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

Abstract

Objective. Interleukin-1 receptor antagonist (IL1Ra) 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 ␣ (TNF␣) 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 TNF␣ 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.Interleukin-1 (IL-1) has been implicated as a mediator of anabolic and catabolic processes in the progression of osteoarthritis (OA). IL-1 is a 17-kd protein that stimulates both synoviocytes and chondrocytes to secrete proteases and other

Synthesis and characterization of a thermally-responsive tumor necrosis factor antagonist

“…This approach maintained both phase transition behavior as well as activity for the drug domain, consistent with results for other ELP-drug conjugates. [32,33] Advantages of this system include acceptable expression levels of chimeric proteins with high drug-to-carrier ratios compared with chemical conjugation. [48][49][50] Purification of genetically engineered ELPs generates proteins of high purity and low endotoxin levels that have been demonstrated to be non-immunogenic, non-pyrogenic, and biocompatible, with endogenous amino acid degradation products.…”

“…The higher fusion protein K D and IC 50 compared against commercial sTNFRII is a trend for other fusion proteins. [33,53] The necessary K D or IC 50 required for this protein to achieve efficacy in the drug delivery concept proposed here depends also on other features of the in vivo system, such as clearance time and mechanism from the targeted space, total amount of protein delivered, and resolubilization kinetics. Caution is important in directly comparing against the commercial sTNFRII because the gene sequence and technique for synthesis and purification are quite different.…”

“…Drugs conjugated with ELPs gain properties of thermally-induced phase transition and also maintain in vitro bioactivity. This has been shown for chemicallyconjugated chemotherapeutics such as doxorubicin, [26] recombinant oligopeptide fusions with cell penetrating peptides [31] and a c-myc oncogene inhibitor, [32] and recombinant protein fusions with interleukin-1 receptor antagonist [33] and other proteins [34,35]. Surfaces coated with an ELP fused to the RGD or fibronectin CS5 cell binding sequence also retain an ability to support in vitro endothelial cell adhesion and spreading.…”

Peptide-based biopolymers in biomedicine and biotechnology

“…In a study by Betre et al, an aggregating ELP was shown to have a 25-fold longer half-life in the injected joint than an equivalent molecular weight protein that remains soluble and does not aggregate [165]. In an accompanying study by Shamji et al, an ELP-IL1Ra fusion displayed a complete recovery of IL1Ra activity, matching that of the commercial antagonist [166]. They also demonstrated the facile collagenase degradation of the ELP domain, which may further promote the therapeutic efficacy of this drug delivery system.…”

Fusion factor

“…3,4 Tropoelastin is the precursor to elastin, a key component of elastic skin fibers. Previous skin remodeling studies have suggested that elastin could be useful in dermal regeneration.…”