Anti-Human CD25 Recombinant Antibody (Daclizumab) (CAT#: TAB-023)

Figure 1 Flow cytometric analysis of peripheral blood lymphocytes of a patient treated with daclizumab.

The dot plots in the first two columns are gated on lymphocytes, and those in the third and fourth columns are gated on CD3+cells. Following treatment, p55 is not detected by anti-CD25 nor by anti-humanized Tac (daclizumab) on CD3+ cells (first and second columns), but the CD3+ cells remain positive for HLA-DR (third column) and faintly positive for p55 using 7G7 (fourth column).

Przepiorka, D., Kernan, N. A., Ippoliti, C., Papadopoulos, E. B., Giralt, S., Khouri, I.,... & Champlin, R. (2000). Daclizumab, a humanized anti-interleukin-2 receptor alpha chain antibody, for treatment of acute graft-versus-host disease. Blood, 95(1), 83-89.

Figure 2 Absolute numbers of cells after treatment with daclizumab.

Note the difference in scale of the y-axis between graphs. (A) Absolute number of lymphocytes (ALC) (solid line), CD3 + 56− (dashed line) and CD3 − 56+(dotted line) cells after treatment with daclizumab. (B) Absolute number of CD3 + 56− cells (solid line), CD3+DR+ cells (dashed line), CD3 + 7G7+ cells (dotted line), and CD3 + 25+ cells (thin line) after treatment with daclizumab.

Przepiorka, D., Kernan, N. A., Ippoliti, C., Papadopoulos, E. B., Giralt, S., Khouri, I.,... & Champlin, R. (2000). Daclizumab, a humanized anti-interleukin-2 receptor alpha chain antibody, for treatment of acute graft-versus-host disease. Blood, 95(1), 83-89.

Figure 3 Change in CEL on brain MRI during daclizumab trial: individual patients (A) and cumulative lesion analysis (B).

(A) Evolution of new CEL on brain MRI during daclizumab trial. All 11 patients are presented. Group average for each time point is calculated from data on 10 MS patients who received 1 mg/kg daclizumab dosing. (B) Cumulative lesion analysis of new and total CEL during daclizumab trial. Number of new (and total) CEL per each month were added together for 10 MS patients and plotted as a cumulative lesion analysis. There is proportional monthly accumulation of CEL in the whole cohort (as evident from the linear relationship), and daclizumab add-on leads to gradual decrease in cumulative CEL (change in slope) that becomes evident after 1.5–2 months of therapy.

Bielekova, B., Richert, N., Howard, T., Blevins, G., Markovic-Plese, S., McCartin, J.,... & Martin, R. (2004). Humanized anti-CD25 (daclizumab) inhibits disease activity in multiple sclerosis patients failing to respond to interferon β. Proceedings of the National Academy of Sciences, 101(23), 8705-8708.

Figure 4 Daclizumab treatment decreases CSF levels of CXCL13 and intrathecal production of IgG.

(A) CSF CXCL13 concentration was measured by ELISA. (B) IgG index as measured by NIH clinical laboratory. Raw data for individual patients measured before (Baseline) and 6.5 months after initiation of daclizumab therapy (Dac Th) are depicted as gray dot and line blots and group data are depicted as box plots with mean highlighted as red and median as black horizontal line.

Perry, J. S., Han, S., Xu, Q., Herman, M. L., Kennedy, L. B., Csako, G., & Bielekova, B. (2012). Inhibition of LTi cell development by CD25 blockade is associated with decreased intrathecal inflammation in multiple sclerosis. Science translational medicine, 4(145), 145ra106-145ra106.

Figure 5 Enhanced intermediate IL-2/IL-15 signaling in daclizumab-treated patients promotes differentiation of ILCs toward functional NK cells (A) Representative pSTAT5 levels of untreated or daclizumab (Dac Th) treated MS patients.

Purified ILCs were stimulated with IL-2 (100IU/ml), IL-7 (10ng/ml), IL-15 (10ng/ml) or no cytokine for 10 minutes (optimal concentrations determined in pilot experiments). ILCs were then immediately fixed and stained for phosphorylated Stat5 production. (B) Group signaling data analogous to (A). (C) Purified c-kit + ILCs cells were cultured for 7 days in media supplemented with SCF and Flt3L (both 10ng/ml) and in the presence of IL-2 (100IU/ml), IL-7 (10ng/ml), IL-15 (10ng/ ml) or no cytokine control. At day 7, cultured cells were stained and analyzed by FACS for the presence of CD56 + NK cells (compared to ex vivo PBMCs of daclizumab-treated MS patients with significant expansion of CD56 bright NK cells). (C) Raw data from a representative experiment and (D) represents group data of the number of CD56 dim NK and CD56 bright NK cells per 1,000 beads. (E) Purified c-kit + ILCs were cultured as in panel C. At day 7, cultured cells were FACS stained for functional NK markers (Perforin, Granzyme A and B; compared to ex vivo PBMC of daclizumab-treated MS patients with significant expansion of CD56 bright NK cells). FACS plots are representative of 4 replications. (F) Also on day 7, cultured cells were incubated for ~16hr with target GFP-tagged K562 cells at a 1:1 effector to target ratio (or K562 cell only control wells). K562 killing was then.

Perry, J. S., Han, S., Xu, Q., Herman, M. L., Kennedy, L. B., Csako, G., & Bielekova, B. (2012). Inhibition of LTi cell development by CD25 blockade is associated with decreased intrathecal inflammation in multiple sclerosis. Science translational medicine, 4(145), 145ra106-145ra106.

Figure 1 Anti-Human CD25 Antibody (TAB-023) in SDS-PAGE

SDS-PAGE analysis of TAB-023 in non-reduced (lane 1) and β-mercaptoethanol-reduced (lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively.

Figure 2 Anti-Human CD25 Antibody (TAB-023) in HPLC

The purity of TAB-023 was greater than 95% as determined by HPLC.