Abstract
Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans-induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.
Highlights
Interleukin-1 (IL-1) is a key mediator of inflammation and immunity
24 h post-injection, the level of IL-1 receptor antagonist (IL-1Ra) in the blood plasma of the anakinra group was undetectable by ELISA; in contrast, a mean of 114.0 Ϯ 34.0 ng/ml IL-1Ra (6.63 Ϯ 1.97 pM) was detected in the PAS600 –IL-1Ra group. This level was measured following the administration of 3.90 nmol of PAS600 –IL-1Ra, which is 25.4% of the mole-equivalent ana
Anakinra is the recombinant form of naturally-occurring IL-1Ra used to treat a broad spectrum of inflammatory diseases [14]; anakinra requires daily administration
Summary
Immunogenicity has not been observed for any PASylated proteins tested in vivo in preclinical models [35, 36] Generating these fusion proteins via transcription/translation of a recombinant gene in a suitable host cell avoids challenges posed by post-translational protein modification strategies like PEGylation, including low functional yields, enhanced hydrophobicity and concerns of immunogenicity, indigestibility, and persistence of the PEG moiety in the body [37,38,39]. Along with these improvements, modulation of the precise length of the PAS moiety (via the number of sequence repeats in the coding gene) allows for fine-tuning of the protein hydrodynamic volume, which correlates with plasma half-life [27].
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