Design and activity evaluation of long-acting IFN-α2a-ELP fusion proteins

Recombinant interferon-α2a (IFN-α2a) has clinically relevant antiviral and antitumor activity, but its short systemic half-life limits durable exposure after intermittent dosing. Elastin-like polypeptides (ELPs) offer a genetically encoded strategy for extending protein residence time through an expected increase in hydrodynamic size and thermally triggered depot formation. We designed two ELP 120 -based IFN-α2a fusion proteins, ELP 120 -IFN-α2a and IFN-α2a-ELP 120 , to compare the effect of fusion orientation on biophysical behavior, receptor engagement, in vitro activity, pharmacokinetics, and antitumor efficacy. The fusion proteins were expressed in Escherichia coli , purified by inverse transition cycling and anion-exchange chromatography, and evaluated using temperature-dependent turbidity, circular dichroism, cell-based receptor binding, HeLa-cell antiproliferative assays, rat pharmacokinetics, and a HeLa xenograft model. Both ELP 120 fusions underwent a sharp phase transition at approximately 32–33 degrees C and retained the major α-helical secondary-structure features of IFN-α2a. ELP fusion reduced apparent receptor binding relative to native IFN-α2a, but IFN-α2a-ELP 120 retained stronger apparent binding than ELP 120 -IFN-α2a, with apparent EC 50 values of 3.97 ± 0.56 nM and 7.06 ± 1.05 nM, respectively. After subcutaneous administration in rats, ELP 120 fusion extended the half-life of IFN-α2a from 1.21 ± 0.21 h to 30.24 ± 3.16 h for ELP 120 -IFN-α2a and 25.81 ± 2.24 h for IFN-α2a-ELP 120 . In the xenograft model, IFN-α2a-ELP 120 produced the lowest final tumor volume despite having a slightly shorter half-life than ELP 120 -IFN-α2a. ELP 120 fusion substantially prolonged IFN-α2a exposure, but activity retention determined the most favorable orientation. IFN-α2a-ELP 120 represents the preferred lead format in this experimental system and warrants further benchmarking against established long-acting interferon platforms.