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Dryad

Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles-primary dataset

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Oct 04, 2023 version files 7.20 MB

Abstract

 

Interleukin-2 (IL-2) had been been engineered up to now by yeast display and in silico rational design. In this article we reshaped IL-2 interface with the IL-2 receptor beta subunit to increase binding affinity between both interacting partners, using phage display. Multiple IL-2 mutated variants were selected from large phage-displayed libraries, showing shared molecular patterns. An accumulation of negative charges in the segment 81-87 of IL-2 primary sequence was observed, as well as the strong preponderance of the replacement I92L. The first feature contributed to an optimized electrostatic complementarity between IL-2 and IL-2 receptor beta chain, resulting in higher affinity and faster association kinetics than the ones of previously reported H9 superkine retrieved from yeast display libraries. The presence of a Leu residue at position 92 was the key molecular determinant for a favourable biophysical profile characterized by high stability and production in mammalian-cell based recombinant systems, and decreased aggregation propensity. The new beta super-binders behaved as potent super agonists, both in vitro and in vivo. The latter scenario showed their better preformance when compared to both non-mutated IL-2 and H9. The current dataset contains source data for graphics showing frequency mutations and charge distribution among unselected variants contained in phage-displayed libraries and selected clones enriched after selection on immobilized beta chain. Data showing the direct comparison between different IL-2 mutated variants produced as Fc-fusion proteins are also presented. The comparison includes the results of beta chain binding assays (ELISA), proliferation and phosphorylation assays in vitro, in vivo expansion of lymphocyte populations and anti-tumor activity in animal models. Taken together, the above described data support the unique features of the new beta super-binders and their potential as immunostimulatory and anti-cancer agents.