Journal: mAbs

Article Title: Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells

doi: 10.1080/19420862.2016.1249079

Figure Lengend Snippet: Increased FcγRIIIa binding of low-and hemi-fucosylated forms of JNJ-61186372. FcγRI (A), FcγRIIa (B), and FcγRIIIa (C) binding by antibodies produced in normal fucose (NF) or low fucose (LF) cell line was assessed by competitive Alpha Screen and compared to a wild type IgG1 control antibody (closed circle). JNJ-61186372 – NF (closed square), JNJ-61186372 – LF (closed up triangle), EGFR x inert arm –LF (closed down triangle), c-Met x inert arm – LF (open up triangle), EGFR (LF) x c-Met (NF) (open down triangle), EGFR (NF) x c-Met (LF) (open diamond), JNJ-61186372 – IgG2σ (open circle). Representative data from 3 replicate experiments is shown.

Article Snippet: His-tagged FcγRI (R&D Systems, cat no. 1257-FC), FcγRIIa (R&D Systems, cat no. 1330-CD/CF) or FcγRIIIa (R&D Systems, cat no. 4325-FC) were captured on nickel chelate acceptor beads (PerkinElmer, cat. no. CUSM0220400EA).

Techniques: Binding Assay, Produced, Control

Journal: mAbs

Article Title: Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells

doi: 10.1080/19420862.2016.1249079

Figure Lengend Snippet: Concentration of antibody (nM) to elicit 50% of maximal FcγR binding activity (IC 50 ). Data presented in mean ± SEM of 2–3 independent experiments. *Difference between JNJ-61186372 - LF and JNJ-61186372 – NF is statistically significant (p value = 0.0001).

Article Snippet: His-tagged FcγRI (R&D Systems, cat no. 1257-FC), FcγRIIa (R&D Systems, cat no. 1330-CD/CF) or FcγRIIIa (R&D Systems, cat no. 4325-FC) were captured on nickel chelate acceptor beads (PerkinElmer, cat. no. CUSM0220400EA).

Techniques: Concentration Assay, Binding Assay, Activity Assay, Significance Assay

Journal: mAbs

Article Title: Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells

doi: 10.1080/19420862.2016.1249079

Figure Lengend Snippet: Fold change to multiple FcγR binding (IC 50 ) of different antibodies to that of JNJ-61186372 – NF.

Article Snippet: His-tagged FcγRI (R&D Systems, cat no. 1257-FC), FcγRIIa (R&D Systems, cat no. 1330-CD/CF) or FcγRIIIa (R&D Systems, cat no. 4325-FC) were captured on nickel chelate acceptor beads (PerkinElmer, cat. no. CUSM0220400EA).

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: FcγR Binding and ADCC Activity of Human IgG Allotypes

doi: 10.3389/fimmu.2020.00740

Figure Lengend Snippet: Characterization of binding strength of IgG allotypes to FcγR. Binding of immunoglobulin G allotypes to (A) FcγRI, (B) FcγRIIa 131H, (C) FcγRIIa 131R, (D) FcγRIIIa 158F and (E) FcγRIIIa 158V as determined by SPR. K D values for each IgG allotype are plotted as bar graphs, in which black bars represent IgG1 allotypes, green bars IgG2 allotypes, blue bars IgG3 allotypes and white bars IgG4 allotypes. The highest antibody concentration (1000 nM/1 × 10 – 6 M) that was used in the SPR measurement is depicted as a dotted line. K D values below this line are unreliable and are represented as >1000 nM. Error bars indicate SEM of ≥2 independent measurements. Individual sensorgrams from which K D values were quantified are displayed in . NK cell mediated killing of (F) bromelain treated RhD+ red blood cells by all anti-RhD allotypes and (G) TNPlated red blood cells by all anti-TNP allotypes at a concentration of 1.25 μg/ml. Percentage ADCC specific killing of red blood cells was measured in triplo and the mean is plotted in a bar graph. NK-cell mediated ADCC by anti-RhD allotypes was measured with NK cells from four individual donors, as shown in . One representative result of four individual experiments is depicted here. To determine significant differences between allotypes within each subclass we used a one-way ANOVA with Sidak’s multiple comparison test, and significant differences are indicated with white asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001. This statistical analysis was performed separately for all allotypes within a subclass and compared to one reference antibody (IgG1*03, IgG2*01, IgG3*01, and IgG4*01).

Article Snippet: Human FcγR constructs FcγRIa (his tag, 10256-H08H-100), FcγRIIa (131His, biotinylated, 10374-H27H1-B-50 and 131Arg, biotinylated, 10374-H27H-B-50), FcγRIIb (biotinylated, 10259-H27H-B-50), and FcγRIIIa (158Phe, biotinylated, 10389-H27H-B-50, and 158Val, biotinylated, 10389-H27H1-B-50) for surface plasmon resonance (SPR) analysis were obtained from Sino Biological (Beijing, China).

