Fig 1.
NKTR-214 delivers a controlled, sustained, and biased signal through the IL2 receptor pathway.
NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of PEG located at the interface of IL2 and IL2Rαβγ. The PEG chains slowly release at physiological pH, creating conjugated-IL2 species with fewer PEG chains and increased bioactivity. Sustained signaling through the heterodimeric IL2 receptor pathway (IL2Rβγ) preferentially activates and expands effector CD8 T and NK cells over Tregs.
Fig 2.
Parameterization of the mathematical model for NKTR-214 dynamics to simulate concentration-time profiles of conjugated-IL2 species derived from NKTR-214 and to describe receptor occupancy of the conjugated-IL2 species at the IL2Rαβγ and IL2Rβγ.
Fig 3.
Release of PEG chains follows first-order kinetics.
A. Free PEG detected at various time points during incubation in 0.5 M sodium phosphate buffer at pH 7.4 and 37°C (circles) fit to first-order kinetic profile (solid line), B. Semi-log plot of non-released PEG in phosphate buffer at pH 7.4 starting from indicated concentrations of NKTR-214 at pH 7.4 and 37°C. C. Arrhenius plot of Ln(K) versus 1/Temp is linear. Calculated Ea = 130 kJ/mole for PEG release, D. Effect of pH and temperature on PEG release rate–an equal volume of 0.5 M sodium phosphate buffer at predetermined pH was added to conjugate to produce the final pH of 6.4 or 7.4.
Fig 4.
The effect of PEGylation is greatest at the alpha-containing IL2 receptors.
Overlay of RU signals normalized by percentage of IL2 response. Each overlay depicts relative response of 1-PEG-IL2 and 2-PEG-IL2 at A. IL2Rα, B. IL2Rαβ, and C. IL2Rβ. Representative data are shown for each analyte at a concentration that is near the respective IL2 Kd concentration per Table 1.
Table 1.
The effect of PEGylation is greatest at the IL2Rαβ complex bound to the SPR chip.
Summary of binding kinetics for IL2 and active conjugated-IL2 species derived from NKTR-214 at the human IL2Rα, IL2Rβ, or IL2Rαβ. IL2Rαβ simulates the heterotrimeric IL2Rαβγ, whereas IL2Rβ simulates the heterodimeric IL2Rβγ.
Fig 5.
A single dose of NKTR-214 leads to sustained exposure.
A. Semi-logarithmic plot of plasma concentration vs. time curves for NKTR-214-RC, NKTR-214-AC, and 1-PEG-IL2 after administration of NKTR-214 B. Semi-logarithmic plot of plasma concentration of IL2 after administration of aldesleukin.
Table 2.
Mean (±SE) plasma PK parameters after administration of NKTR-214 or aldesleukin, each at 0.8 mg/kg, i.v.
Fig 6.
NKTR-214 delivers a controlled and sustained activation signal to the IL2 pathway in vivo measured by pSTAT5 levels in whole peripheral blood.
Mice were treated with a single 0.8 mg/kg dose of either aldesleukin (red) or NKTR-214 (blue) and monitored for up to 240 hours. Peripheral blood was collected at the indicated time points post-dose and pSTAT5 in lymphocyte populations was determined by flow cytometry using antibodies to intracellular pSTAT5 and to extracellular surface marker CD3. N = 5 mice per time point per group.
Fig 7.
Model-calculated concentration–time profile for species of conjugated-IL2 or free-IL2 that could be theoretically generated from a single dose of 0.8 mg/kg NKTR-214.
Where data are available, the model agrees closely with the PK data shown in Table 3. The model calculates that administration of NKTR-214 results in sustained concentrations of conjugated-IL2 with exceedingly low concentrations of free-IL2, consistent with its fast in vivo clearance and slow formation from NKTR-214.
Fig 8.
The model suitably fits to measured NKTR-214 concentration-time profiles.
Summing the concentration-time profile of the individual model-derived conjugated-IL2 fit well to the experimentally determined concentration-time profiles of A. NKTR-214-RC, B. NKTR-214-AC, and C. 1-PEG-IL2, the most active conjugate derived from NKTR-214. Solid lines represent the simulation and individual red symbols represent the experimental data.
Table 3.
Tmax, Cmax, and AUC of the individual species calculated from the model.
Where available, the values from the PK study (Table 2) are shown in parentheses and in the last 2 columns for ease of comparison.
Fig 9.
Conjugated-IL2 species derived from NKTR-214 preferentially occupy IL2Rβγ over IL2Rαβγ.
Percent receptor occupancy calculated by the model from A. 2-PEG-IL2, B. 1-PEG-IL2, and C. free-IL2 derived from NKTR-214, at IL2Rβγ (gray lines) and IL2Rαβγ (green lines) receptor complexes.
Table 4.
Model-simulated values of receptor occupancy for the conjugated-IL2 species and free-IL2 derived from NKTR-214 after a single dose administration.
1-PEG-IL2 and 2-PEG-IL2 are present at significantly higher levels in plasma than IL2. Experimentally, free-IL2 from NKTR-214 is not detectable. The calculation indicates that individually and in sum, the active conjugated-IL2 from NKTR-214 occupy IL2Rβγ to greater extent than IL2Rαβγ.
Fig 10.
Receptor bias is an intrinsic property of NKTR-214.
Simulation of the receptor occupancy percentage over time for therapeutic levels (0.8 mg/kg) of aldesleukin qdx5 (red) or NKTR-214, qd (blue) compared head to head. A. The receptor occupancy area under the curve for IL2Rβγ is 20.8-fold higher after NKTR-214 compared to aldesleukin. B. In contrast, the AUC of IL2Rαβγ is 0.56-fold after NKTR-214 compared to aldesleukin.
Table 5.
Aldesleukin does not achieve a bias in receptor occupancy, regardless of dose or exposure.
Receptor occupancy of a single dose of NKTR-214 compared to several dosing regimens of aldesleukin, including a theoretical sustained release formulation that provides the same measured AUC as NKTR-214. The values are normalized to the AUC of aldesleukin single dose for each receptor complex.
Fig 11.
The model correctly estimates experimental % pSTAT5 activation from the receptor occupancy determination.
Model fit (blue line) of pSTAT5 signaling from NKTR-214 as compared to the observed measured values (symbols).
Fig 12.
NKTR-214 is consistently effective across several established mouse tumor models.
Examples shown below include A. bladder (MBT-2), B. liver (H22), and C. pancreatic (Pan02) for single-agent NKTR-214 compared to vehicle. In all cases, tumors were grown to large size, 80-100mm3, N = 7/group; NKTR-214 0.8 mg/kg q9dx3.*, P < 0.05 (unpaired t-test).