Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 1. Confocal microscopy of Human EGF Receptor 2 (HER2) and trastuzumab (TZM)–AF700 distribution in AU565 cells. (A) Immunofluorescence analysis of HER2 (green; arrows), early endosomal antigen 1 (EEA1), or transferrin receptor (TfR) (red; arrows). Middle slices of z-stacks consisting of 6–8 optical slices are shown. (B) AU565 cells were loaded with 5 µg/mL TZM–AF700 (red) and 5 µg/mL Tf–AF568 (magenta) for 24 h and then processed for immunostaining of HER2 (green) and endosomal markers EEA1 or TfR (magenta). Maximum intensity projections of z-stacks consisting of 6–8 optical slices are shown. Nuclei are visualized with DAPI. Scale bar = 20 µm.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Confocal Microscopy, Immunostaining

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 2. Comparison of intracellular uptake of TZM–AF700 into HER2-positive and HER2-negative cell lines. (A) To test the specificity of HER2-mediated TZM–AF700 binding and internalization, competition experiments were performed. AU565 cells were loaded with 5 µg/mL TZM–AF700 (red) and 5 µg/mL Tf-AF568 (green) in the presence of increasing amounts of unlabeled TZM. Single slice confocal microscopy displays TZM–AF700 (red) and Tf–AF568 (green) binding and internalization into AU565 cells in the presence of increasing 0×, 10×, 50×, and 100× unlabeled TZM. (B) HER2-positive AU565 and HER2-negative MDA-MB-231 and MCF10A cells were subjected to 24 h uptake of 20 µg/mL TZM–AF700 (red) and then processed for anti-HER2 immunostaining (green). Nuclei were visualized with DAPI (blue). Maximum intensity projections of z-stacks consisting of 6–8 optical slices are shown. Confocal images were collected using identical settings on an LSM880 confocal microscope. Scale bar = 20 µm.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Comparison, Binding Assay, Confocal Microscopy, Immunostaining, Microscopy

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 3. TZM Forster Resonance Energy Transfer (FLI-FRET) microscopy (FLIM-FRET) analysis in AU565 cancer cells. (A) The representative time-correlated single photon counting (TCSPC) images of fluorescence intensity and mean lifetime map (τm) in cells treated with TZM-AF700 only [Acceptor/Donor (A:D) = 0:1; donor single-labeled] or with TZM–AF700 plus TZM–AF750 (A:D = 2:1; double-labeled); pseudo-color range = 500–1500 ps. Zoomed regions of interest (ROIs) of both single and double-labeled cells show heterogeneity of fluorescence lifetime of TZM–AF700 within the cells. White arrows indicate x, y coordinates used for the curve fitting using SPCImage. Scale bar = 50 µm. (B) Representative fitting curves and IRF, the fluorescent lifetime decay in the single and double-labeled cells was determined by comparing the fitting of the decay data using both single- and double-exponential decay models, respectively. (C) Fluorescent lifetime distribution in AU565 cells treated with TZM–AF700 (A:D = 0:1) or TZM–AF700 and TZM–AF750 (A:D = 2:1). (D) Quantification of FRET donor (FD%) levels in AU565 cells treated with a near-infrared (NIR) TZM–FRET pair at various A:D ratios. Analysis was performed using 10 distinct pixel coordinates (n = 10) from five independent ROIs; error bars represent confidence interval at 95%. (E) Quantification of TZM–FRET efficiency (E) in relation to A:D ratios. Data presented as mean ± confidence interval at 95%, n = 10.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Förster Resonance Energy Transfer, Microscopy, Labeling

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 4. TZM FLIM-FRET analysis in MDA-MB-231 cancer cells. (A) The representative TCSPC images of fluorescence intensity and mean lifetime map (τm) in cells treated with TZM–AF700 (A:D = 0:1) or with TZM–AF700 plus TZM–AF750 (A:D = 2:1) pseudo-color range = 300–1500 ps. Scale bar = 50 µm. (B) Comparison of fluorescent lifetime distribution in MDA-MB-231 (solid lines) and AU565 cells (dashed lines) treated with TZM–AF700 (A:D = 0:1 red), TZM–AF700 plus TZM–AF750 (A:D = 2:1 black).

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Comparison

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 5. Schematic representation of live small animal NIR wide-field time-resolved macroscopic fluorescence lifetime FRET imaging (MFLI-FRET) for preclinical studies. (A) AU565 tumor xenograft production; (B) Intravenous tail-vein injection of AF700–TZM and/or AF750–TZM; (C) Live small animal imaging using wide-field time-resolved FLI-FRET macroscopy (MFLI-FRET) imager; (D) Ex vivo validation using immunohistochemical (IHC) and H&E staining. Inset top left: illustration of TZM–AF700 and TZM–AF750 ligands. Inset bottom right: illustration of TZM–HER2 FLI-FRET events upon binding of donor- and acceptor-labeled antibodies to HER2 dimer at the surface of cancer cells. DMD: digital micromirror device; CCD: charge-coupled device.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Imaging, Injection, Ex Vivo, Biomarker Discovery, Immunohistochemical staining, Staining, Binding Assay, Labeling

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 6. Whole body quantification of TZM–HER2 engagement via MFLI-FRET in vivo imaging. (A) Mice were injected with 20 µg TZM–AF700 alone (M1) or TZM–AF700 and 40 µg TZM–AF750 and subjected to MFLI-FRET imaging at 48 h p.i. Panels show TZM donor maximum intensity ROIs (both soluble and bound probe) and FRET donor fraction (FD%) map (bound and internalized probe) in the tumors (T), livers (LV), and urinary bladders (UB). (B) Histograms of FD% retrieved for each tumor. Numbers represent mean ±SD. (C) Quantification of FD% in the tumors, bladders and livers. Data presented as box indicating 25–75% pixel values, horizontal and vertical lines indicate mean with ±1.5 SD, respectively.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: In Vivo Imaging, Injection, Imaging

