source  (R&D Systems)

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: ADPs bind with high affinity to recombinant human ADAM8 (rHuADAM8). The half-maximal effective concentration (EC50) obtained in ELISA and binding kinetics (k a , k d , and K D ) obtained through Biacore studies for each ADP are shown.

Article Snippet: Initial pepsin/protease XIII digestion of rHuADAM8 (AA1-497, 1031-AD-020, R&D Systems), LC-MS, and MS/MS data search against human ADAM8 in Mascot revealed 69.7–74% protein coverage, consistent with autocatalytic removal of the PRO.

Techniques: Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

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: ADPs have potent in vitro dual metalloprotease and disintegrin (MP and DI) domain inhibitory activity. ( A ) ADAM8 MP activity was assessed in the presence of each ADP vs. its isotype-matched control IgG by measuring the release of soluble CD23 from the surface of HEK293 cells ectopically overexpressing both ADAM8 and CD23. After overnight antibody (Ab) treatment, conditioned cell media was tested for cleaved CD23 via the detection of its HA-tag in immunoblotting; images were quantified using densitometry. ( B ) ADAM8 DI activity was evaluated in the presence of each ADP vs. control IgG in assays measuring binding of α9β1-Integrin-expressing CHO cells to plates coated with recombinant human ADAM8 (rHuADAM8). Mean MP and DI activity level ± standard deviation (S.D.) from 3 independent experiments is graphed in A and B, respectively. The dashed line represents the level of activity in the presence of MAB1031. Ab-mediated percent inhibition of activity in each case was calculated as a decrease from control IgG levels, which were set to 100%. Mean percent inhibition for each ADP and for MAB1031 ± S.D. is given in red over the respective activity bars.

Article Snippet: Initial pepsin/protease XIII digestion of rHuADAM8 (AA1-497, 1031-AD-020, R&D Systems), LC-MS, and MS/MS data search against human ADAM8 in Mascot revealed 69.7–74% protein coverage, consistent with autocatalytic removal of the PRO.

Techniques: In Vitro, Activity Assay, Control, Western Blot, Binding Assay, Expressing, Recombinant, Standard Deviation, Inhibition

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: ADPs bind to native ADAM8. Flow cytometry was performed using HEK293 cells expressing full-length ADAM8 (HEK-A8) vs. an empty vector control DNA (HEK-EV) and decreasing antibody (Ab) concentrations. Mean Fluorescent Intensity (MFI) indicates extent of binding of each Ab.

Article Snippet: Anti-ADAM8 Abs were made by the hybridoma method [ ] using recombinant human ADAM8 [rHuADAM8] (AD8-H5223, ACRO Biosystems, Newark, DE, USA) injection into Balb/c and SJL mice and fusion of B cells to Sp2/0-Ag14 (RRID: CVCL_2199) myeloma cells.

Techniques: Flow Cytometry, Expressing, Plasmid Preparation, Control, Binding Assay

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: ADPs bind with high affinity to recombinant human ADAM8 (rHuADAM8). The half-maximal effective concentration (EC50) obtained in ELISA and binding kinetics (k a , k d , and K D ) obtained through Biacore studies for each ADP are shown.

Article Snippet: Anti-ADAM8 Abs were made by the hybridoma method [ ] using recombinant human ADAM8 [rHuADAM8] (AD8-H5223, ACRO Biosystems, Newark, DE, USA) injection into Balb/c and SJL mice and fusion of B cells to Sp2/0-Ag14 (RRID: CVCL_2199) myeloma cells.

Techniques: Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

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: 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: Anti-ADAM8 Abs were made by the hybridoma method [ ] using recombinant human ADAM8 [rHuADAM8] (AD8-H5223, ACRO Biosystems, Newark, DE, USA) injection into Balb/c and SJL mice and fusion of B cells to Sp2/0-Ag14 (RRID: CVCL_2199) myeloma cells.

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: ADPs have potent in vitro dual metalloprotease and disintegrin (MP and DI) domain inhibitory activity. ( A ) ADAM8 MP activity was assessed in the presence of each ADP vs. its isotype-matched control IgG by measuring the release of soluble CD23 from the surface of HEK293 cells ectopically overexpressing both ADAM8 and CD23. After overnight antibody (Ab) treatment, conditioned cell media was tested for cleaved CD23 via the detection of its HA-tag in immunoblotting; images were quantified using densitometry. ( B ) ADAM8 DI activity was evaluated in the presence of each ADP vs. control IgG in assays measuring binding of α9β1-Integrin-expressing CHO cells to plates coated with recombinant human ADAM8 (rHuADAM8). Mean MP and DI activity level ± standard deviation (S.D.) from 3 independent experiments is graphed in A and B, respectively. The dashed line represents the level of activity in the presence of MAB1031. Ab-mediated percent inhibition of activity in each case was calculated as a decrease from control IgG levels, which were set to 100%. Mean percent inhibition for each ADP and for MAB1031 ± S.D. is given in red over the respective activity bars.

