# Patent

## Title
Cancer diagnosis and treatment of cancer using anti-ROBO1 antibody

## Abstract

A method for diagnosing cancer comprising detecting ROBO1 protein is disclosed. In addition, a method for treating a disease caused by abnormal cell growth comprising administrating an antibody that binds to ROBO1, as well as a pharmaceutical composition, a cell growth inhibitor and an anticancer agent comprising an antibody that binds to ROBO1 as an active ingredient are disclosed. Further, a method for inducing cell damages in a ROBO1 expressing cell and a method for inhibiting the growth of a ROBO1 expressing cell by bringing a ROBO1 expressing cell into contact with an antibody that binds to ROBO1, are disclosed. Furthermore, a method for monitoring progression of hepatitis by detecting ROBO1 protein is disclosed.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/547,534 filed Apr. 13, 2007, which is a National Phase of International Application No. PCT/JP2005/006838 filed Mar. 31, 2005, which claims benefit designated the United States and was filed on Apr. 6, 2007, which claims the priority benefit of Japanese Patent Application No. 2004-102862 filed Mar. 31, 2004, Japanese Patent Application No. 2004-227899 filed Aug. 8, 2004, and Japanese Patent Application No. 2005-004024 filed Jan. 11, 2005. The disclosure of each of Japanese Patent Application No. 2004-102862, Japanese Patent Application No. 2004-227899, and Japanese Patent Application No. 2005-004024, is hereby incorporated by reference in its entirety.


REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The content of the electronically submitted sequence listing in ASCII text format filed with prior Application No. 11/547,534 on Jul. 24, 2009, is incorporated herein by reference in its entirety.
BACKGROUND
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).
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.
Cancer is the most common cause of death in Japan. Among them, primary hepatocellular cancer is one type of cancer with poor prognosis, representing the third leading cause of death (13%) in male and the fourth (9.0%) in female in 2001 (excerpt from “Population Dynamics Statistics”, Statistics and Information Department, Minster's Secretariat, Ministry of Health, Labour and Welfare). The number of chronic patients caused by viral infection is on the rise year after year and many of them lead to hepatic cirrhosis and then to hepatocellular cancers. Extremely strong demands exist for a diagnostic procedure at an early stage in the progression from hepatic cirrhosis to hepatocellular cancer and for a treatment of hepatocellular cancer. It is believed that without a groundbreaking solution, the number of deaths will follow an increasing trend in the 10 to 15 years to come.
Current hepatocellular cancer diagnostic procedure comprises a comprehensive evaluation based on biochemical data such as the serum value of GOP/GTP, alkaline phosphatase, albumin and the like, or a tumor marker AFP (a-fetoprotein), and diagnostic imaging. Then, if necessary, a small amount of tissue fragment is taken by needle biopsy for pathological judgment to confirm the diagnosis. Currently, tumor markers are mainly used for the diagnosis of hepatocellular cancer. The positive rate of alpha fetoprotein (AFP), which is the most common marker, is 60 to 70 percent in hepatocellular cancer patients, although it is sometimes also positive in chronic liver disease patients or pregnant female. Another hepatic cancer tumor marker PIVKA-II is positive in less than 50 percent of the patient, and the specificity for hepatocellular cancer is thought to be higher than AFP. Mainly these two examinations are currently in practice. In either case, false positive or double negative cases exist, thus a tumor marker with high specificity is needed.
Histological examination of the sample collected by needle biopsy is an important test for confirmed diagnosis of liver diseases. In particular, as the quantity of specimen may be limited, a more definite diagnosis technique is required. It is desired in the art to develop an antibody against an antigen specifically expressed in hepatic cancer to allow for not only pathological characteristics but also identification of hepatocellular cancer from a non-cancer tissue at an early stage.
In the current situation of diagnosis and monitoring of liver disease, progression from inflammation to fibrosis and malignant transformation are diagnosed by examination with multiple markers and examination by biopsy. In many hepatic cancer patients, the progression occurs from viral infection to hepatitis, chronic hepatitis, hepatic cirrhosis and then hepatic cancer. Consequently, a simple method for diagnosis and monitoring of liver diseases will be useful, not only in terms of healthcare economy, but also in mitigating the burden on the patients and in obtaining accurate medical guidelines.
Regarding treatment of hepatocellular cancer, many medical facilities are centered mainly on three types of therapy: surgical removal, transcatheter arterial embolization therapy, and percutaneous ethanol injection therapy. Either method has advantages and disadvantages, and even when transcatheter arterial embolization therapy is selected, which has a relatively broad application range and survival advantages, the rate of complete cure is currently thought to be on the order of 10%. Thus there is a great demand for a novel therapy.
Targeted therapy by monoclonal antibody against cancer specific tumor antigen provides a better outcome in breast cancer and in lymphoma and the like through an action mechanism different from conventional chemotherapy, although no clinical application has done for hepatocellular cancers yet. The action mechanisms of these antibody drugs include antibody dependent cytotoxicity (ADCC) via effector cells and complement-dependent cytotoxicity (CDC) via the complement, agonistic action by the function of the antibody itself, and the neutralization capability of the antibody. Recently molecular therapies have been applied in clinical sites. An antibody drug therapy which applies these molecular therapies and targets to a neoplasm-specifically expressed molecule found on hepatic cancer cells is expected to be developed in the future.
The following are documents related to the present invention: WO99/20764; WO98/48051; WO01/46697; WO03/29488; WO01/00828; WO01/57207; WO01/92581; WO02/04514; WO02/14500; WO02/29103.
SUMMARY OF INVENTION
An object of the present invention is to provide a novel method for diagnosing and treating cancer, as well as a novel cell growth inhibitor and anticancer agent, and to provide a method for diagnosing and monitoring liver disease.
