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.

ACKNOWLEDGEMENTSWe thank CHTN and NDRI tissues and associated pathology reports. We thank Muralidhara Padigaru for genomic mining, Ramakrishnan Sundaram for performing the nude mouse angiogenesis assays; Dinesh Raturi, Pino Luan for protein production and technical assistance. We thank Dr. Stephen Strittmatter, Yale University School of Medicine for his support in providing reagents and also for critical reading of the manuscript. Research PaperRecombinant Semaphorin 6A-1 Ectodomain Inhibits In Vivo Growth Factor and Tumor Cell Line-Induced Angiogenesis ABSTRACTThe Semaphorins are a large family of transmembrane, GPI-anchored and secreted proteins that play an important role in neuronal and endothelial cell guidance. A human gene related to the class 6 Semaphorin family, Semaphorin 6A-1 (Sema 6A-1) was identified by homology-based genomic mining. Recent implication of Sema 3 family members in tumor angiogenesis and our expression analysis of Sema 6A-1 suggested that class 6 Semaphorin might effect tumor neovascularization. The mRNA expression of Sema 6A-1 was elevated in several renal tumor tissue samples relative to adjacent nontumor tissue samples from the same patient. Sema 6A-1 transcript was also expressed in the majority of renal clear cell carcinoma (RCC) cell lines and to a lesser extent in endothelial cells. To test the role of Sema 6A-1 in tumor angiogenesis, we engineered, expressed and purified the Sema 6A-1 soluble extracellular domain (Sema-ECD). The purified Sema-ECD was screened in a variety of endothelial cell-based assays both in vitro and in vivo. In vitro, Sema-ECD blocked VEGF-mediated endothelial cell migration. These effects were explained in part by our observation in endothelial cells that Sema-ECD inhibited VEGF-mediated Src, FAK and ERK phosphorylation. In vivo, mouse Matrigel assays demonstrated that the intraperitoneal administration of recombinant Sema-ECD inhibited both bFGF/VEGF and tumor cell line-induced neovascularization. These findings reveal a novel therapeutic utility for Sema 6A-1 (Sema-ECD) as an inhibitor of growth factor as well as tumor-induced angiogenesis.

SummaryWe have discovered and analysed two novel, linear extrachromosomal double-stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L-dsRNA) had homology with viral RNA-dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activated c-June NH 2 terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C. parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.

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An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.

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Isolates of Cryptosporidium muris and C. serpentis were characterized from different hosts using nucleotide sequence analysis of the rDNA 18S and ITS1 regions, and the heat-shock (HSP-70) gene. Phylogenetic analysis confirmed preliminary evidence that C. muris is not a uniform species. Two distinct genotypes were identified within C. muris ; (1) C. muris genotype A ; comprising bovine and camel isolates of C. muris from different geographical locations, and (2) C. muris genotype B comprising C. muris isolates from mice, a hamster, a rock hyrax and a camel from the same enclosure. These 2 genotypes may represent separate species but further biological and molecular studies are required for confirmation.