Cancer diagnosis and treatment of cancer using anti-robo 1 antibody

#The present invention relates to methods for the diagnosis and treatment of cancer and a method for monitoring progression of hepatitis, as well as to a cell growth inhibitor and an anticancer agent.

#In genetic screening studies of drosophila, ROBO1 has been identified as a molecule regulating the midline crossing of axons, and has been reported to work as a receptor for the Slit protein in subsequent studies (Kidd et al., Cell, 92, 205-215, 1998, Wang et al., Cell, 96, 771-784, 1999, Kidd et al., Cell, 96, 785-794, 1999, Brose et al., 96, 795-806, 1999). In addition, regarding the relationship between ROBO1 and cancer, the chromosome region 3p12 where the human ROBO1 gene is present is highly defective in lung cancer, and expression is suppressed by methylation of the ROBO1 promoter region in breast cancer and kidney cancer, suggesting that ROBO1 gene can be a tumor suppressor gene (Dallol et al., Oncogene, 21, 3020-3028, 2000). Indeed, hyperplasia of tracheal epithelial cells was observed in mice by deleting the first immunoglobulin region present at the N-terminus of ROBO1, which is similar to the minimal defect in the ROBO1 gene identified in lung cancer patients (Xian J et al., PNAS, 98, 15062-15066, 2001). Conversely, it has been reported that ROBO1 was expressed in new blood vessels of cancers, and that increased expression of Slit2, a ligand for ROBO1, on the cancer cells induces cancer neovascularization to direct cancer growth (Wang et al., Cancer Cell, 4, 19-29, 2003).

#Meanwhile, the expression of the Slit2 gene, which is a ligand for ROBO1, is also suppressed in a number of cancer types by methylation or the like, and overexpression of Slit2 or addition of Slit2 induces a growth inhibition and apoptosis in lung cancer, breast cancer and large intestine cancer cells. These observation suggests that Slit2, a ligand for ROBO1, is also thought to be a tumor suppressor gene (Dallol et al., Cancer Research, 62, 5874-5880, 2002, Dallol et al., Cancer Research, 63, 1054-1058, 2003). However, it is not clear in this report through which receptor Slit2 exerts its effect in inhibiting the cell growth, thus the relationship between ROBO1 and cancer has not been absolutely clear.

#FIG. 1 shows the results of ROBO1 gene expression analysis using GeneChip U133. FIG. 1a: ROBO1 gene expression analysis in normal tissues/non-cancer sites; FIG. 1b: ROBO1 gene expression analysis in clinical samples; FIG. 1c: ROBO1 gene expression analysis in cancer cell lines;

#FIG. 2 shows the results of ROBO1 gene expression analysis using GeneChip U95;

#FIG. 3 shows the results of Western analysis of transient expression of full length ROBO1 gene in COS7 cell and HEK293 cell lysates, and the results of Western analysis in the supernatant of the culture thereof;

#The method of the present invention is characterized by the detection of ROBO1 protein. ROBO1 (Roundabout 1) is an axon guidance receptor protein, and its amino acid sequence and the gene sequence coding therefor are disclosed in GenBank ID NM—002941 (SEQ ID NOs: 9 and 10) for variant 1, and GenBank ID NM—133631 (SEQ ID NOs: 11 and 12) for variant 2. In the present invention, ROBO1 protein is meant to include both the full length protein and fragments thereof. A fragment is a polypeptide containing any region of the ROBO1 protein, and may not have the function of the natural ROBO1 protein. Examples of fragments include, but are not limited to, a fragment containing an extracellular region of the ROBO1 protein. The extracellular region of the ROBO1 protein corresponds to positions 1-859 in the amino acid sequence of SEQ ID NO: 11. In addition, the membrane spanning region corresponds to positions 860-880 in the amino acid sequence of SEQ ID NO: 11 (Sundaresan, et al., Molecular and Cellular Neuroscience 11, 29-35, 1998).

#In the present invention, the expression of ROBO1 was found to be enhanced at both the genetic level and the protein level with extremely high frequencies in hepatocellular cancer. In addition, the analysis of clinical samples and cancer cells line of other cancer species suggested that the enhanced expression was shown not only in hepatocellular cancer, but also in lung cancer, breast cancer, uterine cancer, gastric cancer, brain tumor, large intestine cancer and the like. It was also shown that immunohistological diagnosis can be carried out using a monoclonal antibody specific to ROBO1. In addition, ROBO1 was found to be shedded in vivo, and a soluble ROBO1 (sROBO1) was present in the blood of cancer patients, indicating that sROBO1 is useful as a serodiagnosis marker of cancer.

#Detection of ROBO1