Antibody-mediated targeting of TNFR2 activates CD8+ T cells in mice and promotes antitumor immunity.

Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.

Endosialin is expressed in high grade and advanced sarcomas: evidence from clinical specimens and preclinical modeling.

We previously surveyed the expression of endosialin/ CD248/TEM-1 by immunohistochemistry in human clinical specimens of sarcomas and documented expression in tumor cells, stromal cells and vasculature. In the present study, we completed a retrospective analysis of the diagnostic reports available for these same samples in order to identify high-grade and metastatic disease. Our results show that endosialin can be detected in advanced disease. We screened human sarcoma cell lines in vitro for endosialin expression and developed preclinical human xenograft models of disseminated sarcoma. We found that 22 out of 42 human sarcoma cell lines were positive for endosialin with a positive correlation between mRNA and protein levels. When implanted in vivo, endosialin was expressed at all sites of dissemination. These data provide clinical and preclinical evidence that endosialin can be detected in advanced sarcoma. These results demonstrate for the first time that endosialin is a suitable therapeutic target for poor prognosis and advanced disease.

STR/ort mice, a model for spontaneous osteoarthritis, exhibit elevated levels of both local and systemic inflammatory markers.

Osteoarthritis is a common joint disease that currently lacks disease-modifying treatments. Development of therapeutic agents for osteoarthritis requires better understanding of the disease and cost-effective in vivo models that mimic the human disease. Here, we analyzed the joints of STR/ort mice, a model for spontaneous osteoarthritis, for levels of inflammatory and oxidative stress markers and measured serum cytokines to characterize the local and systemic inflammatory status of these mice. Markers of low-grade inflammatory and oxidative stress-RAGE, AGE, S100A4, and HMGB1-were evaluated through immunohistochemistry. Of these, AGE and HMGB1 levels were elevated strongly in hyperplastic synovium, cartilage, meniscus, and ligaments in the joints of STR/ort mice compared with CBA mice, an osteoarthritis-resistant mouse strain. These increases (particularly in the synovium, meniscus, and ligaments) correlated with increased histopathologic changes in the cartilage. Serum analysis showed higher concentrations of several cytokines including IL1ß, IL12p70, MIP1ß, and IL5 in STR/ort mice, and these changes correlated with worsened joint morphology. These results indicate that STR/ort mice exhibited local and systemic proinflammatory conditions, both of which are present in human osteoarthritis. Therefore, the STR/ort mouse model appears to be a clinically relevant and cost-effective small animal model for testing osteoarthritis therapeutics.

Identification of the chondrocyte lineage using microfibril-associated glycoprotein-2, a novel marker that distinguishes chondrocytes from synovial cells.

Methods for the lineage identification of cell or tissue-engineered therapeutics must provide a high degree of performance to confidently distinguish the intended cell type from other lineages that could be present in the finished product. For many applications, these methods also require rapid, high-throughput capability. In this work, methods for the identification of autologous cultured chondrocytes for implantation were investigated. A histological analysis confirmed that fibrous tissue occasionally present in biopsies procured for autologous chondrocyte implantation production comprised synovium. Chondrocyte and synovial cell cultures were then examined using a full transcriptome microarray analysis, which revealed cartilage link protein and microfibril-associated glycoprotein-2 (MAGP2) as the most differentially expressed transcripts between the culture types. Performance characteristics of gene expression assays formed by the analysis of cartilage link protein with normalization to either standard reference genes or to MAGP2 were evaluated. The results demonstrate that the MAGP2-based assay provided superior performance for the purpose of cell culture identification compared to assays using standard reference genes. The selectivity against synovial and heterogeneous samples provided by the novel assay suggests it as an appropriate lineage identification method for cell cultures derived from cartilage.

Improved management of lysosomal glucosylceramide levels in a mouse model of type 1 Gaucher disease using enzyme and substrate reduction therapy.

