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Complement 1q/tumor necrosis factor-related proteins (CTRPs): structure, receptors and signaling
(2023)
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.
Simple Summary
Targeting of CD40 with antibodies attracts significant translational interest. While inhibitory CD40 targeting appears particularly attractive in the field of organ transplantation and for the treatment of autoimmune diseases, stimulatory CD40 targeting is the aim in tumor immunotherapy and vaccination against infectious pathogens. It turned out that lack of FcγR-binding is the crucial factor for the development of safe and well-tolerated inhibitory anti-CD40 antibodies. In striking contrast, FcγR-binding is of great importance for the CD40 stimulatory capacity of the majority of anti-CD40 antibodies. Typically, anti-CD40 antibodies only robustly stimulate CD40 when presented by FcγRs. However, FcγR-binding of anti-CD40 antibodies also triggers unwanted activities such as destruction of CD40 expressing cells by ADCC or ADCP. Based on a brief discussion of the mechanisms of CD40 activation, we give an overview of the ongoing activities in the development of anti-CD40 antibodies under special consideration of attempts aimed at the development of anti-CD40 antibodies with FcγR-independent agonism or FcγR subtype selectivity.
Abstract
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.
Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the in vivo availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior in vivo activity.
Methods
Single-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention and in vitro and in vivo activity. STAR2 (TNC-sc(mu)TNF80 or selective TNF-based agonist of TNF receptor 2), a well-established highly active nonameric TNFR2-specific variant, served as benchmark. NewSTAR2 was assessed in various in vitro and in vivo systems.
Results
STAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparable in vitro activity. The novel domain architecture of NewSTAR2 significantly improved serum retention compared to STAR2, which correlated with efficient binding to FcRn. A single injection of NewSTAR2 enhanced regulatory T cell (Treg) suppressive activity and increased Treg numbers by > 300% in vivo 5 days after treatment. Treg numbers remained as high as 200% for about 10 days. Furthermore, a single in vivo treatment with NewSTAR2 upregulated the adenosine-regulating ectoenzyme CD39 and other activation markers on Tregs. TNFR2-stimulated Tregs proved to be more suppressive than unstimulated Tregs, reducing conventional T cell (Tcon) proliferation and expression of activation markers in vitro. Finally, singular preemptive NewSTAR2 administration five days before allogeneic hematopoietic cell transplantation (allo-HCT) protected mice from acute GvHD.
Conclusions
NewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior in vivo activity exerting powerful protective effects against acute GvHD.
Membrane lymphotoxin-α\(_2\)β is a novel tumor necrosis factor (TNF) receptor 2 (TNFR2) agonist
(2021)
In the early 1990s, it has been described that LTα and LTβ form LTα\(_2\)β and LTαβ\(_2\) heterotrimers, which bind to TNFR1 and LTβR, respectively. Afterwards, the LTαβ\(_2\)–LTβR system has been intensively studied while the LTα\(_2\)β–TNFR1 interaction has been ignored to date, presumably due to the fact that at the time of identification of the LTα\(_2\)β–TNFR1 interaction one knew already two ligands for TNFR1, namely TNF and LTα. Here, we show that LTα\(_2\)β interacts not only with TNFR1 but also with TNFR2. We furthermore demonstrate that membrane-bound LTα\(_2\)β (memLTα\(_2\)β), despite its asymmetric structure, stimulates TNFR1 and TNFR2 signaling. Not surprising in view of its ability to interact with TNFR2, LTα\(_2\)β is inhibited by Etanercept, which is approved for the treatment of rheumatoid arthritis and also inhibits TNF and LTα.
Soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), in contrast to membrane TWEAK and TNF, is only a weak activator of the classical NFκB pathway. We observed that soluble TWEAK was regularly more potent than TNF with respect to the induction of TNF receptor-associated factor 1 (TRAF1), a NFκB-controlled signaling protein involved in the regulation of inflammatory signaling pathways. TNF-induced TRAF1 expression was efficiently blocked by inhibition of the classical NFκB pathway using the IKK2 inhibitor, TPCA1. In contrast, in some cell lines, TWEAK-induced TRAF1 production was only partly inhibited by TPCA1. The NEDD8-activating enzyme inhibitor MLN4924, however, which inhibits classical and alternative NFκB signaling, blocked TNF- and TWEAK-induced TRAF1 expression. This suggests that TRAF1 induction by soluble TWEAK is based on the cooperative activity of the two NFκB signaling pathways. We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity. Oligomerization of soluble TWEAK showed no effect on the dose response of TRAF1 induction, but potentiated the ability of soluble TWEAK to trigger production of the classical NFκB-regulated cytokine IL8. Transfectants expressing soluble TWEAK and membrane TWEAK showed similar induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data indicate that soluble TWEAK may efficiently induce a distinct subset of the membrane TWEAK-targeted genes and argue again for a crucial role of classical NFκB pathway-independent signaling in TWEAK-induced TRAF1 expression. Other TWEAK targets, which can be equally well induced by soluble and membrane TWEAK, remain to be identified and the relevance of the ability of soluble TWEAK to induce such a distinct subset of membrane TWEAK-targeted genes for TWEAK biology will have to be clarified in future studies.
Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4+ T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Treg) but reduced numbers of CD8+ T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8+ T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome.
Die Stimulation des CD95-Todesrezeptors durch seinen natürlichen membranständigen Li-ganden CD95L führt zur kontextabhängigen Aktivierung von sowohl apoptotischen als auch nicht-apoptotischen Signalwegen. Durch Proteolyse wird aus dem membranständigen CD95L löslicher trimerer CD95L freigesetzt. Die Bindung von löslichem trimerem CD95L an CD95 ist nicht ausreichend, um die CD95-Signaltransduktion effizient zu stimulieren. Die Fähigkeit von löslichen CD95L-Trimeren CD95-vermittelte Signalwege robust zu aktivieren kann jedoch durch Oligomerisierung und artifizielle Immobilisierung an eine Oberfläche drastisch gesteigert werden. In dieser Arbeit wurde zunächst bestätigt, dass nur oligomere CD95L-Varianten, die z.B. durch Antikörpervernetzung von N-terminal getaggten rekombinanten CD95L-Varianten oder durch eine gentechnisch erzwungene Hexamerisierung von CD95L-Molekülen erhalten wur-den, in der Lage sind, effizient apoptotische und nicht-apoptotische Signalwege zu aktivieren. Ferner zeigte sich dann, dass die Bindung von löslichen CD95L-Trimeren nicht ausreichend ist, um die Translokation von CD95-Molekülen in detergenzunlösliche „Lipid Raft“- Membrandomänen zu stimulieren. Die „Lipid Raft“-Translokation ist ein zentrales Ereignis bei der CD95-Aktivierung und vor allem für die Induktion der Apoptose bedeutsam. Dabei ist ein selbstverstärkender Prozess aus Caspase-8-Aktivierung und „Lipid Raft“-Assoziation des CD95 von Bedeutung. Um die Interaktion von CD95 und CD95L mit Hilfe von hoch sensitiven zellulären Bindungs-studien analysieren zu können, wurden in dieser Arbeit desweiteren CD95L-Fusionsproteine entwickelt und hergestellt, an welche N-terminal eine Gaussia princeps Luziferase (GpL)- Reporterdomäne gekoppelt ist. So konnte mit den GpL-CD95L-Fusionsproteinen gezeigt werden, dass die Oligomerisierung von CD95L-Trimeren keinen Effekt auf die Ligandenbele-gung des CD95 hat. Dies spricht dafür, dass die höhere spezifische Aktivität von oligomeri-sierten CD95L-Trimeren nicht auf einer Aviditäts-vermittelten Zunahme der apparenten Affi-nität beruht, sondern dies deutet darauf hin, dass die sekundäre Aggregation von sich initial bildenden trimeren CD95L-CD95-Komplexen eine entscheidende Rolle in der CD95-Aktivierung spielt. Durch Scatchard-Analysen zeigte sich ferner, dass trimerer CD95L mit mindestens zwei zellulären Bindungsstellen unterschiedlicher Affinität interagiert. Bindungs-studien mit löslichen monomeren und trimeren GpL-CD95-Rezeptoren an membranständigen CD95L, als auch Inhibitionsstudien ergaben, dass trimerer CD95 weitaus besser an CD95L bindet. Dies legt nahe, dass es sich bei den zuvor beobachteten hoch- und niederaffinen Bindungsstellen für CD95L um monomere bzw. prä-assemblierte CD95-Moleküle handelt. Die GpL-CD95L-Fusionsproteine wurden auch genutzt, um die CD95-Translokation in „Lipid Rafts“ zu analysieren. So wurde trimerer GpL-CD95L als „Tracer“ zur Markierung von inaktiven CD95-Molekülen eingesetzt. Nach Aktivierung der übrigen freien CD95-Moleküle mit hoch aktivem hexameren Fc-CD95L konnte eine Zunahme der inaktiven GpL-CD95L-markierten Rezeptoren in „Lipid Rafts“ beobachtet werden. Offensichtlich stimulieren also aktivierte CD95-Moleküle in „trans“ die Ko-Translokation inaktiver CD95-Rezeptoren in „Lipid Rafts“. Dies bestätigte sich auch in Experimenten mit Transfektanten, die einen chimären CD40-CD95-Rezeptor exprimieren. Letzterer ist nach Stimulation mit CD40L in der Lage, intrazellu-läre CD95-vermittelte Signalwege zu aktivieren. Die Aktivierung von CD95-assoziierten Sig-nalwegen durch Stimulation von endogenem CD95 in CD40-CD95-Transfektanten resultierte nun in der Ko-Translokation von unstimulierten CD40-CD95-Rezeptoren in „Lipid Rafts“. Vice versa zeigte sich die Ko-Translokation von endogenem CD95 nach spezifischer Aktivierung des chimären CD40-CD95-Rezeptors. Schlussendlich erwiesen sich eine funktionsfähige Todesdomäne und die Aktivierung der Caspase-8 als essentiell für die „Lipid Raft“-Assoziation von aktivierten CD95-Molekülen und auch für die durch diese Rezeptorspezies induzierte Ko-Translokation von inaktiven Rezeptoren in „Lipid Rafts“.