Neil James Clarke

Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation.

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations.

Somatostatin receptor 2 knockout/lacZ knockin mice show impaired motor coordination and reveal sites of somatostatin action within the striatum

The peptide somatostatin can modulate the functional output of the basal ganglia. The exact sites and mechanisms of this action, however, are poorly understood, and the physiological context in which somatostatin acts is unknown. Somatostatin acts as a neuromodulator via a family of five 7‐transmembrane G protein‐coupled receptors, SSTR1–5, one of which, SSTR2, is known to be functional in the striatum. We have investigated the role of SSTR2 in basal ganglia function using mice in which Sstr2 has been inactivated and replaced by the lacZ reporter gene. Analysis of Sstr2lacZ expression in the brain by β‐galactosidase histochemistry demonstrated a widespread pattern of expression. By comparison to previously published in situ hybridization and immunohistochemical data, Sstr2lacZ expression was shown to accurately recapitulate that of Sstr2 and thus provided a highly sensitive model to investigate cell‐type‐specific expression of Sstr2. In the striatum, Sstr2 expression was identified in medium spiny projection neurons restricted to the matrix compartment and in cholinergic interneurons. Sstr2 expression was not detected in any other nuclei of the basal ganglia except for a sparse number of nondopaminergic neurons in the substantia nigra. Microdialysis in the striatum showed Sstr2‐null mice were selectively refractory to somatostatin‐induced dopamine and glutamate release. In behavioural tests, Sstr2‐null mice showed normal levels of locomotor activity and normal coordination in undemanding tasks. However, in beam‐walking, a test of fine motor control, Sstr2‐null mice were severely impaired. Together these data implicate an important neuromodulatory role for SSTR2 in the striatum.

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi.

BackgroundStudies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.ResultsWe show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.ConclusionOur results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.

Non invasive imaging assessment of the biodistribution of GSK2849330, an ADCC and CDC optimized anti HER3 mAb, and its role in tumor macrophage recruitment in human tumor-bearing mice

The purpose of this work was to use various molecular imaging techniques to non-invasively assess GSK2849330 (anti HER3 ADCC and CDC enhanced ‘AccretaMab’ monoclonal antibody) pharmacokinetics and pharmacodynamics in human xenograft tumor-bearing mice. Immuno-PET biodistribution imaging of radiolabeled 89Zr-GSK2849330 was assessed in mice with HER3 negative (MIA-PaCa-2) and positive (CHL-1) human xenograft tumors. Dose dependency of GSK2849330 disposition was assessed using varying doses of unlabeled GSK2849330 co-injected with 89Zr-GSK2849330. In-vivo NIRF optical imaging and ex-vivo confocal microscopy were used to assess the biodistribution of GSK2849330 and the HER3 receptor occupancy in HER3 positive xenograft tumors (BxPC3, and CHL-1). Ferumoxytol (USPIO) contrast-enhanced MRI was used to investigate the effects of GSK2849330 on tumor macrophage content in CHL-1 xenograft bearing mice. Immuno-PET imaging was used to monitor the whole body drug biodistribution and CHL-1 xenograft tumor uptake up to 144 hours post injection of 89Zr-GSK2849330. Both hepatic and tumor uptake were dose dependent and saturable. The optical imaging data in the BxPC3 xenograft tumor confirmed the tumor dose response finding in the Immuno-PET study. Confocal microscopy showed a distinguished cytoplasmic punctate staining pattern within individual CHL-1 cells. GSK2849330 inhibited tumor growth and this was associated with a significant decrease in MRI signal to noise ratio after USPIO injection and with a significant increase in tumor macrophages as confirmed by a quantitative immunohistochemistry analysis. By providing both dose response and time course data from both 89Zr and fluorescently labeled GSK2849330, complementary imaging studies were used to characterize GSK2849330 biodistribution and tumor uptake in vivo. Ferumoxytol-enhanced MRI was used to monitor aspects of the immune system response to GSK2849330. Together these approaches potentially provide clinically translatable, non-invasive techniques to support dose optimization, and assess immune activation and anti-tumor responses.

Abstract 1192: Preclinical evaluation of HER3 mutations and rational combinations with AKT inhibition in enhancing anti-tumor activity of HER3 inhibition in gastric cancer

Molecular activating events involving the ERBB RTK family members (EGFR (ERBB1), HER2 (ERBB2), HER3 (ERBB3), HER4 (ERBB4)) drive oncogenesis by inducing proliferation, invasion and survival primarily through RAS/MAPK and PI3K/AKT signaling pathways in several cancers. The clinical successes of HER2 directed therapies are well known in breast cancer and more recently, in gastric cancer. However, resistance develops invariably and gastric cancer continues to be a largely unmet disease necessitating novel therapeutic interventions. Recent reports have highlighted HER3 as an emerging target as it is frequently overexpressed, mutated, preferentially dimerizes with HER2 to activate signaling and is induced as a result of de novo or acquired resistance to PI3K-AKT, MAPK and RTK pathway inhibitors. We explored the utility of an ADCC and CDC enhanced potent anti-HER3 therapeutic antibody (GSK2849330) as a single agent or in combination with a selective small molecule AKT inhibitor (GSK2110183) in patient-derived xenograft (PDX) models of gastric cancer. A panel of 15 HER3 mutant and 4 HER3 wildtype PDX models was screened in vivo for responses to GSK2849330 as measured by% tumor growth inhibition (%TGI). Several models were characterized for other molecular alterations (e.g. HER2, PTEN, HER3, etc.) and represented various subsets of gastric cancer. As a single agent administered at 25mg/kg IP BIW, GSK2849330 was modestly effective (TGI ≥ 50%) in 2/15 mutant and 2/4 wildtype models. While context is likely to matter to drive dependence on HER3, no obvious predictive markers were observed. Furthermore, we evaluated the effect of combination therapy with an AKT inhibitor (GSK2110183) administered at 60mg/kg PO QD in a HER3 wildtype, PTEN deficient model. This resulted in significant durable tumor growth inhibition (∼94% TGI) with improved survival and noticeable tumor regression in a few mice in the combination treatment group relative to either single agent groups. Tumor samples collected at the end of the study showed pronounced pharmacodynamic modulation of p-AKT and p-HER3, demonstrating on target activity of these agents. Taken together, our findings suggest that modest anti-tumor activity was elicited by GSK2849330 as monotherapy in select gastric PDX models with no clear associations between response and HER3 mutations or other known markers. However, robust durable activity was observed upon combination with GSK2110183. To our knowledge, this is the first in vivo evidence supporting the rational combination of a selective AKT inhibitor (GSK2110183) and an anti-HER3 therapeutic antibody (GSK2849330), both of which are actively undergoing clinical trials and warrant further investigation in gastric cancer. Citation Format: Gopi Ganji, Sherry Qin, Crystal Qin, Neil Clarke, Carolyn Buser-Doepner, Christopher Matheny, Rakesh Kumar, Biju Mangatt. Preclinical evaluation of HER3 mutations and rational combinations with AKT inhibition in enhancing anti-tumor activity of HER3 inhibition in gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1192.