Soluble Decoy Receptor Research Articles

Osteoprotegerin Protects against Muscular Dystrophy

Receptor-activator of NF-κB, its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that synchronously regulates bone and skeletal muscle physiopathology still is elusive. Here, we show that muscle cells can produce and secrete osteoprotegerin and pharmacologic treatment of dystrophic mdx mice with recombinant osteoprotegerin muscles. (Recombinant osteoprotegerin-Fc mitigates the loss of muscle force in a dose-dependent manner and preserves muscle integrity, particularly in fast-twitch extensor digitorum longus.) Our data identify osteoprotegerin as a novel protector of muscle integrity, and it potentially represents a new therapeutic avenue for both muscular diseases and osteoporosis.

Novel glycosylated VEGF decoy receptor fusion protein, VEGF-Grab, efficiently suppresses tumor angiogenesis and progression.

Antiangiogenic therapies targeting VEGFA have been commonly used in clinics to treat cancers over the past decade. However, their clinical efficacy has been limited, with drawbacks including acquisition of resistance and activation of compensatory pathways resulting from elevated circulating VEGFB and placental growth factor (PlGF). To bypass these disadvantages, we developed a novel glycosylated soluble decoy receptor fusion protein, VEGF-Grab, that can neutralize VEGFA, VEGFB, and PlGF. VEGF-Grab has the second and third immunoglobulin (Ig)-like domains of VEGF receptor 1 (VEGFR1) fused to IgG1 Fc, with three potential glycosylation sites introduced into the third Ig-like domain of VEGF-Grab by mutagenesis. Compared with VEGF-Trap, VEGF-Grab showed more potent decoy activity against VEGF and PlGF, mainly attributed to the VEGFR1 backbone. Most importantly, the negatively charged O-glycans attached to the third Ig-like domain of VEGFR1 counterbalanced the originally positively charged VEGFR1 backbone, minimizing nonspecific binding of VEGF-Grab to the extracellular matrix, and resulting in greatly improved pharmacokinetic profile. These advancements led to stronger and more durable antiangiogenic, antitumor, and antimetastatic efficacy in both implanted and spontaneous tumor models as compared with VEGF-Trap, while toxicity profiles were comparable with VEGF-Trap. Collectively, our results highlight VEGF-Grab as a promising therapeutic candidate for further clinical drug development.

Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33

Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33

Modulation of Inflammatory Responses After Brain Death and Syngeneic Renal Transplantation By Systemic Overexpression of esRAGE.

Introduction: Donor brain death (BD) adversely affects organ quality. Inflammation mediated by engagement of TLR4 and RAGE may contribute to inferior outcomes. Endogenous secretory (es)RAGE is a soluble decoy receptor which sequesters HMGB1 and other RAGE ligands blocking binding to TLRs 2 and 4, as well as to RAGE. Aim: To determine whether in vivo expression of esRAGE can reduce inflammation in syngeneic KTx from BD donors. Methods: Donor and recipient C57BL/6 mice received 5×1011 VG rAAV-esRAGE or ctrl vector 7 days pre-transplant. Donor kidneys were grafted 1 hr after BD induction, and sampled at d1 and 4 post-Tx. Cellular infiltration, proliferation, and gene expression were determined. Serum and kidneys were also collected 3 hrs post-BD. Results: Serum HMGB1 levels in esRAGE-BD mice (˜10ng/ml) were significantly less than those in untreated BD mice (>25ng/ml, p=0.02). There was a trend towards reduced expression of CXCL10, MCP-1, MIP-2, CCL22, IL-10, IL-6, IL-1β and TNF-α in the kidneys of esRAGE-BD mice, but this was not consistently observed after KTx. Neutrophil and macrophage infiltrate density was substantially reduced by esRAGE (nΦ 9.1±2.0/HPF cf 23.1±6.0, p=0.03; F4/80 27.3±3.2/HPF cf 65.1±9.1, p<0.01).Figure: No Caption available.Proliferation of parenchymal cells (PCNA staining) was enhanced in esRAGE-treated mice (7.6±0.5/HPF cf 4.2±1.2, p=0.05).Figure: No Caption available.Conclusions: esRAGE significantly reduced neutrophil and macrophage infiltration following syngeneic renal Tx from BD donors. RAGE is a binding partner for the integrin MAC-1, and reduced leucocyte infiltration may reflect inhibition of this interaction. esRAGE increased proliferation of renal parenchymal cells after Tx from BD donors, similar to documented effects of soluble RAGE in liver injury. These effects may be translated into improved graft function.