The present inventors discovered that ROBO1 was highly expressed in cancer cells, such as hepatocellular cancer, lung cancer, breast cancer, uterine cancer, gastric cancer, brain tumor, large intestine cancer and the like. In addition, they measured the complement-dependent cytotoxicity (CDC) of the anti-ROBO1 antibody, and found that the anti-ROBO1 antibody had a CDC activity against ROBO1 expressing cells. They also found that the concentration of ROBO1 in blood increased with the progression of liver diseases. From the above observations, the present inventors discovered the effectiveness of the anti-ROBO1 antibody in the diagnosis, prevention and treatment of cancers overexpressing ROBO1, such as hepatocellular cancer, and achieved a method for diagnosing and monitoring liver diseases.
The present invention provides a method for diagnosing cancer comprising detecting ROBO1 protein. In the method of the present invention, preferably the extracellular region of the ROBO1 protein is detected. The method of the present invention is carried out preferably using an antibody that recognizes ROBO1 protein. Preferably, in the method of the present invention, ROBO1 protein in the blood, serum or plasma, or ROBO1 protein isolated from a cell is detected.
In another aspect, the present invention provides a method for diagnosing cancer comprising the steps of:
(a) collecting a sample from a subject; and
(b) detecting ROBO1 protein contained in the collected sample.
In another aspect, the present invention provides a kit for diagnosing cancer, comprising an antibody that binds to ROBO1 protein. Preferably, the cancer is hepatocellular cancer. Also, in the kit of the present invention, the antibody preferably binds to the extracellular region of the ROBO1 protein.
In another aspect, the present invention provides a pharmaceutical composition comprising an antibody that binds to ROBO1 as an active ingredient. The present invention also provides a cell growth inhibitor comprising an antibody that binds to ROBO1 as an active ingredient. The present invention also provides an anticancer agent comprising an antibody that binds to ROBO1 as an active ingredient. Preferably, the antibody that binds to ROBO1 has cytotoxicity. The cancer is preferably a hepatocellular cancer.
In another aspect, the present invention provides a method for treating a disease caused by abnormal cell growth, comprising administrating to a patient in need of such treatment a pharmaceutical composition comprising an antibody that binds to ROBO1 as an active ingredient. The present invention also provides a method for treating cancer, comprising administrating to a patient in need of such treatment a pharmaceutical composition comprising an antibody that binds to ROBO1 as an active ingredient. Preferably, the cancer is hepatocellular cancer.
In another aspect, the present invention provides a method for inducing cell damages in a ROBO1 expressing cell by bringing a ROBO1 expressing cell into contact with an antibody that binds to ROBO1. The present invention also provides a method for inhibiting the growth of a ROBO1 expressing cell by bringing a ROBO1 expressing cell into contact with an antibody that binds to ROBO1. Preferably, the antibody that binds to ROBO1 has cytotoxicity. Preferably, the ROBO1 expressing cell is a cancer cell.
In still another aspect, the present invention provides an antibody that binds to ROBO1 and has cytotoxicity against a ROBO1 expressing cell.
In another aspect, the present invention provides a kit for monitoring the progression of hepatitis comprising an anti-ROBO1 antibody. Preferably, the anti-ROBO1 antibody specifically recognizes ROBO1. Preferably, the kit of the present invention predicts the progression from hepatitis or hepatic cirrhosis to hepatic cancer. In a preferred embodiment, the kit of the present invention contains a first anti-ROBO1 antibody immobilized on a support and a second anti-ROBO1 antibody labeled with a labeling substance.
In still another aspect, the present invention provides a method for monitoring the progression of hepatitis by measuring ROBO1 in a test sample. Preferably, the ROBO1 in the test sample is measured using an anti-ROBO1 antibody. Preferably, the anti-ROBO1 antibody specifically recognizes ROBO1. Preferably, the test sample is blood, serum or plasma. Preferably, the monitoring method of the present invention predicts the progression from hepatitis or hepatic cirrhosis to hepatic cancer. In a preferred embodiment, the monitoring method of the present invention is carried out using a first anti-ROBO1 antibody immobilized on a support and a second anti-ROBO1 antibody labeled with a labeling substance.



DESCRIPTION OF DRAWINGS
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 COST cell and HEK293 cell lysates, and the results of Western analysis in the supernatant of the culture thereof;
FIG. 4 shows the results of Western analysis of the hepatic cancer cell line lysate using anti-ROBO1 monoclonal antibody A7241A;
FIG. 5 shows the results of immunohistological staining analysis of hepatocellular cancer paraffin preparations using anti-ROBO1 monoclonal antibody A7241A;
FIG. 6 shows the results of Western analysis of soluble ROBO1 in patient serum using anti-ROBO1 monoclonal antibody A7241A;
FIG. 7 shows the results of Western analysis of lysate of HEK293 cell forced to express the full length ROBO1 gene using sROBO1-immunized rabbit serum;
FIG. 8 shows the results of FACS analysis of lysate of HEK293 cell forced to express the full length ROBO1 gene using sROBO1-immunized rabbit serum;
FIG. 9 shows the results of FACS analysis of lysate of HepG2 cell using sROBO1-immunized rabbit serum;
FIG. 10 shows a standard curve for ROBO1 measurement by enzyme immunoassay using rabbit anti-sROBO1 antibody;
FIG. 11 shows the correlation between ROBO1 concentration and the severity of liver diseases;
FIG. 12 shows the variation in ROBO1 concentration of liver disease patients;
FIG. 13 shows the results of measurement of CDC activity of anti-ROBO1 antiserum against ROBO1-expressing HEK293 cell;
FIG. 14 shows the results of measurement of CDC activity of anti-ROBO1 monoclonal antibody against ROBO1-expressing HEK293 cell;
FIG. 15 shows the results of measurement of CDC activity of anti-ROBO1 monoclonal antibody against ROBO1-expressing Alexander cell; and
FIG. 16 shows the results of measurement of ADCC activity of anti-ROBO1 monoclonal antibody against ROBO1-expressing HEK293 cell.



DETAILED DESCRIPTION OF INVENTION
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
ROBO1 protein detected in the present invention is preferably human ROBO1 protein, but any ROBO1 may be used in the invention, including, but is not limited to, canine ROBO1, feline ROBO1, mouse ROBO1 and hamster ROBO1.
In the present invention, detection may be quantitative or non-quantitative. Examples of non-quantitative detection include measurement as to merely whether ROBO1 protein is present, measurement as to whether a given quantity or more ROBO1 protein is present, measurement comparing the amount of ROBO1 protein with other sample (for instance, control sample). Examples of quantitative detection include measurement of ROBO1 protein concentration, measurement of ROBO1 protein quantity, and the like.
Test samples are not particularly limited as long as they may contain ROBO1 protein, and are preferably those collected from the bodies of living organisms such as mammals, more preferably those collected from humans. Specific examples of test samples include, for instance, blood, interstitial tissue fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph, saliva, urine and the like, preferably blood, serum, or plasma. Preferably, the test samples used in the present invention also include those derived from the original test samples, such as the culture solution of cells collected from the body of a living organism.
The cancer to be diagnosed is not particularly limited and may be any cancer, including hepatic cancer, pancreatic cancer, lung cancer, large intestine cancer, breast cancer, kidney cancer, brain tumor, uterine cancer, lung cancer, gastric cancer, prostate gland cancer, leukemia, lymphoma and the like. Hepatic cancer is preferred, and hepatocellular cancer is more preferred.
In the present invention, when ROBO1 protein is detected in a test sample, and the amount of ROBO1 protein detected is determined to be higher than a negative control or a healthy subject, the subject is determined as having cancer or as having high potentiality to develop cancer.
In addition, progression of a liver disease can be monitored by measuring the concentration of ROBO1 protein in a patient having the liver disease.
A preferred embodiment of the diagnosis method of the present invention is detection of ROBO1 protein released from cells and present in blood. Particularly preferably, a fragment containing the extracellular region of the ROBO1 protein is detected.
Method for detecting ROBO1 protein contained in a test sample is not limited, but preferably include, detection by an immunological method using an anti-ROBO1 antibody. Examples of immunological methods include, for instance, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, immunoprecipitation method, immunonephelometry, Western blot, immunostaining, immunodiffusion method and the like, preferably enzyme immunoassay, and particularly preferably enzyme-linked immunosorbent assay (ELISA) (for instance, sandwich ELISA). The immunological methods such as ELISA can be carried out by those skilled in the art according to well known methods.
For instance, general detection of ROBO1 protein in a test sample using an anti-ROBO1 antibody may be carried out by immobilizing an anti-ROBO1 antibody on a support, adding a test sample, incubating the sample to bind ROBO1 protein to the anti-ROBO1 antibody, washing, and detecting the ROBO1 protein bound to the support via the anti-ROBO1 antibody.
Examples of supports used for immobilizing anti-ROBO1 antibody in the present invention may include, for instance, insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicon resin, polystyrene resin, polyacrylamide resin, nylon resin and polycarbonate resin, and insoluble supports such as glass. The support may be used in the form of beads or plates. In case of beads, a column may be filled with the beads. In case of plates, multi-well plate (96-well multi-well plate or the like), biosensor chip and the like can be used. For binding the anti-ROBO1 antibody to the support, conventional binding methods may be used, such as chemical bond or physical adsorption. Commercially available supports may be used for these purposes.
Binding of anti-ROBO1 antibody to ROBO1 protein is conventionally carried out in a buffer solution, such as phosphate buffer solution, Tris buffer solution, citric acid buffer solution, borate buffer solution, carbonate buffer solution, and the like. In addition, regarding incubation conditions, for instance, incubation for 1 hour to 24 hours at 4° C. to room temperature is carried out under conventionally used conditions. The procedure may optionally contain a washing step using a buffer solution containing a surfactant such as Tween 20 or the like, as long as it does not prevent binding of the anti-ROBO1 antibody to ROBO1 protein.
In the ROBO1 protein detection method of the present invention, a control sample may be prepared in addition to the test sample to be tested for ROBO1 protein. Examples of control samples include a negative control sample that does not contain ROBO1 protein, and a positive control sample that contains ROBO1 protein. In this case, the ROBO1 protein can be detected in the test sample by comparison with results obtained from the negative control sample without ROBO1 protein, and results obtained from the positive control sample with ROBO1 protein. In addition, a series of control samples with increment in concentration is prepared and a standard curve is established from the detection result for each control sample. ROBO1 protein contained in a test sample may be quantitatively determined from the numerical value for the test sample based on the standard curve.
In a preferred embodiment, the ROBO1 protein bound to the support via the anti-ROBO1 antibody is detected using an anti-ROBO1 antibody labeled with a labeling substance. For instance, a test sample is brought into contact with an anti-ROBO1 antibody immobilized on a support, and after washing, ROBO1 is detected using a labeled antibody that specifically recognizes the ROBO1 protein.
The labeling of an anti-ROBO1 antibody can be carried out by generally known methods. Labeling substances well known to those skilled in the art may be used, for example, fluorescent dyes, enzymes, coenzymes, chemiluminescent substances. Examples of the labeling substance include radioisotopes (32P, 14C, 125I, 3H, 131I and the like), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase, microperoxidase, biotin and the like. Preferably, when using biotin as a labeling substance, a biotinylated antibody is added and then avidin conjugated with an enzyme such as alkaline phosphatase is added. Well known methods can be used for preparing a conjugation of the labeling substance and the anti-ROBO1 antibody, such as, the glutaraldehyde method, the maleimide method, the pyridyl disulphide method and the periodic acid method.
In a specific example, a solution containing anti-ROBO1 antibody is added to a support, such as a plate having the anti-ROBO1 antibody immobilized onto the support. After washing the plate, it is blocked with, for instance, BSA, gelatine, albumin or the like, to prevent non-specific binding of proteins. The plate is washed again, and a test sample is added to the plate. After incubation, the plate is washed, and a labeled anti-ROBO1 antibody is added. After an adequate incubation, the plate is washed and the labeled anti-ROBO1 antibody remaining on the plate is detected. The detection can be carried out by methods well known to those skilled in the art. For instance, in the case of labeling by a radioactive substance, it may be detected by liquid scintillation or the RIA method. In the case of labeling by an enzyme, a substrate is added and the enzymatic modification of the substrate, for instance color development, can be detected using a photometer. Examples of substrates include 2,2-azinobis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS), 1,2-phenylene diamine(ortho-phenylene diamine), 3,3′,5,5′-tetramethylbenzin (TMB) and the like. In the case of a fluorescent substance, it may be detected with a spectrofluorimeter.
In a particularly preferred embodiment of the present invention, ROBO1 protein is detected using a biotin-labeled anti-ROBO1 antibody and avidin.
In a specific example, a solution containing anti-ROBO1 antibody is added to a support to immobilize the anti-ROBO1 antibody to the support, such as a plate. After washing the plate, it is blocked with, for instance, BSA or the like, to prevent non-specific binding of proteins. The plate is washed again, and a test sample is added to the plate. After incubation, the plate is washed, and a biotinylated anti-ROBO1 antibody is added. After an adequate incubation, the plate is washed, and avidin conjugated with an enzyme such as alkaline phosphatase or peroxidase is added. After incubation, the plate is washed, a substrate corresponding to the enzyme conjugated to avidin is added, and the ROBO1 protein is detected with the enzymatic modification of the substrate as the indicator.
In another preferred embodiment of the present invention, ROBO1 protein may be detected using one or more species of a primary antibody that specifically recognizes the ROBO1 protein, and one or more species of a secondary antibody that specifically recognizes the primary antibody.
For instance, a test sample is brought into contact with one or more species of an anti-ROBO1 antibody immobilized on a support, incubated and washed. Then the ROBO1 protein bound is detected with a primary anti-ROBO1 antibody and one or more species of a secondary antibody that specifically recognizes the primary antibody. In this case, the secondary antibody is preferably labeled with a labeling substance.
In another embodiment of the present invention, ROBO1 protein is detected using agglutination reaction. In this method, ROBO1 can be detected using a carrier sensitized with an anti-ROBO1 antibody. Any carriers may be used as carriers to be sensitized with the antibody, as long as they are insoluble, do not provoke non-specific reactions, and are stable. For instance, carriers include latex particles, bentonite, collodion, kaolin, immobilized sheep red blood cell and the like, preferably latex particles. For instance, polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyl toluene latex particles may be used as latex particles. Polystyrene latex particles is preferred. The sensitized particles are mixed with a sample and stirred for a predetermined length of time. Since the higher the concentration of anti-ROBO1 antibody contained in the sample, the larger the degree of agglutination of the particles become, ROBO1 can be detected by direct observation of the agglutination. Also the turbidity due to agglutination may be measured with a spectrophotometer.
In another embodiment of the present invention, ROBO1 protein may be detected using a biosensor that employs, for instance, the surface plasmon resonance phenomenon. A biosensor that utilizes surface plasmon resonance phenomenon is able to detect protein-protein interaction in real time as a surface plasmon resonance signal, with a small amount of protein without labeling. For instance, binding of ROBO1 protein to anti-ROBO1 antibody can be detected using a biosensor such as the BIAcore (manufactured by Amersham Biosciences). In a specific example, a test sample is brought into contact with a sensor chip where an anti-ROBO1 antibody has been immobilized, and ROBO1 protein binding to anti-ROBO1 antibody can be detected as a variation in the resonance signal.
The detection method of the present invention may be automated using a variety of automatic examination apparatus, allowing examination for a number of samples to be carried out at once.
It is also an object of the present invention to provide a diagnosis drug or kit for detecting ROBO1 protein in a test sample for the diagnosis of cancer. Such a diagnosis drug or kit contains at least an anti-ROBO1 antibody. If the diagnosis drug or kit is based on an EIA method, such as the ELISA method, a carrier for immobilizing the antibody may be included, and the antibody may be pre-bound to the carrier. If the diagnosis drug or kit is based on an agglutination method using a carrier such as latex, a carrier with adsorbed antibody may be included. In addition, the kit may suitably contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for processing a sample, and the like.
Preparation of Anti-ROBO1 Antibody
The anti-ROBO1 antibody used in the present invention specifically binds to ROBO1 protein, regardless of the origin, type (monoclonal, polyclonal) and shape thereof. Well known antibodies such as mouse antibodies, rat antibodies, human antibodies, chimeric antibodies, and humanized antibodies may be used in the invention. The antibody may be a polyclonal antibody, but a monoclonal antibody is preferred.
The anti-ROBO1 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using well known means. In particular, monoclonal antibodies that are derived from a mammal are preferred as the anti-ROBO1 antibody used in the present invention. Monoclonal antibodies derived from a mammal include those produced by hybridoma, and those produced by a host that has been transformed with an expression vector containing the antibody gene by a genetic engineering method.
A monoclonal antibody-producing hybridoma can be prepared basically using well known techniques, in the following way. An animal is immunized with ROBO1 as a sensitizing antigen according to a conventional immunization method. Immunocytes from the animal is fused with a well known parental cell by a conventional cell fusion method, and screening for an antibody-producing monoclonal cell by a conventional screening method.
Specifically, a monoclonal antibody may be prepared in the following way. First, ROBO1 is expressed and used as a sensitizing antigen to generate antibodies. The gene/amino acid sequence of ROBO1 is disclosed in the GenBank Accession Number BF059159 (NM_133631). The gene coding for ROBO1 is inserted into a well known expression vector system and transformed a suitable host cell. The human ROBO1 protein of interest is purified from the host cell or the culture supernatant by a well known method. Alternatively, natural ROBO1 may also be purified and used.
Next, the purified ROBO1 protein is used as a sensitizing antigen. Alternatively, a partial peptide from ROBO1 can also be used as a sensitizing antigen. In this case, the partial peptide may be obtained by chemical synthesis based on the amino acid sequence of the human ROBO1, or by expression of a portion of the ROBO1 gene inserted into an expression vector, or by degradation of the natural ROBO1 with a protease. The region and size of ROBO1 to be used as the partial peptide is not limited.
The type of mammals to be immunized with the sensitizing antigen are not particularly limited but is preferably selected based on the compatibility with the parental cell used for cell fusion. In general, rodents, for instance, mouse, rat and hamster, or rabbit, monkey and the like are used.
An animal is immunized with the sensitizing antigen according to a well known method. In general, a mammal is immunize by injecting the sensitizing antigen intraperitoneally or subcutaneously into the mammal. Specifically, a sensitizing antigen is suitably diluted and suspended in PBS (Phosphate-Buffered Saline), physiological saline or the like, and mixed with a suitable amount of conventional adjuvant, for instance Freund complete adjuvant as desired. A mammal is administered with the emulsion several times every 4 to 21 days. A suitable carrier may also be used with the sensitizing antigen during immunization. If a partial peptide with a particularly small molecular is used as the sensitizing antigen, it is desirable to conjugate the peptide with a carrier protein such as albumin and keyhole limpet hemocyanin before immunization.
After a mammal is immunized as described above and the increase in the desired antibody level in the serum is observed, the immunocytes are taken out from the mammal and are subjected to cell fusion. Preferred immunocytes include, in particular, the spleen cells.
A mammalian myeloma cell may also be used as a parent cell for cell fusion with the immunocyte. Preferably, known variety cell lines are used as the myeloma cell such as P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F. et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978) 148, 313-323), and R210 (Galfre, G. et al., Nature (1979) 277, 131-133).
The cell fusion of the immunocyte and the myeloma cell may be effected principally according to a known method such as a method of Kohler and Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).
More specifically, the cell fusion is carried out in a conventional nutritional medium in the presence of, for example, a cell fusion-promoting agent. The cell fusion-promoting agent include, for example, polyethyleneglycol (PEG), Sendai virus (HVJ) or the like. An auxiliary agent such as dimethylsulfoxide can also be used to increase the fusion efficiency as needed.
The ratio of the number of the immunocyte to the myeloma cell to be used may be appropriately determined. For example, the number of the immunocyte is preferred to be set at 1 to 10 times that of the myeloma cell. The culture medium to be used in the above-mentioned cell fusion includes culture media suitable for the growth of the above-mentioned myeloma cell line, for example, RPMI 1640 culture medium and MEM culture medium, and a standard culture medium which is used for this type of cell culture. A serum supplement such as fetal calf serum (FCS) may be used in combination.
In cell fusion, predetermined number of the immunocytes and myeloma cells are thoroughly mixed in the culture medium, a PEG solution previously heated to about 37° C. (for example, an average molecular weight of about 1000 to 6000) is added at a concentration of 30 to 60% (w/v) and mixed to form a desired fusion cell (hybridoma). Then, the process of sequential addition of an appropriate culture medium, centrifugation and removal of a supernatant is repeated to remove the cell fusion agent and those which are undesirable for the growth of the hybridoma.
The resulting hybridoma is then selected by culturing it in a standard selection culture medium such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). The cultivation in the above-mentioned HAT culture medium is continued for sufficient time (usually from several days to several weeks) so that cells other than the desired hybridoma (non-fused cells) will die. Then, a hybridoma that produces a desired antibody is screened and monocloned by a standard limiting dilution method.
Note that the antibody that recognizes ROBO1 can also be prepared using the method described in International Publication WO03/104453.
A desired antibody may be screened and monocloned by a known screening method based on an antigen-antibody reaction. For example, an antigen is bound to a support such as beads made of polystyrene or the like or a commercially available 96-well microtiter plate, then a culture supernatant of hybridoma is added. After the support is washed, an enzyme-labeled secondary antibody or the like is added to determine whether or not a desired antibody reacting with the sensitizing antigen is contained in the culture supernatant. The hybridoma that produces a desired antibody can be cloned by a limiting dilution method or the like. The antigen used for immunization may be used in the screening procedure.
In addition to the method where an animal other than human is immunized with an antigen to obtain a hybridoma, it is also possible to sensitize a human lymphocyte in vitro with ROBO1, and the resulting sensitized lymphocyte is fused with a human myeloma cell having the ability to divide permanently, whereby a desired human antibody having the activity of binding to ROBO1 can be obtained (see JP-B-1-59878). Alternatively, ROBO1 is administered to a transgenic animal having the repertoire of all the genes for human antibody to obtain a cell producing the anti-ROBO1 antibody. The cell is immortalized and a human antibody against ROBO1 may be obtained from the immortalized cell (see International Patent Application Nos. WO 94/25585, WO 93/12227, WO 92/03918 and WO 94/02602).
The thus prepared hybridoma that produces a monoclonal antibody can be subcultured in a standard culture medium, or can be stored for a long period of time in liquid nitrogen.
In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a standard method and an antibody is obtained as the culture supernatant. Alternatively, the hybridoma is administered to and grown in a mammal compatible with the hybridoma and an antibody is obtained from the ascites of the mammal. The former method is suitable for obtaining high-purity antibodies, whereas the latter is suitable for mass production of antibodies.
According to the present invention, a recombinant monoclonal antibody produced by genetic engineering techniques can also be used as a monoclonal antibody. The antibody gene is cloned from the hybridoma, incorporated into an appropriate vector and introduced into the host cell to produce a recombinant-type monoclonal antibody (see, for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990). Specifically, mRNA encoding the variable (V) region of the anti-ROBO1 antibody is isolated from the hybridoma producing the anti-ROBO1 antibody. The isolation of mRNA is carried out by a known method such as guanidine ultracentrifugation (Chirgwin, J. M. et al. Biochemistry (1979) 18, 5294-5299) or the AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159) to prepare total RNA, and then a desired mRNA is prepared by using an mRNA Purification Kit (manufactured by Pharmacia). Alternatively, mRNA can be directly prepared by using a QuickPrep mRNA Purification Kit (manufactured by Pharmacia).
cDNA coding for the V region of the antibody is synthesized from the resulting mRNA by using a reverse transcriptase. The synthesis of the cDNA is carried out by using, for example, AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Kogyo). Alternatively, cDNA may be synthesized and amplified by the 5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002, Belyaysky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using a 5′-Ampli FINDER RACE Kit (manufactured by Clontech), PCR and the like.
The desired DNA fragment is purified from the resulting PCR product and ligated with a vector DNA. Then a recombinant vector is constructed therefrom and introduced into E. coli or the like, and a colony is selected, whereby a desired recombinant vector is prepared. The nucleotide sequence of the desired DNA is checked by a known method such as the dideoxy nucleotide chain termination method. Once the desired DNA encoding the V region of the anti-ROBO1 antibody is obtained, and the DNA is incorporated into an expression vector containing DNA encoding the constant region (C region) of a desired antibody.
In order to produce the anti-ROBO1 antibody to be used in the present invention, the antibody gene is incorporated into an expression vector so as to be expressed under the control of the expression regulatory region, for example, an enhancer or a promoter. Subsequently, a host cell is transformed with the expression vector, and the antibody is expressed in the cell.
The antibody gene may be expressed in the cell by separately introducing DNAs encoding the heavy chain (H chain) and the light chain (L chain) of the antibody into expression vectors and co-transforming a host cell with the vectors; or by introducing DNAs encoding the H chain and the L chain into a single expression vector and transforming a host cell with the vector (see WO 94/11523).
When an antibody gene is isolated and introduced into a suitable host to produce an antibody, a combination of suitable host and expression vector can be used. Eucaryotic cells to be used as a host include animal cells, plant cells and fungal cells. Known animal cells include (1) mammalian cells, for instance, CHO, COS, myeloma, BHK (baby hamster kidney), HeLa and Vero, (2) amphibian cells, for instance, Xenopus laevis oocyte, or (3) insect cells, for instance, sf9, sf21, Tn5 and the like. Known plant cells include the Nicotiana genus, for instance, those derived from Nicotiana tabacum, which is grown in callus culture. Known fungal cells include yeast, for instance, the Saccharomyces genus such as Saccharomyces serevisiae, filamentous fungus, for instance, the Aspergillus genus such as Aspergillus niger, and the like. When using a prokaryotic cell, a production system using bacterial cell are available. Known bacterial cells include Escherichia coli (E. coli) and Bacillus subtilis. The target antibody gene is introduced into these cells by transformation, and the antibody may be obtained by culturing the transformed cells in vitro.
In addition to the above host cells, a transgenic animal can be used for the production of a recombinant antibody. For example, an antibody gene is inserted into the middle of a gene encoding a protein produced specifically into milk (such as goat β-casein) to prepare a fusion gene. A DNA fragment containing the fusion gene comprising the antibody gene is injected into a goat's embryo, which is then introduced into a female goat. A desired antibody can be obtained from milk produced by a transgenic goat which is born from the goat that had received the embryo or offspring thereof. To increase the amount of milk containing the desired antibody produced by the transgenic goat, an appropriate hormone may be administered to the transgenic goat (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
In the present invention, an artificially modified recombinant antibody, for instance, a chimeric antibody, a humanized antibody, can be used with the aim of decreasing heterologous antigenicity against human. These modified antibodies can be prepared using a known method. A chimeric antibody is an antibody comprising the variable regions on the heavy chain and the light chain of an antibody from a mammal other than human, such as mouse, and the constant regions on the heavy chain and light chain from a human antibody. It is obtained by ligating the DNA coding for the variable region of the mouse antibody and the DNA coding for the constant region of the human antibody, and incorporating into an expression vector, and introducing a host for antibody production.
C regions from the human antibody is used as the C region in the chimeric antibody or the humanized antibody. For example, Cγ1, Cγ2, Cγ3 or Cγ4 can be used for the H chain, and Cκ or Cλ can be used for the L chain. The C region of the human antibody may be modified in order to improve the stability of the antibody itself or the production process.
A chimeric antibody is composed of the variable region of an antibody derived from a non-human mammal and the constant region derived from a human antibody. On the other hand, a humanized antibody is composed of the complementarity determining region of an antibody derived a non-human mammal, and the framework region and the constant region derived from a human antibody. Since the antigenicity of the humanized antibody is expected to be reduced in human body, the humanized antibody is useful as an active ingredient of a therapeutic agent of the present invention.
A humanized antibody, also referred to as a “reshaped humane antibody”, is obtained by grafting the complementarity determining region (CDR) of an antibody from a non-human mammal, such as a mouse, into the complementarity determining region of a human antibody. Specifically, a DNA sequence designed to ligate a mouse antibody CDR to the framework region (FR) of a human antibody is synthesized by PCR using as primers several oligonucleotides constructed to have overlapping portions at the ends of both CDR and FR.
The obtained DNA is ligated with the DNA coding for the constant region of the human antibody, then incorporated into an expression vector, which is introduced into and expressed by a host to obtain the antibody (see European Patent EP 239400 and International Publication WO 96/02576).
The framework region of the human antibody to be ligated via the CDR is selected such that the complementarity determining region will form a favorable antigen-binding site. As necessary, amino acids in the framework region of an antibody variable region may be substituted, so that the complementarity determining region of a reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).
In addition, a method for obtaining a human antibody is also known. For instance, a human lymphocyte is sensitized with a desired antigen or a cell expressing the desired antigen in vitro, and sensitized lymphocyte is fused with a human myeloma cell, for instance U266, to obtain the desired human antibody capable of binding to the antigen (refer to Japanese Patent Publication No. H1-59878). In addition, a transgenic animal having the entirety of the repertoire of human antibody genes can be immunized with the desired antigen to obtain the desired human antibody (refer to International Publication WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735). In addition, a technique where a human antibody is selected by panning from a human antibody library is also known. For instance, the variable region of the human antibody is expressed as a single chain antibody (scFv) on the surface of a phage by the phage display method, and a phage binding to the antigen is selected. The gene of the selected phage is analyzed to determine the sequence of the DNA coding for the variable region of the human antibody binding to the antigen. Once the DNA sequence of the scFv binding to the antigen is determined, a suitable expression vector containing the sequence can be prepared to produce the human antibody. These methods are well known, and described in International Publication WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388.
The antibody to be used in the present invention is not limited to the whole antibody molecule and may be a fragment of the antibody or a modified fragment thereof as long as it binds to ROBO1, including a divalent antibody and a monovalent antibody. Examples of the fragment of the antibody include Fab, F(ab′)2, Fv, Fab/c having one Fab and a full Fc, and a single chain Fv (scFv) where the Fv of the H chain and the L chain are linked via an appropriate linker.
Specifically, an antibody is treated with an enzyme such as papain or pepsin to provide a fragment of the antibody. Alternatively, a gene encoding such an antibody fragment is constructed and introduced into an expression vector, and the antibody fragment is expressed in a suitable host cell (see, for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc., Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc., Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-669, Bird, R. E. et al., TIBTECH (1991) 9, 132-137).
The scFv can be obtained by linking the H chain V region and the L chain V region of an antibody. In the scFv, the H chain V region and the L chain V region are preferably linked via a linker, preferably a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H chain V region and the L chain V region in scFv may be derived from any antibody described as an antibody in this specification. For example, any single chain peptide having 2 to 25 amino acid residues may be used as the peptide linker for ligating the V regions. DNA encoding scFv can be obtained by amplifying a fragment by PCR using as a template a DNA portion encoding all or a desired amino acid sequence of the sequences of DNA encoding the H chain or the H chain V region of the above-mentioned antibody and DNA encoding the L chain or the L chain V region of the above-mentioned antibody with a primer pair that defines the both ends thereof. Then the fragment is amplified with a combination of DNA encoding a peptide linker portion and a primer pair which defines both ends to be ligated to the H chain and the L chain. Once DNA encoding scFv is prepared, an expression vector containing the DNA and a host cell transformed with the expression vector can be obtained according to a standard method. The scFv can be obtained from such a host according to a standard method. These antibody fragments can be produced in a host by obtaining the gene thereof in the same manner as described above and by allowing it to be expressed.
A modified antibody conjugated with any of a variety of molecules such as polyethylene glycol (PEG) can also be used in the invention. It is also possible to conjugate the antibody with a cytotoxic agent, such as a radioisotope, a chemotherapeutic agent and a cell-derived cytotoxin. Such a modified antibody can be obtained by chemically modifying the antibody obtained as above. Methods of modifying an antibody have already been established in the art. The term “antibody” in the present invention also encompasses such a modified antibody.
Further, the antibody to be used in the present invention may be a bispecific antibody. The bispecific antibody may have antigen-binding sites that recognize different epitopes on the ROBO1 molecule. Alternatively, one of which may recognize ROBO1, and the other may recognize a cytotoxic agent, such as a radioactive substance, a chemotherapeutic agent or a cell-derived toxin. In this case, the cytotoxic agent can directly act on a cell expressing ROBO1 to specifically damage the tumor cells to inhibit the proliferation of the tumor cells. The bispecific antibody can also be produced by ligating an HL pair of two types of antibodies, or by fusing hybridomas producing different monoclonal antibodies to provide a fusion cell producing the bispecific antibody. Furthermore, the bispecific antibody can also be produced by genetic engineering techniques.
Antibodies can be expressed from the antibody gene constructed as described above by a known method. In the case of a mammalian cell, the gene can be expressed by operably linking a conventional useful promoter, an antibody gene to be expressed and a poly A signal at the 3′-downstream of the gene. A promoter/enhancer includes, for example, a human cytomegalovirus immediate early promoter/enhancer.
Further, examples of the promoter/enhancer used for expressing antibodies to be used in the present invention include, for example, viral promoter/enhancers such as retrovirus, polyoma virus, adenovirus and simian virus 40 (SV40), mammalian promoter/enhancers such as human elongation factor 1α (HEF1α).
Antibodies can be readily expressed by the method of Mulligan et al. (Nature (1979) 277, 108) when SV40 promoter/enhancer is used, and by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322) when HEF1α promoter/enhancer is used.
In the case of E. coli, the gene can be expressed by operably linking a conventional useful promoter, a signal sequence for antibody secretion and an antibody gene to be expressed. A promoter includes, for example, lacZ promoter and araB promoter. The gene can be expressed by the method of Ward et al. (Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427) when the lacZ promoter is used, and by the method of Better et al. (Science (1988) 240, 1041-1043) when the araB promoter is used.