Techniques: Binding Assay, Concentration Assay, Comparison

Journal: Frontiers in Immunology

Article Title: FcγR Binding and ADCC Activity of Human IgG Allotypes

doi: 10.3389/fimmu.2020.00740

Figure Lengend Snippet: FcγR avidity measurements of IgG3 allotypes. The avidity of RBCs opsonized with various IgG3 allotypes to FcγR as determined by cSPR. Avidity measurements to FcγRIIa 131R, FcγRIIa 131H, FcγRIIIa 158F, FcγRIIIa 158V were determined for seven IgG3 allotypes (*01, *04, *12, *14, *16, *17, *18) and one IgG3 mutant expressing a L291 (*12/*14). To compare between allotypes we calculated area under the curve (AUC) values from the Total/Sedimentation (T/S) ratios that are plotted in at a specific receptor density and RBC opsonization concentration. Thus, avidity measurements to (A) FcγRIIa 131R at a receptor density of 30 nM and opsonization concentration of 0.625 μg/ml, (B) FcγRIIa 131H at a receptor density of 30 nM and opsonization concentration of 2.5 μg/ml, (C) FcγRIIIa 158F at a receptor density of 30 nM and opsonization concentration of 0.625 μg/ml and (D) FcγRIIIa 158V at a receptor density of 10 nM and opsonization concentration of 1.25 μg/ml. (E) Binding strength to an anti-kappa nanobody (density of 1 nM and opsonization concentration of 0.625 μg/ml) was determined simultaneously to confirm equal RBC opsonization levels with each allotype. Error bars indicate SD of ≥2 independent measurements. In all graphs the amino acid at residue 291 (single-letter code) for each antibody is indicated at the x -axis below the IgG3 allotype number, where 291-mutated antibodies are displayed with a red letter. Statistical comparison between antibodies were performed using a one way ANOVA analysis with Sidak’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human FcγR constructs FcγRIa (his tag, 10256-H08H-100), FcγRIIa (131His, biotinylated, 10374-H27H1-B-50 and 131Arg, biotinylated, 10374-H27H-B-50), FcγRIIb (biotinylated, 10259-H27H-B-50), and FcγRIIIa (158Phe, biotinylated, 10389-H27H-B-50, and 158Val, biotinylated, 10389-H27H1-B-50) for surface plasmon resonance (SPR) analysis were obtained from Sino Biological (Beijing, China).

Techniques: Mutagenesis, Expressing, Sedimentation, Concentration Assay, Binding Assay, Residue, Comparison

Journal: Cell Reports

Article Title: Anti-SARS-CoV-2 antibodies elicited by COVID-19 mRNA vaccine exhibit a unique glycosylation pattern

doi: 10.1016/j.celrep.2021.110114

Figure Lengend Snippet: IgG Fab and Fc responses to BNT162b2 SARS-CoV-2 vaccine (A) Experimental scheme. Blood was collected before receiving any vaccine dose (baseline, n = 23), at 2 weeks after the first vaccine dose (pre-boost, n = 127), and at 5 weeks (post-boost, n = 127). (B) RBD-binding IgG levels at baseline, pre-boost, and post-boost. Dotted red line indicates threshold for positivity. (C) Correlation between anti-RBD IgG titers following the first vaccine dose and following the second dose (n = 127, non-parametric Spearman’s correlation). (D) Anti-RBD IgG subclass distribution at the pre-boost and post-boost time points. Kruskal-Wallis test was used with Dunn’s post hoc test to correct for multiple comparisons. (E) (IgG1 + IgG3):(IgG2 + IgG4) ratio of sera anti-RBD IgG subclasses. Pre-boost, n = 123; post-boost, n = 127. – (F) Scheme of the IgG Fc glycan structure. The N-glycan is attached at the Asn297 position of each IgG heavy chain. The dashed line indicates the conserved heptasaccharide core, which may have the indicated saccharide extensions. (G) Fc glycosylation patterns of IgG1 in vaccinated individuals, determined by mass spectrometry. Shown are the total IgGs produced at the pre-boost time point (n = 59) and anti-RBD IgGs of participants who had an IgG1 response at pre-boost (n = 12) and at post-boost (n = 39). Detected glycan structures are shown in Figure S2 . (H) Ratios between RBD-specific IgGs binding to activating (FcγRIIa + FcγRIIIa) versus inhibitory (FcγRIIb) receptors at each time point (pre-boost, n = 39; post-boost, n = 59; also see Figure S1 ). Data are presented as scatterplots indicating individual measurements (dots); black line represents the mean; error bars represent standard deviations (SDs). Unless otherwise mentioned, unpaired 2-sided Mann-Whitney U test was used to evaluate the differences between groups. p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: the human recombinant receptors FcγRIIA , Sino Biological , Cat# 10374-H08H.

Techniques: Binding Assay, Mass Spectrometry, Produced, MANN-WHITNEY

Journal: Cell Reports

Article Title: Anti-SARS-CoV-2 antibodies elicited by COVID-19 mRNA vaccine exhibit a unique glycosylation pattern

doi: 10.1016/j.celrep.2021.110114

Figure Lengend Snippet:

Article Snippet: the human recombinant receptors FcγRIIA , Sino Biological , Cat# 10374-H08H.

Techniques: Isolation, Recombinant, Magnetic Beads, Mass Spectrometry, Construct, Plasmid Preparation, Software