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 7. IHC ex vivo validation confirms TZM accumulation in the cancer cells both positive for HER2 and HER3. Consecutive sections of tumors M1–M3 and tumor from untreated mouse were processed for H&E staining, anti-HER2, anti-HER3, and anti-TZM immunohistochemical staining. NovaRED was used as peroxidase substrate (brown stain), tissue was counterstained with methyl green. Scale bar = 100 µm.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Ex Vivo, Biomarker Discovery, Staining, Immunohistochemical staining

Journal: Analytical biochemistry

Article Title: Antibody drug quantitation in coexistence with anti-drug antibodies on nSMOL bioanalysis.

doi: 10.1016/j.ab.2017.11.002

Figure Lengend Snippet: Fig. 1. ADA interference of Trastuzumab bioa- nalysis using nSMOL and ELISA. Concentrations of Trastuzumab coexistent with indicated ratios of ADA against Trastuzumab (0.5 μg/mL) were obtained by nSMOL (A) or ELISA (B). HCA168, HCA176, and HCA177 are human monoclonal antibodies. MAB11130 is mouse monoclonal antibody.

Article Snippet: Anti-Trastuzumab antibodies (HCA169, HCA176, and HCA177) and anti-Bevacizumab antibodies (HCA182 and HCA185) were purchased from Bio-Rad (Raleigh, NC).

Techniques: Enzyme-linked Immunosorbent Assay, Bioprocessing

Journal: Analytical biochemistry

Article Title: Antibody drug quantitation in coexistence with anti-drug antibodies on nSMOL bioanalysis.

doi: 10.1016/j.ab.2017.11.002

Figure Lengend Snippet: Fig. 3. Acid dissociation effect on quantification of ADA-binding Trastuzumab (A) or Bevacizumab (B) using nSMOL. Concentrations of nonacidified- and acidified- treated plasma samples were obtained by nSMOL.

Article Snippet: Anti-Trastuzumab antibodies (HCA169, HCA176, and HCA177) and anti-Bevacizumab antibodies (HCA182 and HCA185) were purchased from Bio-Rad (Raleigh, NC).

Techniques: Binding Assay, Clinical Proteomics

Journal: Analytical biochemistry

Article Title: Antibody drug quantitation in coexistence with anti-drug antibodies on nSMOL bioanalysis.

doi: 10.1016/j.ab.2017.11.002

Figure Lengend Snippet: Fig. 4. Quantification of ErbB2 binding Trastuzumab using nSMOL and ELISA. Concentrations of Trastuzumab coexistent with indicated ratios of ErbB2 were obtained by nSMOL (●) or ELISA (◇).

Article Snippet: Anti-Trastuzumab antibodies (HCA169, HCA176, and HCA177) and anti-Bevacizumab antibodies (HCA182 and HCA185) were purchased from Bio-Rad (Raleigh, NC).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A The four subclasses of Ab25. The constant domains of the heavy chains are depicted in different colors. B The Y-axis shows percentage of IgG+ bacteria. The K D values (nM) with a 95% confidence interval (CI) are shown in each graph with statistical significance up to 99% CI compared to IgG1 due to non-overlapping CIs. C MOP 50 curves of the subclasses, bacteria were opsonized with 15 μg/mL of IgG. The Y-axis shows the percentage of bacteria+ THP-1 cells, while X-axis depicts the ratio of bacteria to phagocytes added (MOP). The MOP 50 values with a 95% CI are shown in each graph with statistical significance shown up to 99% CI compared to IgG1. The data points from ( B ) to ( C ) are from N = 3 independent experiments. D Shows the MOP 50 curves of all the antibody treatments in one graph. Statistical significance compared to IgG1 is ** for IgG3 (99% CI) * for IgG2 and IgG4 (95% CI). E Individual MOP 50 values from the N = 3 independent experiments. F Percentage of THP-1 cells with internalized bacteria across an MOP range. G Amount of bacteria phagocytosed by the whole THP-1 cell population measured as median MFI of bacterial signal across MOP range. H Phagocytosis score for each mAb treatment across MOP 200-12.5. In ( F )–( H ) statistically significant fold-change differences between IgG3 and IgG1 are highlighted in each graph. I , J Ex vivo phagocytosis with neutrophils and monocytes, respectively, at MOP 25. For ( I ) and ( J ), repeated measures one-way ANOVA was used with Dunnett’s post hoc test after multiple comparisons and compared against IgG3. The data points in ( B )–( J ) represent the mean value, and the error bars are in SEM. The phagocytosis score seen in ( I ) and ( J ) is normalized to Ab25 IgG1 for each experiment. In ( F )–( J ) N = 6 independent experiments were performed. R 2 = 0.99 for all non-linear regression curves. Statistical analyses for ( F )–( H ) were done by comparing IgG1 with one-way ANOVA multiple comparisons corrected by Dunnett’s correction test. * denotes p -value < 0.05, ** denotes p -value < 0.01, and p -value > 0.05 is ns. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Bacteria, Ex Vivo

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A The average distance between the center of mass of the M1 protein and the Fc domain of the IgG, IgG3, and IgGh 62 was measured over the three replicates of each system (reported in pink and cyan, respectively). The shades correspond to standard deviations, and the initial distances are shown with dashed lines. B The center of mass of the Fc domain of IgG1 (in blue), IgG3 (in red), and IgGh 62 (green) are depicted at every snapshot of the simulations. The intensity of the colors represents the population of each point. The center of mass of M1 is shown with a gray circle at the origin. The individual distribution of points are reported for C IgG1-M1, D IgG3-M1, and E IgGh 62 -M1 simulations. F The angle changes formed between the Fc domain and protein M1 are reported for IgG1 (in pink), IgG3 (in cyan), and IgGh 62 (green) when both Fabs contact the protein M1. The average values are shown with lines. The vectors forming each angle are depicted in the inset. G The most representative conformation of the M1-IgG3 is reported when dual-Fab binding was observed. The IgG-M1 models in the figure are the starting models for the MD simulation. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Binding Assay

