Interaction of TWEAK with Fn14 leads to the progression of fibrotic liver disease by directly modulating hepatic stellate cell proliferation.

Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) have been associated with liver regeneration in vivo. To further investigate the role of this pathway we examined their expression in human fibrotic liver disease and the effect of pathway deficiency in a murine model of liver fibrosis. The expression of Fn14 and TWEAK in normal and diseased human and mouse liver tissue and primary human hepatic stellate cells (HSCs) were investigated by qPCR, western blotting and immunohistochemistry. In addition, the levels of Fn14 in HSCs following pro-fibrogenic and pro-inflammatory stimuli were assessed and the effects of exogenous TWEAK on HSCs proliferation and activation were studied in vitro. Carbon tetrachloride (CCl4 ) was used to induce acute and chronic liver injury in TWEAK KO mice. Elevated expression of both Fn14 and TWEAK were detected in acute and chronic human liver injury, and co-localized with markers of activated HSCs. Fn14 levels were low in quiescent HSCs but were significantly induced in activated HSCs, which could be further enhanced with the profibrogenic cytokine TGFß in vitro. Stimulation with recombinant TWEAK induced proliferation but not further HSCs activation. Fn14 gene expression was also significantly up-regulated in CCl4 models of hepatic injury whereas TWEAK KO mice showed reduced levels of liver fibrosis following chronic CCl4 injury. In conclusion, TWEAK/Fn14 interaction leads to the progression of fibrotic liver disease via direct modulation of HSCs proliferation, making it a potential therapeutic target for liver fibrosis.

TWEAK-Fn14 Signaling Activates Myofibroblasts to Drive Progression of Fibrotic Kidney Disease.

The identification of the cellular origins of myofibroblasts has led to the discovery of novel pathways that potentially drive myofibroblast perpetuation in disease. Here, we further investigated the role of innate immune signaling pathways in this process. In mice, renal injury-induced activation of pericytes, which are myofibroblast precursors attached to endothelial cells, led to upregulated expression of TNF receptor superfamily member 12a, also known as fibroblast growth factor-inducible 14 (Fn14), by these cells. In live rat kidney slices, administration of the Fn14 ligand, TNF-related weak inducer of apoptosis (TWEAK), promoted pericyte-dependent vasoconstriction followed by pericyte detachment from capillaries. In vitro, administration of TWEAK activated and differentiated pericytes into cytokine-producing myofibroblasts, and further activated established myofibroblasts in a manner requiring canonical and noncanonical NF-κB signaling pathways. Deficiency of Fn14 protected mouse kidneys from fibrogenesis, inflammation, and associated vascular instability after in vivo injury, and was associated with loss of NF-κB signaling. In a genetic model of spontaneous CKD, therapeutic delivery of anti-TWEAK blocking antibodies attenuated disease progression, preserved organ function, and increased survival. These results identify the TWEAK-Fn14 signaling pathway as an important factor in myofibroblast perpetuation, fibrogenesis, and chronic disease progression.

Pathological activation of canonical nuclear-factor κB by synergy of tumor necrosis factor α and TNF-like weak inducer of apoptosis in mouse acute colitis.

Tumor necrosis factor (TNF)-α is a major effector in various inflammatory conditions. TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily that promotes inflammatory tissue damage through its receptor, FGF-inducible molecule 14 (Fn14). Since both TWEAK and TNF-α have been shown to mediate pathological responses through inter-dependent or independent pathways by in vitro, the potential interplay of these pathways was investigated in a mouse colitis model. Acute colitis was induced by rectal injection of trinitrobenzene sulfonic acid (TNBS), with administration of control IgG, TNF receptor (TNFR)-Ig chimeric protein, anti-TWEAK monoclonal antibody, or the combination of TNFR-Ig and anti-TWEAK antibody. On day 4, disease severity was evaluated and gene expression profiling was analyzed using whole colon tissue. NF-κB activation was investigated with Western blot. Levels of transcript of TWEAK, Fn14 and NF-κB-related molecules were measured in purified colon epithelial cells (ECs). As a result, activation of the canonical (p50/RelA), but not noncanonical (p100/RelB)-mediated pathway was the hallmark of inflammatory responses in this model. Inflammation induced upregulation of Fn14 only in ECs but not in other cell types. Combination treatment of TNFR-Ig and anti-TWEAK antibody synergistically reduced disease severity in comparison with the control antibody or single agent treatment. Gene expression profile of the colon indicated downregulation of canonical NF-κB pathway with combination treatment. In conclusion, synergistic activation of canonical NF-κB by TWEAK and TNF-α is critical for the induction of inflammatory tissue damage in acute inflammation.

TWEAK signals through JAK-STAT to induce tumor cell apoptosis.

The TWEAK receptor Fn14 (TNFRSF12), a member of the TNF Receptor superfamily, can mediate many processes, including apoptosis. Fn14 agonists have therefore been the subject of interest as potential cancer therapeutics. In cell culture experiments, interferon gamma (IFNγ) is typically required for induction of apoptotic activity by either TWEAK or Fn14 agonistic antibodies in most cell lines. We have investigated the mechanism of IFNγ signaling and the role of JAK-STAT signaling in TWEAK/Fn14-mediated tumor cell killing. We found that IFNγ-mediated enhancement of tumor cell killing is JAK-STAT dependent, as JAK inhibitors block IFNγ-dependent TWEAK induced apoptosis. Exposure of tumor cells to IFNγ results in an increase in Fn14 expression on the cell surface, which may be a mechanism by which IFNγ induces sensitivity to TWEAK. In a reciprocal fashion, we observed that IFNγ receptor levels increase in response to TWEAK treatment in WiDr cells. Significantly, we found that TWEAK alone can induce STAT1 phosphorylation in WiDr tumor cells. Moreover, TWEAK induction of tumor cell apoptosis in WiDr cells in the absence of IFNγ is mediated by the JAK-STAT pathway. Correspondingly, we show that treatment of tumor bearing mice with mBIIB036, an Fn14 agonistic antibody, results in STAT1 phosphorylation in the tumors. Notably, the level of STAT1 phosphorylation appears to correlate with the degree of tumor growth inhibition by BIIB036 in vivo. Additionally, in WiDr cells, TWEAK induces a soluble factor, which we have identified as IFNß, capable of independently inducing STAT1 phosphorylation when transferred to naïve cells. Finally, either IFNα or IFNß can partially substitute for IFNγ in sensitizing tumor cells to Fn14 agonists. In summary, we show that TWEAK/Fn14 can signal through the JAK-STAT pathway to induce IFNß, and that the ability of TWEAK to induce tumor cell apoptosis is mediated by JAK-STAT signaling. We also demonstrate that IFNγ enhancement of TWEAK/FN14-mediated tumor cell death is JAK-dependent and may occur by IFNγ-dependent upregulation of Fn14 on tumor cells. These findings may have implications for the appropriately targeted clinical development of Fn14 agonists as anti-cancer therapy.

Attenuated liver fibrosis in the absence of B cells.

Analysis of mononuclear cells in the adult mouse liver revealed that B cells represent as much as half of the intrahepatic lymphocyte population. Intrahepatic B cells (IHB cells) are phenotypically similar to splenic B2 cells but express lower levels of CD23 and CD21 and higher levels of CD5. IHB cells proliferate as well as splenic B cells in response to anti-IgM and LPS stimulation in vitro. VDJ gene rearrangements in IHB cells contain insertions of N,P region nucleotides characteristic of B cells maturing in the adult bone marrow rather than in the fetal liver. To evaluate whether B cells can have an impact on liver pathology, we compared CCl4-induced fibrosis development in B cell-deficient and wild-type mice. CCl4 caused similar acute liver injury in mutant and wild-type mice. However, following 6 weeks of CCl4 treatment, histochemical analyses showed markedly reduced collagen deposition in B cell-deficient as compared with wild-type mice. By analyzing mice that have normal numbers of B cells but lack either T cells or immunoglobulin in the serum, we established that B cells have an impact on fibrosis in an antibody- and T cell-independent manner.

Recombinant ST2 boosts hepatic Th2 response in vivo.

Excessive scarring or fibrosis is a common feature of a wide spectrum of diseases characterized by an exaggerated Th2 response. The TLR/IL-1 receptor (IL-1R)-related protein ST2 is expressed in a membrane-bound form selectively by Th2 cells and was shown to be indispensable for some in vivo Th2 responses. ST2 was also found to block TLR signaling. We addressed the impact of the ST2 pathway on fibrogenesis using a mouse model of hepatic injury and fibrosis induced by carbon tetrachloride (CCl(4)). We showed that cytokine production by intrahepatic lymphocytes from CCl(4)-injured liver is abrogated in the absence of TLR-4. Interfering with the ST2 pathway using an ST2-Fc fusion protein accelerated and enhanced hepatic fibrosis, paralleled by the increasing ex vivo secretion of Th2 cytokines IL-4, -5, -10, and -13 by intrahepatic lymphocytes of ST2-Fc-treated, CCl(4)-gavaged mice. Absence of IL-4/13 signaling in IL-4Ralpha-deficient mice obliterated this ST2-Fc effect on fibrogenesis. Moreover, depletion of CD4(+) T cells abrogated ST2-Fc-enhanced Th2 cytokines and accelerated fibrosis. Thus, ST2-Fc caused overproduction of Th2 cytokines by intrahepatic CD4(+) T cells, possibly by modifying TLR-4 signaling in injured liver. This ST2-Fc-driven Th2 response exacerbated CCl(4)-induced hepatic fibrosis.

Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing.

Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA)-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo.

Development of an Fn14 agonistic antibody as an anti-tumor agent.

TWEAK, a TNF family ligand with pleiotropic cellular functions, was originally described as capable of inducing tumor cell death in vitro. TWEAK functions by binding its receptor, Fn14, which is up-regulated on many human solid tumors. Herein, we show that intratumoral administration of TWEAK, delivered either by an adenoviral vector or in an immunoglobulin Fc-fusion form, results in significant inhibition of tumor growth in a breast xenograft model. To exploit the TWEAK-Fn14 pathway as a therapeutic target in oncology, we developed an anti-Fn14 agonistic antibody, BIIB036. Studies described herein show that BIIB036 binds specifically to Fn14 but not other members of the TNF receptor family, induces Fn14 signaling, and promotes tumor cell apoptosis in vitro. In vivo, BIIB036 effectively inhibits growth of tumors in multiple xenograft models, including colon (WiDr), breast (MDA-MB-231), and gastric (NCI-N87) tumors, regardless of tumor cell growth inhibition response observed to BIIB036 in vitro. The anti-tumor activity in these cell lines is not TNF-dependent. Increasing the antigen-binding valency of BIB036 significantly enhances its anti-tumor effect, suggesting the contribution of higher order cross-linking of the Fn14 receptor. Full Fc effector function is required for maximal activity of BIIB036 in vivo, likely due to the cross-linking effect and/or ADCC mediated tumor killing activity. Taken together, the anti-tumor properties of BIIB036 validate Fn14 as a promising target in oncology and demonstrate its potential therapeutic utility in multiple solid tumor indications.

Stability engineering of scFvs for the development of bispecific and multivalent antibodies.

Single-chain Fvs (scFvs) are commonly used building blocks for creating engineered diagnostic and therapeutic antibody molecules. Bispecific antibodies (BsAbs) hold particular interest due to their ability to simultaneously bind and engage two distinct targets. We describe a technology for producing stable, scalable IgG-like bispecific and multivalent antibodies based on methods for rapidly engineering thermally stable scFvs. Focused libraries of mutant scFvs were designed using a combination of sequence-based statistical analyses and structure-, and knowledge-based methods. Libraries encoding these designs were expressed in E. coli and culture supernatants-containing soluble scFvs screened in a high-throughput assay incorporating a thermal challenge prior to an antigen-binding assay. Thermally stable scFvs were identified that retain full antigen-binding affinity. Single mutations were found that increased the measured T(m) of either the V(H) or V(L) domain by as much as 14 degrees C relative to the wild-type scFv. Combinations of mutations further increased the T(m) by as much as an additional 12 degrees C. Introduction of a stability-engineered scFv as part of an IgG-like BsAb enabled scalable production and purification of BsAb with favorable biophysical properties.

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR.

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTbetaR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti-LTbetaR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a fulllength anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.