Journal: Vaccines

Article Title: Development of a Macrophage-Based ADCC Assay

doi: 10.3390/vaccines9060660

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Trastuzumab and Rituximab antibodies were purchased from Invivogen: anti-HER2-Tra-hIgG1 (her2tra-mab1), anti-hCD20-hIgG1 (hcd20-mab1), anti-hCD20-hIgG1NQ (hcd20-mab12), anti-hCD20-hIgG2 (hcd20-mab2), anti-hCD20-hIgG3 (hcd20-mab3), anti-hCD20-IgG4 (hcd20-mab4), anti-hCD20-mIgG1 (hcd20-mab9), and anti-CD20-mIgG2a (hcd20-mab10). mIgG2b isotype control clone MPC-11 (559530) was purchased from BD.

Techniques:

Journal: Vaccines

Article Title: Development of a Macrophage-Based ADCC Assay

doi: 10.3390/vaccines9060660

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Trastuzumab and Rituximab antibodies were purchased from Invivogen: anti-HER2-Tra-hIgG1 (her2tra-mab1), anti-hCD20-hIgG1 (hcd20-mab1), anti-hCD20-hIgG1NQ (hcd20-mab12), anti-hCD20-hIgG2 (hcd20-mab2), anti-hCD20-hIgG3 (hcd20-mab3), anti-hCD20-IgG4 (hcd20-mab4), anti-hCD20-mIgG1 (hcd20-mab9), and anti-CD20-mIgG2a (hcd20-mab10). mIgG2b isotype control clone MPC-11 (559530) was purchased from BD.

Techniques:

Journal: Frontiers in Immunology

Article Title: Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

doi: 10.3389/fimmu.2019.00704

Figure Lengend Snippet: IgA mediates higher tumor cell lysis than IgG with human neutrophils. (A–D) Specific lysis of tumor cells by isolated human neutrophils (E:T = 40:1) using a 4 h 51 Cr release assay. Anti-CD20 Abs were used for Ramos and EL4-CD20, anti-HER2 Abs were used for SK-BR-3 and Ba/F3-HER2, anti-EGFR Abs were used for A431 and A1207 target cells. (A–C) Specific lysis of indicated target cells using a broad titration range of IgA and IgG antibodies. (D) Antibody concentrations were identical (10 μg/mL) for IgG and IgA antibodies against all target cells except for Ramos (13.3 μg/mL) and A1207 cells (1 μg/mL). Ctrl indicates condition without antibody (or 0.001 μg/mL IgG1-anti-CD20 for Ramos cells). One representative graph is shown for n = 4–6 independent experiments with different healthy donors. p < 0.001: *** , p < 0.0001: **** , unpaired Student's t test. (E) Stills from live cell microscopy of adhered A431-HER2 cells (calcein labeled) and neutrophils in the presence of 5 μg/mL anti-HER2 IgA2 or IgG (Trastuzumab) and TO-PRO-3 (red fluorescence), time in minutes, image acquisition every 30 s for 1.5 h.

Article Snippet: Antibodies: to target human CD20, rituximab (Roche), anti-CD20-IgA1 (invivogen, hcd20-mab6), or in-house made mAbs ( ) were used.

Techniques: Lysis, Isolation, Release Assay, Titration, Microscopy, Labeling, Fluorescence

Journal: Frontiers in Immunology

Article Title: Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

doi: 10.3389/fimmu.2019.00704

Figure Lengend Snippet: Blocking FcγRIIIb only marginally improves tumor cell lysis. (A) Quantitative expression of FcγR and FcαRI on human neutrophils as analyzed by flow cytometry (Qifikit), n = 6–11 healthy donors. (B) 3 h 51 Cr release assays using EL4-CD20 with anti-CD20 IgA2 or IgG1 (rituximab), SK-BR-3 with anti-HER2 IgA1 or IgG1 and A431 with anti-EGFR IgA2 or IgG1, all at 10 ug/mL. 3G8 F(ab′) 2 fragments (1 ug/mL final concentration) were added 15 min prior to start of the ADCC assays. Statistics: one-way ANOVA with Tukey's multiple comparison test, p < 0.05: * , p < 0.01: ** , p < 0.0001: **** . Ctrl indicates no antibody added.

Article Snippet: Antibodies: to target human CD20, rituximab (Roche), anti-CD20-IgA1 (invivogen, hcd20-mab6), or in-house made mAbs ( ) were used.

Techniques: Blocking Assay, Lysis, Expressing, Flow Cytometry, Concentration Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

doi: 10.3389/fimmu.2019.00704

Figure Lengend Snippet: Neutrophils display similar binding characteristics to IgA- or IgG-associated surfaces. (A) Example of relative binding of a donor of which calcein labeled neutrophils were allowed to associate to anti-CD20 IgA2 or IgG1 (clone UMAB001) coated 96 well plate. Remaining calcein fluorescence was measured after repeating wash steps. Calcein fluorescence before the first wash was set 100%. Ctrl indicates that no antibody was used during coating. (B–D) As in A , but data of 6–9 different donors at wash 10 are combined per target being anti-CD20 IgA2 or IgG1 (clone UMAB001) or anti-HER2 IgA2 or IgG1. Each color indicates data from the same donor. Statistics: two-way ANOVA with Tukey's multiple comparison test, p < 0.0001: **** . (E) Examples images of neutrophils binding to albumin, anti-CD20 IgA, or IgG coated Dynabeads (ratio beads:neutrophils = 5:1). Rosettes are defined as cells binding 5 or more beads. (F,G) Quantification of rosettes of anti-CD20- or anti-HER2-coated Dynabeads with neutrophils. One representative of n = 3 individual experiments is shown. Differences were not significant according to one-way ANOVA with Tukey's multiple comparison test. (H) Binding traces for calcein labeled neutrophils binding to anti-CD20 IgA- (red) or IgG- (black) opsonized Daudi cells. One representative of n = 5 experiments is shown. (I) Binding association and (J) average half-life of the Daudi:neutrophils complex pooled from 5 different donors. Statistics: paired Student's t -test.

Article Snippet: Antibodies: to target human CD20, rituximab (Roche), anti-CD20-IgA1 (invivogen, hcd20-mab6), or in-house made mAbs ( ) were used.

Techniques: Binding Assay, Labeling, Fluorescence, Comparison

Journal: Frontiers in Immunology

Article Title: Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

doi: 10.3389/fimmu.2019.00704

Figure Lengend Snippet: Strong p-ERK signal in neutrophils elicited by IgA. (A,B) Time-dependent increase and decrease of p-ERK signal in neutrophils after exposure to anti-CD20 or anti-HER2 IgA2- or IgG1-coated Dynabeads. Actin detection was performed on the same blot after destroying the p-ERK or total ERK signal. (C,D) Quantification of signaling defined as the ratio of normalized p-ERK/actin over normalized total ERK/actin. Data was normalized against the 0 min time point. (E) 4 h 51 Cr release assays using SK-BR-3 with anti-HER2 IgA2 or IgG1, both at 1 ug/mL. Where indicated, pharmacological inhibitors of signaling factors were included (wortmannin at 0.5 μM, Ly294002 and U0126 both at 20 μM). Statistics: two-way ANOVA with Tukey's multiple comparisons test p < 0.05: * , p < 0.01: ** , p < 0.001: *** , p < 0.0001: **** .

Article Snippet: Antibodies: to target human CD20, rituximab (Roche), anti-CD20-IgA1 (invivogen, hcd20-mab6), or in-house made mAbs ( ) were used.

Techniques: