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From the beginning of research on receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), agonistic antibodies have been used to stimulate TNFRSF receptors in vitro and in vivo. Indeed, CD95, one of the first cloned TNFRSF receptors, was solely identified as the target of cell death-inducing antibodies. Early on, it became evident from in vitro studies that valency and Fcγ receptor (FcγR) binding of antibodies targeting TNFRSF receptors can be of crucial relevance for agonistic activity. TNFRSF receptor-specific antibodies of the IgM subclass and secondary cross-linked or aggregation prone dimeric antibodies typically display superior agonistic activity compared with dimeric antibodies. Likewise, anchoring of antibodies to cell surface-expressed FcγRs potentiate their ability to trigger TNFRSF receptor signaling. However, only recently has the relevance of oligomerization and FcγR binding for the in vivo activity of antibody-induced TNFRSF receptor activation been straightforwardly demonstrated in vivo. This review discusses the crucial role of oligomerization and/or FcγR binding for antibody-mediated TNFRSF receptor stimulation in light of current models of TNFRSF receptor activation and especially the overwhelming relevance of these issues for the rational development of therapeutic TNFRSF receptor-targeting antibodies. [ABSTRACT FROM AUTHOR]

Constitutively active PI3K catalytic subunit α (PIK3CA) interfered with apoptosis induction downstream of death receptor-signaling complex formation allowing robust caspase-8 activation without triggering the execution steps of apoptosis. In mutant PIK3CA-expressing cells, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95L stimulated nuclear factor kappaB (NFκB) activation, invasion, and transition to an amoeboid-like morphology. NFκB activation and adoption of amoeboid shape were inhibited by caspase-8 knockdown or FLIP-S expression, but only the cell morphology alterations required caspase-8 activity. Furthermore, we identified caspase-8-mediated, caspase-3-independent cleavage of the protein kinase rho-associated, coiled-coil containing protein kinase 1 as a novel mechanism for acquiring amoeboid shape and enhanced invasiveness in response to TRAIL and CD95L. Taken together, we provide evidence that mutated PIK3CA converts the ‘tumor surveillance’ activity of cancer cell-expressed death receptors and caspase-8 toward tumor promotion. [ABSTRACT FROM AUTHOR]

Soluble TNF-like weak inducer of apoptosis (TWEAK) trimers induce, in a variety of cell lines, translocation of cytosolic tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) to a triton X-100-insoluble compartment without changes in the total cellular TRAF2 content. TWEAK-induced TRAF2 translocation is paralleled by a strong increase in nuclear factor kappaB 2 (NFκB2)/p100 processing to p52, indicating that TRAF2 redistribution is sufficient for activation of the alternative NFκB pathway. In accordance with the crucial role of TRAF2 in proinflammatory, anti-apoptotic TNF receptor-1 (TNFR1) signaling, we observed that TWEAK-primed cells have a reduced capacity to activate the classical NFκB pathway or JNK (cJun N-terminal kinase) in response to TNF. Furthermore, TWEAK-primed cells are sensitized for the TNFR1-mediated induction of apoptotic and necrotic cell death. Notably, the expression of the NFκB-regulated, TRAF2-interacting TRAF1 protein can attenuate TWEAK-induced depletion of the triton X-100-soluble TRAF2 fraction and improve TNFR1-induced NFκB signaling in TWEAK-primed cells. Taken together, we demonstrate that soluble TWEAK desensitizes cells for proinflammatory TNFR1 signaling and thus identify TWEAK as a modifier of TNF signaling. [ABSTRACT FROM AUTHOR]

Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity.Cell Death and Differentiation (2007) 14, 2021–2034; doi:10.1038/sj.cdd.4402213; published online 17 August 2007 [ABSTRACT FROM AUTHOR]

To achieve tumor cell-restricted activation of CD95, we developed a CD95L fusion protein format, in which CD95L activity is only unmasked upon antibody-mediated binding to tumor cells and subsequent processing by tumor-associated proteases, such as matrix metalloproteases (MMPs) and urokinase plasminogen activator (uPA). On target-negative, but MMP- and uPA-expressing HT1080 tumor cells, the CD95L prodrugs were virtually inactive. On target antigen-expressing HT1080 cells, however, the CD95L prodrugs showed an apoptotic activity comparable to soluble CD95L artificially activated by crosslinking. CD95 activation by the CD95L prodrugs was preceded by prodrug processing. Apoptosis was blocked by inhibitors of MMPs or uPA and by neutralizing antibodies recognizing the targeted cell surface antigen or the CD95L moiety of the prodrugs. In a xenotransplantation tumor model, local application of the prodrug reduced the growth of target antigen-expressing, but not antigen-negative tumor cells, verifying targeted CD95L prodrug activation in vivo.Cell Death and Differentiation (2007) 14, 765–774. doi:10.1038/sj.cdd.4402051; published online 20 October 2006 [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF) prodrugs are fusion proteins comprised of an N-terminal single-chain antibody variable fragment (scFv) targeting a TNF effector and a C-terminal TNF receptor (TNFR)1-derived inhibitor module. Introduction of matrix metalloproteinase (MMP)-2 recognition motifs between TNF and the TNFR1 fragment allowed activation by recombinant MMP-2 and MMP-expressing HT1080 cells. Processing by endogeneous MMPs required specific membrane binding of the TNF prodrug via the targeting scFv, ensuring strictly antigen-dependent activation. Interestingly, TNF bioactivity of the processed prodrug was ∼1000-fold higher upon scFv-mediated targeting, and signaled juxtatropic cell death also to antigen-negative cells. Microscopical analyses of TNFR2 clustering and TNF receptor-associated factor 2 recruitment at contact sites to adjacent cells revealed the formation of stable TNFR complexes by target-bound, processed prodrug, resembling the increased signal capacity of natural, membrane-expressed TNF. MMP-2-sensitive TNF prodrugs represent novel cytokine-based reagents for targeted cancer therapy, which should be exploitable for MMP-overexpressing tumors.Cell Death and Differentiation (2006) 13, 273–284. doi:10.1038/sj.cdd.4401735; published online 29 July 2005 [ABSTRACT FROM AUTHOR]

A single mouse click on the topic tumor necrosis factor (TNF) in PubMed reveals about 50 000 articles providing one or the other information about this pleiotropic cytokine or its relatives. This demonstrates the enormous scientific and clinical interest in elucidating the biology of a molecule (or rather a large family of molecules), which began now almost 30 years ago with the description of a cytokine able to exert antitumoral effects in mouse models. Although our understanding of the multiple functions of TNF in vivo and of the respective underlying mechanisms at a cellular and molecular level has made enormous progress since then, new aspects are steadily uncovered and it appears that still much needs to be learned before we can conclude that we have a full comprehension of TNF biology. This review shortly covers some general aspects of this fascinating molecule and then concentrates on the molecular mechanisms of TNF signal transduction. In particular, the multiple facets of crosstalk between the various signalling pathways engaged by TNF will be addressed. [ABSTRACT FROM AUTHOR]

Inhibition of CD95/Fas activation is currently under clinical investigation as a therapy for glioblastoma multiforme and preclinical studies suggest that disruption of the CD95-CD95L interaction could also be a strategy to treat inflammatory and neurodegenerative disorders. Besides neutralizing anti-CD95L/FasL antibodies, mainly CD95ed-Fc, a dimeric Fc fusion protein of the extracellular domain of CD95 (CD95ed), is used to prevent CD95 activation. In view of the fact that full CD95 activation requires CD95L-induced CD95 trimerization and clustering of the resulting liganded CD95 trimers, we investigated whether fusion proteins of the extracellular domain of CD95 with a higher valency than CD95ed-Fc have an improved CD95L-neutralization capacity. We evaluated an IgG1(N297A)-based tetravalent CD95ed fusion protein which was obtained by replacing the variable domains of IgG1(N297A) with CD95ed (CD95ed-IgG1(N297A)) and a hexavalent variant obtained by fusion of CD95ed with a TNC-Fc(DANA) scaffold (CD95ed-TNC-Fc(DANA)) promoting hexamerization. The established N297A and DANA mutations were used to minimize FcγR binding of the constructs under maintenance of neonatal Fc receptor (FcRn) binding. Size exclusion high-performance liquid chromatography indicated effective assembly of CD95ed-IgG1(N297A). More important, CD95ed-IgG1(N297A) was much more efficient than CD95ed-Fc in protecting cells from cell death induction by human and murine CD95L. Surprisingly, despite its hexavalent structure, CD95ed-TNC-Fc(DANA) displayed an at best minor improvement of the capacity to neutralize CD95L suggesting that besides valency, other factors, such as spatial organization and agility of the CD95ed domains, play also a role in neutralization of CD95L trimers by CD95ed fusion proteins. More studies are now required to evaluate the superior CD95L-neutralizing capacity of CD95ed-IgG1(N297A) in vivo., (© 2024 The Author(s). PROTEINS: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Most anti-CD40 antibodies show robust agonism only upon binding to FcγR + cells, such as B cells, macrophages, or DCs, but a few anti-CD40 antibodies display also strong intrinsic agonism dependent on the recognized epitope and/or isotype. It is worth mentioning, however, that also the anti-CD40 antibodies with intrinsic agonism can show a further increase in agonistic activity when bound by FcγR-expressing cells. Thus, conventional antibodies appear not to be sufficient to trigger the maximum possible CD40 activation independent from FcγR-binding. We proved here the hypothesis that oligomeric and oligovalent anti-CD40 antibody variants generated by genetic engineering display high intrinsic, thus FcγR-independent, agonistic activity. We generated tetra-, hexa- and dodecavalent variants of six anti-CD40 antibodies and a CD40-specific nanobody. All these oligovalent variants, even when derived of bivalent antagonistic anti-CD40 antibodies, showed strongly enhanced CD40 agonism compared to their conventional counterparts. In most cases, the CD40 agonism reached the maximum response induced by FcγR-bound anti-CD40 antibodies or membrane CD40L, the natural engager of CD40. In sum, our data show that increasing the valency of anti-CD40 antibody constructs by genetic engineering regularly results in molecules with high intrinsic agonism and level out the specific limitations of the parental antibodies.

Tumor necrosis factor-α (TNFα) is a master cytokine which induces expression of chemokines and adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in endothelial cells to initiate the vascular inflammatory response. In this study, we identified neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, as a modulator of TNFα-induced inflammatory response of endothelial cells. NRP1 shRNA expression suppressed TNFα-stimulated leukocyte adhesion and expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs). Likewise, it reduced TNFα-induced phosphorylation of MAPK p38 but did not significantly affect other TNF-induced signaling pathways, such as the classical NFκB and the AKT pathway. Immunofluorescent staining demonstrated co-localization of NRP1 with the two receptors of TNF, TNFR1 and TNFR2. Co-immunoprecipitation further confirmed that NRP1 was in the same protein complex or membrane compartment as TNFR1 and TNFR2, respectively. Modulation of NRP1 expression, however, neither affected TNFR levels in the cell membrane nor the receptor binding affinities of TNFα. Although a direct interface between NRP1 and TNFα/TNFR1 appeared possible from a protein docking model, a direct interaction was not supported by binding assays in cell-free microplates and cultured cells. Furthermore, TNFα was shown to downregulate NRP1 in a time-dependent manner through TNFR1-NFκB pathway in HUVECs. Taken together, our study reveals a novel reciprocal crosstalk between NRP1 and TNFα in vascular endothelial cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Wang, Anany, Füllsack, Huo, Dutta, Ji, Hoeppner, Kilari, Misra, Caulfield, Vander Kooi, Wajant and Mukhopadhyay.)