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: The interaction network at the interface of Fab domain and protein M1. The frequency of A Hbonds and B salt bridges formed in every replicate of the IgG1-M1 and IgG3-M1 simulations is reported in red and blue shades, respectively. The interactions are also shown on the structure of C IgG1-M1, D IgG3-M1, and E IgGh 62 -M1 with black lines for both Hbonds and Salt-bridges. The residues involved in the interactions are shown as spheres. The two chains of protein M1 are colored in light and dark purple, and the VH (variable heavy), CH1 (constant heavy domain 1), VL (variable light), and CL (constant domain light chain) domains are shown in light green, dark green, pink, and red, respectively. The IgG-M1 models in the figure are the starting models for the MD simulations. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques:

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A Schematic illustration of hinge-modified variants with Ab25 variable domains, IgG1 backbone but different segments of IgG3 hinge (17, 32, 47 or 62 amino acids). B IgG binding to live SF370 bacteria at different concentrations. C % of phagocytes associating (FITC + ) with bacteria at different MOP’s, while D shows the same but with % internalized and E shows the normalized bacterial signal and F shows the phagocytosis score. In C – F IgG1, IgGh 17, 32, and, 47 hinge variants are assessed. G Shows the phagocytosis score at different MOP’s for the 62 hinge-variant, 47-variant and IgG1. In B-F 15 ug/mL of mAbs was used. The mean is shown in all panels, and the error bars are SEM. The phagocytosis score in E – F is normalized to Ab25 IgG1’s phagocytosis score for each individual experiment ( N = 4 independent experiments). Data in B – G are from N = 4 independent experiments. Experiments in G were done separately from those at B-F. All statistical comparisons were made against the IgGh 47 variant. The statistical test was one-way ANOVA with multiple comparisons corrected by Dunnett’s post hoc test. **** denotes P -value < 0.0001, *** denotes P -value < 0.001, ** denotes P -value < 0.01, * denotes P -value < 0.05 and P -value > 0.05 is ns. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Modification, Binding Assay, Bacteria, Variant Assay

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A Schematic of IgG1, IgG3, and the IgGh 47 . The constant domains of the heavy chains are depicted in different colors: blue for IgG1 and red for IgG3. B MOP 50 curves of the subclasses, bacteria were opsonized with 15 ug/mL of IgG. The Y-axis shows the percentage of bacteria+ THP-1 cells, while the X-axis depicts the ratio of bacteria to phagocytes added (MOP). The MOP 50 values with a 95% CI are shown in each graph, and fold differences over IgG1 (non-overlapping 95% CI) to show statistical significance. The data points are from N = 3 independent experiments. C Shows the MOP 50 curves of all the antibody treatments in one graph, control is IgG1-isotype. C Individual MOP 50 values from N = 3 independent experiments. D Percentage of THP-1 cells with internalized bacteria across the MOP range. E Amount of bacteria that are phagocytosed by the whole THP-1 cell population measured as median MFI of FITC-A (bacterial signal, Oregon Green). F , G Phagocytosis score for each antibody treatment is shown with MOP on the X-axis for F , while in G , the MOP is 200 except for internalization when the MOP is 100. H-I shows Ex vivo phagocytosis with neutrophils and monocytes at MOP 25. The graphs depict %association (normalized to Ab25 IgG1), % internalization, bacterial signal (normalized to Ab25 IgG1) and phagocytosis score (normalized to Ab25 IgG1). For C – G , statistical analysis was done by comparing the treatments to IgG1 with one-way ANOVA multiple comparisons corrected by Dunnett’s post hoc correction test. For H and I , repeated measures one-way ANOVA was used and compared against IgGh 47 . *** denotes P -value < 0.001, ** denotes P -value < 0.01, * denotes P -value < 0.05 and P -value > 0.05 is ns.* denotes P -value < 0.05 and P -value > 0.05 is ns. The data points in ( B )–( I ) represent the mean value, and the error bars are in SEM. In ( D )–( G ), N = 5 independent experiments were performed, while in ( H )–( I ), data was acquired from N = 6 independent experiments and unique donors ( N = 6). Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Bacteria, Control, Ex Vivo

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A Schematic of the animal experiment: pre-treatment 0.4 mg of antibody treatment or PBS 6 hours before infection with AP1, made using Biorender. Animals were sacrificed 24 hours post-infection, and organs and blood were harvested for analysis. B Shows pooled data from two independent experiments of 5 mice in each cohort (total n = 10 mice). Bacterial load (CFU/g) in each organ was determined by serial dilution and viable count determination after overnight incubation (undetectable bacteria was labeled as 14 CFU/g). C shows the percentage of mice positive for bacteria in each respective organ for all treatments with statistical significant differences (or non-significance) compared with IgGh 47 seen above each treatment from test in ( E ). D Cytokine levels (pg/ mL) in plasma were measured using a cytometric bead array at two different occasions analyzing 5 mice/condition at time ( N = 10). E Kaplan-Meier curves are depicted in E for PBS vs. IgGh 47 , IgGh 47 vs. IgG3, and IgGh 47 vs. IgG1 comparisons. The Y-axis shows the probability of bacterial dissemination (adverse outcome) with log-rank (Mantel-Cox) tests used to determine statistical significance, which are also shown for the PBS comparison in the table in ( C ). F – H Ab25-derived peptides identified in the plasma of antibody-treated mice challenged with GAS. Plasma was collected at 24 h postinfection (n = 5 animals/condition; treatments corresponded to Ab25 with an IgG1, IgG3, or IgGh 47 and the peptides were identified by Protein G pulldowns, trypsin digestion and mass spectrometric analysis. Log2 intensity of peptides derived from F the heavy chain, G light chain and H hinge regions of the monoclonal antibodies. In B and D , the median is shown. Statistical analysis was performed, comparing the treatments to the IgGh 47 in B , by Kruskal-Wallis with multiple comparisons and Dunn’s correction test. ** denotes P -value < 0.01, * denotes P < 0.05 and P -value > 0.05 is ns across the figure. In E , log-rank (Mantel-Cox) test was performed, in F – H one-way ANOVA was done with Dunnett’s post hoc test to correct for multiple comparisons. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Infection, Serial Dilution, Incubation, Bacteria, Labeling, Clinical Proteomics, Comparison, Derivative Assay, Bioprocessing

