Journal: FEBS Open Bio

Article Title: CCR4‐IL2 bispecific immunotoxin is more effective than brentuximab for targeted therapy of cutaneous T‐cell lymphoma in a mouse CTCL model

doi: 10.1002/2211-5463.13625

Figure Lengend Snippet: In vitro binding affinity of CCR4‐IL2 IT versus brentuximab. (A) Flow cytometry binding affinity analysis of Alexa Fluor 488–labeled CCR4‐IL2 IT or brentuximab and human CD25 + CCR4 + CD30 + Hut102/6TG cells. Fluorescein‐mouse antihuman/rat CCR4 mAb, FITC‐mouse antihuman CD25 mAb, and PE‐mouse antihuman CD30 mAb were included as positive controls. The data are representative of three individual experiments. (B–C) K D comparison of CCR4‐IL2 IT and brentuximab. K D was determined using nonlinear regression analysis of the flow cytometry data with a saturation binding equation ( graphpad prism 9.4.1 ). The MFI was plotted over a wide range of concentrations of the Alexa Fluor 488–labeled CCR4‐IL2 IT or brentuximab. Nonlinear regression analysis was based on the eq. Y = B max × X /( K D + X ), where Y = MFI at a given concentration of Alexa Fluor 488–labeled brentuximab or CCR4‐IL2 IT after subtracting the background, X = the concentration of the Alexa Fluor 488–labeled brentuximab or CCR4‐IL2 IT, and B max = the maximum specific binding in the same units as Y . CCR4‐IL2 IT, C–C chemokine receptor type 4 interleukin 2 bispecific immunotoxin; FITC, fluorescein isothiocyanate; K D , dissociation constant; mAb, monoclonal antibody; MFI, median fluorescence intensity; PE, phycoerythrin.

Article Snippet: A dissociation constant ( K D ) determination was performed using nonlinear regression analysis of the flow cytometry data with a saturation binding equation ( graphpad prism 9.4.1 , GraphPad Software, San Diego, CA, USA).

Techniques: In Vitro, Binding Assay, Flow Cytometry, Labeling, Comparison, Concentration Assay, Fluorescence