assay, Activity Assay, In Vitro, Activation Assay, Mlr Assay, Incubation, Control, Purification

Journal: mAbs

Article Title: Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

doi: 10.1080/19420862.2021.1954136

Figure Lengend Snippet: Antitumor and pharmacodynamic activity of dostarlimab monotherapy in a humanized mouse model of non–small cell lung cancer. (a) Study schema: dostarlimab or IgG4 isotype control (200 µg/mouse, twice weekly) was dosed to established A549 tumors (80–120 mm 3 ) grown as xenografts in humanized NOG-EXL mice (Taconic), and (b) antitumor activity and (c) pharmacodynamic effects in the tumor. Human CD4 + , CD8 + , NK/NKT, and activated CD8 + (GZMB, CD8 + ) cells in the tumors were quantified by flow cytometry as percentage of human CD45 + cells. * p < .05 (unpaired t test). PD-1: programmed cell death protein 1; CD-8: cluster of differentiation-8; GZMB: Granzyme B; IgG: immunoglobulin G; NK: natural killer; T reg : regulatory T cells

Article Snippet: Binding of dostarlimab to FcγRI (Cat#1257-FC-050), FcγRIIa (Cat#9595-CD-050), FcγRIIb (Cat#1875-CD-050), FcγRIIIa (Cat#4325-FC-050), and FcγRIIIb (Cat#1597-FC-050; all from R&D Systems) was assessed by SPR using Biacore T200 and Biacore 4000 instruments in which the appropriate Fcγ receptors were immobilized to a Biacore CM5 sensor chip via amine coupling.

Techniques: Activity Assay, Control, Flow Cytometry

Journal: mAbs

Article Title: Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

doi: 10.1080/19420862.2021.1954136

Figure Lengend Snippet: Antitumor activity of dostarlimab monotherapy in a humanized mouse model of breast cancer. (a) Study schema; dostarlimab or IgG4 isotype control (200 μg/mouse, twice weekly) was dosed to already established MDA-MB-436 tumors (80–120 mm 3 ) grown as xenografts in humanized NOG-EXL mice (Taconic), and (b) antitumor activity. PD-1: programmed cell death protein 1; IgG: immunoglobulin G

Article Snippet: Binding of dostarlimab to FcγRI (Cat#1257-FC-050), FcγRIIa (Cat#9595-CD-050), FcγRIIb (Cat#1875-CD-050), FcγRIIIa (Cat#4325-FC-050), and FcγRIIIb (Cat#1597-FC-050; all from R&D Systems) was assessed by SPR using Biacore T200 and Biacore 4000 instruments in which the appropriate Fcγ receptors were immobilized to a Biacore CM5 sensor chip via amine coupling.

Techniques: Activity Assay, Control

Journal: mAbs

Article Title: Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

doi: 10.1080/19420862.2021.1954136

Figure Lengend Snippet: Concentration–time profiles of single-dose dostarlimab. Mean serum concentration–time profiles of dostarlimab after single intravenous infusion of dostarlimab to male and female cynomolgus monkeys. Concentration–time profiles were plotted through the first post-dose time point that was below the limit of quantification, if applicable. The lower limit of quantification was 31.0 ng/mL

Article Snippet: Binding of dostarlimab to FcγRI (Cat#1257-FC-050), FcγRIIa (Cat#9595-CD-050), FcγRIIb (Cat#1875-CD-050), FcγRIIIa (Cat#4325-FC-050), and FcγRIIIb (Cat#1597-FC-050; all from R&D Systems) was assessed by SPR using Biacore T200 and Biacore 4000 instruments in which the appropriate Fcγ receptors were immobilized to a Biacore CM5 sensor chip via amine coupling.