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A Illustration depicting a SARS-CoV-2 virion with the spike protein trimer antigen and the three different clones Ab11, Ab36 and Ab77 which were made into IgGh 47 from an original IgG1. Schematic made using Biorender. B MOP curves with MOP 50 and 95% CI are in brackets for the different mAbs, with statistically significant differences (non-overlapping 95%CI) between the two mAbs are highlighted (*). C Phagocytosis score across the different MOPs. D , E Depicts association and phagocytosis score at MOP 30. Statistical comparison was made by comparing the IgG1 with that of IgGh 47 version with Mann-Whitney U two-tailed test. Across the figure mean is shown and error bars are SEM.*** denotes P -value < 0.001, ** denotes P -value < 0.01 * denotes P < 0.05 and P -value > 0.05 is ns. Positive control is DuomAb IgG3, and negative control is Xolair IgG1. In B-C N = 3 independent experiments were performed while at D and E N = 4 independent experiments were performed. Source data is provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Clone Assay, Comparison, MANN-WHITNEY, Two Tailed Test, Positive Control, Negative Control

Journal: ACS sensors

Article Title: Detecting Disruption of HER2 Membrane Protein Organization in Cell Membranes with Nanoscale Precision.

doi: 10.1021/acssensors.3c01437

Figure Lengend Snippet: Figure 6. Normalized mean fluorescent intensity of SKBR3 cells stained with (a, b) 10 μg/mL Fluorescein-HER2 Ab and (d, e) 10 μg/mL TPE- HER2 Ab after treatment with a range of concentrations of Trastuzumab (0 μg/mL to 100 μg/mL) for (a, d) 24 h and (b, e) different time intervals of 2, 8, and 24 h all in one plot. The data is collected in triplicate, and on average, each data point consists of 30 SKBR3 cells. (*p < 0.05 and **p < 0.01). Schematic of HER2-overexpressing SKBR3 cell membrane indicating the fluorescent response of (c) Fluorescein-HER2 Ab and (f) TPE-HER2 Ab stained HER2 proteins after Trastuzumab treatment.

Article Snippet: After reaching the ideal cell confluency on day 3, different concentrations of Trastuzumab (Selleckchem, A2007) in McCoy media (from 0 to 100 μg/mL) were prepared by serial dilution.

Techniques: Staining, Membrane

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: ERBB2 knockout inhibits autophagy in ERBB2-positive SKBR3 breast cancer cells. ( A ) Knockout of ERBB2 was performed by a double-nickase Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system and demonstrated by Western blotting. Autophagy was measured by Western blotting LC3-II and p62. Chloroquine (CQ, 20 µM) was used to block lysosomal degradation for autophagic flux measurement (the same hereafter). No AA, amino acid starvation. Anti-actin beta (ACTB) was used as a loading control (the same hereafter). Uncropped Western blot images and quantification of the protein band intensities were shown in . ( B ) Autophagy was measured by demonstrating the mRFP-LC3 puncta in cells and counting the numbers of mRFP-LC3 puncta per cell. Data represent or demonstrate the results from at least three independent experiments (the same hereafter). More example images of mRFP-LC3 puncta are shown in the . *, p < 0.5; **, p < 0.01; ***, p < 0.001 (the same hereafter). NIC- Con , control cells; NIC- ERBB2 , ERBB2 knockout cells.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Knock-Out, CRISPR, Western Blot, Blocking Assay, Control

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Overexpression of ERBB2 promotes autophagy in ERBB2-negative and -ERBB2-positive breast cancer cells. Western blotting demonstration of ERBB2 overexpression in the ERBB2-negative MDA-MB-231 cells and the ERBB2-positive MCF7 cells ( A ). ERBB2 overexpression increased the basal levels of autophagy ( B ) and amino acid starvation (No AA)-induced levels of autophagy ( C ). Autophagy was measured by Western blotting LC3-II in the absence and presence of CQ. Uncropped Western blot images and quantification of the protein band intensities were shown in .

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Over Expression, Western Blot

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Effects of lapatinib on autophagy in NIH3T3 cells expressing EGFR or ERBB2. ( A ) Western blotting showed the expression of EGFR and ERBB2. ( B ) Effects of EGFR and ERBB2 on basal autophagy. ( C ) Effects of lapatinib (1 µM) on autophagy in cells expressing vector alone, EGFR or ERBB2. Autophagy was measured by Western blotting LC3-II and SQSTM1/p62 in the absence and presence of CQ. Uncropped Western blot images and quantification of the protein band intensities were shown in . *, p < 0.5; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Plasmid Preparation

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: ERBB2 regulates the expression of autophagy genes. ( A ) Bioinformatics analysis of the mRNA levels of different autophagy genes (ATG genes) in ERBB2-positive and -negative human breast tumor tissues. The data were extracted from the Cancer Genome Atlas (TCGA) database. RE, relative mRNA expression of an autophagy gene in ERBB2-positive breast tumors compared to that in ERBB2-negative breast tumors. The mRNA expression was quantified by RNA-seq by Expectation–Maximization (RSEM) followed by log2 transformation to estimate the gene-level transcription. ( B ) Western blotting demonstration of the effects of ERBB2 overexpression and knockout on protein expression of autophagy proteins ULK1, FIP200 (RB1CC1), BECN1, ATG7, ATG5, and ATG12. Overexpression of ERBB2 in MDA-MB-231 and MCF7 cells and ERBB2 knockout in SKBR3 cells are demonstrated in A and A, respectively. Expression of ATG5 or ATG12 was demonstrated by the level of ATG12–ATG5 complex since it is well known that ATG5 and ATG12 form a conjugate in the form of ATG12–ATG5 during autophagy. Uncropped Western blot images and quantification of the protein band intensities are shown in . NIC- Con , control cells; NIC- ERBB2 , ERBB2 knockout cells.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, RNA Sequencing, Transformation Assay, Western Blot, Over Expression, Knock-Out, Control

