Development and characterization of a fusion protein between thermally responsive elastin-like polypeptide and interleukin-1 receptor antagonist: Sustained release of a local antiinflammatory therapeutic

Objective. Interleukin-1 receptor antagonist (IL-1Ra) has been evaluated for the intraarticular treatment of osteoarthritis. Such administration of proteins may have limited utility because of their rapid clearance and short half-life in the joint. The fusion of a drug to elastin-like polypeptides (ELPs) promotes the formation of aggregating particles that form a "drug depot" at physiologic temperatures, a phenomenon intended to prolong the presence of the drug. The purpose of this study was to develop an injectable drug depot composed of IL-1Ra and ELP domains and to evaluate the properties and bioactivity of the recombinant ELP-IL-1Ra fusion protein. Methods. Fusion proteins between IL-1Ra and 2 distinct sequences and molecular weights of ELP were overexpressed in Escherichia coli. Environmental sensitivity was demonstrated by turbidity and dynamic light scattering as a function of temperature. IL-1Ra domain activity was evaluated by surface plasmon resonance, and in vitro antagonism of IL-1-mediated lymphocyte and thymocyte proliferation, as well as IL-1-induced tumor necrosis factor α (TNFα) expression and matrix metalloproteinase 3 (MMP-3) and ADAMTS-4 messenger RNA expression in human intervertebral disc fibrochondrocytes. IL-1Ra immunoreactivity was assessed before and after proteolytic degradation of the ELP partner. Results. Both fusion proteins underwent supramolecular aggregation at subphysiologic temperatures and slowly resolubilized at 37°C. Interaction with IL-1 receptor was slower in association but equivalent in dissociation as compared with the commercial antagonist. Anti-IL-1 activity was demonstrated by inhibition of lymphocyte and thymocyte proliferation and by decreased TNFα expression and ADAMTS-4 and MMP-3 transcription by fibrochondrocytes. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity as the commercial IL-1Ra. This peptide was more bioactive against lymphocyte proliferation, nearly equivalent to the commercial antagonist. Conclusion. The ELP-IL-1Ra fusion protein proved to retain the characteristic ELP inverse phasetransitioning behavior as well as the bioactivity of the IL-1Ra domain. This technology represents a novel drug carrier designed to prolong the presence of bioactive peptides following intraarticular delivery.