Tumor necrosis factor (TNF) was named for its ability to induce tumor regression. TNF-α is the first identified member of the TNF superfamily, followed by lymphotoxin-α (LT-α)/TNF-β. These two cytotoxic factors exhibit 50% amino acid sequence homology and bind to the same receptors. Based on sequence homology with TNF-α, additional members of the TNFSF have been identified. The Tumor Necrosis Factor Superfamily (TNFSF) comprises 19 ligands that can interact with 29 members of the TNF receptor superfamily (TNFRSF)^[1].

Figure 1. TNF Superfamily Ligand-Receptor Interactions and Downstream Signaling Pathways^[2].

TNF Superfamily

Most TNFSF members are trimeric type II transmembrane proteins, whose C-terminal extracellular regions contain a characteristic TNF homology domain (THD). The unique structural features of TNFSF ligands and their receptors enable them to directly engage signaling pathways involved in cell proliferation, survival, and differentiation. These proteins are primarily expressed by immune cells and control various cellular functions, including immune responses and inflammation^[3-4].

TNF-α – The Core Member of TNF Ligands

TNF-α is mainly produced by activated macrophages, monocytes, T cells (particularly Th1 and CD8⁺ T cells), NK cells, mast cells, and neutrophils, with its secretion markedly induced by lipopolysaccharides (LPS) and IL-1.

TNF-α exists in two forms: membrane-bound TNF-α (mTNF-α) and soluble TNF-α (sTNF-α). Both forms are biologically active as trimers but may elicit different, and sometimes opposing biological effects.

mTNF-α is expressed on the cell surface as a type II transmembrane protein and signals through TNFR2 receptor to promote eproliferation, survival, immune regulation, and tissue repair. In contrast, sTNF-α is generated by the metalloproteinases (such as TACE)-mediated cleavage of mTNF-α and signals via the TNFR1 receptor to drive pro-inflammatory, pro-apoptotic, and necrotic responses^[5-6].

Figure 2. Downstream Signaling Pathways of Tumor Necrosis Factor-α (TNF-α)^[6].

TNF-α and Immune-Related Diseases

Abnormal production of TNF-α and TNF receptor signaling is associated with the pathogenesis of various inflammatory diseases, including rheumatoid arthritis (RA), Crohn's disease, atherosclerosis, psoriasis, and cancer.

TNF-α exhibits both anti-cancer and pro-cancer effects in the tumor microenvironment (TME). It can induce tumor cell apoptosis by directly regulating the activation, function, and survival of leukocytes during cancer progression. However, due to its high toxicity, this effect is usually localized. More commonly and significantly, TNF-α exerts a pro-tumorigenic role. Endogenous TNF-α, persistently produced by tumor stroma and the TME, has been reported to induce tumor angiogenesis and promote cancer progression. Moreover, TNF-α induces the expression of adhesion molecules such as intracellular adhesion molecules (ICAM-1, VCAM-1) and E-selectin in liver sinusoidal endothelial cells, facilitating tumor invasion and metastasis^[8-9].

Apart from cancer, TNF-α plays a pivotal role in the immune milieu of rheumatoid arthritis (RA). It has been shown to exert significant and widespread effects on the function of regulatory T cells (Treg) and to directly induce the activation of osteoclasts, leading to bone erosion, which is a hallmark of chronic inflammation and joint destruction in RA. TNF-α inhibitors are cornerstone biologic agents in RA therapies, significantly improving symptoms and signs, suppressing radiographic progression (including bone erosion and joint space narrowing), and achieving clinical remission in a subset of patients^[10].

To date, several specific antibodies targeting TNF-α have been approved by the FDA, including Infliximab, Adalimumab, Golimumab, Certolizumab pegol, and Etanercept. These drugs hold significant value in the treatment of various human diseases; however, their use is challenged by issues such as ineffectiveness in some patients, increased risk of immunosuppression, and a higher chance of infections^[7,11].

Other Important Members of the TNF Ligands Family

TNF-β (also known as Lymphotoxin-α, LT-α) is primarily produced by lymphocytes. It shares the same receptorsas TNF-α—TNFR1/2, and exhibits similar functions, albeit with lower potency. Notably, TNF-β has another significant role in forming the LTα1β2 heterotrimer with LT-β, which binds to the LTβR receptor. This interaction promotes the structural development and maintenance of secondary lymphoid organs^[12].

