Journal: Molecular oncology

Article Title: Treg depletion in non-human primates using a novel diphtheria toxin-based anti-human CCR4 immunotoxin

doi: 10.1016/j.molonc.2015.11.008

Figure Lengend Snippet: In vitro binding and depletion analysis of the anti‐human CCR4 immunotoxins to monkey CCR4+ PBMC. (A) Flow cytometry binding analysis of the anti‐human CCR4 immunotoxins to CCR4+ cells within monkey PBMC. Monkey PBMC was stained with the biotinylated anti‐human CCR4 immunotoxin as primary staining and PE‐conjugated streptavidin as secondary staining. First panel: monovalent anti‐human CCR4 immunotoxin [DT390‐scFv (1567)]; second panel: bivalent anti‐human CCR4 immunotoxin [DT390‐BiscFv (1567)]; third panel: single‐chain foldback diabody anti‐human CCR4 immunotoxin. Monkey PBMC with only the secondary staining (PE‐conjugated streptavidin) served as the negative control and human CCR4 fluorescein mAb (clone#205410) as the CCR4 positive control, mouse IgG2B fluorescein for the isotype control of human CCR4 fluorescein mAb (clone#205410), PerCp anti‐human CD4 mAb (clone# L200) as CD4 positive control, PerCp mouse IgG1 κ (clone# MOPC‐21) for the isotype control of PerCp anti‐human CD4 mAb (clone# L200). Biotin‐labeled porcine CD3‐εγ (Peraino et al., 2012) was included as a negative control for background due to protein biotinylation. (B) In vitro depletion of the CCR4+ cells within monkey PBMC using the anti‐human CCR4 immunotoxins. Monkey PBMC was incubated with the unlabeled anti‐human CCR4 immunotoxin at 37 °C for 48 h and analyzed by flow cytometry using PE anti‐human CCR4 mAb (clone# L291H4). First panel: monovalent anti‐human CCR4 immunotoxin [DT390‐scFv (1567)]; second panel: bivalent anti‐human CCR4 immunotoxin [DT390‐BiscFv (1567)]; third panel: single‐chain foldback diabody anti‐human CCR4 immunotoxin. PE anti‐human CCR4 antibody (clone #L291H4) for the CCR4 positive control, PE‐mouse IgG1, κ as isotype control of PE‐anti‐human CCR4 mAb (clone# L291H4), APC anti‐human CD4 mAb (clone# L200) for the CD4 positive control, APC mouse IgG1 κ (clone# MOPC‐21) for the isotype control of APC anti‐human CD4 mAb (clone# L200). DT390 (100 nM) was included as negative depleter control. Propidium iodide was added before running the FACS machine for gating the living cells. All control cells in Figure 2B were also incubated at 37 °C for 48 h. (C) Flow cytometry binding analysis of the anti‐human CCR4 immunotoxins to the Foxp3+CCR4+ monkey PBMC. Monkey PBMC was stained with Alexa Fluor© 647 anti‐human Foxp3 mAb (clone# 150D) and biotinylated anti‐human CCR4 immunotoxin. First panel: monovalent anti‐human CCR4 immunotoxin [DT390‐scFv(1567)]; second panel: bivalent anti‐human CCR4 immunotoxin [DT390‐BiscFv(1567)]; third panel: single‐chain foldback diabody anti‐human CCR4 immunotoxin. Monkey PBMC with only the secondary staining (PE‐conjugated streptavidin) served as the negative control, human CCR4 fluorescein mAb (clone#205410) as the CCR4 positive control, mouse IgG2B fluorescein for the isotype control of human CCR4 fluorescein mAb (clone#205410), Alexa Fluor© 647 anti‐human Foxp3 mAb (clone# 150D) for the Foxp3 positive control, Alex Fluor 647 Mouse IgG1 κ (clone# MOPC‐21) for the isotype control of Alexa Fluor© 647 anti‐human Foxp3 mAb (clone# 150D). Biotin‐labeled porcine CD3‐εγ (Peraino et al., 2012) was included as a negative control for background due to protein biotinylation. All Figure 2C analysis was CD4 gated. All of the data (A‐C) are representative of three individual experiments.

Article Snippet: Human/rat CCR4 fluorescein mAb (clone 205410, cat# FAB1567F), human/rat CCR4 PE mAb (clone 205410, cat# FAB1567P) and mouse IgG2B fluorescein isotype control (clone# 133303, cat# IC0041F) were purchased from R&D Systems.

Techniques: In Vitro, Binding Assay, Flow Cytometry, Staining, Negative Control, Positive Control, Control, Labeling, Incubation