PEGylation Strategy for Improving the Pharmacokinetic and Antitumoral Activity of the IL-2 No-alpha Mutein

Background: In a previous work, an IL-2R beta gamma biased mutant derived from human IL-2 and called IL-2no alpha, was designed and developed. Greater antitumor effects and lower toxicity were observed compared to native IL-2. Nevertheless, mutein has some disadvantages, such as a very short half-life of about 9-12 min, propensity for aggregation, and solubility problems.Objective: In this study, PEGylation was employed to improve the pharmacokinetic and antitumoral properties of the novel protein.Methods: Pegylated IL-2no alpha was characterized by polyacrylamide gel electrophoresis, size exclusion chromatography, in vitro cell proliferation and in vivo cell expansion bioassays, and pharmacokinetic and antitumor studies.Results: IL-2no alpha-conjugates with polyethylene glycol (PEG) of 1.2 kDa, 20 kDa, and 40 kDa were obtained by classical acylation. No significant changes in the secondary and tertiary structures of the modified protein were detected. A decrease in biological activity in vitro and a significant improvement in half-life were observed, especially for IL-2no alpha-PEG20K. PEGylation of IL-2no alpha with PEG20K did not affect the capacity of the mutant to induce preferential expansion of T effector cells over Treg cells. This pegylated IL-2no alpha exhibited a higher antimetastatic effect compared to unmodified IL-2no alpha in the B16F0 experimental meta- stases model, even when administered at lower doses and less frequently.Conclusion: PEG20K was selected as the best modification strategy, to improve the blood circulation time of the IL-2no alpha with a superior antimetastatic effect achieved with lower doses.