Background: Selective TNFR2 activation can be used to treat immune pathologies by activating and expanding regulatory T-cells (Tregs) but may also restore anti-tumour immunity by co-stimulating CD8 + T-cells. Oligomerized TNFR2-specific TNF mutants or anti-TNFR2 antibodies can activate TNFR2 but suffer either from poor production and pharmacokinetics or in the case of anti-TNFR2 antibodies typically from the need of FcγR binding to elicit maximal agonistic activity. Methods: To identify the major factor(s) determining FcγR-independent agonism of anti-TNFR2 antibodies, we systematically investigated a comprehensive panel of anti-TNFR2 antibodies and antibody-based constructs differing in the characteristics of their TNFR2 binding domains but also in the number and positioning of the latter. Results: We identified the domain architecture of the constructs as the pivotal factor enabling FcγR-independent, thus intrinsic TNFR2-agonism. Anti-TNFR2 antibody formats with either TNFR2 binding sites on opposing sites of the antibody scaffold or six or more TNFR2 binding sites in similar orientation regularly showed strong FcγR-independent agonism. The affinity of the TNFR2 binding domain and the epitope recognized in TNFR2, however, were found to be of only secondary importance for agonistic activity. Conclusion: Generic design principles enable the generation of highly active bona fide TNFR2 agonists from nearly any TNFR2-specific antibody., Competing Interests: Competing Interests: The University of Würzburg has filed a patent application for “Novel TNFR2 binding molecules” with H.W., A.B., T.S, J.M. and L.V.R. as inventors. Furthermore, University of Würzburg receives funding from Dualyx NV and H.W. and A.B. are consultants of Dualyx NV., (© The author(s).)

Ischemic stroke often leaves survivors with permanent disabilities and therapies aimed at limiting detrimental inflammation and improving functional outcome are still needed. Tumor necrosis factor (TNF) levels increase rapidly after ischemic stroke, and while signaling through TNF receptor 1 (TNFR1) is primarily detrimental, TNFR2 signaling mainly has protective functions. We therefore investigated how systemic stimulation of TNFR2 with the TNFR2 agonist NewSTAR2 affects ischemic stroke in mice. We found that NewSTAR2 treatment induced changes in peripheral immune cell numbers and transiently affected microglial numbers and neuroinflammation. However, this was not sufficient to improve long-term functional outcome after stroke in mice., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Tumor necrosis factor (TNF) receptor 1 (TNFR1), TNFR2 and fibroblast growth factor-inducible 14 (Fn14) belong to the TNF receptor superfamily (TNFRSF). From a structural point of view, TNFR1 is a prototypic death domain (DD)-containing receptor. In contrast to other prominent death receptors, such as CD95/Fas and the two TRAIL death receptors DR4 and DR5, however, liganded TNFR1 does not instruct the formation of a plasma membrane-associated death inducing signaling complex converting procaspase-8 into highly active mature heterotetrameric caspase-8 molecules. Instead, liganded TNFR1 recruits the DD-containing cytoplasmic signaling proteins TRADD and RIPK1 and empowers these proteins to trigger cell death signaling by cytosolic complexes after their release from the TNFR1 signaling complex. The activity and quality (apoptosis versus necroptosis) of TNF-induced cell death signaling is controlled by caspase-8, the caspase-8 regulatory FLIP proteins, TRAF2, RIPK1 and the RIPK1-ubiquitinating E3 ligases cIAP1 and cIAP2. TNFR2 and Fn14 efficiently recruit TRAF2 along with the TRAF2 binding partners cIAP1 and cIAP2 and can thereby limit the availability of these molecules for other TRAF2/cIAP1/2-utilizing proteins including TNFR1. Accordingly, at the cellular level engagement of TNFR2 or Fn14 inhibits TNFR1-induced RIPK1-mediated effects reaching from activation of the classical NFκB pathway to induction of apoptosis and necroptosis. In this review, we summarize the effects of TNFR2- and Fn14-mediated depletion of TRAF2 and the cIAP1/2 on TNFR1 signaling at the molecular level and discuss the consequences this has in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Siegmund, Zaitseva and Wajant.)

The cytokine TNF signals via two distinct receptors, TNF receptor 1 (TNFR1) and TNFR2, and is a central mediator of various immune-mediated diseases. Indeed, TNF-neutralizing biologic drugs have been in clinical use for the treatment of many inflammatory pathological conditions, including various rheumatic diseases, for decades. TNF has pleiotropic effects and can both promote and inhibit pro-inflammatory processes. The integrated net effect of TNF in vivo is a result of cytotoxic TNFR1 signalling and the stimulation of pro-inflammatory processes mediated by TNFR1 and TNFR2 and also TNFR2-mediated anti-inflammatory and tissue-protective activities. Inhibition of the beneficial activities of TNFR2 might explain why TNF-neutralizing drugs, although highly effective in some diseases, have limited benefit in the treatment of other TNF-associated pathological conditions (such as graft-versus-host disease) or even worsen the pathological condition (such as multiple sclerosis). Receptor-specific biologic drugs have the potential to tip the balance from TNFR1-mediated activities to TNFR2-mediated activities and enable the treatment of diseases that do not respond to current TNF inhibitors. Accordingly, a variety of reagents have been developed that either selectively inhibit TNFR1 or selectively activate TNFR2. Several of these reagents have shown promise in preclinical studies and are now in, or approaching, clinical trials., (© 2023. Springer Nature Limited.)

Fibroblast growth factor (FGF)-inducible 14 (Fn14) activates the classical and alternative NFκB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) signaling pathway but also enhances tumor necrosis factor (TNF)-induced cell death. Fn14 expression is upregulated in non-hematopoietic cells during tissue injury and is also often highly expressed in solid cancers. In view of the latter, there were and are considerable preclinical efforts to target Fn14 for tumor therapy, either by exploiting Fn14 as a target for antibodies with cytotoxic activity (e.g. antibody-dependent cellular cytotoxicity (ADCC)-inducing IgG variants, antibody drug conjugates) or by blocking antibodies with the aim to interfere with protumoral Fn14 activities. Noteworthy, there are yet no attempts to target Fn14 with agonistic Fc effector function silenced antibodies to unleash the proinflammatory and cell death-enhancing activities of this receptor for tumor therapy. This is certainly not at least due to the fact that anti-Fn14 antibodies only act as effective agonists when they are presented bound to Fcγ receptors (FcγR). Thus, there are so far no antibodies that robustly and selectively engage Fn14 signaling without triggering unwanted FcγR-mediated activities. In this study, we investigated a panel of variants of the anti-Fn14 antibody 18D1 of different valencies and domain architectures with respect to their inherent FcγR-independent ability to trigger Fn14-associated signaling pathways. In contrast to conventional 18D1, the majority of 18D1 antibody variants with four or more Fn14 binding sites displayed a strong ability to trigger the alternative NFκB pathway and to enhance TNF-induced cell death and therefore resemble in their activity soluble (TNF)-like weak inducer of apoptosis (TWEAK), one form of the natural occurring ligand of Fn14. Noteworthy, activation of the classical NFκB pathway, which naturally is predominately triggered by membrane-bound TWEAK but not soluble TWEAK, was preferentially observed with a subset of constructs containing Fn14 binding sites at opposing sites of the IgG scaffold, e.g. IgG1-scFv fusion proteins. A superior ability of IgG1-scFv fusion proteins to trigger classical NFκB signaling was also observed with the anti-Fn14 antibody PDL192 suggesting that we identified generic structures for Fn14 antibody variants mimicking soluble and membrane-bound TWEAK., Competing Interests: The University of Würzburg filed a patent describing novel Fn14 agonists with authors OZ, CO and HW as co-inventors. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zaitseva, Hoffmann, Löst, Anany, Zhang, Kucka, Wiegering, Otto and Wajant.)

Staphylococcus aureus is the leading cause of skin and soft tissue infections and is a major health burden due to the emergence of antibiotic-resistant strains. To address the unmet need of alternative treatments to antibiotics, a better understanding of the protective immune mechanisms against S. aureus skin infection is warranted. Here, we report that tumor necrosis factor (TNF) promoted protection against S. aureus in the skin, which was mediated by bone marrow-derived immune cells. Furthermore, neutrophil-intrinsic TNF receptor (TNFR) signaling directed immunity against S. aureus skin infections. Mechanistically, TNFR1 promoted neutrophil recruitment to the skin, whereas TNFR2 prevented systemic bacterial dissemination and directed neutrophil antimicrobial functions. Treatment with a TNFR2 agonist showed therapeutic efficacy against S. aureus and Pseudomonas aeruginosa skin infections, which involved increased neutrophil extracellular trap formation. Our findings revealed nonredundant roles for TNFR1 and TNFR2 in neutrophils for immunity against S. aureus and can be therapeutically targeted for protection against bacterial skin infections.

Many new immunotherapeutic approaches aim on the stimulatory targeting of receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) using antibodies with intrinsic or conditional agonism. There is an initial need to characterize corresponding TNFRSF receptor (TNFR)-targeting antibodies with respect to affinity, ligand binding, receptor activation and the epitope recognized. Here, we report a collection of simple and matched protocols enabling the detailed investigation of these aspects by help of Gaussia princeps luciferase (GpL) fusion proteins and analysis of interleukin-8 (IL8) production as an easily measurable readout of TNFR activation. In a first step, the antibodies and antibody variants of interest are transiently expressed in human embryonal kidney 293 cells, either in non-modified form or as fusion proteins with GpL as a reporter domain. The supernatants containing the antibody-GpL fusion proteins can then be used without further purification in cell-free and/or cellular binding studies to determine affinity. Similarly, binding studies with mutated TNFR variants enable the characterization of the antibody binding site within the TNFR ectodomain. Furthermore, in cellular binding studies with GpL fusion proteins of soluble TNFL molecules, the ability of the non-modified antibody variants to interfere with TNFL-TNFR interaction can be analyzed. Last but not least, we describe a protocol to determine the intrinsic and the Fc gamma receptor (FcγR)-dependent agonism of anti-TNFR antibodies which exploits i) the capability of TNFRs to trigger IL8 production in tumor cell lines lacking expression of FcγRs and ii) vector- and FcγR-transfected cells, which produce no or only very low amounts of human IL8. The presented protocols only require standard molecular biological equipment, eukaryotic cell culture and plate readers for the quantification of luminescent and colorimetric signals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zaitseva, Anany, Wajant and Lang.)

Adiponectin and the other 15 members of the complement 1q (C1q)/tumor necrosis factor (TNF)-related protein (CTRP) family are secreted proteins composed of an N-terminal variable domain followed by a stalk region and a characteristic C-terminal trimerizing globular C1q (gC1q) domain originally identified in the subunits of the complement protein C1q. We performed a basic PubMed literature search for articles mentioning the various CTRPs or their receptors in the abstract or title. In this narrative review, we briefly summarize the biology of CTRPs and focus then on the structure, receptors and major signaling pathways of CTRPs. Analyses of CTRP knockout mice and CTRP transgenic mice gave overwhelming evidence for the relevance of the anti-inflammatory and insulin-sensitizing effects of CTRPs in autoimmune diseases, obesity, atherosclerosis and cardiac dysfunction. CTRPs form homo- and heterotypic trimers and oligomers which can have different activities. The receptors of some CTRPs are unknown and some receptors are redundantly targeted by several CTRPs. The way in which CTRPs activate their receptors to trigger downstream signaling pathways is largely unknown. CTRPs and their receptors are considered as promising therapeutic targets but their translational usage is still hampered by the limited knowledge of CTRP redundancy and CTRP signal transduction.

