Agonistic anti-DCIR antibody inhibits ITAM-mediated inflammatory signaling and promotes immune resolution.

DC inhibitory receptor (DCIR) is a C-type lectin receptor selectively expressed on myeloid cells, including monocytes, macrophages, DCs, and neutrophils. Its role in immune regulation has been implicated in murine models and human genome-wide association studies, suggesting defective DCIR function associates with increased susceptibility to autoimmune diseases such as rheumatoid arthritis, lupus, and Sjögren's syndrome. However, little is known about the mechanisms underlying DCIR activation to dampen inflammation. Here, we developed anti-DCIR agonistic antibodies that promote phosphorylation on DCIR's immunoreceptor tyrosine-based inhibitory motifs and recruitment of SH2 containing protein tyrosine phosphatase-2 for reducing inflammation. We also explored the inflammation resolution by depleting DCIR+ cells with antibodies. Utilizing a human DCIR-knock-in mouse model, we validated the antiinflammatory properties of the agonistic anti-DCIR antibody in experimental peritonitis and colitis. These findings provide critical evidence for targeting DCIR to develop transformative therapies for inflammatory diseases.

Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase.

Efficient targeting of therapeutic reagents to tissues and cell types of interest is critical to achieving therapeutic efficacy and avoiding unwanted side effects due to offtarget uptake. To increase assay efficiency and reduce the number of animals used per experiment during preclinical development, we used a combination of direct fluorescence labeling and confocal microscopy to simultaneously examine the biodistribution of two therapeutic proteins, Cerezyme and Ceredase, in the same animals. We show that the fluorescent tags do not interfere with protein uptake and localization. We are able to detect Cerezyme and Ceredase in intact cells and organs and demonstrate colocalization within target cells using confocal microscopy. In addition, the relative amount of protein internalized by different cell types can be quantified using cell type-specific markers and morphometric analysis. This approach provides an easy and straightforward means of assessing the tissue and cell type-specific biodistribution of multiple protein therapeutics in target organs using a minimal number of animals.

Homeostatic role of transforming growth factor-beta in the oral cavity and esophagus of mice and its expression by mast cells in these tissues.

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor; its overexpression has been implicated in many diseases, making it a desirable target for therapeutic neutralization. In initial safety studies, mice were chronically treated (three times per week) with high doses (50 mg/kg) of a murine, pan-neutralizing, anti-TGF-beta antibody. Nine weeks after the initiation of treatment, a subset of mice exhibited weight loss that was concurrent with decreased food intake. Histopathology revealed a unique, nonneoplastic cystic epithelial hyperplasia and tongue inflammation, as well as dental dysplasia and epithelial hyperplasia and inflammation of both the gingiva and esophagus. In an effort to determine the cause of this site-specific pathology, we examined TGF-beta expression in these tissues and saliva under normal conditions. By immunostaining, we found higher expression levels of active TGF-beta1 and TGF-beta3 in normal tongue and esophageal submucosa compared with gut mucosal tissues, as well as detectable TGF-beta1 in normal saliva by Western blot analysis. Interestingly, mast cells within the tongue, esophagus, and skin co-localized predominantly with the TGF-beta1 expressed in these tissues. Our findings demonstrate a novel and restricted pathology in oral and esophageal tissues of mice chronically treated with anti-TGF-beta that is associated with basal TGF-beta expression in saliva and by mast cells within these tissues. These studies illustrate a previously unappreciated biological role of TGF-beta in maintaining homeostasis within both oral and esophageal tissues.

Characterization of in vitro antimurine thymocyte globulin-induced regulatory T cells that inhibit graft-versus-host disease in vivo.

Antithymocyte/antilymphocyte globulins are polyclonal antihuman T-cell antibodies used clinically to treat acute transplant rejection. These reagents deplete T cells, but a rabbit antihuman thymocyte globulin has also been shown to induce regulatory T cells in vitro. To examine whether antithymocyte globulin-induced regulatory cells might be functional in vivo, we generated a corresponding rabbit antimurine thymocyte globulin (mATG) and tested its ability to induce regulatory cells in vitro and whether those cells can inhibit acute graft-versus-host disease (GVHD) in vivo upon adoptive transfer. In vitro, mATG induces a population of CD4(+)CD25(+) T cells that express several cell surface molecules representative of regulatory T cells. These cells do not express Foxp3 at either the protein or mRNA level, but do show suppressive function both in vitro and in vivo when adoptively transferred into a model of GVHD. These results demonstrate that in a murine system, antithymocyte globulin induces cells with suppressive activity that also function in vivo to protect against acute GVHD. Thus, in both murine and human systems, antithymocyte globulins not only deplete T cells, but also appear to generate regulatory cells. The in vitro generation of regulatory cells by anti-thymocyte globulins could provide ad-ditional therapeutic modalities for immune-mediated disease.

Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease.

Recombinant human glucocerebrosidase (imiglucerase, Cerezyme) is used in enzyme replacement therapy for Gaucher disease. Complex oligosaccharides present on Chinese hamster ovary cell-expressed glucocerebrosidase (GCase) are enzymatically remodeled into a mannose core, facilitating mannose receptor-mediated uptake into macrophages. Alternative expression systems could be used to produce GCase containing larger oligomannose structures, offering the possibility of an improvement in targeting to macrophages. A secondary advantage of these expression systems would be to eliminate the need for carbohydrate remodeling. Here, multiple expression systems were used to produce GCase containing primarily terminal oligomannose, from Man2 to Man9. GCase from these multiple expression systems was compared to Cerezyme with respect to affinity for mannose receptor and serum mannose-binding lectin (MBL), macrophage uptake, and intracellular half-life. In vivo studies comparing clearance and targeting of Cerezyme and the Man9 form of GCase were carried out in a Gaucher mouse model (D409V/null). Mannose receptor binding, macrophage uptake, and in vivo targeting were similar for all forms of GCase. Increased MBL binding was observed for all forms of GCase having larger mannose structures than those of Cerezyme, which could influence pharmacokinetic behavior. These studies demonstrate that although alternative cell expression systems are effective for producing oligomannose-terminated glucocerebrosidase, there is no biochemical or pharmacological advantage in producing GCase with an increased number of mannose residues. The display of alternative carbohydrate structures on GCase expressed in these systems also runs the risk of undesirable consequences, such as an increase in MBL binding or a possible increase in immunogenicity due to the presentation of non-mammalian glycans.

Measurement of lysosomal glucocerebrosidase activity in mouse liver using a fluorescence-activated cell sorter assay.

Lysosomal acid beta-glucocerebrosidase hydrolyzes glucocerebroside to glucose ceramide. Patients diagnosed with Gaucher disease, however, lack this enzyme, leading to the accumulation of glucocerebroside in tissue macrophages within multiple organs. Such patients can receive enzyme replacement therapy during which a human placental-derived or recombinant form of acid beta-glucocerebrosidase is targeted to the macrophages. As part of evaluating the effectiveness of such therapies, currently available methodologies for measuring acid beta-glucocerebrosidase activity are primarily conducted in cultured cell lines or tissue culture. However, these in vitro assays are limited by their ability to evaluate the efficacy of in vivo acid beta-glucocerebrosidase replacement therapy in animal models. In particular, there is an unmet need to simultaneously define cellular localization and evaluate enzyme activity following treatment in vivo. In addition, results of commonly used fluorescent-based assays for enzyme activity are difficult to compare day to day and/or across laboratories due to the variability inherent in flow cytometric measurement. In this article, we describe a reproducible and consistent quantitative method for the combined measurement of fluorescein intensity from enzyme-substrate conversion and cell localization by phenotype-specific phycoerythrin-antibody staining. Following infusion of recombinant human acid beta-glucocerebrosidase in mice, nonparenchymal cells are prepared from the livers of treated and control animals. Acid beta-glucocerebrosidase activity is measured in molecules of equivalent soluble fluorophore units within Kupffer cell populations as defined by phenotype-specific monoclonal antibodies. This assay should be applicable to investigations of other Gaucher disease treatments in both human and animal models.