Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer.

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: Figure 1. ADPs bind to five epitope clusters on ADAM8. Diagram indicating the epitope clusters for ADP binding on human ADAM8, and their partial overlap identified based on epitope binning using competitive ELISA.

Article Snippet: As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed.

Techniques: Binding Assay, Competitive ELISA

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer.

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: Figure 4. ADPs with in vivo anti-tumor activity bind to the ADAM8 DI. (A) ADP2, ADP3, or ADP13 binding to HEK293 cells expressing full-length ADAM8 (HEK-A8) vs. remnant form ADAM8 (HEK-REM) was assessed by flow cytometry; HEK293 cells expressing empty vector DNA (HEK-EV) were used as a negative control. Representative histograms of three independent runs are shown. (B) Schematic representation of the ADAM8 constructs used in part A, with domain information, amino acid (AA) numbers, and immunogen used for ADP generation indicated. The broad epitope region for ADP2, ADP3, and ADP13 binding to ADAM8, identified by the flow cytometry analysis in part A, is indicated (striped box). ADAM8 domains: PRO—prodomain; MP—metalloproteinase; DI—disintegrin; CRD—cysteine-rich; ELD—EGF-like; TM—transmembrane; CYTO—cytoplasmic. (C) Three-dimensional model of the predicted ADAM8 extracellular structure (residues 195-647,

Article Snippet: As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed.

Techniques: In Vivo, Activity Assay, Binding Assay, Expressing, Flow Cytometry, Plasmid Preparation, Negative Control, Construct

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer.

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: Figure 5. Amino acids (AAs) within the ADAM8 DI mediating ADP2 and ADP13 binding. (A) AA residues important for ADP2 and ADP13 binding to ADAM8 were identified using alanine (ALA) scanning mutagenesis plus flow cytometry. Mean binding reactivity (in duplicate samples) of ADP2 or ADP13 antigen−binding fragments (Fabs) to ADAM8 protein mutated at the indicated residues (mutation) within the MP and DI vs. binding of a positive control ADAM8 Ab (Control A8 Ab), which binds outside the MP and DI regions and is therefore unaffected, is presented as a percentage of binding to wild−type (WT) ADAM8. The range of binding reactivity (maximum–minimum) in each case is indicated in parentheses. AAs identified as critical for binding (i.e., those for which Control A8 Ab binding was >70% of WT but test Ab binding was <20% of WT binding) are shown in red boxes. Blue boxes show residues of secondary importance, i.e., AAs in close proximity to critical residues whose mutation led to a substantial (although not <20% of WT) reduction in binding. Epitope AAs for ADP2 (B) and ADP13 (C) Fab binding, identified through mutagenesis, are indicated on a crystal structure model of the ADAM8 ectodomain based on the structure of vascular apoptosis−inducing protein−1.

Article Snippet: As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed.

Techniques: Binding Assay, Mutagenesis, Flow Cytometry, Positive Control, Control