Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: N-glycan analysis of the anti-PD-L1 variants αPDL1 WT and αPDL1 GE

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques:

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody shows an enhanced binding to FcγRIIIa. A competitive FcγRIIIa AlphaLISA was performed for the three anti-PD-L1 variants. Thereby the test antibody competes with antibody-conjugated acceptor beads for binding to FcγRIIIa-conjugated donor beads. The chemiluminescent signal as a result of close proximity of the donor and acceptor beads was plotted against increasing concentrations of αPDL1 NG (open circles), αPDL1 WT (gray triangles), and αPDL1 GE (black squares). Statistics: mean and SD were plotted in the graph. Data are representative of two independent experiments.

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody and its normal and non-glycosylated counterparts show comparable antigen binding characteristics. The three anti-PD-L1 variants αPDL1 NG (open circles), αPDL1 WT (gray triangle), and αPDL1 GE (black squares) were tested for PD-L1 antigen binding and their capacity to block interaction with PD-L1 ligands in enzyme-linked immunosorbent assays (ELISA). (A) PD-L1 antigen binding ELISA. OD 450–620 values were plotted against increasing concentrations of test antibody to assess binding to plate-bound human PD-L1. (B) Competitive ELISA measuring binding of soluble programmed death 1 to plate-bound PD-L1 in presence of test antibody. OD4 450–620 values were plotted against increasing concentrations of test antibody. (C) Competitive ELISA measuring binding of soluble CD80 to plate-bound PD-L1 in presence of test antibody. OD 450–620 values were plotted against increasing concentrations of test antibody. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments.

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Competitive ELISA

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces strongest NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) against PD-L1 + cancer cells, but none against B cells and monocytes. (A) The NK cell line KHYG-1-CD16aV as effector cells was incubated with europium-loaded PD-L1 + DU-145 cancer cells as target cells in an effector to target ratio of 10:1 in the presence of increasing concentrations of αPDL1 NG (white circles), αPDL1 WT (gray triangles), or αPDL1 GE (black squares) for 5 h to determine the lysis of target cells in an in vitro cytotoxicity assay. The percentage of specific target cell lysis is plotted against the antibody concentration used. The dashed line indicates % of lysis in the medium control. (B,C) The NK cell line KHYG-1-CD16aV as effector cells were incubated with calcein-labeled primary B cells (B) or monocytes (C) as target cells in an effector to target ratio of 10:1 in the presence of increasing concentrations of αPDL1 WT (gray bars) or αPDL1 GE (black bars) for 4 h to determine the killing of target cells in a flow cytometry based in vitro cytotoxicity assay. The relative frequency of dead 7-AAD + of calcein + target cells was plotted against the antibody concentration used. As a positive control (striped bars) either obinutuzumab (αCD20) was used to induce B cell lysis or staurosporine was used to induce monocyte lysis. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments. Significance was tested against the medium control (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. Abbreviation: ns., not significant).

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Incubation, Lysis, In Vitro, Cytotoxicity Assay, Concentration Assay, Control, Labeling, Flow Cytometry, Positive Control

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces strong CD8 T cell activation in a mixed leukocyte reaction. The three anti-PD-L1 variants αPDL1 NG (open circles), αPDL1 WT (gray triangles), and αPDL1 GE (black squares) were tested for their effect on T cell activation in a mixed leukocyte reaction (MLR). The medium control (black crosses) served as a negative control. T cells as responder cells were isolated from a single healthy donor (donor A). Monocyte-derived dendritic cells as stimulator cells were generated from different healthy donors. (A) IL-2 enzyme-linked immunosorbent assays on day 2 of MLR. The determined concentrations of IL-2 in the culture supernatants were plotted. (B) The activation status of CD8 and CD4 T cells in the MLR was determined on day 5 by flow cytometric analysis. The relative frequencies of CD25 + and CD137 + in CD8 and CD4 T cells were plotted. (C) The proliferation of CFSE-labeled CD8 T cells in the MLR was determined on day 5 by CFSE dilution measured by flow cytometric analysis. Representative plots of CD25 expression and CFSE signal intensity of CD8 T cells are shown. Statistics for (A,B) : besides individual data points ( n = 8 for IL-2 secretion, n = 16 for CD25 expression, and n = 7 for CD137), mean and SEM were plotted in all graphs (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. Abbreviation: ns, not significant).

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Activation Assay, Control, Negative Control, Isolation, Derivative Assay, Generated, Labeling, Expressing

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces increased CD8 T cell activation in presence of cancer cells. The three anti-PD-L1 variants αPDL1 NG (light gray bar), αPDL1 WT (dark gray bar), and αPDL1 GE (black bar) were tested for their effect on T cell activation (donor A) in allogeneic mixed leukocyte reaction (MLRs) in absence or presence of the cancer cell lines HSC-4 (horizontal stripes), ZR-75-1 (plaid), and Ramos (vertical stripes). MLR without addition of test antibody (medium; white bar) served as negative control. The relative frequencies of CD25 + in CD8 T cells were plotted. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments. Significance was tested against the medium control without presence of cancer wells (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001).

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Activation Assay, Negative Control, Control

Journal: EMBO Molecular Medicine

Article Title: Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

doi: 10.15252/emmm.202114182

Figure Lengend Snippet: BW5147 reporter cells stably expressing human FcγR‐ζ chain chimeras or BW5147 parental cells (grey/dashed) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry. Bar graphs show means of three independent experiments performed in technical duplicates. Error bars = SD. FCS coating of an ELISA microtiter plate allows for suspension of subsequently added IgG. Plate bound IgG was quantified via ELISA. Bar graph shows means of one experiment performed in technical triplicates. Error bars = SD. Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X‐Axis shows sample concentration determined by antibody molarity. Y‐Axis shows FcγR activation determined by reporter cell mouse IL‐2 production (OD 450 nm). Two independent experiments performed in technical duplicates. Error bars = SD. Schematic of used assay setups. BW5147 reporter cells expressing chimeric human FcγR receptors express endogenous CD69 or secrete mouse IL‐2 in response to FcγR activation by clustered IgG. sICs are generated using mAbs and multivalent antigens. sIC suspension requires pre‐blocking of an ELISA plate using PBS supplemented with 10% FCS (FCS coat, grey‐dashed).

Article Snippet: FcγR‐specific mAbs were obtained from Stem Cell technologies (CD16: clone 3G8; CD32: IV.3).

Techniques: Stable Transfection, Expressing, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, Activation Assay, Generated, Blocking Assay

Journal: EMBO Molecular Medicine

Article Title: Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

doi: 10.15252/emmm.202114182

Figure Lengend Snippet: Ultra‐pure antigen (Ag, TNF‐α) mixed with therapy‐grade mAb (infliximab, Ifx) was used to generate sICs. X‐Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. Immobilized IgG (Rtx) or immobilized FcγR‐specific mAbs served as positive controls. Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown. Left panel: IL‐2 quantification 16 h after reporter cell activation. Background (blank) was subtracted (dashed line). IL‐2 was measured via anti‐IL‐2 ELISA (A 450nm ) and IL‐2 concentrations were calculated from an IL‐2 standard. Right panel: Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry. Mean florescence intensity (MFI) were normalized to untreated cells (ctrl.) and are presented as fold‐change increase.

Article Snippet: FcγR‐specific mAbs were obtained from Stem Cell technologies (CD16: clone 3G8; CD32: IV.3).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

doi: 10.15252/emmm.202114182

Figure Lengend Snippet: Two different multivalent ultra‐pure antigens (Ag) mixed with respective therapy‐grade mAbs were used to generate sICs as indicated for each set of graphs (top to bottom). IC pairs: mepolizumab (Mpz) and rhIL‐5; bevacizumab (Bvz) and rhVEGFA. X‐Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. FcγR responses were normalized to immobilized rituximab (Rtx) at 1 µg/well (set to 1) and a medium control (set to 0). Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown.

Article Snippet: FcγR‐specific mAbs were obtained from Stem Cell technologies (CD16: clone 3G8; CD32: IV.3).

Techniques: