Purpose: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to identify melanoma-associated cell surface molecules and to develop as well as preclinically test immunotherapeutic reagents designed to exploit such targets. Experimental Design and Results: By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma. The CR011monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011alone did not inhibit the growth of melanoma cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg. Conclusion: These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma.

No abstract

These two controlled studies involving a total of 3150 Thai children provide evidence that astroviruses are a common cause of viral gastroenteritis. Astroviruses were found in association with gastroenteritis more frequently than were enteric adenoviruses, and with nearly half the frequency of rotaviruses.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.

Consumption of raw shellfish has long been known to be associated with individual cases and sporadic outbreaks of enteric illness. However, during 1982, outbreaks of gastroenteritis associated with eating raw shellfish reached epidemic proportions in New York State. Between May 1 and December 31, there were 103 well-documented outbreaks in which 1017 persons became ill: 813 cases were related to eating clams, and 204 to eating oysters. The most common symptoms were diarrhea, nausea, abdominal cramps, and vomiting. Incubation periods were generally 24 to 48 hours long, and the duration of illness was 24 to 48 hours. Bacteriologic analyses of stool and shellfish specimens did not reveal a causative agent. Norwalk virus was implicated as the predominant etiologic agent by clinical features of the illness and by seroconversion and the formation of IgM antibody to Norwalk virus in paired serum samples from persons in five (71 percent) of seven outbreaks in which testing was done. In addition, Norwalk virus was identified by radioimmunoassay in clam and oyster specimens from two of the outbreaks. Determining the source of the shellfish was not always possible, but northeastern coastal waters were implicated. The magnitude, persistence, and widespread nature of these outbreaks raise further questions about the safety of consuming raw shellfish.

No abstract

An enzyme-linked immunosorbent assay (ELISA), based on monoclonal antibodies to the astrovirus group antigen, was designed for the detection of astroviruses in stools of patients with gastroenteritis. Compared to immune electron microscopy used as the standard test, the sensitivity of the astrovirus ELISA was 91% (31/34) and the specificity was 96% (54/56). All five of the known astrovirus serotypes could be detected in 16 samples on which serotyping was done. In tests on 155 stools containing other enteric viruses, including adenoviruses, rotaviruses, caliciviruses, Hawaii virus, Snow Mountain virus, and Norwalk virus (30, 20, 70, 24, 4, and 7 samples, respectively), only 3 were positive in the astrovirus ELISA. The combined specificity for all astrovirus immune electron microscopy-negative samples was 98% (206/211). The results demonstrate that the new ELISA provides a sensitive and specific means for the diagnosis of astrovirus gastroenteritis.

Cultivation of human astroviruses in human embryonic kidney or LLCMK2 cell cultures was corroborated for four of the five serotypes originally reported (types 1, 2, 4, and 5). By using type-specific rabbit antisera and immunofluorescence of virus-infected cells, we readily distinguished between serotypes of astrovirus; however, these serotypes showed a high degree of cross-reactivity by enzyme-linked immunoassay, a result indicating the presence of a group antigen. We prepared monoclonal antibodies to astrovirus type 2 antigen and selected them on the basis of group antigen reactivity. The antibodies were reactive with the four astrovirus serotypes that we could cultivate, as well as with the Marin County strain of astrovirus. A previously reported cell-cultivated astrovirus type 3 also reacted with the monoclonal antibodies. These monoclonal antibodies, and the finding of group reactivity among the human astroviruses, should facilitate studies on the importance of these viruses as agents of viral gastroenteritis.