Extended Data Fig. 3: Binding of anti- CD25NIB, RG6292 and Daclizumab to mouse, human and cynomolgus CD25 positive cells. ADCC and ADCP capacity of RG6292.
For binding experiments with RG6292 and Daclizumab, SU-DHL1 tumor cells (human CD25+, (a)) and HSC-F cells (cynomolgus CD25+, (b)) were used. To quantify binding of anti-CD25NIB, splenocytes were isolated of spleens resected from female C57BL6-Foxp3tm1Flv/J mice (c) Cells were incubated with indicated serial dilutions of the test antibody detected then by fluorescently labeled 2nd antibody against human and mouse Fcγ, respectively. Living mouse Treg cells (Aqua−, mRFP+ singlets) and tumor cells (Aqua−, singlets), respectively, were gated and the mean fluorescence intensity of the secondary antibody was plotted. EC50 values were calculated by as described in the data analysis section in materials and methods. Shown are technical duplicates of one representative experiment out of several independent ones conducted (n>2). (d) RG6292 (and the fully fucosylated version RG6292 (FF)) depleted via ADCC in-vitro differentiated Treg cells using purified, IL-2 activated NK cells. Shown are technical duplicates of one representative experiment out of several independent ones conducted (n>2). (e) RG6292 and RG6292 (FF) mediated ADCP of in-vitro differentiated Treg cells when co-cultured with MCSF differentiated macrophages. Flow cytometric analysis was performed to determine percentage of phagocytosis. Shown are technical duplicates of one representative experiment out of several independent ones conducted (n>2). (f) Schematics of binder selection.