Article Snippet: Anti-ADAM8 Abs were made by the hybridoma method [ ] using recombinant human ADAM8 [rHuADAM8] (AD8-H5223, ACRO Biosystems, Newark, DE, USA) injection into Balb/c and SJL mice and fusion of B cells to Sp2/0-Ag14 (RRID: CVCL_2199) myeloma cells.

Techniques: In Vitro, Activity Assay, Control, Western Blot, Binding Assay, Expressing, Recombinant, Standard Deviation, Inhibition

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: 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, including MP, DI, CRD, and ELD) using the crystal structure of ADAM22 as template and Swiss-model software (2003). Regions of ADP2, ADP3, and ADP13 binding, including overlapping sequences, identified through hydrogen/deuterium exchange–mass spectrometry (HDX-MS) analysis are indicated. MP with active catalytic site, DI with integrin-binding region, and hypervariable region (HVR) of CDR are shown.

Article Snippet: Anti-ADAM8 Abs were made by the hybridoma method [ ] using recombinant human ADAM8 [rHuADAM8] (AD8-H5223, ACRO Biosystems, Newark, DE, USA) injection into Balb/c and SJL mice and fusion of B cells to Sp2/0-Ag14 (RRID: CVCL_2199) myeloma cells.

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

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: 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: Anti-ADAM8 Abs were made by the hybridoma method [ ] using recombinant human ADAM8 [rHuADAM8] (AD8-H5223, ACRO Biosystems, Newark, DE, USA) injection into Balb/c and SJL mice and fusion of B cells to Sp2/0-Ag14 (RRID: CVCL_2199) myeloma cells.

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

Journal: Cancer Cell International

Article Title: ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients

doi: 10.1186/s12935-023-03024-3

Figure Lengend Snippet: Development of an anti-ADAM8 ADP2 IHC assay and breast CCM. A ADAM8 protein levels in breast lines. Protein extracts (30 μg) from breast lines MCF10A, SUM149, MB-231 and MB-231-LUC, and control lines HEK-A8 and HEK-EV, grown in 2D or 3D as indicated, were subjected to immunoblotting with an anti-ADAM8 antibody (LS-C20181), which detected precursor (Proform) and active ADAM8; β-actin was used as loading control. A representative blot of two independent experiments with similar results is shown. To create a breast CCM with a gradient of active ADAM8, MCF10A-2D, MB-231-2D and MB-231-3D were selected; HEK-EV-2D and HEK-A8-2D were included as negative and positive controls, respectively. B ADPs did not detect ADAM8 in breast lines under IHC conditions optimized for the commercial anti-human ADAM8 LS-B4068 antibody. CCM slides were subjected to IHC using conditions for the rabbit LS-B4068 antibody and either LS-B4068 or our newly developed ADP antibodies. Representative images of staining in HEK-A8-2D and MB-231-3D cells are shown. Pink color is due to Matrigel used (only) in this early CCM. C–E ADP2 under ADP-optimized conditions provides superior ADAM8 detection compared to LS-B4068. CCM IHC staining was performed using the final optimized conditions for the ADP mAbs and either our top diagnostic mAb ADP2 (1:100) or IgG2b (1:100) isotype-matched control for non-specific staining ( C ). The CCM breast lines (red box) displayed ADAM8 levels consistent with those seen in Western blotting (Part A ). Values given below were determined by densitometry of immunoblots with extracts from independent cultures loaded at various concentrations to ensure a linear range of quantitation, and are presented relative to MCF10A (set to 1.0). IHC of the CCM with LS-B4068 (1:50) and its optimal conditions shows weaker staining than that observed with ADP2 ( D ). IHC of CCM with LS-B4068 (1:100) using ADP conditions shows poorer staining than with its optimal conditions ( E ). Images are at 40× magnification. MB-231, MDA-MB-231

Article Snippet: Competition studies were carried out using recombinant human ADAM8 (rHuA8) protein purchased from ACRO Biosystems [amino acids (AA) 17-497, AD8-H5223, Newark, DE, USA] and from R&D Systems (AA1-497, 1031-AD, Minneapolis, MN, USA).

Techniques: Control, Western Blot, Staining, Immunohistochemistry, Diagnostic Assay, Quantitation Assay

