In search of a small molecule agonist of the relaxin receptor RXFP1 for the treatment of liver fibrosis.

The peptide hormone human relaxin-2 (H2-RLX) has emerged as a potential therapy for cardiovascular and fibrotic diseases, but its short in vivo half-life is an obstacle to long-term administration. The discovery of ML290 demonstrated that it is possible to identify small molecule agonists of the cognate G-protein coupled receptor for H2-RLX (relaxin family peptide receptor-1 (RXFP1)). In our efforts to generate a new medicine for liver fibrosis, we sought to identify improved small molecule functional mimetics of H2-RLX with selective, full agonist or positive allosteric modulator activity against RXFP1. First, we confirmed expression of RXFP1 in human diseased liver. We developed a robust cellular cAMP reporter assay of RXFP1 signaling in HEK293 cells transiently expressing RXFP1. A high-throughput screen did not identify further specific agonists or positive allosteric modulators of RXFP1, affirming the low druggability of this receptor. As an alternative approach, we generated novel ML290 analogues and tested their activity in the HEK293-RXFP1 cAMP assay and the human hepatic cell line LX-2. Differences in activity of compounds on cAMP activation compared with changes in expression of fibrotic markers indicate the need to better understand cell- and tissue-specific signaling mechanisms and their disease-relevant phenotypes in order to enable drug discovery.

CCR8+FOXp3+ Treg cells as master drivers of immune regulation.

The current study identifies CCR8+ regulatory T cells (Treg cells) as drivers of immunosuppression. We show that in human peripheral blood cells, more than 30% of Treg up-regulate CCR8 following activation in the presence of CCL1. This interaction induces STAT3-dependent up-regulation of FOXp3, CD39, IL-10, and granzyme B, resulting in enhanced suppressive activity of these cells. Of the four human CCR8 ligands, CCL1 is unique in potentiating Treg cells. The relevance of these observations has been extended using an experimental model of multiple sclerosis [experimental autoimmune encephalomyelitis, (EAE)] and a stabilized version of mouse CCL1 (CCL1-Ig). First, we identified a self-feeding mechanism by which CCL1 produced by Treg cells at an autoimmune site up-regulates the expression of its own receptor, CCR8, on these cells. Administration of CCL1-Ig during EAE enhanced the in vivo proliferation of these CCR8+ regulatory cells while inducing the expression of CD39, granzyme B, and IL-10, resulting in the efficacious suppression of ongoing EAE. The critical role of the CCL1-CCR8 axis in Treg cells was further dissected through adoptive transfer studies using CCR8-/- mice. Collectively, we demonstrate the pivotal role of CCR8+ Treg cells in restraining immunity and highlight the potential clinical implications of this discovery.

Novel Bispecific Domain Antibody to LRP6 Inhibits Wnt and R-spondin Ligand-Induced Wnt Signaling and Tumor Growth.

UNLABELLED: Aberrant WNT signaling is associated with the formation and growth of numerous human cancer types. The low-density lipoprotein receptor-related protein 6 (LRP6) is the least redundant component of the WNT receptor complex with two independent WNT ligand-binding sites. Using domain antibody (dAb) technology, a bispecific antibody (GSK3178022) to LRP6 was identified that is capable of blocking stimulation in the presence of a range of WNT and R-spondin (RSPO) ligands in vitro GSK3178022 was also efficacious in reducing WNT target gene expression in vivo, in both cancer cell line and patient-derived xenograft models, and delays tumor growth in a patient-derived RSPO fusion model of colorectal cancer. IMPLICATIONS: This article demonstrates the inhibition of a key oncogenic receptor, intractable to mAb inhibition due to multiple independent ligand interaction sites, using an innovative dAb approach. Mol Cancer Res; 14(9); 859-68. ©2016 AACR.

The immune synapse clears and excludes molecules above a size threshold.

Natural killer cells assess target cell health via interactions at the immune synapse (IS) that facilitates signal integration and directed secretion. Here we test whether the IS also functions as a gasket. Quantitative fluorescence microscopy of nanometer-scale dextrans within synapses formed by various effector-target cell conjugates reveal that molecules are excluded in a size-dependent manner at activating synapses. Dextran sized ≤4 nm move in and out of the IS, but access is significantly reduced (by >50%) for dextran sized 10-13 nm, and dextran ≥32 nm is almost entirely excluded. Depolymerization of F-actin abrogated exclusion. Unexpectedly, larger-sized dextrans are cleared as the IS assembles in a zipper-like manner. Monoclonal antibodies are also excluded from the IS but smaller single-domain antibodies are able to penetrate. Therefore, the IS can clear and exclude molecules above a size threshold, and drugs designed to target synaptic cytokines or cytotoxic proteins must fit these dimensions.

Inhibition of proliferative retinopathy by the anti-vascular agent combretastatin-A4.

Retinal neovascularization occurs in a variety of diseases including diabetic retinopathy, the most common cause of blindness in the developed world. There is accordingly considerable incentive to develop drugs that target the aberrant angiogenesis associated with these conditions. Previous studies have shown that a number of anti-angiogenic agents can inhibit retinal neovascularization in a well-characterized murine model of ischemia-induced proliferative retinopathy. Combretastatin-A4 (CA-4) is an anti-vascular tubulin-binding agent currently undergoing clinical evaluation for the treatment of solid tumors. We have recently shown that CA-4 is not tumor-specific but elicits anti-vascular effects in nonneoplastic angiogenic vessels. In this study we have examined the capacity of CA-4 to inhibit retinal neovascularization in vivo. CA-4 caused a dose-dependent inhibition of neovascularization with no apparent side effects. The absence of vascular abnormalities or remnants of disrupted neovessels in retinas of CA-4-treated mice suggests an anti-angiogenic mechanism in this model, in contrast to the anti-vascular effects observed against established tumor vessels. Importantly, histological and immunohistochemical analyses indicated that CA-4 permitted the development of normal retinal vasculature while inhibiting aberrant neovascularization. These data are consistent with CA-4 eliciting tissue-dependent anti-angiogenic effects and suggest that CA-4 has potential in the treatment of nonneoplastic diseases with an angiogenic component.