Abteilung für Molekulare Innere Medizin (in der Medizinischen Klinik und Poliklinik II)
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- Abteilung für Molekulare Innere Medizin (in der Medizinischen Klinik und Poliklinik II) (68)
- Medizinische Klinik und Poliklinik II (7)
- Graduate School of Life Sciences (5)
- Julius-von-Sachs-Institut für Biowissenschaften (5)
- Theodor-Boveri-Institut für Biowissenschaften (5)
- Klinik und Poliklinik für Allgemein-, Viszeral-, Gefäß- und Kinderchirurgie (Chirurgische Klinik I) (4)
- Klinik und Poliklinik für Mund-, Kiefer- und Plastische Gesichtschirurgie (3)
- Klinik und Poliklinik für Nuklearmedizin (3)
- Lehrstuhl für Tissue Engineering und Regenerative Medizin (3)
- Neurologische Klinik und Poliklinik (3)
Sonstige beteiligte Institutionen
The TRAF-binding receptor CD40 belongs to the TNFR superfamily and is broadly expressed on healthy cells, mainly on antigen-presenting cells, but also on other immune cells and non-immune cells. CD40 is bound by its ligand CD40L, which is essential for a wide range of immunological responses by inducing or inhibiting different pathways that are essential for a variety of cellular processes, including immune activation and maturation. (1,2) Dysregulated CD40 signalling has been implicated in inflammatory diseases, such as hyper-IgM syndrome, psoriasis, and cancer. (3–6) Due to its broad expression across various tumour types, it can serve as a tumour-associated antigen and has therefore been proposed as a target for antibodies for cancer treatment. (2,7,8)
Agonistic anti-CD40 antibodies have been demonstrated to induce anti-tumoural immune responses as well as therapeutic immunity. (2) Furthermore, prolonged stimulation of CD40 in tumour cells in vitro has been shown to decrease proliferation, increase expression of cytotoxic TNFSFLs and induce apoptosis. (9,10) Their effect on anti-tumoral responses has been well studied and anti-tumoral responses by DC maturation and suppression of malignant growth of B-cells have been confirmed and were found to induce cell death in tumours in vitro. (11–14)
Many agonistic anti-CD40 antibodies specifically have been reported to require secondary crosslinking by binding to either activating or inhibitory FcγRs to be agonistic in vitro, while in vivo studies have indicated inhibitory FcƴR2B expression as critical factor. (15–17) However, FcƴR independent agonism has also been reported for anti-CD40 antibodies. (18,19) While agonistic anti-CD40 IgG1, IgG3 and IgG4 antibodies have been shown to display FcƴR dependent agonism, agonistic anti-CD40 IgG2 antibodies have shown to display FcƴR independent agonism. Conversion of anti-CD40 IgG1 antibodies into IgG2 has also been shown to convert the antibody’s agonism into FcƴR independent agonism. (20)
To overcome FcƴR dependency, bispecific antibody fusion proteins containing a scFv as anchoring domain allowing for crosslink independent of FcƴR binding have been designed before. This approach has been found to display strong agonism for other antibody fusion proteins when bound to both targets, with response levels resembling that of FcƴR bound antibodies. (21,22)
The relevance of antibody isotype and idiotype for FcƴR-dependent agonism as well as the relevance of valency and antibody oligomerization for FcƴR-independent agonism were investigated in this study on a panel of different anti-CD40 antibodies. Several clinically investigated anti-CD40 antibodies (ADC-1013(23), APX005M(24), ChiLob7.4(25) and CP-870,893(26)) and one preclinical antibody (G28.5(27,28)) were considered. Selected antibodies were then cloned onto an IgG1, IgG1(N297A), IgG2 and IgG4 backbone. The IgG1(N297A) isotype is an IgG1 antibody with a point mutation (N297A) that is known to strongly reduce binding to FcƴR1, while reducing the binding affinity to FcƴR2B to undetectable levels. (29,30) In this work it is demonstrated that the investigated anti-CD40 antibody variants across different isotypes activate both the classical and alternative NFκB pathway by stimulating U2OS cells in an FcƴR dependent manner. Stimulation in the presence of both human FcƴRs as well as murine FcƴRs resulted in CD40 stimulation. A difference in binding competition was observed for the various anti-CD40 IgG1 antibodies, but no indication of a CRD-dependent mechanism responsible for their agonistic activity was found. Moreover, this FcƴR dependency could be overcome by creation of tetravalent antibody fusion proteins.
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.
In the initiation phase of acute graft-versus-host disease (aGvHD), CD4+ T cells are activated by hematopoietic antigen presenting cells in secondary lymphoid organs whereas in effector phase by non-hematopoietic cells in the small intestine. We hypothesized that alloreactive CD4+ T cells primarily home to the secondary lymphoid organs subsequent to allogeneic hematopoietic cell transplantation in the initiation phase of aGvHD and are activated by the non-hematopoietic lymph node stromal cells via MHC class II. To test this hypothesis, we employed CD4+ T cell-dependent major mismatch aGvHD mouse model to study this correlation.
Upon analyzing the early events following allo-HCT with bioluminescence imaging, flow cytometry and whole-mount light sheet fluorescence microscopy, we found that allogeneic T cells exclusively home to the spleen, lymph nodes and the Peyer’s patches and not to the intestinal lamina propria in the initiation phase of aGvHD. Utilizing mice devoid of partial or complete hematopoietic antigen presentation we could show allogeneic CD4+ T cells activation in the lymphoid organs of MHCIIΔCD11c and MHCIIΔ BM chimeric mice early after allo-HCT. MHCIIΔ BM chimeras failure of thymic negative selection and developing tissue wasting disease upon syn-HCT deemed them unsuitable to study non-hematopoietic antigen presentation in aGvHD. To overcome this challenge, we generated MHCIIΔVav1 mice that lack MHC class II expression on all hematopoietic cells. MHCIIΔVav1 mice were susceptible to aGvHD and LNSCs from these animals activated allogeneic CD4+ T cells in mixed lymphocyte reaction. Likewise, mesenteric lymph nodes from CD11c.DTR mice surgically transplanted into a MHCIIΔ mouse could activate CD4+ T cells in vivo, clearly demonstrating LNSCs as non-hematopoietic APCs of the lymphoid organs.
We specifically target lymph node stromal cell subsets via the Cre/loxP system, we employed single cell RNA sequencing and selected Ccl19 and VE-Cadherin to specifically target the fibroblastic reticular cells and endothelial cells of the lymph nodes respectively. In MHCIIΔCcl19 mice, alloreactive CD4+ T cells activation was discreetly reduced in the initiation phase of aGvHD whereas absence of MHCII on fibroblastic reticular cells resulted in hyper-activation of allogeneic CD4+ T cells leading to poor survival. This phenotype was modulated by the regulatory T cells that were able to rescue H2-Ab1fl mice but not the MHCIIΔCcl19 subsequent to GvHD.
Knock-out of MHCII on endothelial cells MHCIIΔVE Cadherin, resulted only in modest reduction of CD4+ T cells activation in the initiation phase of GvHD, conversely MHCIIΔVE Cadherin mice showed a protective phenotype compared against littermates H2-Ab1fl mice in long-term survival. Furthermore, to pin-point endothelial cells MHCII antigen presentation we generated MHCIIΔVE Cadherin ΔVav1 animals devoid of antigen presentation in both endothelial and hematopoietic compartments. LNSCs from MHCIIΔVE Cadherin ΔVav1 were unable to activate alloreactive CD4+ T cells in mixed lymphocyte reaction.
Altogether, we demonstrate for the first time that MHC class II on the lymph node stromal cells plays a crucial role in the modulation of allogeneic CD4+ T cells in the initiation and later in the effector phase of graft-versus-host-disease.
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.
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.
Despite the great success of TNF blockers in the treatment of autoimmune diseases and the identification of TNF as a factor that influences the development of tumors in many ways, the role of TNFR2 in tumor biology and its potential suitability as a therapeutic target in cancer therapy have long been underestimated. This has been fundamentally changed with the identification of TNFR2 as a regulatory T-cell (Treg)-stimulating factor and the general clinical breakthrough of immunotherapeutic approaches. However, considering TNFR2 as a sole immunosuppressive factor in the tumor microenvironment does not go far enough. TNFR2 can also co-stimulate CD8\(^+\) T-cells, sensitize some immune and tumor cells to the cytotoxic effects of TNFR1 and/or acts as an oncogene. In view of the wide range of cancer-associated TNFR2 activities, it is not surprising that both antagonists and agonists of TNFR2 are considered for tumor therapy and have indeed shown overwhelming anti-tumor activity in preclinical studies. Based on a brief summary of TNFR2 signaling and the immunoregulatory functions of TNFR2, we discuss here the main preclinical findings and insights gained with TNFR2 agonists and antagonists. In particular, we address the question of which TNFR2-associated molecular and cellular mechanisms underlie the observed anti-tumoral activities of TNFR2 agonists and antagonists.
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.
Conventional bivalent IgG antibodies targeting a subgroup of receptors of the TNF superfamily (TNFSF) including fibroblast growth factor-inducible 14 (anti-Fn14) typically display no or only very limited agonistic activity on their own and can only trigger receptor signaling by crosslinking or when bound to Fcγ receptors (FcγR). Both result in proximity of multiple antibody-bound TNFRSF receptor (TNFR) molecules, which enables engagement of TNFR-associated signaling pathways. Here, we have linked anti-Fn14 antibodies to gold nanoparticles to mimic the “activating” effect of plasma membrane-presented FcγR-anchored anti-Fn14 antibodies. We functionalized gold nanoparticles with poly-ethylene glycol (PEG) linkers and then coupled antibodies to the PEG surface of the nanoparticles. We found that Fn14 binding of the anti-Fn14 antibodies PDL192 and 5B6 is preserved upon attachment to the nanoparticles. More importantly, the gold nanoparticle-presented anti-Fn14 antibody molecules displayed strong agonistic activity. Our results suggest that conjugation of monoclonal anti-TNFR antibodies to gold nanoparticles can be exploited to uncover their latent agonism, e.g., for immunotherapeutic applications.
Purpose
Knowledge on Ruxolitinib exposure in patients with graft versus host disease (GvHD) is scarce. The purpose of this prospective study was to analyze Ruxolitinib concentrations of GvHD patients and to investigate effects of CYP3A4 and CYP2C9 inhibitors and other covariates as well as concentration-dependent effects.
Methods
262 blood samples of 29 patients with acute or chronic GvHD who were administered Ruxolitinib during clinical routine were analyzed. A population pharmacokinetic model obtained from myelofibrosis patients was adapted to our population and was used to identify relevant pharmacokinetic properties and covariates on drug exposure. Relationships between Ruxolitinib exposure and adverse events were assessed.
Results
Median of individual mean trough serum concentrations was 39.9 ng/mL at 10 mg twice daily (IQR 27.1 ng/mL, range 5.6-99.8 ng/mL). Applying a population pharmacokinetic model revealed that concentrations in our cohort were significantly higher compared to myelofibrosis patients receiving the same daily dose (p < 0.001). Increased Ruxolitinib exposure was caused by a significant reduction in Ruxolitinib clearance by approximately 50%. Additional comedication with at least one strong CYP3A4 or CYP2C9 inhibitor led to a further reduction by 15% (p < 0.05). No other covariate affected pharmacokinetics significantly. Mean trough concentrations of patients requiring dose reduction related to adverse events were significantly elevated (p < 0.05).
Conclusion
Ruxolitinib exposure is increased in GvHD patients in comparison to myelofibrosis patients due to reduced clearance and comedication with CYP3A4 or CYP2C9 inhibitors. Elevated Ruxolitinib trough concentrations might be a surrogate for toxicity.