Background: Tumor necrosis factor-alpha (TNF-α) is a master cytokine involved in a variety of inflammatory and neurological diseases, including Alzheimer's disease (AD). Therapies that block TNF-α proved ineffective as therapeutic for neurodegenerative diseases, which might be explained by the opposing functions of the two receptors of TNF (TNFRs): while TNFR1 stimulation mediates inflammatory and apoptotic pathways, activation of TNFR2 is related to neuroprotection. Despite the success of targeting TNFR2 in a transgenic AD mouse model, research that better mimics the human context is lacking., Objective: The aim of this study is to investigate whether stimulation of TNFR2 with a TNFR2 agonist is effective in activating human TNFR2 and attenuating AD neuropathology in the J20xhuTNFR2-k/i mouse model., Methods: Transgenic amyloid-β (Aβ)-overexpressing mice containing a human extracellular TNFR2 domain (J20xhuTNFR2-k/i) were treated with a TNFR2 agonist (NewStar2). After treatment, different behavioral tests and immunohistochemical analysis were performed to assess different parameters, such as cognitive functions, plaque deposition, synaptic plasticity, or microglial phagocytosis., Results: Treatment with NewStar2 in J20xhuTNFR2-k/i mice resulted in a drastic decrease in plaque load and beta-secretase 1 (BACE-1) compared to controls. Moreover, TNFR2 stimulation increased microglial phagocytic activity, leading to enhanced Aβ clearance. Finally, activation of TNFR2 rescued cognitive impairments and improved synaptic plasticity., Conclusion: Our findings demonstrate that activation of human TNFR2 ameliorates neuropathology and improves cognitive functions in an AD mouse model. Moreover, our study confirms that the J20xhuTNFR2-k/i mouse model is suitable for testing human TNFR2-specific compounds.

Inhibitory targeting of the CD40L-CD40 system is a promising therapeutic option in the field of organ transplantation and is also attractive in the treatment of autoimmune diseases. After early complex results with neutralizing CD40L antibodies, it turned out that lack of Fcγ receptor (FcγR)-binding is the crucial factor for the development of safe inhibitory antibodies targeting CD40L or CD40. Indeed, in recent years, blocking CD40 antibodies not interacting with FcγRs, has proven to be well tolerated in clinical studies and has shown initial clinical efficacy. Stimulation of CD40 is also of considerable therapeutic interest, especially in cancer immunotherapy. CD40 can be robustly activated by genetically engineered variants of soluble CD40L but also by anti-CD40 antibodies. However, the development of CD40L-based agonists is biotechnologically and pharmacokinetically challenging, and anti-CD40 antibodies typically display only strong agonism in complex with FcγRs or upon secondary crosslinking. The latter, however, typically results in poorly developable mixtures of molecule species of varying stoichiometry and FcγR-binding by anti-CD40 antibodies can elicit unwanted side effects such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) of CD40 expressing immune cells. Here, we summarize and compare strategies to overcome the unwanted target cell-destroying activity of anti-CD40-FcγR complexes, especially the use of FcγR type-specific mutants and the FcγR-independent cell surface anchoring of bispecific anti-CD40 fusion proteins. Especially, we discuss the therapeutic potential of these strategies in view of the emerging evidence for the dose-limiting activities of systemic CD40 engagement.