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: The Kaplan–Meier plots of the relapse-free survival (RFS)-based survival probability of patients with ERBB2-positive and -negative breast tumors. The plots were downloaded from the Kaplan–Meier Plotter website ( http://kmplot.com/analysis/index.php?p=service&cancer=breast (accessed on 19 November 2019)). ( A ) Data of patients with ATG12 expression ( ATG12 mRNA) in breast tumors. ( B ) Data of patients with MAP1LC3B (LC3B) expression ( MAP1LC3B mRNA) in breast tumors. ( C ) Data of patients with BECN1 expression ( BECN1 mRNA) in breast tumors.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Autophagy is ATG12-dependent in ERBB2-positive and -negative breast cancer cells. Autophagy was measured by Western blotting LC3-II in the absence and presence of CQ. ( A ) Western blotting demonstration of siRNA knockdown of ATG12 in the ERBB2-positive SKBR3 breast cancer cells and ATG12 overexpression in the ERBB2-negative MDA-MB-231 breast cancer cells. ( B ) ATG12 knockdown decreased the basal level and amino acid starvation (No AA)-induced level of autophagy in SKBR3 cells. ( C ) ATG12 overexpression increased the basal level and amino acid starvation (No AA)-induced level of autophagy in MDA-MB-231 cells. Uncropped Western blot images and quantification of the protein band intensities are shown in .

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Western Blot, Knockdown, Over Expression

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: ERBB2 promotion of autophagy inhibits cell death induced by chemotherapy drug Taxol in breast cancer cells. ( A ) ERBB2 knockout decreased autophagy induced by Taxol (paclitaxel, 10 µM) in SKBR3 cells. Autophagy was measured by Western blotting LC3-II in the absence and presence of NH 4 Cl (30 mM). ERBB2 knockout is demonstrated in A. ( B ) Cell death induced by Taxol was increased by ERBB2 knockout or ATG12 knockdown in SKBR3 cells. Knockdown of ATG12 and the related effect on autophagy are demonstrated in A,B. ( C ) ATG12 overexpression increased Taxol-induced autophagy in MDA-MB-231 cells. Overexpression of ERBB2 is demonstrated in A. Autophagy was measured by Western blotting LC3-II in the absence and presence of NH 4 Cl. ( D ) Cell death induced by Taxol was decreased by overexpression of ERBB2 or ATG12 in MDA-MB-231 cells. ATG12 overexpression and the related effect on autophagy are demonstrated in A,C. NH 4 Cl, instead of CQ, was chosen to measure Taxol-induced autophagy because CQ can induce higher cell death than NH 4 Cl ( and unpublished data). Uncropped Western blot images and quantification of the protein band intensities were shown in . NIC- Con , control cells; NIC- ERBB2 , ERBB2 knockout cells. *, p < 0.5; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Knock-Out, Western Blot, Knockdown, Over Expression, Control

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Effects of ERBB2 antibody on ATG12 expression, autophagy, and cell death. An anti-human ERBB2 antibody (Ab) (research-grade trastuzumab biosimilar) was used to treat SKBR3 cells. ( A ) ERBB2 Ab treatment reduced the protein levels of ERBB2 and ATG12. ( B ) Real-time PCR experiments showed that ERBB2 Ab treatment decreased ATG12 mRNA level. ( C ) Immunoprecipitation (IP) demonstration of the interaction between ERBB2 and ATG12. ( D ) ERBB2 Ab treatment inhibited autophagy at the basal and amino acid starvation (No AA)-induced levels. Autophagy was measured by Western blotting LC3-II in the absence and presence of CQ. ( E ) ERBB2 Ab treatment increased cell death without and with Taxol treatment. Uncropped Western blot images and quantification of the protein band intensities are shown in . *, p < 0.5; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: A graphic model for ERBB2 promotion of autophagy and its effect on cancer treatment and outcome to patients. ERBB2 expression upregulates the protein levels autophagy-related (ATG) proteins ULK1, FIP200, ATG5, ATG7, and ATG12 increasing ATG12-dependent autophagy. This leads to treatment resistance of breast cancer and a worse outcome for patients with ERBB2-positive breast cancer. Treatment with ERBB2 antibody (Herceptin or others) causes degradation of ERBB2 and downregulation of ATG12 resulting in autophagy inhibition.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Inhibition

Journal: bioRxiv

Article Title: Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

doi: 10.1101/2021.12.12.472296

Figure Lengend Snippet: (a) Confocal microscopy images of perfused tumoroid MVNs. The arrows indicate focal leaks. The scale bar is 400 μm. (b) Permeability to fluorescent dextran, Trastuzumab, and Cetuximab of tumoroid MVNs compared to control MVNs and (c) effective permeability of those MVNs as a function of applied intravascular pressure; n = 3. (d) Schematic diagram of computation model of the MVNs (left), and model results of interstitial fluid pressure, IFP , as a function of changes in vascular hydraulic conductivity, L p , and matrix permeability, k . (e) Confocal microscopy image of vascular HA in control and SKBR3 MVNs. (f) Gene expression of HA-associated proteins for ECs; n = 3. (g) Quantification of vascular HA concentration in 1 mm biopsies centered 2 mm from the tumoroids in the MVN devices. (h) Effective permeability of MVNs treated with HA-ase, resulting in increased filtration and hydraulic conductivity; n = 3. Significance assessed by one-way ANOVA after confirming a normal distribution of the data; p < 0.05 *, p < 0.01 **, p < 0.001 ***.

Article Snippet: Permeability of fluorescent molecules in the MVNs was measured by confocal microscopy and image analysis with the software ImageJ as previously described , using fluorescein isothiocyanate (FITC, #F4274, Sigma Aldrich), dextran (FITC-conjugated, 70 kDa, 0.1 mg mL −1 , #FD70, Sigma Aldrich), Trastuzumab (Alexa Fluor 488-conjugated, #FAB9589G, 0.1 mg mL −1 , R&D Systems), and Cetuximab (Alex Fluor 647-conjugated, #FAB9577R, 0.1 mg mL −1 , R&D Systems).