SP0213 Clinical and Pathogenetic Aspects of Autoinflammatory Syndromes

Some 15 years ago, the term autoinflammatory syndrome has been coined to delineate a group of disorders in which patients suffer from recurrent or chronic inflammation without an obvious exogenous...

A novel decoy receptor fusion protein for FGF-2 potently inhibits tumour growth.

Background:Antiangiogenic therapies have been proven effective in cancer treatment. Fibroblast growth factor-2 (FGF-2) has been functionally implicated in tumour angiogenesis and is an important target of antiangiogenic therapies. The aim of this work was to develop a novel FGF-2 inhibitor for cancer therapy.Methods:Eleven fusion proteins were developed by fusing various truncated extracellular regions of FGFR1 with the Fc region of IgG1. The optimal decoy receptor fusion protein with the highest binding affinity for FGF-2 was identified by an FGF-2-binding assay and its potential antitumour effects were investigated.Results:We obtained a soluble decoy receptor fusion protein with the highest binding activity for FGF-2, named FGF-Trap. Fibroblast growth factor-Trap significantly abolished FGF-2-stimulated activation of FGF signalling as demonstrated by its suppression of FGF-2-mediated phosphorylation of Erk1/2 and Akt, upregulation of cyclins D1 and E and the increase in mRNA levels of vascular endothelial growth factor R1 and R2 (VEGFR1 and VEGFR2). Furthermore, FGF-Trap effectively suppressed FGF-2-induced proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro. Most importantly, FGF-Trap potently inhibited tumour growth and angiogenesis in Caki-1 and A549 xenograft models in vivo.Conclusions:Fibroblast growth factor-Trap potently inhibits tumour growth by blocking FGF-2 signalling pathways and could be an effective therapeutic agent for cancer patients.

Intravitreal aflibercept (Eylea(®)): a review of its use in patients with macular oedema secondary to central retinal vein occlusion.

Aflibercept is a fully human, recombinant fusion protein that acts as a soluble decoy receptor for vascular endothelial growth factor (VEGF) family members, including VEGF-A, VEGF-B and placental growth factor (P1GF), thereby inhibiting downstream signalling mediated by these ligands. Aflibercept binds all isoforms of VEGF-A with high affinity, and a markedly higher affinity than that of ranibizumab or bevacizumab. A formulation of aflibercept developed specifically for intravitreal injection (Eylea(®)) is approved for use in several countries for the treatment of patients with macular oedema secondary to central retinal vein occlusion (CRVO). In clinical trials (GALILEO and COPERNICUS) in patients with this condition, intravitreal aflibercept 2 mg every month improved best corrected visual acuity (BCVA), as measured by the proportion of study eyes with a gain of ≥15 Early Treatment Diabetic Retinopathy Study letters from baseline, significantly more than sham injections at week 24 (primary analysis). The significant improvements achieved with intravitreal aflibercept compared with sham in the first 6 months were maintained in the second 6 months with as-needed (prn) dosing and monthly monitoring. Continued prn dosing with a reduced monitoring frequency was associated with decreased improvements. More data are needed to confirm the optimal monitoring frequency for use with prn dosing, subsequent to initial monthly injections, in order to maintain long-term efficacy. Intravitreal aflibercept was generally well tolerated in clinical trials and there is little potential for systemic drug accumulation. Thus, intravitreal aflibercept is an effective and generally well tolerated agent that extends the options available for the treatment of macular oedema secondary to CRVO.