Journal: Cancer Cell International

Article Title: ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients

doi: 10.1186/s12935-023-03024-3

Figure Lengend Snippet: The ADP2 IHC assay detects a range of ADAM8 levels with high specificity and reproducibility. A ADP2 staining is specific for ADAM8 in competition analyses. ADP2 was pre-incubated overnight at 4 °C at 1:1000 dilution in the absence or presence of 1×, 10× or 100× molar equivalents of purified recombinant human ADAM8 protein (rHuA8), and used in IHC of HEK-A8-2D and MB-231-3D cells. IgG2b was employed as isotype control. ADP2 staining was reduced in a dose-dependent manner in the presence of rHuA8, demonstrating the assay’s specificity for ADAM8. B ADP2 has excellent range and linearity of ADAM8 detection. IHC was performed using slides of the breast CCM and a range of ADP2 dilutions from 1:50 to 1:120,000 under the optimized ADP staining conditions vs isotype control IgG2b at 1:50. ADP2 detected both very low and very high levels of ADAM8 in a dose-dependent manner. C ADP2 detects ADAM8 in PDX tumor tissue samples at levels that are within the range of the CCM. IHC was performed using ADP2 at dilutions of 1:50, 1:100 and 1:500 vs isotype control IgG2b at 1:50 and ADAM8-expressing TNBC PDX samples 5998, 3561, and 4849 vs the breast CCM. All 3 PDX samples displayed strong ADAM8 staining that was in the range of that seen in the breast lines of the CCM, indicating the latter is suitable for scoring of more complex tissue samples. Representative images of staining with ADP2 1:50 dilution, which was identified as optimal for tissue sample analysis, are shown. D ADP2 reproducibly detects ADAM8 in PDX tumor tissue samples. Consecutively cut slide sets of each PDX were processed on different days (3–4 days apart), and demonstrated equal staining, indicating the reproducibility of the ADP2 IHC assay. Representative images of PDX3561 are shown. Images are at 40× magnification. MB-231, MDA-MB-231

Article Snippet: Competition studies were carried out using recombinant human ADAM8 (rHuA8) protein purchased from ACRO Biosystems [amino acids (AA) 17-497, AD8-H5223, Newark, DE, USA] and from R&D Systems (AA1-497, 1031-AD, Minneapolis, MN, USA).

Techniques: Staining, Incubation, Purification, Recombinant, Control, Expressing

Journal: Cancer Cell International

Article Title: ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients

doi: 10.1186/s12935-023-03024-3

Figure Lengend Snippet: ADAM8 is expressed in a third of all breast cancers

Article Snippet: Competition studies were carried out using recombinant human ADAM8 (rHuA8) protein purchased from ACRO Biosystems [amino acids (AA) 17-497, AD8-H5223, Newark, DE, USA] and from R&D Systems (AA1-497, 1031-AD, Minneapolis, MN, USA).

Techniques:

Journal: Cancer Cell International

Article Title: ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients

doi: 10.1186/s12935-023-03024-3

Figure Lengend Snippet: ADAM8 is expressed in all breast cancer subtypes. Freshly cut consecutive TMA slides containing breast cancer patient primary tumor samples were subjected to IHC with ADP2 vs isotype-matched control IgG2b at 1:50 dilutions. H&E staining was used to confirm tissue quality and exclude depleted samples. A Representative images of ADP2-stained TNBC samples with increasing ADAM8 expression and associated H-scores are shown. Staining with IgG2b was negative as expected (not shown). Upper panels: whole core images, scale bar: 200 μm; lower panels: magnified images of same samples, scale bar: 50 μm. B Representative images of ADP2 IHC staining for ADAM8 in breast cancer patient samples of non-TNBC HR−/HER2+, HR+/HER2− and HR+/HER2+ subtypes. Specimens with high H-scores (e.g., 250–300) are shown. Upper panels: whole core images, scale bar: 200 μm; lower panels: magnified images of same samples, scale bar: 50 μm

Article Snippet: Competition studies were carried out using recombinant human ADAM8 (rHuA8) protein purchased from ACRO Biosystems [amino acids (AA) 17-497, AD8-H5223, Newark, DE, USA] and from R&D Systems (AA1-497, 1031-AD, Minneapolis, MN, USA).

Techniques: Control, Staining, Expressing, Immunohistochemistry

Journal: Cancer Cell International

Article Title: ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients

doi: 10.1186/s12935-023-03024-3

Figure Lengend Snippet: Summary of patient demographic and disease characteristics

Article Snippet: Competition studies were carried out using recombinant human ADAM8 (rHuA8) protein purchased from ACRO Biosystems [amino acids (AA) 17-497, AD8-H5223, Newark, DE, USA] and from R&D Systems (AA1-497, 1031-AD, Minneapolis, MN, USA).

Techniques: Significance Assay

Journal: Cancer Cell International

Article Title: ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients

doi: 10.1186/s12935-023-03024-3

Figure Lengend Snippet: High ADAM8 expression identifies a group of HR+/HER2− breast cancer patients with poor survival. A 10-year Cox proportional hazards model for patients with the HR+/HER2− breast cancer subtype (n = 300 patients) was run using SAS 9.4 software to allow for age and race adjusted survival analysis, along with an adjusted survival curve based on ADAM8 expression levels from the ADP2 IHC assay. Survival was calculated as time from surgery until death or censored at time from surgery until last follow-up; if a patient survived beyond 10 years, they were censored at 10 years. Hazard Ratios with 95% confidence intervals (CI) and significance values of the parameter estimates using Wald’s Chi-Square test are presented. H-scores of 200 to 300, obtained through the ADP2 IHC assay, identified a group of patients at risk of poor outcome

Article Snippet: Competition studies were carried out using recombinant human ADAM8 (rHuA8) protein purchased from ACRO Biosystems [amino acids (AA) 17-497, AD8-H5223, Newark, DE, USA] and from R&D Systems (AA1-497, 1031-AD, Minneapolis, MN, USA).

Techniques: Expressing, Software