Techniques: Confocal Microscopy, Permeability, Control, Gene Expression, Concentration Assay, Filtration

Journal: bioRxiv

Article Title: Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

doi: 10.1101/2021.12.12.472296

Figure Lengend Snippet: (a) Effective permeability of MDA-MB-468 MVNs with intravascular pressure as a result of different treatments targeting stromal HA; n = 3. (b) Normalized MVN concentration of dextran near the tumoroids to that at a distance > 5mm, as measured by proxy of fluorescence intensity, as a result of treatment with HA-ase, a CD44 blocking antibody (“CD44 block”), and a TGFβ blocking antibody (“TGFβ block”), and (c) representative confocal images of treated tumoroid (circles) MVNs perfused with dextran. (d) Quantification of CD44 isoforms and TGFβ in 1 mm biopsies centered at tumoroids in the MVN devices, and (e) quantification of stromal HA concentrations in the tumoroids as a result of the stromal HA-targeting treatments. (f) Confocal microscopy images of SKBR3 tumoroid cryosections showing increased Trastuzumab penetration after HA degradation. (g) Cell death in the tumoroids as a function of combined treatment with Trastuzumab + Cetuximab (“mAB”) with different targeting strategies for stromal HA, as measured by proxy of fluorescent SYTOX intensity. Significance assessed by one-way ANOVA after confirming a normal distribution of the data; p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Article Snippet: Permeability of fluorescent molecules in the MVNs was measured by confocal microscopy and image analysis with the software ImageJ as previously described , using fluorescein isothiocyanate (FITC, #F4274, Sigma Aldrich), dextran (FITC-conjugated, 70 kDa, 0.1 mg mL −1 , #FD70, Sigma Aldrich), Trastuzumab (Alexa Fluor 488-conjugated, #FAB9589G, 0.1 mg mL −1 , R&D Systems), and Cetuximab (Alex Fluor 647-conjugated, #FAB9577R, 0.1 mg mL −1 , R&D Systems).

Techniques: Permeability, Concentration Assay, Fluorescence, Blocking Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

doi: 10.1101/2021.12.12.472296

Figure Lengend Snippet: (a) Confocal microscopy image of cell death signal in an MCF7 tumoroid in the MVN device after treatment with Trastuzumab and Cetuximab (“mAB”). (b) Change in size of TC line tumoroids after combination mAB treatment with stromal HA-targeting therapeutic strategies. (c) Normalized cell death signal in TC line tumoroids in well plates treated with Trastuzumab (“T”), Cetuximab (“C”), or both (“T+C”). The concentration indicted is in μg mL −1 . Significance assessed by one-way ANOVA; p < 0.5 *, p < 0.001 ***, p < 0.0001 ****.

Article Snippet: Permeability of fluorescent molecules in the MVNs was measured by confocal microscopy and image analysis with the software ImageJ as previously described , using fluorescein isothiocyanate (FITC, #F4274, Sigma Aldrich), dextran (FITC-conjugated, 70 kDa, 0.1 mg mL −1 , #FD70, Sigma Aldrich), Trastuzumab (Alexa Fluor 488-conjugated, #FAB9589G, 0.1 mg mL −1 , R&D Systems), and Cetuximab (Alex Fluor 647-conjugated, #FAB9577R, 0.1 mg mL −1 , R&D Systems).

Techniques: Confocal Microscopy, Concentration Assay

Journal: bioRxiv

Article Title: Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

doi: 10.1101/2021.12.12.472296

Figure Lengend Snippet: (a) Projected confocal microscopy images of patient-derived tumoroids show high levels of vascularization and different levels of cancer vascular colonization. (b) Effective permeability of patient-derived tumoroid MVNs with intravascular pressure; n = 3. (c) Quantification of stromal HA concentrations in the tumoroids as a result of stromal and vascular HA-targeting treatments, and (d) resulting cell death in the tumoroids when the treatments are administered in combination with Trastuzumab and Cetuximab, as measured by proxy of fluorescent SYTOX intensity. Significance assessed by one-way ANOVA after confirming a normal distribution of the data; p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Article Snippet: Permeability of fluorescent molecules in the MVNs was measured by confocal microscopy and image analysis with the software ImageJ as previously described , using fluorescein isothiocyanate (FITC, #F4274, Sigma Aldrich), dextran (FITC-conjugated, 70 kDa, 0.1 mg mL −1 , #FD70, Sigma Aldrich), Trastuzumab (Alexa Fluor 488-conjugated, #FAB9589G, 0.1 mg mL −1 , R&D Systems), and Cetuximab (Alex Fluor 647-conjugated, #FAB9577R, 0.1 mg mL −1 , R&D Systems).

Techniques: Confocal Microscopy, Derivative Assay, Permeability

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A The four subclasses of Ab25. The constant domains of the heavy chains are depicted in different colors. B The Y-axis shows percentage of IgG+ bacteria. The K D values (nM) with a 95% confidence interval (CI) are shown in each graph with statistical significance up to 99% CI compared to IgG1 due to non-overlapping CIs. C MOP 50 curves of the subclasses, bacteria were opsonized with 15 μg/mL of IgG. The Y-axis shows the percentage of bacteria+ THP-1 cells, while X-axis depicts the ratio of bacteria to phagocytes added (MOP). The MOP 50 values with a 95% CI are shown in each graph with statistical significance shown up to 99% CI compared to IgG1. The data points from ( B ) to ( C ) are from N = 3 independent experiments. D Shows the MOP 50 curves of all the antibody treatments in one graph. Statistical significance compared to IgG1 is ** for IgG3 (99% CI) * for IgG2 and IgG4 (95% CI). E Individual MOP 50 values from the N = 3 independent experiments. F Percentage of THP-1 cells with internalized bacteria across an MOP range. G Amount of bacteria phagocytosed by the whole THP-1 cell population measured as median MFI of bacterial signal across MOP range. H Phagocytosis score for each mAb treatment across MOP 200-12.5. In ( F )–( H ) statistically significant fold-change differences between IgG3 and IgG1 are highlighted in each graph. I , J Ex vivo phagocytosis with neutrophils and monocytes, respectively, at MOP 25. For ( I ) and ( J ), repeated measures one-way ANOVA was used with Dunnett’s post hoc test after multiple comparisons and compared against IgG3. The data points in ( B )–( J ) represent the mean value, and the error bars are in SEM. The phagocytosis score seen in ( I ) and ( J ) is normalized to Ab25 IgG1 for each experiment. In ( F )–( J ) N = 6 independent experiments were performed. R 2 = 0.99 for all non-linear regression curves. Statistical analyses for ( F )–( H ) were done by comparing IgG1 with one-way ANOVA multiple comparisons corrected by Dunnett’s correction test. * denotes p -value < 0.05, ** denotes p -value < 0.01, and p -value > 0.05 is ns. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Bacteria, Ex Vivo