Fas Ligand (FasL, also known as CD95L) is mainly expressed on activated T cells, particularly cytotoxic T cells, and NK cells. Upon binding to its receptor Fas on target cells, FasL induces apoptosis in various cell types such as activated lymphocytes, virus-infected cells, and tumor cells. This process is crucial for maintaining immune tolerance and peripheral immune homeostasis^[12].

TNF-related apoptosis-inducing ligand (TRAIL) exists in both soluble and membrane-bound forms. It binds to pro-apoptotic death receptors—DR4 and DR5 and anti-apoptotic decoy receptors—DcR1, DcR2, and OPG. TRAIL selectively induces apoptosis in tumor cells and transformed cells while exhibiting minimal toxicity to normal cells. This property makes TRAIL an important target in tumor immunotherapy^[13].

CD40 Ligand is primarily expressed on the surface of activated CD4+ T cells. It binds to CD40 on the surface of B cells, dendritic cells, macrophages, and other immune cells, playing a crucial role in B cell activation and the immune response. This interaction is essential for promoting antibody production and facilitating effective adaptive immunity^[14].

RANKL/OPGL exists in both soluble and membrane-bound forms. It is predominantly expressed by osteoblasts and activated T cells. RANKL binds to the receptor RANK, inducing differentiation, activation, and survival of osteoclasts, which makes it a key regulator of bone resorption (bone destruction). Additionally, RANKL can bind to OPG (decoy receptor), which serves as an important bone-protective factor by antagonizing the effects of RANKL^[15].

B-cell activating factor (BAFF) is primarily produced by myeloid cells such as macrophages, dendritic cells (DCs), and neutrophils. BAFF is a soluble factor that can bind to various receptors including BAFFR, TACI, and BCMA. It promotes B cell survival, maturation, and the maintenance of homeostasis. Abnormal BAFF signaling has been closely associated with autoimmune diseases, such as systemic lupus erythematosus (SLE), as well as certain B cell lymphomas^[16].

TNF Receptor Superfamily

The TNF receptor superfamily (TNFRSF) is characterized by one to six cysteine-rich domains (CRDs) in their extracellular regions, which are involved in ligand binding and receptor homodimerization. With the exception of BAFFR, BCMA, TACI, and EDA2R, which are type III transmembrane proteins, the majority of TNFRSF members are either type I transmembrane proteins or soluble decoy receptors. Based on structural similarities and functions, TNFRSF can be categorized into three classes:

• 1) Receptors with a cytoplasmic Death Domain (DD): These receptors promote the activation of caspases, leading to apoptosis;
• 2) Receptors lacking an intracellular DD: These receptors interact with members of the TNF receptor-associated factor (TRAF) family to activate signaling pathways such as NF-κB and MAPK, thereby regulating inflammation, cell survival, or death;
• 3) Receptors lacking a cytoplasmic domain or acting as decoy receptors: These receptors do not have intrinsic signaling capabilities and act by sequestering bind ligands without triggering intracellular signaling^[17-19].

Product Experiment Validation

Loaded Frexalimab (HY-P99625) on AHC2 biosensor, can bind CD40L/CD154/TRAP Protein, Human (HY-P70609) with an affinity constant of 4.117E-10 M as determined in BLI assay.

HY-P70609 CD40L/CD154/TRAP Protein, Human (HEK293)

Measured in a cytotoxicity assay using L-929 mouse fibroblast cells in the presence of the metabolic inhibitor actinomycin D. The ED50 for this effect is 62.25 pg/mL.

HY-P7058 TNF-alpha/TNFSF2 Protein, Human

Measured by its ability to inhibit Fas Ligand-induced apoptosis of Jurkat human acute T cell leukemia cells. The ED50 for this effect is 2.506 μg/mL in the presence of 2 ng/mL Human Fas Ligand.

HY-P70329 Fas/CD95 Protein, Human (HEK293, His)

Measured by its ability to induce osteoclast differentiation of RAW 264.7 mouse monocyte/macrophage cells. The ED50 for this effect is 19.69 ng/mL.

HY-P73386 RANKL/TNFSF11 Protein, Human (HEK293)