Techniques: Concentration Assay

Journal: mAbs

Article Title: Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

doi: 10.1080/19420862.2021.1954136

Figure Lengend Snippet: Mean pharmacokinetic parameters for treatment groups of dostarlimab after single-dose intravenous infusion to monkeys

Article Snippet: Binding of dostarlimab to FcγRI (Cat#1257-FC-050), FcγRIIa (Cat#9595-CD-050), FcγRIIb (Cat#1875-CD-050), FcγRIIIa (Cat#4325-FC-050), and FcγRIIIb (Cat#1597-FC-050; all from R&D Systems) was assessed by SPR using Biacore T200 and Biacore 4000 instruments in which the appropriate Fcγ receptors were immobilized to a Biacore CM5 sensor chip via amine coupling.

Techniques:

Journal: mAbs

Article Title: Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

doi: 10.1080/19420862.2021.1954136

Figure Lengend Snippet: Mean pharmacokinetic parameters for treatment groups of dostarlimab after intravenous infusion to monkeys at day 1 and day 22 in 1-month repeat-dose toxicology study

Article Snippet: Binding of dostarlimab to FcγRI (Cat#1257-FC-050), FcγRIIa (Cat#9595-CD-050), FcγRIIb (Cat#1875-CD-050), FcγRIIIa (Cat#4325-FC-050), and FcγRIIIb (Cat#1597-FC-050; all from R&D Systems) was assessed by SPR using Biacore T200 and Biacore 4000 instruments in which the appropriate Fcγ receptors were immobilized to a Biacore CM5 sensor chip via amine coupling.

Techniques:

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet: Activated FcγRI is retained in intracellular compartments described by IRAP (A–C) DC2.4 cells expressing FcγRI-GFP (green) were incubated with anti-FcγRI (clone 10.1) and cross-linked with anti-mouse IgG (blue) at 4°C. After removal of excess antibodies, the cells were shifted for the indicated time points at 37°C, fixed and stained for EEA1 (red) (A), IRAP (red) (B) or LAMP1 (red) (C). The pictures show representative images from three independent experiments and the graphs show colocalization between FcγRI and the endocytic markers. Each dot represents a cell. Data are represented as mean ± SEM. Scale bars = 5 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Expressing, Incubation, Staining

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet: Activated FcγRI recruits signaling partners at the level of IRAP endosomes (A–C) DC2.4 cells expressing FcγRI-GFP (green) were incubated with human IgG and anti-human IgG at 4°C. After removal of excess antibodies, cells were shifted for the indicated time points at 37°C, fixed and stained for: pSrc (red) and IRAP (gray) (A), LAT (red) and IRAP (gray) (B), or PLCγ (red) and IRAP (gray) (C). The pictures show representative images from three independent experiments and the graphs show colocalization between the indicated signalosome component and IRAP. Each dot represents a cell. Scale bars = 5 μm. (D) The graph shows the colocalization between FcγRI and IRAP that was quantified in the conditions (A), (B) and (C). (E) The interaction of FcγRI with Syk was measured by FLIM experiments and the graph shows the GFP lifetime for cells expressing only GFP, both GFP and mCherry as soluble proteins and the cells expressing both FcγRI-GFP and Syk-mCherry in steady state and after the receptor crosslinking for indicated time points. In all the graphs, each dot represents a cell. Data are represented as mean ± SEM. Scale bars = 5 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Expressing, Incubation, Staining

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet: IRAP deletion affects the endosomal recruitment of active Syk to FcγRI (A and B) Wild-type (WT) or IRAP-deficient (KO) BM-DCs were incubated with anti-mouse FcγRI (clone AT152-9), followed by crosslinking with non-labeled anti-rat IgG at 4°C. After removal of excess antibodies, the cells were shifted for the indicated time points at 37°C, fixed and stained for endogenous FcγRI (green) and γ-chain (red) in (A) and for γ-chain (green) and Syk phosphorylated on Tyrosine 352 (pSyk) (red) in (B). The pictures show representative images from three independent experiments and the graphs show colocalization between FcγRI and the γ-chain (A) and colocalization between the γ-chain and phosphorylated, active Syk (B). Each dot represents a cell. Data are represented as mean ± SEM. Scale bars = 5 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Incubation, Labeling, Staining

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet:

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Produced, Western Blot, Recombinant, Lysis, Isolation, Enzyme-linked Immunosorbent Assay, Cloning, Software