Abstract
The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signaling that cause collateral damage to protective signaling cascades carrying the potential for unwanted side effects. The variable domains of heavy-chain only antibodies (HCAbs) discovered in Camelidae are stable and display to be fully functional in antigen-binding against variable targets, which seem to be attractive candidates for the next-generation biologic drug study. The purpose of our study was to establish a simple prokaryotic expression system for large-scale expression, purification, and refolding of the recombinant anti-tumor necrosis factor α (TNF-α) fusion protein (FVH1-1) from inclusion bodies. Over 95% purity of the recombinant anti-TNF-α fusion proteins was obtained by just one purification step in our developed prokaryotic expression system, while the results of surface plasmon resonance (SPR) established the high-efficiency potent binding ability of FVH1-1 to human TNF-α. The counteraction of TNF-α cytotoxic effect experiment on the mouse fibroblast fibrosarcoma cell line (L929) confirmed that the expressed FVH1-1 were able to selectively and highly combine with human recombinant TNF-α (hTNF-α) in vitro. Western blot results showed that FVH1-1 can inhibit the activation of caspase-9 and PARP, which are the apoptotic signaling pathway proteins activated by hTNF-α. Meanwhile, lysosome autophagy signaling pathways stimulated by hTNF-α were inhibited by FVH1-1, which down-regulated the expression of LC3II/LC3I and up-regulated the expression of P62, indicating that the autophagy linked with TNF-α-induced apoptosis in response to rheumatoid arthritis. The results of the AIA rat model experiment presented that FVH1-1 can reduce the degree of joint swelling and inflammatory factors to a certain extent in vivo.
Highlights
Tumor necrosis factor α (TNF-α), a multifunctional immunomodulatory molecule secreted by stimulated mononuclear macrophages and some other cells in vivo, can bind to the cytomembrane receptor, leading to the local aggregation of immune effector cells or the death of target cells [1,2]
The present study aimed to explore the therapeutic effect of the recombinant anti-TNF-α fusion proteins (FVH1-1), obtained by our developed prokaryotic expression system, on adjuvant-induced arthritis (AIA) rats, and elucidated its underlying mechanism of autophagy action related to inflammatory mediator TNF-α in rheumatoid arthritis
The presence of the recombinant protein in the induced E. coli cells was confirmed by a Western blot using His-tag specific antibody that allows visualization of His-tagged fusion proteins
Summary
Tumor necrosis factor α (TNF-α), a multifunctional immunomodulatory molecule secreted by stimulated mononuclear macrophages and some other cells in vivo, can bind to the cytomembrane receptor, leading to the local aggregation of immune effector cells or the death of target cells [1,2]. The excessive inflammatory responses lead to the pathological processes of multiple diseases, such as metabolic disease, autoimmune diseases, and chronic inflammation [3]. Anti-cytokine therapies and immune checkpoint inhibitors, representing antagonists or inhibitors of signaling cascades related to be pathogenic in a particular disease state, strongly improve the treatment of autoimmune diseases and cancer [5]. Many currently used therapeutic antibodies can neutralize TNF-α and negatively regulate the activity of TNF-α in vivo [7]. When such anti-TNF-α antibodies are applied systemically, it is difficult for them to separate pathogenic signaling from physiological signaling, leading to limited clinical efficacy and unwanted side effects [5]. A more cost-effective alternative anti-TNF-α therapeutic antibody that can target the related cognate antigens only in the particular organ or cell lineage is, urgently required
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