Fig. 5: PEG length modulates H16 exposure and pharmacodynamics in NHP.

a Pegylation of H16 with 30 kDa or 50 kDa linear mPEG retains bias towards signaling in Treg ex vivo. NHP PBMC were stimulated in triplicate with concentration series of rhIL-2, H16-30 kDa or H16-50 kDa. Treated cell populations were analyzed using multi-color flow cytometry to detect and quantify pSTAT5 in different cell subsets. The plots shown represent the average pSTAT5 fluorescence intensity, fit to a baseline restrained 4 parameter logistic regression and normalized to maximum signal to facilitate comparison of potency, with error bars representing SEM. b, c A single subcutaneous dose, estimated to reach EC50 at the target Treg cell, of H16-30 kDa (orange) or H16-50 kDa (purple) was administered to cynomolgus monkeys. b Peripheral blood samples were collected at the indicated times post-dose and analyzed for H16 level using ELISA (see Methods). c Peripheral blood samples were collected at the indicated times, and multi-color flow cytometry was used to quantify Treg (CD4 + CD25+ FoxP3 +) expansion; data shown as Treg percent in WBC and CD4 + T cells (total peripheral blood cells at each time point; data points represent the mean from three individual animals with error bars corresponding to SEM. CD4 cluster of differentiation 4, CD8 cluster of differentiation 8, CD25 cluster of differentiation 25, EC50 half-maximal effective concentration, ELISA enzyme-linked immunosorbent assay, FoxP3 forkhead box protein 3, kDa kilo Dalton, MFI median fluorescence intensity, NHP non-human primate, NK natural killer, PBMC peripheral blood mononuclear cells, PEG polyethylene glycol, pSTAT5 phosphorylated Signal Transducer and Activator of Transcription 5, SEM standard error of mean, Treg regulatory T cell, WBC white blood cell.