Vascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein 1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1␤ and tumor necrosis factor-␣ treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis.

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.

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We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.

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.

The methylation of vertebrate DNA at the 5-position of approximately 3-10% of its cytosine residues occurs in a sequence-specific and tissue-specific manner and has been implicated in the control of transcription. How these differences are established and how they mediate the initiation or maintenance of transcription are unknown. DNA methylation might also have other roles, such as modulating DNA replication, transposition, DNA repair or chromosome configuration. These other roles suggested for DNA methylation would be consistent with the finding that tissue-specific differences in methylation of certain gene regions, highly repeated satellite DNA sequences and whole genomes often do not correlate with transcriptional activity. For DNA methylation to modulate the expression, maintenance or duplication of chromosomes, there should be effector macromolecules, presumably proteins, which specifically recognize 5-methylcytosine (m5C) residues in DNA. We describe here the first identification of a mammalian protein that binds preferentially to m5C-rich DNA sequences.

Phosphorylation of pp6wcsrc by p34c2 at three amino-proximal serine/threonine residues is temporally correlated with, but insufficient for, mitotic activation of c-Src kinase. The direct cause of activation during mitosis appears to be temporally correlated partial dephosphorylation of Tyr-527, a residue whose phosphorylation strongly suppresses pp60c-src activity. Site-directed mutagenesis of the serine/threonine phosphorylation sites blocks half the mitosisspecific decrease in Tyr-527 phosphorylation and half the increase in pp60-src kinase activity. We conclude that p34cdc2 partially activates pp6Wcsrc by a two-step process in which its serine/threonine phosphorylations either sensitize pp6Oe-sr' to a Tyr-527 phosphatase or desensitize it to a Tyr-527 kinase.Furthermore, additional events, independent of these p34-2 mediated phosphorylations, participate in mitotic activation of pp6Ocerc.p34cdc2, a critical cell cycle regulator (see refs. 1 and 2 for reviews), is activated at the onset of mitosis as a serine/ threonine protein kinase by an intricate sequence of dephosphorylation and phosphorylation events and by its association with cyclin proteins. Activated p34cdc2 is thought to initiate a cascade of protein kinases and, possibly, phosphatases that results in mitosis-specific cytostructural rearrangements such as cell rounding, nuclear envelope breakdown, and mitotic spindle formation. p34cdc2 phosphorylates chicken pp60c-src, the product of the chicken c-src protooncogene, in vitro at residues . These same sites are phosphorylated during mitosis in genetically modified NIH 3T3 mouse fibroblasts that overexpress chicken pp60c-src (3, 4). Thr-34, Ser-72, and their flanking p34cdc2 substrate-consensus sequences are conserved in frog, chicken, mouse, and human pp60c-src (5-8) and are phosphorylated during mitosis at least in mouse (unpublished results) and human (9) pp60c-src. The mitosisspecific phosphorylations (MSPs) ofpp60c-src in cultured cells retard its electrophoretic mobility and are associated with a 2-to 9-fold increase in specific kinase activity (4, 10,11). Both these mitosis-specific effects disappear as fibroblasts exit mitosis (4).These facts suggest that pp60c-src may participate in the transduction of p34cdc2-initiated signals and mitotic protein kinase cascades (see ref. 12 for review). However, phosphorylation of pp60c-src by p34cdc2 does not stimulate its specific kinase activity (3,9). This suggests that some other mitosisspecific event is involved in the activation. This event appears to be partial dephosphorylation of Tyr-527 (10, 11), a residue that is highly (>90%) phosphorylated in wild-type (wt) pp60c-src in unsynchronized cells (13). Complete inhibition of Tyr-527 phosphorylation causes a 10-to 30-fold stimulation of pp60c-src specific kinase activity (see ref. 14 for review), so the 2-to 3-fold mitotic increase in kinase activity observed in our system could be caused by a 10-20o decrease in Tyr-527 phosphorylation. The small magnitude of this predicted change has preclude...

Short-term anti-tumor effects and long-term effects (complete regression) were observed with CR011-vcMMAE, but not with the reference agents. These results suggest that CR011-vcMMAE may provide therapeutic benefit in malignant melanoma.