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A The average distance between the center of mass of the M1 protein and the Fc domain of the IgG, IgG3, and IgGh 62 was measured over the three replicates of each system (reported in pink and cyan, respectively). The shades correspond to standard deviations, and the initial distances are shown with dashed lines. B The center of mass of the Fc domain of IgG1 (in blue), IgG3 (in red), and IgGh 62 (green) are depicted at every snapshot of the simulations. The intensity of the colors represents the population of each point. The center of mass of M1 is shown with a gray circle at the origin. The individual distribution of points are reported for C IgG1-M1, D IgG3-M1, and E IgGh 62 -M1 simulations. F The angle changes formed between the Fc domain and protein M1 are reported for IgG1 (in pink), IgG3 (in cyan), and IgGh 62 (green) when both Fabs contact the protein M1. The average values are shown with lines. The vectors forming each angle are depicted in the inset. G The most representative conformation of the M1-IgG3 is reported when dual-Fab binding was observed. The IgG-M1 models in the figure are the starting models for the MD simulation. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Binding Assay

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: The interaction network at the interface of Fab domain and protein M1. The frequency of A Hbonds and B salt bridges formed in every replicate of the IgG1-M1 and IgG3-M1 simulations is reported in red and blue shades, respectively. The interactions are also shown on the structure of C IgG1-M1, D IgG3-M1, and E IgGh 62 -M1 with black lines for both Hbonds and Salt-bridges. The residues involved in the interactions are shown as spheres. The two chains of protein M1 are colored in light and dark purple, and the VH (variable heavy), CH1 (constant heavy domain 1), VL (variable light), and CL (constant domain light chain) domains are shown in light green, dark green, pink, and red, respectively. The IgG-M1 models in the figure are the starting models for the MD simulations. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques:

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A Schematic illustration of hinge-modified variants with Ab25 variable domains, IgG1 backbone but different segments of IgG3 hinge (17, 32, 47 or 62 amino acids). B IgG binding to live SF370 bacteria at different concentrations. C % of phagocytes associating (FITC + ) with bacteria at different MOP’s, while D shows the same but with % internalized and E shows the normalized bacterial signal and F shows the phagocytosis score. In C – F IgG1, IgGh 17, 32, and, 47 hinge variants are assessed. G Shows the phagocytosis score at different MOP’s for the 62 hinge-variant, 47-variant and IgG1. In B-F 15 ug/mL of mAbs was used. The mean is shown in all panels, and the error bars are SEM. The phagocytosis score in E – F is normalized to Ab25 IgG1’s phagocytosis score for each individual experiment ( N = 4 independent experiments). Data in B – G are from N = 4 independent experiments. Experiments in G were done separately from those at B-F. All statistical comparisons were made against the IgGh 47 variant. The statistical test was one-way ANOVA with multiple comparisons corrected by Dunnett’s post hoc test. **** denotes P -value < 0.0001, *** denotes P -value < 0.001, ** denotes P -value < 0.01, * denotes P -value < 0.05 and P -value > 0.05 is ns. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Modification, Binding Assay, Bacteria, Variant Assay

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A Schematic of IgG1, IgG3, and the IgGh 47 . The constant domains of the heavy chains are depicted in different colors: blue for IgG1 and red for IgG3. B MOP 50 curves of the subclasses, bacteria were opsonized with 15 ug/mL of IgG. The Y-axis shows the percentage of bacteria+ THP-1 cells, while the X-axis depicts the ratio of bacteria to phagocytes added (MOP). The MOP 50 values with a 95% CI are shown in each graph, and fold differences over IgG1 (non-overlapping 95% CI) to show statistical significance. The data points are from N = 3 independent experiments. C Shows the MOP 50 curves of all the antibody treatments in one graph, control is IgG1-isotype. C Individual MOP 50 values from N = 3 independent experiments. D Percentage of THP-1 cells with internalized bacteria across the MOP range. E Amount of bacteria that are phagocytosed by the whole THP-1 cell population measured as median MFI of FITC-A (bacterial signal, Oregon Green). F , G Phagocytosis score for each antibody treatment is shown with MOP on the X-axis for F , while in G , the MOP is 200 except for internalization when the MOP is 100. H-I shows Ex vivo phagocytosis with neutrophils and monocytes at MOP 25. The graphs depict %association (normalized to Ab25 IgG1), % internalization, bacterial signal (normalized to Ab25 IgG1) and phagocytosis score (normalized to Ab25 IgG1). For C – G , statistical analysis was done by comparing the treatments to IgG1 with one-way ANOVA multiple comparisons corrected by Dunnett’s post hoc correction test. For H and I , repeated measures one-way ANOVA was used and compared against IgGh 47 . *** denotes P -value < 0.001, ** denotes P -value < 0.01, * denotes P -value < 0.05 and P -value > 0.05 is ns.* denotes P -value < 0.05 and P -value > 0.05 is ns. The data points in ( B )–( I ) represent the mean value, and the error bars are in SEM. In ( D )–( G ), N = 5 independent experiments were performed, while in ( H )–( I ), data was acquired from N = 6 independent experiments and unique donors ( N = 6). Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Bacteria, Control, Ex Vivo

