Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.

Agonist antibodies that activate cellular receptors are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their potent activation. This can be achieved using antibodies that recognize two unique epitopes on the same receptor and mediate receptor superclustering. However, identifying compatible pairs of antibodies to generate biepitopic antibodies (also known as biparatopic antibodies) for activating TNF receptors typically requires animal immunization and is a laborious and unpredictable process. Here, we report a simple method for systematically identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses off-the-shelf, receptor-specific IgG antibodies, which lack intrinsic (Fc-gamma receptor-independent) agonist activity, to first block their corresponding epitopes. Next, we perform selections for single-chain antibodies from human nonimmune libraries that bind accessible epitopes on the same ectodomains using yeast surface display and fluorescence-activated cell sorting. The selected single-chain antibodies are finally fused to the light chains of IgGs to generate human tetravalent antibodies that engage two different receptor epitopes and mediate potent receptor activation. We highlight the broad utility of this approach by converting several existing clinical-stage antibodies against TNF receptors, including ivuxolimab and pogalizumab against OX40 and utomilumab against CD137, into biepitopic antibodies with highly potent agonist activity. We expect that this widely accessible methodology can be used to systematically generate biepitopic antibodies for activating other receptors in the TNF receptor superfamily and many other receptors whose activation is dependent on strong receptor clustering.

Tumor necrosis factor (TNF) is an important cytokine that can regulate a variety of cellular responses by binding tumor necrosis factor receptor (TNFR). We studied whether the TNF of Eriocheir sinensis can regulate hemocyte proliferation. The results showed that the EsTNF and EsTNFR were constitutively expressed in all tested tissues, including the heart, hepatopancreas, muscles, gills, stomachs, intestines, and hemocytes. We found that low levels of EsTNF and EsTNFR transcripts were present in hemocytes. The gene expression levels were significantly increased in the hemocytes after being stimulated by Staphylococcus aureus or Vibrio parahaemolyticus. We also found some genes related to cell proliferation were expressed at a higher level in pulsing rTNF-stimulated hemocytes compared with the control group. We also knocked down the EsTNFR gene with RNAi technology. The results showed that the expression level of these genes related to cell proliferation was significantly down-regulated compared with the control group when the TNF does not bind TNFR. We used Edu technology to repeat the above experiments and the results were similar. Compared with the control group, the hemocytes stimulated by rTNF showed more significant proliferation, and the proliferation rate was significantly down-regulated after knocking down the EsTNFR gene. Therefore, we indicate that TNF binding TNFR can affect the proliferation of E. sinensis hemocytes, which might be manifested by affecting the expression of some proliferation-related genes.

In this study, we examined CD4 T cell activation using various stimuli in pediatric MOGAD patients (n = 4, untreated remission samples) and healthy controls (n = 5), to understand how both antigen-specific and bystander mechanisms contribute to CD4 T cell activation in MOGAD. TNFα, IL6, and MOG peptide pool were found to activate NF-κB or STAT3 pathways by measuring the expression of regulators (A20, IκBα) and phosphorylated subunits (phospho-p65 and phospho-STAT3) using immunolabeling. Prednisolone reversed activation of both NF-κB and STAT3 and increased the expression of A20 and IκBα. TNFR blocking partially reversed NF-κB activation in certain CD4 T cell subsets, but did not effect STAT3 activation. We observed that activation of NF-κB and STAT3 in response to various stimuli behaves mostly same in MOGAD (remission) and HC. IL6 stimulation resulted in higher STAT3 phosphorylation in MOGAD patients at 75 min, specifically in central and effector memory CD4 T cells (with unadjusted p-values). These findings suggest the potential therapeutic targeting of NF-κB and STAT3 pathways in MOGAD. Further investigation is needed to validate the significance of extended STAT3 phosphorylation and its correlation with IL6 receptor blocker treatment response.

Astrocytes contribute to chronic neuroinflammation in a variety of neurodegenerative diseases, including Parkinson\'s disease (PD), the most common movement disorder. However, the precise role of astrocytes in neuroinflammation remains incompletely understood. Herein, we show that regulator of G-protein signaling 5 (RGS5) promotes neurodegenerative process through augmenting astrocytic tumor necrosis factor receptor (TNFR) signaling. We found that selective ablation of Rgs5 in astrocytes caused an inhibition in the production of cytokines resulting in mitigated neuroinflammatory response and neuronal survival in animal models of PD, whereas overexpression of Rgs5 had the opposite effects. Mechanistically, RGS5 switched astrocytes from neuroprotective to pro-inflammatory property via binding to the receptor TNFR2. RGS5 also augmented TNFR signaling-mediated pro-inflammatory response by interacting with the receptor TNFR1. Moreover, interrupting RGS5/TNFR interaction by either RGS5 aa 1-108 or small molecular compounds feshurin and butein, suppressed astrocytic cytokine production. We showed that the transcription of astrocytic RGS5 was controlled by transcription factor early B cell factor 1 whose expression was reciprocally influenced by RGS5-modulated TNF signaling. Thus, our study indicates that beyond its traditional role in G-protein coupled receptor signaling, astrocytic RGS5 is a key modulator of TNF signaling circuit with resultant activation of astrocytes thereby contributing to chronic neuroinflammation. Blockade of the astrocytic RGS5/TNFR interaction is a potential therapeutic strategy for neuroinflammation-associated neurodegenerative diseases.

Costimulatory receptors on immune cells represent attractive targets for immunotherapy given that these molecules can increase the frequency of individual protective immune cell populations and their longevity, as well as enhance various effector functions. 4-1BB, a member of the TNF receptor superfamily, also known as CD137 and TNFRSF9, is one such molecule that is inducible on several cell types, including T cells and NK cells. Preclinical studies in animal models have validated the notion that stimulating 4-1BB with agonist reagents or its natural ligand could be useful to augment conventional T cell and NK cell immunity to protect against tumor growth and against viral infection. Additionally, stimulating 4-1BB can enhance regulatory T cell function and might be useful in the right context for suppressing autoimmunity. Two human agonist antibodies to 4-1BB have been produced and tested in clinical trials for cancer, with variable results, leading to the production of a wealth of second-generation antibody constructs, including bi- and multi-specifics, with the hope of optimizing activity and selectivity. Here, we review the progress to date in agonism of 4-1BB, discuss the complications in targeting the immune system appropriately to elicit the desired activity, together with challenges in engineering agonists, and highlight the untapped potential of manipulating this molecule in infectious disease and autoimmunity.

BACKGROUND: Ectodysplasin-A (EDA), a skin-specific TNF ligand, interacts with its membrane receptor EDAR to trigger EDA signaling in skin appendage formation. Gene mutations in EDA signaling cause Anhidrotic/Hypohidrotic Ectodermal Dysplasia (A/HED), which affects the formation of skin appendages including hair, teeth, and several exocrine glands.
RESULTS: We report that EDA triggers the translocation of its receptor EDAR from a cytosolic compartment into the plasma membrane. We use protein affinity purification to show that upon EDA stimulation EDAR associates with SNAP23-STX6-VAMP1/2/3 vesicle trafficking complexes. We find that EDA-dependent PKA activation is critical for the association. Notably, either of two HED-linked EDAR mutations, T346M and R420W, prevents EDA-induced EDAR translocation; and both EDA-induced PKA activation and SNAP23 are required for Meibomian gland (MG) growth in a skin appendage model.
CONCLUSIONS: Overall, in a novel regulatory mechanism, EDA increases plasma membrane translocation of its own receptor EDAR, augmenting EDA-EDAR signaling in skin appendage formation. Our findings also provide PKA and SNAP23 as potential targets for the intervention of HED.

The tumor necrosis factor superfamily (TNFSF) and their receptors (TNFRSF) are important regulators of the immune system, mediating proliferation, survival, differentiation, and function of immune cells. As a result, their targeting for immunotherapy is attractive, although to date, under-exploited. In this review we discuss the importance of co-stimulatory members of the TNFRSF in optimal immune response generation, the rationale behind targeting these receptors for immunotherapy, the success of targeting them in pre-clinical studies and the challenges in translating this success into the clinic. The efficacy and limitations of the currently available agents are discussed alongside the development of next generation immunostimulatory agents designed to overcome current issues, and capitalize on this receptor class to deliver potent, durable and safe drugs for patients.

The tumor necrosis factor alpha (TNF-α)-TNF-α receptor (TNFR) interaction plays a central role in the pathogenesis of various autoimmune diseases, particularly rheumatoid arthritis, and is therefore considered a key target for drug discovery. However, natural compounds that can specifically block the TNF-α-TNFR interaction are rarely reported. (-)-Epigallocatechin-3-gallate (EGCG) is the most active, abundant, and thoroughly investigated polyphenolic compound in green tea. However, the molecular mechanism by which EGCG ameliorates autoimmune arthritis remains to be elucidated. In the present study, we found that EGCG can directly bind to TNF-α, TNFR1, and TNFR2 with similar μM affinity and disrupt the interactions between TNF-α and TNFR1 and TNFR2, which inhibits TNF-α-induced L929 cell death, blocks TNF-α-induced NF-κB activation in 293-TNF-α response cell line, and eventually leads to inhibition of TNF-α-induced NF-κB signaling pathway in HFLS and MH7A cells. Thus, regular consumption of EGCG in green tea may represent a potential therapeutic agent for the treatment of TNF-α-associated diseases.

The capacity to induce tumour-cell specific apoptosis represents the most unique feature of the TNF receptor (TNFR) family member CD40. Recent studies on the signalling events triggered by its membrane-presented ligand CD40L (mCD40L) in normal and malignant epithelial cells have started to unravel an exquisite context and cell type specificity for the functional effects of CD40. Here, we demonstrate that, in comparison to other carcinomas, mCD40L triggered strikingly more rapid apoptosis in colorectal carcinoma (CRC) cells, underpinned by its ability to entrain two concurrently operating signalling axes. CD40 ligation initially activates TNFR-associated factor 3 (TRAF3) and subsequently NADPH oxidase (NOX)/Apoptosis signal-regulating kinase 1 (ASK1)-signalling and induction of reactive oxygen species (ROS) to mediate p38/JNK- and ROS-dependent cell death. At that point, p38/JNK signalling directly activates the mitochondrial pathway, and triggers rapid induction of intracellular TNF-related apoptosis-inducing ligand (TRAIL) that signals from internal compartments to initiate extrinsic caspase-10-asscociated apoptosis, leading to truncated Bid (tBid)-activated mitochondrial signalling. p38 and JNK are essential both for direct mitochondrial apoptosis induction and the TRAIL/caspase-10/tBid pathway, but their involvement follows functional hierarchy and temporally controlled interplay, as p38 function is required for JNK phosphorylation. By engaging both intrinsic and extrinsic pathways to activate apoptosis via two signals simultaneously, CD40 can accelerate CRC cell death. Our findings further unravel the multi-faceted properties of the CD40/mCD40L dyad, highlighted by the novel TNFR crosstalk that accelerates tumour cell-specific death, and may have implications for the use of CD40 as a therapeutic target.