Blockade of receptor activator of nuclear factor‐κB signaling greatly improves SERCA activity and contractility in atrophic and dystrophic skeletal muscles (731.7)

Receptor‐activator of nuclear factor‐κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are members of the tumor necrosis factor (TNF) superfamily that control osteoclast differentiation, bone remodelling and osteoporosis. Since bone and skeletal muscle physiopathology occurs synchronously, we tested whether RANK/RANKL pathway is involved in muscle dysfunction following different muscle wasting conditions. Contractile properties of EDL muscles were obtained following 14d of denervation in mice specifically deficient in RANK skeletal muscle or in dystrophic mdx mice treated daily for 10d with OPG [0.3mg/kg], the natural inhibitor of RANKL. The activity and expression of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were determined in denervated or dystrophic muscles. RANK muscle deletion did not prevent atrophy but greatly reduced the losses of force production and SERCA activity in denervated EDL muscles. Force production of EDL muscles also increased by 140% in OPG‐treated relative to PBS‐treated mdx mice while the treatment with OPG prevented the loss of the fast twitch SERCA1 protein content in dystrophic mice. Together, these data indicate that the deletion or blockage of RANK/RANKL pathway has inotropic effects and increases SERCA activity and expression in fast‐twitch skeletal muscles opening promising new avenues for several muscle diseases.

Quadriceps muscle strength and its relationship to radiographic knee osteoarthritis

Quadriceps muscle strength and its relationship to radiographic knee osteoarthritis

Hydrogen Sulfide

Vascular calcification is the major cause of cardiovascular morbidity and mortality in patients with type 2 diabetes mellitus, chronic kidney disease, and in aging patients.1 Considerable progress has been made in the past 2 decades in understanding the molecular mechanisms of vascular calcification.2–4 Regardless of the morphology and location, most evidence indicates that vascular calcification involves an organized process recapitulating many cellular and molecular events that govern skeletal bone formation. Although a large body of evidence shows that osteoblastic and osteochonrocytic cells contribute to vascular calcification, it remains unclear how osteoclasts are differentiated from their precursors and how osteoclasts play a role in calcium reabsorption in calcifying arteries. Osteoclasts develop from monocytic precursors of the hematopoietic lineage, and 2 distinct signaling systems are both necessary and sufficient for the early differentiation into multinucleated osteoclasts as demonstrated by osteopetrotic mice models with loss-of-function mutation of either gene.5–7 One is a signaling system that involves macrophage colony-stimulating factor (MCSF) and its receptor c-fms, and the other involves receptor activator of nuclear factor κB (RANK) ligand (RANKL), RANK, and osteoprotegerin (OPG), a soluble decoy receptor for RANKL.8–10 MCSF and RANKL induce osteoclast differentiation, fusion, and maturation on binding to its specific receptors, c-fms and RANK, respectively, on the surface of preosteoclastic monocytes. Increased production of RANKL in atherosclerotic lesions, particularly in endothelial cells and vascular smooth muscle cells (SMCs), has been well documented.11–13 See accompanying article on page 626 Studies of …

Role of aflibercept in the treatment of advanced colorectal cancer

SUMMARY Colorectal cancer is the third most common cancer in men, representing 10% of cases, and the second most common cancer in women, representing 9.4% of cases, worldwide. It is estimated that 50–60% of patients will develop metastases during the course of their disease, while 15–25% will present with synchronous metastatic lesions, reducing the probability of 5-year survival to 12%. Colon cancer represents the fourth most common cause of death from cancer worldwide and the second most common cause in developed countries. First-line therapy of advanced or metastatic colorectal carcinoma (mCRC) usually consists of the administration of oxaliplatin or irinotecan in combination with leucovorin and 5-fluorouracil. In the first- or second-line settings, monoclonal antibodies can be added to chemotherapy. Bevacizumab (anti-VEGF monoclonal antibody) is utilized with fluoropyrimidine-containing regimens in combination with oxaliplatin or irinotecan. Cetuximab can be administered in combination with irinotecan or as a single agent in patients who have wild-type KRAS. Panitumumab has also been approved for third-line single-agent therapy in KRAS wild-type patients and recently in first- and second-line therapy in combination with chemotherapy. There are no standard second-line treatments of mCRC specifically used in combination with FOLFIRI, and no drugs have been approved for patients with prior bevacizumab treatment. Aflibercept is a new angiogenesis inhibitor with a unique mechanism of action. Aflibercept is a recombinant fusion protein consisting of VEGF-binding portions from extracellular domains of the human VEGFR1 and 2 fused to the Fc portion of human IgG1. Aflibercept acts as a soluble decoy receptor that binds to VEGF-A with higher affinity than its native receptors, as well as the related ligands PIGF and VEGF-B. Aflibercept interferes with the biological actions of VEGF by ‘trapping’ VEGF and preventing it from interacting with its receptors on endothelial cells, blocking the activation of VEGFRs and the subsequent proliferation of endothelial cells; this results in inhibition of the growth of new vessels. The role of aflibercept in the management of mCRC was studied in the VELOUR trial, which showed that its addition to the FOLFIRI regimen resulted in a survival benefit, giving a statistically significant log-rank test with p = 0.0032 (hazard ratio: 0.817; 95.34% CI: 0.713–0.937). This benefit was supported by subgroup and sensitivity analyses, the increased median progression-free survival, and improved response rates observed in the aflibercept arm. Aflibercept toxicity is acceptable, manageable and within the established range of other VEGF inhibitors.

Anti-Interleukin-1 Therapy in the Management of Gout

Gout is the most common inflammatory arthritis in humans. Current treatment options to control the pain and inflammation of acute and chronic gout include nonsteroidal anti-inflammatory drugs, colchicine, and corticosteroids. However, patients are commonly unresponsive to, intolerant of, or have contraindications to current treatments. Interleukin-1 (IL-1), a proinflammatory cytokine, plays a major role in mediating gouty inflammation. This role of IL-1 has led investigators to explore a new class of anti-inflammatory drugs that inhibit IL-1 signal transduction. IL-1 inhibitors currently in trials for gout include anakinra, rilonacept, and canakinumab. Anakinra is an IL‑1 receptor antagonist that inhibits the activity of both IL‑1α and IL‑1β, rilonacept is a soluble decoy receptor and canakinumab is an anti‑IL‑1β monoclonal antibody. In case cohorts, anakinra was found to be efficacious in combating acute gout pain and inflammation, whereas rilonacept has been found to be efficacious for reducing the risk of recurrent attacks. Canakinumab has been shown to be efficacious in both reducing pain and inflammation in acute attacks, and for reducing the risk of recurrent attacks. All three IL-1 inhibitors are generally well tolerated. This article reviews the current IL-1 inhibitors and the results of trials in which they have been tested for the management of acute and chronic gouty inflammation.

An expanding role for interleukin-1 blockade from gout to cancer.

There is an expanding role for interleukin (IL)-1 in diseases from gout to cancer. More than any other cytokine family, the IL-1 family is closely linked to innate inflammatory and immune responses. This linkage is because the cytoplasmic segment of all members of the IL-1 family of receptors contains a domain, which is highly homologous to the cytoplasmic domains of all toll-like receptors (TLRs). This domain, termed "toll IL-1 receptor (TIR) domain," signals as does the IL-1 receptors; therefore, inflammation due to the TLR and the IL-1 families is nearly the same. Fundamental responses such as the induction of cyclo-oxygenase type 2, increased surface expression of cellular adhesion molecules and increased gene expression of a broad number of inflammatory molecules characterizes IL-1 signal transduction as it does for TLR agonists. IL-1β is the most studied member of the IL-1 family because of its role in mediating autoinflammatory disease. However, a role for IL-1α in disease is being validated because of the availability of a neutralizing monoclonal antibody to human IL-1α. There are presently three approved therapies for blocking IL-1 activity. Anakinra is a recombinant form of the naturally occurring IL-1 receptor antagonist, which binds to the IL-1 receptor and prevents the binding of IL-1β as well as IL-1α. Rilonacept is a soluble decoy receptor that neutralizes primarily IL-1β but also IL-1α. Canakinumab is a human monoclonal antibody that neutralizes only IL-1β. Thus, a causal or significant contributing role can be established for IL-1β and IL-1α in human disease.

A myostatin and activin decoy receptor enhances bone formation in mice

Myostatin is a member of the bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) super-family of secreted differentiation factors. Myostatin is a negative regulator of muscle mass as shown by increased muscle mass in myostatin deficient mice. Interestingly, these mice also exhibit increased bone mass suggesting that myostatin may also play a role in regulating bone mass. To investigate the role of myostatin in bone, young adult mice were administered with either a myostatin neutralizing antibody (Mstn-mAb), a soluble myostatin decoy receptor (ActRIIB-Fc) or vehicle. While both myostatin inhibitors increased muscle mass, only ActRIIB-Fc increased bone mass. Bone volume fraction (BV/TV), as determined by microCT, was increased by 132% and 27% in the distal femur and lumbar vertebrae, respectively. Histological evaluation demonstrated that increased BV/TV in both locations was attributed to increased trabecular thickness, trabecular number and bone formation rate. Increased BV/TV resulted in enhanced vertebral maximum compressive force compared to untreated animals. The fact that ActRIIB-Fc, but not Mstn-mAb, increased bone volume suggested that this soluble decoy receptor may be binding a ligand other than myostatin, that plays a role in regulating bone mass. This was confirmed by the significant increase in BV/TV in myostatin deficient mice treated with ActRIIB-Fc. Of the other known ActRIIB-Fc ligands, BMP3 has been identified as a negative regulator of bone mass. However, BMP3 deficient mice treated with ActRIIB-Fc showed similar increases in BV/TV as wild type (WT) littermates treated with ActRIIB-Fc. This result suggests that BMP3 neutralization is not the mechanism responsible for increased bone mass. The results of this study demonstrate that ActRIIB-Fc increases both muscle and bone mass in mice. Therefore, a therapeutic that has this dual activity represents a potential approach for the treatment of frailty.

The soluble Decoy Receptor 3 is regulated by a PI3K-dependent mechanism and promotes migration and invasion in renal cell carcinoma

BackgroundOverexpression of Decoy Receptor 3 (DcR3), a soluble member of the tumor necrosis factor receptor superfamily, is a common event in several types of cancer. In renal cell carcinoma (RCC), DcR3 overexpression is associated with lymph node and distant metastasis as well as a poor prognosis. However, the functional role and regulation of DcR3 expression in RCC is so far unknown.MethodsModulation of DcR3 expression by siRNA and ectopic gene expression, respectively, was performed in ACHN and 769-P RCC cell lines. Functional effects of a modulated DcR3 expression were analyzed with regard to migration, invasion, adhesion, clonogenicity, and proliferation. Furthermore, quantitative RT-PCR and immunoblot analyses were performed to evaluate the expression of downstream mediators of DcR3. In further experiments, luciferase assays, quantitative RT-PCR and immunoblot analyses were applied to study the regulation of DcR3 expression in RCC. Additionally, an ex vivo tissue slice culture technique combined with immunohistochemistry was used to study the regulation of DcR3 expression in human RCC specimens.ResultsHere, we show that DcR3 promotes adhesion, migration and invasiveness of RCC cells. The DcR3-dependent increase in cellular invasiveness is accompanied with an up-regulation of integrin alpha 4, matrixmetalloproteinase 7 and urokinase plasminogen activator (uPA). Further, we identified a signaling pathway regulating DcR3 expression in RCC. Using in vitro experiments as well as an ex vivo RCC tissue slice culture model, we demonstrate that expression of DcR3 is regulated in a PI3K/AKT-dependent manner involving the transcription factor nuclear factor of activated T-cells (NFAT).ConclusionsTaken together, our results identify DcR3 as a key driver of tumor cell dissemination and suggest DcR3 as a promising target for rational therapy of RCC.

Model-based rational design of an oncolytic virus with improved therapeutic potential

Oncolytic viruses are complex biological agents that interact at multiple levels with both tumour and normal tissues. Antiviral pathways induced by interferon are known to have a critical role in determining tumour cell sensitivity and normal cell resistance to infection with oncolytic viruses. Here we pursue a synthetic biology approach to identify methods that enhance antitumour activity of oncolytic viruses through suppression of interferon signalling. On the basis of the mathematical analysis of multiple strategies, we hypothesize that a positive feedback loop, established by virus-mediated expression of a soluble interferon-binding decoy receptor, increases tumour cytotoxicity without compromising normal cells. Oncolytic rhabdoviruses engineered to express a secreted interferon antagonist have improved oncolytic potential in cellular cancer models, and display improved therapeutic potential in tumour-bearing mice. Our results demonstrate the potential of this methodology in evaluating potential caveats of viral immune-evasion strategies and improving the design oncolytic viruses.

Inhibitory effect of soluble EP2 receptor on ovarian tumor growth in nude mice and utility of TMPRSS4 as a combinatorial molecular target

We have previously reported that FuEP2/Ex2, a soluble decoy receptor for PGE2, suppresses tumor growth in an orthotopic xenograft model. To examine whether it has further uses, we examined the effect of FuEP2/Ex2 in an intraperitoneal metastasis model of ovarian cancer cells. We established FuEP2/Ex2-expressing ovarian cancer cells (SKOV/ip-FuEP2/Ex2) and injected them intraperitoneally into female nude mice. Mice injected with SKOV/ip-FuEP2/Ex2 had no ascitic fluid and showed smaller tumor lesions compared to mice injected with vector control cells, with decreased microvessel density and M2 macrophages. To identify molecular targets for combination treatment, we conducted cDNA microarray analysis and found three genes encoding enzyme [matrix metalloproteinase-7 (MMP-7), transmembrane protease serin 4 (TMPRSS4) and cytocrome P450 1B1 (CYP1B1)] to be upregulated in SKOV/ip-FuEP2/Ex2-derived tumors. Administration of TMPRSS4 inhibitor further reduced tumor weight and decreased the number of Ki-67‑positive cells in SKOV/ip-FuEP2/Ex2-injected mice. These data indicate a possible EP-targeting strategy using FuEP2/Ex2 in the treatment of ovarian cancer and suggest that dual targeting of EP-mediated signaling and TMPRSS4 may enhance therapeutic value.

Assessing DcR3 expression in relation to survivin and other prognostic factors in B cell non-Hodgkin’s lymphoma

The soluble decoy receptor 3 (DcR3) is a member of the tumor necrosis factor receptor superfamily whose overexpression has been observed in several human malignancies. Survivin is one of the inhibitors of apoptosis proteins that are thought to play an important role in the pathogenesis of malignancies. We aimed to evaluate the expression of DcR3 in relation to survivin in B cell non-Hodgkin`s lymphoma (NHL) and then we focused on patients with diffuse large B cell non-Hodgkin's lymphoma (DLBCL) (50 cases) and correlated DcR3 expression with survivin expression and other prognostic parameters. Fifteen subjects with reactive lymphoid hyperplasia were included as controls. The expression of DcR3 and survivin were analyzed by immunohistochemistry on formalin-fixed paraffin-embedded lymph node sections from 80 cases of B cell NHL and 15 controls. Bone marrow biopsy sections of patients were also immunostained with the previous markers. DcR3 expression was found in 32.5% of B cell NHL patients versus 6.7% of controls (p <0.001) and was associated with the aggressive/highly aggressive subtypes. DcR3 was strongly expressed in 30% of DLBCL patients, where it was associated with survivin expression, high international prognostic index (IPI), the presence of extra nodal disease, ECOG performance status >1, reduced remission rates and shorter event-free survival. The expression of survivin was 40% in B cell NHL patients versus 13.3% in the control group (p <0.001). The expression of survivin in aggressive/highly aggressive B cell NHL was significantly higher than that in indolent B cell NHL. Survivin expression has been detected in 44% of the DLBCL patients and was associated with their clinical stage and shorter event-free survival (p = 0.026). Bone marrow biopsy sections from DLBCL patients showed significant DcR3 and survivin over expressions in sections with infiltration by lymphoma cells than sections with no infiltration. DcR3 expression was associated with other prognostic factors including survivin, reduced remission rates, and shorter event-free survival. Survivin is closely related to aggressive/highly aggressive subtypes of B cell NHL and is associated with shorter event-free survival. Both DcR3 and survivin expressions on bone marrow sections can be of help in diagnosing bone marrow infiltration.