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A Schematic of the animal experiment: pre-treatment 0.4 mg of antibody treatment or PBS 6 hours before infection with AP1, made using Biorender. Animals were sacrificed 24 hours post-infection, and organs and blood were harvested for analysis. B Shows pooled data from two independent experiments of 5 mice in each cohort (total n = 10 mice). Bacterial load (CFU/g) in each organ was determined by serial dilution and viable count determination after overnight incubation (undetectable bacteria was labeled as 14 CFU/g). C shows the percentage of mice positive for bacteria in each respective organ for all treatments with statistical significant differences (or non-significance) compared with IgGh 47 seen above each treatment from test in ( E ). D Cytokine levels (pg/ mL) in plasma were measured using a cytometric bead array at two different occasions analyzing 5 mice/condition at time ( N = 10). E Kaplan-Meier curves are depicted in E for PBS vs. IgGh 47 , IgGh 47 vs. IgG3, and IgGh 47 vs. IgG1 comparisons. The Y-axis shows the probability of bacterial dissemination (adverse outcome) with log-rank (Mantel-Cox) tests used to determine statistical significance, which are also shown for the PBS comparison in the table in ( C ). F – H Ab25-derived peptides identified in the plasma of antibody-treated mice challenged with GAS. Plasma was collected at 24 h postinfection (n = 5 animals/condition; treatments corresponded to Ab25 with an IgG1, IgG3, or IgGh 47 and the peptides were identified by Protein G pulldowns, trypsin digestion and mass spectrometric analysis. Log2 intensity of peptides derived from F the heavy chain, G light chain and H hinge regions of the monoclonal antibodies. In B and D , the median is shown. Statistical analysis was performed, comparing the treatments to the IgGh 47 in B , by Kruskal-Wallis with multiple comparisons and Dunn’s correction test. ** denotes P -value < 0.01, * denotes P < 0.05 and P -value > 0.05 is ns across the figure. In E , log-rank (Mantel-Cox) test was performed, in F – H one-way ANOVA was done with Dunnett’s post hoc test to correct for multiple comparisons. Source data are provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Infection, Serial Dilution, Incubation, Bacteria, Labeling, Clinical Proteomics, Comparison, Derivative Assay, Bioprocessing

Journal: Nature Communications

Article Title: The hinge-engineered IgG1-IgG3 hybrid subclass IgGh 47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

doi: 10.1038/s41467-024-47928-8

Figure Lengend Snippet: A Illustration depicting a SARS-CoV-2 virion with the spike protein trimer antigen and the three different clones Ab11, Ab36 and Ab77 which were made into IgGh 47 from an original IgG1. Schematic made using Biorender. B MOP curves with MOP 50 and 95% CI are in brackets for the different mAbs, with statistically significant differences (non-overlapping 95%CI) between the two mAbs are highlighted (*). C Phagocytosis score across the different MOPs. D , E Depicts association and phagocytosis score at MOP 30. Statistical comparison was made by comparing the IgG1 with that of IgGh 47 version with Mann-Whitney U two-tailed test. Across the figure mean is shown and error bars are SEM.*** denotes P -value < 0.001, ** denotes P -value < 0.01 * denotes P < 0.05 and P -value > 0.05 is ns. Positive control is DuomAb IgG3, and negative control is Xolair IgG1. In B-C N = 3 independent experiments were performed while at D and E N = 4 independent experiments were performed. Source data is provided as a Source Data file.

Article Snippet: To generate heavy chain plasmids of the different subclasses, pVITRO1-Trastuzumab-IgG2 (Addgene plasmid #61884; RRID: Addgene_61884), IgG3 (Addgene plasmid #61885; RRID:Addgene_61885), and IgG4 (Addgene plasmid #61887; RRID:Addgene_61887) was a gift from Andrew Beavils lab .

Techniques: Clone Assay, Comparison, MANN-WHITNEY, Two Tailed Test, Positive Control, Negative Control

Journal: Nature Communications

Article Title: Unlocking the potential of allogeneic Vδ2 T cells for ovarian cancer therapy through CD16 biomarker selection and CAR/IL-15 engineering

doi: 10.1038/s41467-023-42619-2

Figure Lengend Snippet: a – g In vitro ADCC assay. Three human ovarian cancer cell lines (OVCAR3-FG, OVCAR8-FG, and SKOV3-FG) and four effector cells (Vδ2T, MCAR-Vδ2T, MCAR15-Vδ2T, and MCAR-T as a benchmark control) were included in the study. The same CD16 Hi donor PBMCs were used to generate all 4 types of effector cells. a Experimental design. Data were collected at 24 h after co-culture. Anti-HER2 Ab: monoclonal antibody trastuzumab. b FACS detection of HER2 expression on the indicated tumor cell lines. c Killing of OVCAR3-FG tumor cells by Vδ2T cells in the presence of titrated amounts of either isotype control or anti-HER2 Ab ( n = 3). Tumor cell killing data of ( d ) OVCAR3-FG, ( e ) OVCAR8-FG, and ( f ) SKOV3-FG in the presence or absence of anti-HER2 Ab (anti-HER2 Ab concentration = 0.1 μg/mL; E:T ratio = 0.5:1; n = 3). g ELISA measurements of IFN-γ production at 24 h in the presence or absence of anti-HER2 Ab (anti-HER2 Ab concentration = 0.1 μg/mL; E:T ratio = 1:1; n = 3). h , i In vitro repeated tumor cell challenge assay studying ADCC. h Experimental design. Effector cells were mixed with tumor cells and rechallenged every 3 days. Tumor cell killing data was measured at 24 h post co-culture with or without the addition of anti-HER2 antibody (anti-HER2 Ab concentration = 0.1 μg/mL; E:T ratio = 2:1; n = 3). i KO OVCAR3-FG tumor cell killing data collected over time. Representative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by Student’s t test ( c ) or by one-way ANOVA ( d – g ). Sour c e data and exact p values are provided as a Source Data file.

Article Snippet: InVivo SIM anti-human HER2 (Trastuzumab Biosimilar, Cat. SIM0005) was purchased from BioXCell.

Techniques: In Vitro, ADCC Assay, Control, Co-Culture Assay, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay