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.

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.

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.

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.

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.

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.

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.

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.

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.

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.