Identification of a new cell therapy candidate reveals immunological specialization and robust therapeutic efficacy against high-mortality sepsis (P4212)

Abstract Sepsis is an aggressive inflammatory syndrome, killing 7.3 million people per year. In mouse and human systems, we have identified highly differentiated, immune-specialized myofibroblasts called Fibroblastic Reticular Cells (FRCs) as a new cell therapy candidate for sepsis. One injection of ex-vivo expanded allogeneic FRCs significantly reduced mortality when administered at therapeutic timepoints. When administered 4 hrs after cecal ligation and puncture (CLP) in mice, FRCs prevented peritonitis progressing to blood sepsis, reducing deaths by 50%, while mesenchymal stromal cells (MSCs) were ineffective. FRCs also reduced lung pathology and lowered systemic concentrations of pathogenic cytokines. Rarely for putative sepsis therapies, we found that when FRCs were administered very late, as mice approached euthanasia endpoints (16 hrs), survival was still extremely robust: 44% of FRC-treated mice survived compared to 9% of controls. Production of the prominent vasodilator and immunosuppressant NOS2 was a primary molecular mechanism. Human FRCs showed strong similarities to mouse FRCs and exhibited desirable traits for a cell therapy candidate, including low variance between unrelated donors. Importantly, human FRCs constitutively secrete high levels of soluble cytokine decoy receptors against major mediators of sepsis. Together these data describe a novel anti-inflammatory cell type and provide robust pre-clinical evidence for therapeutic efficacy in multiple models of sepsis.

Fibroblast Growth Factor Inducible (Fn14)-specific Antibodies Concomitantly Display Signaling Pathway-specific Agonistic and Antagonistic Activity

Fn14 is a therapeutic target in various diseases. Anti-Fn14 antibodies activate the alternative NFκB pathway but not other Fn14-related activities induced by soluble or membrane-bound TWEAK. FcγR-bound anti-Fn14 antibodies, however, activate the full spectrum of Fn14-associated activities. Anti-Fn14 antibodies elicit agonistic activities differing from those of the natural Fn14 ligand TWEAK. These findings influence the rationale of designing Fn14-targeted therapies. The Fn14-specific monoclonal antibodies PDL192 and P4A8, which are under consideration in clinical trials, showed no agonistic activity with respect to IL8 production and cell death induction. However, oligomerization with protein G or binding to Fcγ receptors converted both anti-Fn14 antibodies into potent agonists. TNF-like weak inducer of apoptosis (TWEAK), the ligand of Fn14, occurs naturally in two forms with partly different signaling capabilities, as a membrane-bound ligand and as a soluble trimeric molecule. Although membrane TWEAK strongly triggers all Fn14-associated pathways, soluble TWEAK predominately triggers the alternative nuclear factor κB (NFκB) pathway and enhances TNF-induced cell death but has only a poor effect on the classical NFκB pathway and chemokine production. Thus, the oligomerized and FcγR-bound anti-Fn14 mAbs mimicked the activity of membrane TWEAK. Notably, both anti-Fn14 antibodies significantly triggered p100 processing, the hallmark of the alternative NFκB pathway, and therefore resembled soluble TWEAK. In contrast to the latter, however, the anti-Fn14s showed no effect on TNF receptor 1-induced cell death and P4A8 even blocked the corresponding TWEAK response. Thus, we showed that Fn14 antibodies display an alternative NFκB pathway-specific agonistic activity but fail to phenocopy other activities of soluble TWEAK, whereas oligomerized or FcγR-bound Fn14 antibodies fully mimic the activity of membrane TWEAK. In view of the trivalent nature of the TWEAK-Fn14 interaction, this suggests that the alternative NFκB pathway is uniquely responsive already to Fn14 dimerization enabling antibodies to elicit an unnatural response pattern distinct from that of the naturally occurring Fn14 ligands.