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
BACKGROUND: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method. RESULTS: Here, we present a comprehensive molecular and immunological characterization of 1E10 produced by the two different production processes in order to determine the impact of the manufacturing process in vaccine performance. We observed differences in glycosylation pattern, charge heterogeneity and structural stability between in vivo-produced 1E10 and bioreactor-obtained 1E10. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models. CONCLUSIONS: Changes in 1E10 primary structure like glycosylation; asparagine deamidation and oxidation affected 1E10 structural stability but did not affect the immune response elicited in mice and chickens when compared to 1E10 produced in mice.
