Fig 1.
Structural models of IL-6, IL-6R and ZDC scaffold.
A. Crystal structure of IL-6R (cyan tube) bound to IL-6 (blue surface model). Cα-Cβ vectors of interacting residues of IL-6R with IL-6 were used as a query. B. A model of ZDC (cyan tube) that matches Cα-Cβ vectors of IL-6R bound to IL-6 (blue surface model). C. Guided by the helical nature of the ZDC scaffold, residues (red) that were mutated in phage display and medicinal chemistry to optimize potency are shown. Underlined residue numbers are those that were a component of the initial random library. The remaining residues were explored during optimization with the goal of expanding the initial binding footprint of ZDC, including extending the termini (residues not shown).
Table 1.
Peptide Sequences and Anti-IL-6 Activity of Key Peptides in the Development of PN-2921.
Fig 2.
Determination of PEGylation site and effect of PEGylation on IC50 and plasma exposure.
A. Ribbon diagram of ZDC (in cyan) bound to IL-6. Tolerated PEGylation sites on ZDC are indicated in green, and the final PEGylation site, position 29, is labeled. B. Five positions on the non-binding exoface of PN-2171 (sequence shown in S1 Table) were scanned with Lys, Nε-acetylated Lys (Lys-Ac), and LysPEG20L for their effect on IL-6 induced pSTAT3 in U937 cells. IC50 values are averages of n = 2 determinations. C. Effect of PEG molecular weight on the activity of peptides. PN-2519 is the non-PEGylated parent, PN-2520 contains a PEG40Br, PN-2566 contains PEG30L and PN-2567 contains a PEG20Br, all PEGylated at position 29. D. PK analysis in rats of peptide analogs with different molecular weight PEG moieties. In this experiment, the non-PEGylated comparator is PN-2365 (a PN-2519 analog with K26, K29). Peptides were administered by SC injection at 0.23 μmol/kg and plasma samples were taken at the indicated time points.
Fig 3.
PN-2921 demonstrates potent anti-IL-6 activity in vitro.
A. In vitro potency of PN-2729 and PN-2921 was determined by the ability to inhibit IL-6-induced pSTAT3 in U937 cells. Peptides were incubated with IL-6 for 2.5 h before cell treatment. The observed IC50 values in this representative experiment were 1.2 nM for PN-2729 and 0.4 nM for PN-2921. B. PN-2921 dose-dependently right-shifted the IL-6-induced pSTAT3 concentration response curve in U937 cells. C. PN-2921 decreased IL-6-induced pSTAT3 in monocytes. Peptides or vehicle control were incubated with IL-6 for 2.5 h at 37°C and then added to freshly collected heparinized human whole blood (pooled from six donors) for 15 min at 37°C. pSTAT3 was quantitated by flow cytometry. D. PN-2921 inhibited rhIL-6-induced proliferation in mouse B9 cells. B9 cells were incubated with rhIL-6 and varying concentrations of PN-2921 for 72 h at 37°C, with 0.2 μCi/well of 3H-thymidine present for the last 18 h.
Table 2.
Summary of inhibitory Activity of PN-2921.
Table 3.
Inhibitory Activity of PN-2921 Against IL-6 From Other Species.
Fig 4.
PN-2921 demonstrates long-lived exposure in multiple species.
PN-2921 was dosed in male ICR mice, male SD rats and male cynomolgus monkeys by IV and SC routes. Both mouse and rats were dosed at 0.23 μmol/kg, while monkeys were dosed at 0.1 μmol/kg. Plasma samples were collected for 168 h (7 d) for both rats and mice, while monkey plasma samples were collected for 336 h (14 d). Samples were analyzed by LC-MS/MS. Mouse and rat data were normalized to 0.1 μmol/kg for the purpose of comparison to monkey.
Fig 5.
PN-2921 decreases hIL-6-induced SAA levels in mice.
Balb/c mice were injected with either vehicle, the indicated dose of PN-2921, or a control anti-hIL-6 mAb. After 24 h, mice were challenged with recombinant human IL-6. After an additional 4 h, mice were sacrificed and whole blood was collected. SAA levels were determined from serum by using the mouse SAA ELISA. Statistical analysis was performed using 2-way ANOVA. *** indicates p<0.001.
Table 4.
Single Dose PK parameters of PN-2921 in Mice, Rats, and Monkeys.
Fig 6.
PN-2921 inhibits IL-6-induced CRP and SAA levels in cynomolgus monkey.
Cynomolgus monkeys were treated SC with vehicle, and a second group of six male cynomolgus monkeys were treated SC with 2.3 μmol/kg of PN-2921 at 24 h prior to SC administration of 0.6 μg/kg IL-6. Blood samples were collected immediately prior to IL-6 administration and at 24 h post IL-6. Resultant plasma samples were assayed for CRP and SAA levels by ELISA, and plasma PN-2921 levels by LC-MS/MS. Statistical analysis was performed using 2-way ANOVA.