Objective This phase II, multicenter, single-arm, two-stage study in platinum-resistant, advanced epithelial ovarian or primary peritoneal cancer evaluated the efficacy, safety, and tolerability of weekly single-agent volociximab. Pharmacokinetic/pharmacodynamic (PK/PD) studies were also performed. Methods Sixteen patients were enrolled in Stage 1. Volociximab was administered at 15 mg/kg IV qwk until progression of disease or drug intolerability. Tumor response was assessed every 8 weeks. Serum samples for PK or whole blood for the evaluation of circulating tumor cells, endothelial cells, and endothelial progenitor cells were obtained on Days 1, 8, 15, 29, and 50. Ascites from one patient was collected for volociximab concentration analysis. Archived tumor tissue was analyzed by immunohistochemistry (IHC) for α5 integrin expression. Results Safety data are available on all 16 patients; 14 were evaluable for efficacy. One patient had stable disease at 8 weeks. The remaining 13 progressed on treatment. Twelve patients (75%) experienced study-related adverse events (AEs); the most common (≥20%) were headache and fatigue. Three patients experienced possible study-related serious AEs (SAEs): reversible posterior leukoencephalopathy syndrome, pulmonary embolism, and hyponatremia. Peak serum concentrations of volociximab increased 2–3 fold from Day 1 to Day 50. Clinically relevant trough levels were achieved (>150 µg/mL). IHC analysis of archived tumor sections showed low-to-moderate expression of α5 integrin on all ovarian cancer tissue evaluated. Conclusion Despite insufficient clinical activity in this refractory patient population to continue the study, weekly volociximab was well tolerated, and the gained understanding of the mechanism of action of volociximab will inform future development efforts.

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Tumor necrosis factor (TNF) receptor 6 (TR6), also called decoy receptor 3 (DcR3) or M68, is a member of the TNF receptor family that is produced as a secreted protein (3,25,35). Similar to other members of the family, TR6 binds with high affinity to multiple ligands, including Fas ligand (FasL), LIGHT, and TL1A (18,25,35). Since TR6 lacks a transmembrane domain, it can function as an inhibitor by competing with the signal-transducing receptor for the ligand. The recombinant protein was indeed able to inhibit in vitro and in vivo FasL-induced apoptosis (5, 25). Because of TR6 overexpression in a substantial number of tumors (23, 28), it has been postulated that the decoy receptor promotes the survival of tumor cells by helping them to escape FasL-dependent cell death (3, 25). In addition, several studies have suggested a role for TR6 in immunity, through the modulation of T-cell responses. TR6 was found to inhibit the interaction of TL1A, a T-cell costimulatory cytokine, with its receptor DR3 (18). Soluble protein was also shown to downmodulate the cytotoxic activity of T lymphocytes and ameliorate heart allograft rejection in mice, possibly by interfering with LIGHT/TR2 binding (36). In another study, solid-phase TR6 was reported to stimulate proliferation and cytokine production in T lymphocytes by reverse signaling through LIGHT (32). Lastly, TR6 was reported to modulate dendritic cell maturation (10).Little is known about the regulation of TR6 expression in nonmalignant cells. TR6 mRNA is expressed in endothelial cells and keratinocytes (16,35), and the protein was detected in epithelial cells of the colon (3). The mRNA was also detected at low levels in other healthy human tissues, such as stomach, lymph node, lung, and spleen tissues (3). The involvement of TR6 in immune responses raised the possibility that the expression of the protein may be regulated in cells of the immune system. Therefore, the aim of our study was to investigate the expression and release of TR6 by immune cells. MATERIALS AND METHODSReagents. Cytokines were purchased from PeproTech (Rocky Hill, N.J.); Staphylococcus aureus Cowan I, PD98059, SB203580, herbimycin A, and wortmannin were purchased from Calbiochem (San Diego, Calif.); phytohemagglutinin (PHA), lipopolysaccharide (LPS), and lipoteichoic acids (LTA) were purchased from Sigma-Aldrich (St. Louis, Mo.); and CD40 ligand (CD40L) and a TNF-␣ enzyme-linked immunosorbent assay (ELISA) kit were purchased from R&D Systems (Minneapolis, Minn.).Cell cultures. Monocytes were obtained from human peripheral blood mononuclear cells (PBMC) by centrifugation of leukopheresis preparations (BRT Inc., Baltimore, Md.) through Histopaque gradients (Sigma-Aldrich) followed by counterflow centrifugal elutriation. Cell purity was greater than 92%. Cells were cultured in complete medium consisting of RPMI 1640 medium (GIBCO BRL, Rockville, Md.) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 50 g of gentamicin (Biofluids Inc., Rockville, Md.)/ml. Myeloid dendritic cel...

Vascular endothelial cells (EC) participate in the process of bone formation through the production of factors regulating osteoclast differentiation and function. In this study, we report the selective expression in primary human microvascular EC of Osteostat/TNF superfamily 18, a ligand of the TNF superfamily. Osteostat protein is detectable in human microvascular EC and is highly up-regulated by IFN-alpha and IFN-beta. Moreover, an anti-Osteostat antibody strongly binds to the vascular endothelium in human tissues, demonstrating that the protein is present in the EC layers surrounding blood vessels. Functional in vitro assays were used to define Osteostat involvement in osteoclastogenesis. Both recombinant and membrane-bound Osteostat inhibit differentiation of osteoclasts from monocytic precursor cells. Osteostat suppresses the early stage of osteoclastogenesis via inhibition of macrophage colony-stimulating factor-induced receptor activator of NF-kappaB (RANK) expression in the osteoclast precursor cells. This effect appears to be specific for the differentiation pathway of the osteoclast lineage, because Osteostat does not inhibit lipopolysaccharide-induced RANK expression in monocytes and dendritic cells, or activation-induced RANK expression in T cells. These findings demonstrate that Osteostat is a novel regulator of osteoclast generation and substantiate the major role played by the endothelium in bone physiology.

The advent of time‐of‐flight mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system to simultaneous interrogate a multitude of parameters and gain a better understanding of immunologic data from clinical trial samples. Here we describe the development and validation of a 33‐marker mass cytometry workflow for measuring gastrointestinal (GI) trafficking peripheral blood mononuclear cells (PBMCs) in patients with celiac disease (CeD). This panel builds upon identification of well‐characterized immune cells and expands to include markers modulated in response to gluten challenge in patients with CeD. The CeD panel was optimized and validated according to accepted industry practice for validation of flow cytometry assays and builds upon established sample processing workflows for mass cytometry studies. Several critical parameters were evaluated during the assay development phase of this study including optimization of the sample processing steps, antibody specificity, and ensuring the panel as a whole performed to expectation. The panel was then validated using a fit‐for‐purpose approach tailored to the intended use of the data in the clinical trial. Validation included assessment of analytical parameters essential to understanding the reliability and robustness of the CeD panel such as intra‐assay precision, inter‐assay precision, inter‐operator precision and sample processing stability. Together, this validated mass cytometry workstream provides robust and reproducible high‐dimensional analysis of human peripheral blood immune cells to characterize patient samples from clinical trials.