TITLEEngineering a single-agent cytokine-antibody fusion that selectively expands regulatory T cells for autoimmune disease therapy. AUTHORS

Interleukin-2 (IL-2) has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (comprised of IL-2 receptor-  [IL-2R  ], IL-2R  , and common  [  c ]). IL-2R  , which is highly expressed on regulatory T (T Reg ) cells regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 antibody preferentially biases cytokine activity toward T Reg cells through 1:1 molar ratio of antibody fragment Cells fixed formaldehyde 1.5% Permeabilization of cells 100% and permeabilized cells twice FACS 7.2 0.1% bovine serum and with anti-STAT5 pY694 (BD Biosciences) diluted in FACS buffer 2 hr Cells were then washed twice in FACS and MFI was determined on a CytoFLEX flow cytometer (Beckman-Coulter). Dose-response curves were fitted to a logistic model and half-maximal effective concentrations (EC 50 s) were calculated using GraphPad Prism data analysis software after subtraction of the mean fluorescence intensity (MFI) of unstimulated cells and normalization to the maximum signal intensity. Experiments were conducted in triplicate and performed three times with similar results. After two washes in Permeabilization Buffer (eBioscience), T Reg cells were stained with FITC-conjugated anti-mouse/rat Foxp3 mAb (eBioscience, clone FJK-16s) for 1 hour on ice. Two final washes were conducted in Permeabilization Buffer and cells were resuspended in FACS buffer for flow cytometric analysis on an LSRII (BD Biosciences). Data were analyzed using FlowJo X software (Tree Star). Ratios of the absolute numbers of T Reg cells to CD8 + effector T cells are presented. Statistical significance was determined by two-tailed unpaired Student’s t -test. Experiments were performed three times with similar results. IL-2R  , T Reg s (CD3 + CD4 + Foxp3 + ) - V500 Horizon®-conjugated anti-CD3 (BD Biosciences, clone 500A2), PerCP-conjugated anti-CD4 (BD Biosciences, clone RM4-5), PE-conjugated anti-mouse/rat Foxp3 (eBioscience, clone FJK-16s), and APC-conjugated anti-IL-2R  mAbs (eBioscience, clone PC61.5); MP CD8 + T cells (CD3 + CD8 + CD44 + IL-2R  + ) - V500 Horizon®-conjugated anti-CD3, PerCP-Cy5.5-conjugated anti-CD8 (eBioscience, clone 53-6.72), APC-conjugated anti-CD44 (eBioscience, clone IM7), PE-conjugated anti-IL-2R  (eBioscience, clone 5H4), and eFluor 450®-conjugated anti-IL-2R  (eBioscience, clone PC61.5) mAbs; and NK cells (CD3 - CD49b + CD161) - V500 Horizon®-conjugated anti-CD3, PE-conjugated anti-CD49b (eBioscience, clone DX5), APC-conjugated anti-CD161 (eBioscience, clone PK136), and eFluor 450®-conjugated anti-IL-2R  mAbs. Fixable Viability Dye eFluor™ 780 (eBioscience) was used to assess live cells. Data were analyzed using FlowJo X software (Tree Star). Relative number of cells and mean fluorescence intensity (MFI) of IL-2R  are presented for each cohort in all four immune cell subsets. Statistical significance was determined by two-tailed unpaired Student’s t -test. The experiment was performed three times with similar results.