Fibroblast growth factor-inducible 14 (Fn14) is a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) and is activated by its ligand TNF-like weak inducer of apoptosis (TWEAK). The latter occurs as a homotrimeric molecule in a soluble and a membrane-bound form. Soluble TWEAK (sTWEAK) activates the weakly inflammatory alternative NF-κB pathway and sensitizes for TNF-induced cell death while membrane TWEAK (memTWEAK) triggers additionally robust activation of the classical NF-κB pathway and various MAP kinase cascades. Fn14 expression is limited in adult organisms but becomes strongly induced in non-hematopoietic cells by a variety of growth factors, cytokines and physical stressors (e.g., hypoxia, irradiation). Since all these Fn14-inducing factors are frequently also present in the tumor microenvironment, Fn14 is regularly found to be expressed by non-hematopoietic cells of the tumor microenvironment and most solid tumor cells. In general, there are three possibilities how the tumor-Fn14 linkage could be taken into consideration for tumor therapy. First, by exploitation of the cancer associated expression of Fn14 to direct cytotoxic activities (antibody-dependent cell-mediated cytotoxicity (ADCC), cytotoxic payloads, CAR T-cells) to the tumor, second by blockade of potential protumoral activities of the TWEAK/Fn14 system, and third, by stimulation of Fn14 which not only triggers proinflammtory activities but also sensitizes cells for apoptotic and necroptotic cell death. Based on a brief description of the biology of the TWEAK/Fn14 system and Fn14 signaling, we discuss the features of the most relevant Fn14-targeting biologicals and review the preclinical data obtained with these reagents. In particular, we address problems and limitations which became evident in the preclinical studies with Fn14-targeting biologicals and debate possibilities how they could be overcome., Competing Interests: The University of Wuerzburg filed a patent describing novel Fn14 agonists with OZ, CO, and HW as co-inventors. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zaitseva, Hoffmann, Otto and Wajant.)

Targeted therapies using biopharmaceuticals are of growing clinical importance in disease treatment. Currently, there are several limitations of protein-based therapeutics (biologicals), including suboptimal biodistribution, lack of stability, and systemic side effects. A promising approach to overcoming these limitations could be a therapeutic cell-loaded 3D construct consisting of a suitable matrix component that harbors producer cells continuously secreting the biological of interest. Here, the recombinant spider silk proteins eADF4(C16), eADF4(C16)-RGD, and eADF4(C16)-RGE have been processed together with HEK293 producer cells stably secreting the highly traceable reporter biological TNFR2-Fc-GpL, a fusion protein consisting of the extracellular domain of TNFR2, the Fc domain of human IgG1, and the luciferase of Gaussia princeps as a reporter domain. eADF4(C16) and eADF4(C16)-RGD hydrogels provide structural and mechanical support, promote HEK293 cell growth, and allow fusion protein production by the latter. Bioink-captured HEK293 producer cells continuously release functional TNFR2-Fc-GpL over 14 days. Thus, the combination of biocompatible, printable spider silk bioinks with drug-producing cells is promising for generating implantable 3D constructs for continuous targeted therapy.

Tumor necrosis factor-α (TNF-α) is a pleiotropic, proinflammatory cytokine related to different neurodegenerative diseases, including Alzheimer's disease (AD). Although the linkage between increased TNF-α levels and AD is widely recognized, TNF-α-neutralizing therapies have failed to treat AD. Previous research has associated this with the antithetic functions of the two TNF receptors, TNF receptor 1, associated with inflammation and apoptosis, and TNF receptor 2 (TNFR2), associated with neuroprotection. In our study, we investigated the effects of specifically stimulating TNFR2 with a TNFR2 agonist (NewStar2) in a transgenic Aβ-overexpressing mouse model of AD by administering NewStar2 in two different ways: centrally, via implantation of osmotic pumps, or systemically by intraperitoneal injections. We found that both centrally and systemically administered NewStar2 resulted in a drastic reduction in amyloid β deposition and β-secretase 1 expression levels. Moreover, activation of TNFR2 increased microglial and astrocytic activation and promoted the uptake and degradation of Aβ. Finally, cognitive functions were also improved after NewStar2 treatment. Our results demonstrate that activation of TNFR2 mitigates Aβ-induced cognitive deficits and neuropathology in an AD mouse model and indicates that TNFR2 stimulation might be a potential treatment for AD.