Journal: Molecular Oncology

Article Title: A novel diphtheria toxin‐based bivalent human EGF fusion toxin for treatment of head and neck squamous cell carcinoma

doi: 10.1002/1878-0261.12919

Figure Lengend Snippet: In vitro binding affinity and efficacy analysis of the mono‐EGF‐IT and bi‐EGF‐IT to the human EGFR + HNSCC cell line. (A) Binding affinity analysis of the mono‐EGF‐IT and bi‐EGF‐IT to the human EGFR + HNSCC cell line, Cal27, using flow cytometry. Anti‐human EGFR mAb was used as a positive control, and biotinylated anti‐murine PD‐1 immunotoxin served as a negative background control for protein biotinylation. The data are representative of three individual experiments. (B) K D determination of the human EGF fusion toxins for Cal27 cells using flow cytometry and nonlinear least‐squares fitting. The MFI was plotted over a wide range of biotinylated mono‐EGF‐IT or bi‐EGF‐IT concentrations. Nonlinear regression was based on the equation Y = B max × X /( K D + X ), where Y = MFI at the given biotinylated fusion toxin concentration after subtracting the background, X = biotinylated fusion toxin concentration, and B max = the maximum specific binding in the same units as Y . (C) Analysis of the blocking of anti‐human EGFR mAb binding to the human EGFR + HNSCC cell line, Cal27, by mono‐EGF‐IT and bi‐EGF‐IT using flow cytometry. (D) The percentage inhibition of anti‐human EGFR mAb binding to Cal27 cells is plotted versus the concentration of binding competitor (mono‐EGF‐IT or bi‐EGF‐IT). The data are representative of three individual experiments. (E) In vitro efficacy of the mono‐EGF‐IT and bi‐EGF‐IT in the human EGFR + HNSCC cell line, Cal27 determined by the CellTiter‐Glo® Luminescent Cell Viability Assay (red line: mono‐EGF‐IT group; green line: bi‐EGF‐IT group; blue line: anti‐murine PD‐1 immunotoxin group as the negative control). Y‐axis: percent inhibition of cell viability determined by the number of viable cells based on the quantification of ATP. X‐axis: fusion toxin concentration. Cycloheximide (1.25 mg·mL −1 ) was used as a positive control. The negative control wells contained cells without fusion toxin. Data are from three individual experiments. Error bars indicate SD.

Article Snippet: Biotin‐labeled anti‐human EGFR mAb (Cat #555997; BD Pharmingen) was then added to each tube at a final concentration of 0.36 n m , and the cell suspensions were incubated at 4 °C in the dark for 30 min.

Techniques: In Vitro, Binding Assay, Flow Cytometry, Positive Control, Concentration Assay, Blocking Assay, Inhibition, Cell Viability Assay, Negative Control

Journal: Molecular Oncology

Article Title: A novel diphtheria toxin‐based bivalent human EGF fusion toxin for treatment of head and neck squamous cell carcinoma

doi: 10.1002/1878-0261.12919

Figure Lengend Snippet: In vitro binding affinity and efficacy analysis of the mono‐EGF‐IT and bi‐EGF‐IT to the human EGFR + HNSCC cell line. (A) Binding affinity analysis of the mono‐EGF‐IT and bi‐EGF‐IT to the human EGFR + HNSCC cell line, Cal27, using flow cytometry. Anti‐human EGFR mAb was used as a positive control, and biotinylated anti‐murine PD‐1 immunotoxin served as a negative background control for protein biotinylation. The data are representative of three individual experiments. (B) K D determination of the human EGF fusion toxins for Cal27 cells using flow cytometry and nonlinear least‐squares fitting. The MFI was plotted over a wide range of biotinylated mono‐EGF‐IT or bi‐EGF‐IT concentrations. Nonlinear regression was based on the equation Y = B max × X /( K D + X ), where Y = MFI at the given biotinylated fusion toxin concentration after subtracting the background, X = biotinylated fusion toxin concentration, and B max = the maximum specific binding in the same units as Y . (C) Analysis of the blocking of anti‐human EGFR mAb binding to the human EGFR + HNSCC cell line, Cal27, by mono‐EGF‐IT and bi‐EGF‐IT using flow cytometry. (D) The percentage inhibition of anti‐human EGFR mAb binding to Cal27 cells is plotted versus the concentration of binding competitor (mono‐EGF‐IT or bi‐EGF‐IT). The data are representative of three individual experiments. (E) In vitro efficacy of the mono‐EGF‐IT and bi‐EGF‐IT in the human EGFR + HNSCC cell line, Cal27 determined by the CellTiter‐Glo® Luminescent Cell Viability Assay (red line: mono‐EGF‐IT group; green line: bi‐EGF‐IT group; blue line: anti‐murine PD‐1 immunotoxin group as the negative control). Y‐axis: percent inhibition of cell viability determined by the number of viable cells based on the quantification of ATP. X‐axis: fusion toxin concentration. Cycloheximide (1.25 mg·mL −1 ) was used as a positive control. The negative control wells contained cells without fusion toxin. Data are from three individual experiments. Error bars indicate SD.

Article Snippet: The biotin‐labeled anti‐human EGFR monoclonal antibody (mAb) (Cat #555997; BD PharMingen, San Jose, CA, USA) was used as a positive control at a final concentration of 36 n m . Negative control cells were stained only with streptavidin (SA)‐PE, at a final concentration of 1.5 ng·µL −1 .

Techniques: In Vitro, Binding Assay, Flow Cytometry, Positive Control, Concentration Assay, Blocking Assay, Inhibition, Cell Viability Assay, Negative Control