Fig 1: Binding activity to human or cynomolgus CD25 and direct competitive binding characteristics of BA9 and BT942. (a) BA9 and BT942 specifically bind to SU-DHL-1 cells determined by flow cytometry. Data were presented as Mean ± SEM. (b) BA9 and BT942 specifically bind to HEK293T-CD25 cells determined by flow cytometry. Data were presented as mean ± SEM. (c,d) Binding kinetics of BA9 (c) or BT942 (d) for Human CD25 were measured in a surface plasmon resonance (SPR) assay with BIAcore. Experiments were performed in triplicate. (e,f) Cross-reactivity with cynomolgus CD25 by BA9 (e) and BT942 (f) were measured in a surface plasmon resonance (SPR) assay with BIAcore. Experiments were performed in triplicate. (g,h) Direct competitive binding characteristics of BA9 and BT942 was performed using in-tandem format binning assay. SA sensors immobilized with biotinylated human CD25 were saturated with the first antibody or PBST, then exposed to the second antibody or PBST. Data of KD were presented as mean ± SD.
Fig 2: IL-2/IL-2 receptor blocking activity of BA9, BT942 and Daclizumab. (a) Characterization of anti-CD25 antibodies compared to isotype antibody in respect to blocking IL-2 signaling in a STAT5 phosphorylation assay using human PBMCs. PBMCs were co-cultured with 10 µg/mL antibody for 30 min, then 10 U/mL IL2 was added and cultured for 10 min. Isotype is a human IgG1 with kappa light chain (Crown Bio, C0001-2). Data were presented as mean ± SEM. (b) BA9 and BT942 do not alter activation and proliferation of CD8+ and CD4+ T cells in vitro IL-2 signaling by T cell activation assay. Antibodies were tested for 72 h at 10 µg/mL. Data were presented as mean ± SEM.
Fig 3: Antitumor effect of BA9 and BT942 in B-hIL2RA mice in MC38 tumor model for Early phase and Late phase treatment. (a) Early phase treatment: mean tumor volume of MC38 tumors in B-hIL2RA mice treated with vehicle versus anti-CD25 antibody (n = 8/group). Randomly, B-hIL2RA mice were grouped by weight and treated with anti-CD25 (10 mg/kg, i.p., twice a week). MC38 cells (5 × 105 cells) were injected s.c. into mice in the next day (day 0). (b) Late phase treatment: mean tumor volume of MC38 tumors in B-hIL2RA mice treated with vehicle versus anti-CD25 (n = 8). MC38 cells (5 × 105 cells) were injected subcutaneous into B-hIL2RA mice (day 0). Antibody treatments (10 mg/kg, i.p., twice a week) were started when tumors had grown to 50–60 mm3 (day 5). P values were calculated using two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).
Fig 4: Cryo-EM structure of CD25-BT942 Fab complex. (a) The cryo-EM map of CD25-BT942 Fab complex. CD25 is colored in yellow, and the light chain and heavy chain of BT942 Fab is shown as cyan and green density, respectively. (b) The atomic model of CD25-BT942 Fab complex. Color codes are as in (a). The left and right panels show two different views of this complex. (c) BT942 Fab and IL-2 bind with CD25 in opposite direction according to the superposition of the CD25-BT942 Fab cryo-EM structure with the IL-2 quaternary signaling complex structure (PDB: 2B5I). The color codes of CD25-BT942 Fab complex are as in (a), with IL-2Rß and IL-2R? shown as a grey transparent surface. IL-2 is shown in red to highlight its orientation. The boxed area represents the interface shown in (d) and (e). (d,e) The close-up views of the interface between CD25 and BT942 Fab showing the epitopes on CD25 (grey labels) and paratopes on BT942 (green labels). The hydrogen bonds are depicted as yellow dashed lines.
Fig 5: ADCC activity of BA9 and BT942. (a,b) ADCC was evaluated for BA9 and BT942 by a reporter bioassay. The target cells were SU-DHL-1 (a) or 293T-CD25 (b), the effector cells were Jurkat cells from Promega. BA9 can induce higher ADCC activity to both cell lines than BT942. Experiments were performed in duplicate. Data were presented as mean ± SEM. (c,d) ADCC was evaluated for BA9, BT942 and daclizumab by PBMC-mediated cytotoxicity. The target cells were SU-DHL-1 (c) or HEK293T-CD25 cells (d), the effector cells were PBMC. BA9 has higher ADCC activity than BT942. Experiments were performed in duplicate. Data were presented as mean ± SEM. (e,f) ADCC against activated CD8+T cells was evaluated for BA9 and BT942. The target cells were activated CD8+T cells. ADCC was evaluated by a reporter bioassay and the effector cells were Jurkat cells (e). ADCC was evaluated by PBMC-mediated cytotoxicity and the effector cells were PBMC (f). Experiments were performed in duplicate. Data were presented as mean ± SEM.
Supplier Page from Sino Biological, Inc. for Human IL2Ra / CD25 Protein (His Tag)