ABSTRACT
The neutralization of tumor necrosis factor alpha (TNFα) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNFα-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNFα and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNFα-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNFα was assessed. By microscale thermophoresis, the KD value for the interaction was 4.17 ± 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNFα-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNFα-antagonist agent.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Collagen/genetics , Endotoxins/antagonists & inhibitors , Etanercept/pharmacology , Receptors, Tumor Necrosis Factor, Type II/genetics , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Cell Survival/drug effects , Collagen/metabolism , Endotoxins/metabolism , Endotoxins/toxicity , Etanercept/chemistry , Etanercept/metabolism , Gene Expression , HEK293 Cells , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , Protein Engineering/methods , Protein Multimerization , Receptors, Tumor Necrosis Factor, Type II/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/toxicityABSTRACT
TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS-PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10-9â M and >10-8â M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.
Subject(s)
Adalimumab/chemistry , Etanercept/chemistry , Tumor Necrosis Factor-alpha , Humans , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/chemistryABSTRACT
Etanercept is a recombinant fusion protein approved for the treatment of TNF-α mediated diseases such as rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, and ankylosing spondylitis. Herein, we present an evaluation of the physicochemical and biological properties of a biosimilar etanercept and its reference product followed by a clinical study in patients diagnosed with RA intended to demonstrate comparability of their immunomodulatory activity. Identity analyses showed a total correspondence of the primary and higher-order structure between the two products. In regard to intrinsic heterogeneity, both products showed to be highly heterogenous; however the biosimilar etanercept exhibited similar charge and glycan heterogeneity intervals compared to the reference product. Apoptosis inhibition assay also showed that, despite the high degree of heterogeneity exhibited by both products, no significant differences exist in their in vitro activity. Finally, the clinical assessment conducted in RA-diagnosed patients did not show significant differences in the evaluated pharmacodynamic markers of both products. Collectively, the results from the comparability exercise provide convincing evidence that the evaluated biosimilar etanercept can be considered an effective alternative for the treatment of RA.