Gaucher disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (acid beta-glucosidase), with consequent cellular accumulation of glucosylceramide (GL-1). The disease is managed by intravenous administrations of recombinant glucocerebrosidase (imiglucerase), although symptomatic patients with mild to moderate type 1 Gaucher disease for whom enzyme replacement therapy (ERT) is not an option may also be treated by substrate reduction therapy (SRT) with miglustat. To determine whether the sequential use of both ERT and SRT may provide additional benefits, we compared the relative pharmacodynamic efficacies of separate and sequential therapies in a murine model of Gaucher disease (D409V/null). As expected, ERT with recombinant glucocerebrosidase was effective in reducing the burden of GL-1 storage in the liver, spleen, and lung of 3-month-old Gaucher mice. SRT using a novel inhibitor of glucosylceramide synthase (Genz-112638) was also effective, albeit to a lesser degree than ERT. Animals administered recombinant glucocerebrosidase and then Genz-112638 showed the lowest levels of GL-1 in all the visceral organs and a reduced number of Gaucher cells in the liver. This was likely because the additional deployment of SRT following enzyme therapy slowed the rate of reaccumulation of GL-1 in the affected organs. Hence, in patients whose disease has been stabilized by intravenously administered recombinant glucocerebrosidase, orally administered SRT with Genz-112638 could potentially be used as a convenient maintenance therapy. In patients naïve to treatment, ERT followed by SRT could potentially accelerate clearance of the offending substrate.

Investigation of the mechanism of action of alemtuzumab in a human CD52 transgenic mouse model.

Alemtuzumab is a humanized monoclonal antibody against CD52, an antigen found on the surface of normal and malignant lymphocytes. It is approved for the treatment of B-cell chronic lymphocytic leukaemia and is undergoing Phase III clinical trials for the treatment of multiple sclerosis. The exact mechanism by which alemtuzumab mediates its biological effects in vivo is not clearly defined and mechanism of action studies have been hampered by the lack of cross-reactivity between human and mouse CD52. To address this issue, a transgenic mouse expressing human CD52 (hCD52) was created. Transgenic mice did not display any phenotypic abnormalities and were able to mount normal immune responses. The tissue distribution of hCD52 and the level of expression by various immune cell populations were comparable to those seen in humans. Treatment with alemtuzumab replicated the transient increase in serum cytokines and depletion of peripheral blood lymphocytes observed in humans. Lymphocyte depletion was not as profound in lymphoid organs, providing a possible explanation for the relatively low incidence of infection in alemtuzumab-treated patients. Interestingly, both lymphocyte depletion and cytokine induction by alemtuzumab were largely independent of complement and appeared to be mediated by neutrophils and natural killer cells because removal of these populations with antibodies to Gr-1 or asialo-GM-1, respectively, strongly inhibited the activity of alemtuzumab whereas removal of complement by treatment with cobra venom factor had no impact. The hCD52 transgenic mouse appears to be a useful model and has provided evidence for the previously uncharacterized involvement of neutrophils in the activity of alemtuzumab.

A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease.

An approach to treating Gaucher disease is substrate inhibition therapy which seeks to abate the aberrant lysosomal accumulation of glucosylceramide. We have identified a novel inhibitor of glucosylceramide synthase (Genz-112638) and assessed its activity in a murine model of Gaucher disease (D409V/null). Biochemical characterization of Genz-112638 showed good potency (IC(50) approximately 24nM) and specificity against the target enzyme. Mice that received drug prior to significant accumulation of substrate (10 weeks of age) showed reduced levels of glucosylceramide and number of Gaucher cells in the spleen, lung and liver when compared to age-matched control animals. Treatment of older mice that already displayed significant amounts of tissue glucosylceramide (7 months old) resulted in arrest of further accumulation of the substrate and appearance of additional Gaucher cells in affected organs. These data indicate that substrate inhibition therapy with Genz-112638 represents a viable alternate approach to enzyme therapy to treat the visceral pathology in Gaucher disease.

AAV8-mediated expression of glucocerebrosidase ameliorates the storage pathology in the visceral organs of a mouse model of Gaucher disease.

BACKGROUND: Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL-1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL-1 was used to evaluate adeno-associated viral (AAV)-mediated gene therapy. METHODS: A recombinant AAV8 serotype vector bearing human GC (hGC) was administered intravenously to the mice. The levels of hGC in blood and tissues were determined, as were the effects of gene transfer on the levels of GL-1. Histopathological evaluation was performed on liver, spleen and lungs. RESULTS: Vector administration to pre-symptomatic Gaucher mice resulted in sustained hepatic secretion of hGC at levels that prevented GL-1 accumulation and the appearance of Gaucher cells in the liver, spleen and lungs. AAV administration to older mice with established disease resulted in normalization of GL-1 levels in the spleen and liver and partially reduced that in the lung. Analysis of the bronchoalveolar lavage fluid (BALF) from treated mice showed significant correction of the abnormal cellularity and cell differentials. No antibodies to the expressed hGC were detected following a challenge with recombinant enzyme suggesting the animals were tolerized to human enzyme. CONCLUSIONS: These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in a Gaucher mouse model, and thus support the continued consideration of this vector as an alternative approach to treating Gaucher disease.