TNF-α and signaling

TNF-α is the most widely studied pleiotropic cytokine of the tumor necrosis factor superfamily. As an important cytokine in the innate immune response, TNF-α plays a key role in the direct host defense against invading microorganisms before the activation of the adaptive immune system. It is primarily produced by the activation of membrane-bounded pattern recognition molecules such as toll-like receptors by macrophages, which detect common bacterial cell surface products. TNF-α is also produced by several other pro-inflammatory cells, including monocytes, dendritic cells, B cells, CD4+ cells, neutrophils, mast cells, and eosinophils, as well as structural cells (i.e., fibroblasts, epithelial cells, and smooth muscle cells).

TNF-α is initially a biologically active, 26KD membrane-anchored precursor protein (mTNF-α) that is subsequently cleaved by TNF-α converting enzyme (TACE) to form a 17KD free protein. These proteins form biologically active homotrimers that act on the ubiquitously expressed TNF-α receptors 1 and 2. This receptor-ligand interaction results in intracellular signaling without internalizing the complex, and the phosphorylation of IkBa, which activates the nuclear factor-kB (p50-p65) heterodimer, which further interacts with DNA chromatin structure to increase the transcription of pro-inflammatory genes such as IL-1B, IL-6, IL-8, and TNF-α. The activating response of TNF-α is balanced by the shedding of the extracellular domain of the TNF-α receptor.

The biological mechanism and signal transduction of TNF-α

TNF-α and CRS

TNF-α is a major pleiotropic mediator of acute and chronic systemic inflammatory responses, regulating both apoptosis and proliferation, and promoting the production of other chemokines and cytokines. It is also involved in a range of physiological processes, such as antitumor responses, control of inflammation, and immune system homeostasis. TNF-α is also one of the most important pro-inflammatory cytokines in the innate immune response. The TNF-α signaling pathway plays a crucial role in cytokine release storm (CRS), and dysregulation of TNF-α signaling can trigger CRS. Elevation of TNF-α may accelerate viral infection, organ damage, and block cytokine-mediated inflammatory cell death signaling pathways which may benefit COVID-19 patients or other infectious diseases and limit tissue damage.

Anti-TNF-α therapy for CRS following COVID-19 infection

Some studies have pointed out that the adoption of anti-TNF therapy may be limited by its potential side effects. It is particularly important to distinguish patients with poor prognosis and improve clinical outcomes from patients who can recover on their own and find the treatment time for COVID-19 patients, so as to minimize potential risks associated with immunosuppression.

Drugs targeting TNF or its receptors in clinical studies of COVID-19 patients

TNF-α Antibodies

As we mentioned before, TNF-α is a powerful pro-inflammatory cytokine with pleiotropic effects on multiple cell types and plays an important role in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis (RA). A variety of anti-TNF drugs have been successfully used to treat chronic inflammatory diseases such as Crohn's disease, RA, ankylosing spondylitis (AS), and psoriasis. The clinical efficacy of different anti-TNF drugs may be due to their different effects on transmembrane TNF-α cells.

Different anti-TNF-α monoclonal antibodies

－ In terms of structure, Infliximab, Adalimumab, and Golimumab are monoclonal antibodies against human TNF-α. Infliximab is a human-mouse chimeric anti-TNF-α monoclonal antibody, consisting of a murine variable region and a human variable region IgG1 constant region. Adalimumab and Golimumab are fully humanized anti-TNF-α monoclonal antibodies. Etanercept is composed of two extracellular parts Human TNF-R2 (p75 TNF receptor) linked to the Fc part (CH2 and CH3 domains) of human IgG1. Certolizumab is an anti-TNFα- Fab fragment of IgG1 mAb, the hinge region is covalently linked to two 20KDa polyethylene glycol cross-linked chains, lacking the Fc fragment

－ Their binding properties are different, Infliximab can bind both monomeric and trimeric forms of soluble TNF-α, while Etanercept can only bind the trimer. Infliximab forms stable complexes with soluble TNF-α, whereas Etanercept forms relatively unstable complexes. Each Infliximab molecule can bind to two TNF-α molecules, and three Infliximab molecules can bind to the TNF-α homotrimer. In contrast, Etanercept is thought to form a 1:1 complex with TNF-α trimers。

Verification Data

Monoclonal Anti-TNF-alpha antibody, Mouse IgG1 (13B8).Cat. No. TNA-Y58

Immobilized Human TNF-alpha, His Tag (active trimer) (Cat. No. TNA-H5228) at 2 μg/mL (100 μL/well) can bind Monoclonal Anti-TNF-alpha antibody, Mouse IgG1 (13B8) (Cat. No. TNA-Y58) with a linear range of 0.3-20 ng/mL (QC tested).

Protocol

Monoclonal Anti-TNF-alpha antibody, Mouse IgG1 (13B8) (Cat. No. TNA-Y58) captured on CM5 chip via anti-mouse antibodies surface can bind Human TNF-alpha, His Tag (active trimer) (Cat. No. TNA-H5228) with an affinity constant of 0.323 nM as determined in a SPR assay (Biacore 8K).

Protocol

Monoclonal Anti-TNF-alpha antibody, Mouse IgG1 (16H5) Cat. No. TNA-Y59

Immobilized Human TNF-alpha, His Tag (active trimer) (Cat. No. TNA-H5228) at 2 μg/mL (100 μL/well) can bind Monoclonal Anti-TNF-alpha antibody, Mouse IgG1 (16H5) (Cat. No. TNA-Y59) with a linear range of 0.001-0.078 μg/mL (QC tested).

Protocol

Monoclonal Anti-TNF-alpha antibody, Mouse IgG1 (16H5) (Cat. No. TNA-Y59) captured on CM5 chip via anti-mouse antibodies surface can bind Human TNF-alpha, His Tag (active trimer) (Cat. No. TNA-H5228) with an affinity constant of 0.274 nM as determined in a SPR assay (Biacore 8K).

Protocol

Related Reading

＞＞[Newly Launched] Three anti-IFN γ antibodies in ACRO’s “mAb Matrix”

References

1. Christopher Brightling, Mike Berry, Yassine Amrani. Targeting TNF-α: A novel therapeutic approach for asthma. Journal of Allergy and Clinical Immunology (2008). https://doi.org/10.1016/j.jaci.2007.10.028.

2. Guo Y, Hu K, Li Y, Lu C, Ling K, Cai C, Wang W, Ye D. Targeting TNF-α for COVID-19: Recent Advanced and Controversies (2022). Front. Public Health. https://doi.org/10.3389/fpubh.2022.833967

3. Takahiko Horiuchi, Hiroki Mitoma, Shin-ichi Harashima, Hiroshi Tsukamoto, Terufumi Shimoda. Transmembrane TNF-α: structure, function and interaction with anti-TNF agents. Rheumatology (2010). https://doi.org/10.1093/rheumatology/keq031