A signal sequence for antibody secretion may be used for producing the antibody in the periplasm of E. coli, such as pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379). After isolating the antibody produced in the periplasm, the antibody is appropriately refolded for use.
A replication origin may be derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV). To amplify the gene copy number in a host cell system, the expression vector may contain as a selection marker the aminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene, the E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene or the like.
Any expression system, for example, a eukaryotic cell or a prokaryotic cell can be used for producing the antibody to be used in the present invention. Examples of the eukaryotic cell include established animals cells such as mammalian cells, insect cells, filamentous fungus cells, and yeast cells and the like. Examples of the prokaryotic cell include bacteria cells such as E. coli cells.
The antibody to be used in the present invention is preferably expressed in a mammalian cell such as a CHO, COS, myeloma, BHK, Vero, or Hela cell.
Subsequently, the transformed host cell is cultured in vitro or in vivo to produce a desired antibody. The host cell may be cultured according to a known method. For example, DMEM, MEM, RPM11640 and IMDM can be used as a culture medium, and a serum supplement such as fetal calf serum (FCS) may be used in combination.
The thus expressed and produced antibody can be purified using known methods conventionally applied in protein purification. For example, the antibody can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column, chromatography columns, besides the above-mentioned, filters, ultra filtration, salting-out, dialysis and the like (Antibodies A Laboratory Manual, Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
The antigen binding activity of the antibody may be measured by a known method (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988) by, for instance, ELISA (enzyme linked immunosorbent assay), EIA (enzyme immuno assay), RIA (radioimmuno assay) or immunofluorescence.
Pharmaceutical Composition
In another aspect, the invention features a pharmaceutical composition comprising an antibody that binds to ROBO1 as an active ingredient. In addition, the present invention features a cell growth inhibitor, in particular an anticancer agent, comprising an antibody that binds to ROBO1 as an active ingredient.
In the present invention, the term “comprising an antibody that binds to ROBO1 as an active ingredient” means comprising an anti-ROBO1 antibody as a major active component, and is not meant to restrict the content ratio of the anti-ROBO1 antibody.
The antibody contained in the cell growth inhibitor of the present invention is not particularly limited, as long as it binds to ROBO1. Preferably, it is an antibody that binds specifically to ROBO1, and more preferably, it is an antibody that has cytotoxicity. In addition, the antibody used in the present invention may be an antibody with a modified glycosyl chain. It is known that cytotoxicity of an antibody can be increased by modifying its glycosyl chain. For instance, antibodies with modified glycosylation (WO99/54342 and the like), antibodies that are deficient in fucose added to the glycosyl chain (WO00/61739, WO02/31140 and the like)), antibodies having a glycosyl chain with a bisecting GlcNAc (WO02/79255 and the like) are known as those having modified glycosyl chain.
In the present invention, for instance, the cytotoxity includes antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and the like. In the present invention, CDC activity means a cytotoxicity caused by the complement system. ADCC activity means that when a specific antibody attaches to the cell surface antigen of the target cell, an Fcγ receptor carrier cell (immune cell, etc.) binding to the Fc portion thereof via the Fcγ receptor damages the target cell.
An anti-ROBO1 antibody can be tested for its ADCC activity or CDC activity by well known methods (for instance, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993) and the like).
Specifically, first, effector cells, complement solution, and target cells are prepared.
(1) Preparation of Effector Cells
Spleen is extirpated from a CBA/N mouse or the like, and spleen cells are separated in RPM11640 culture medium (manufactured by GIBCO). After washing in the same culture medium containing 10% fetal bovine serum (FBS, manufactured by HyClone), the cells are adjusted at a concentration of 5×106/ml, to prepare effector cells.
(2) Preparation of Complement Solution
Baby Rabbit Complement (manufactured by CEDARLANE) is diluted 10-fold in a culture medium (manufactured by GIBCO) containing 10% FBS, to prepare a complement solution.
(3) Preparation of Target Cell
Cells expressing ROBO1 (cells transformed with a gene coding for ROBO1, hepatic cancer cells, lung cancer cells, breast cancer cells, uterine cancer cells, gastric cancer cells, large intestine cancer cells, and the like) are radioactively labeled by incubating with 0.2 mCi of sodium chromate-51Cr (manufactured by Amersham Pharmacia Biotech) in a DMEM culture medium containing 10% FBS for one hour at 37° C. After radioactive labeling, cells are washed three times with RPM11640 culture medium containing 10% FBS, and adjusted at a concentration of 2×105/ml to prepare the target cells.
Next, the ADCC activity or the CDC activity is measured. In the case of ADCC activity measurement, 50 μl each of target cell and anti-ROBO1 antibody are added to a 96-well U bottom plate (manufactured by Beckton Dickinson), and incubated on ice for 15 minutes. Thereafter, 100 μl of effector cell are added and incubated in a carbon dioxide incubator for 4 hours. The final concentration of antibody is 0 or 10 μg/ml. After the culture, 100 μl of supernatant is collected, and the radioactivity is measured with a gamma counter (COBRAII AUTO-GAMMA, MODEL D5005, manufactured by Packard Instrument Company). The cytotoxicity (%) can be determined according to the equation:

(A−C)/(B−C)×100

wherein A represents the radioactivity (cpm) in a sample, B represents the radioactivity (cpm) in a sample where 1% NP-40 (manufactured by Nakarai) has been added, and C represents the radioactivity (cpm) of a sample containing the target cells only.
Meanwhile, in the case of CDC activity measurement, 50 μl each of target cell and anti-ROBO1 antibody are added to a 96-well flat-bottomed plate (manufactured by Becton Dickinson), and incubated on ice for 15 minutes. Thereafter, 100 μl of complement solution is added, and incubated in a carbon dioxide incubator for 4 hours. The final concentration of antibody is 0 or 3 μg/ml. After the culture, 100 μl of supernatant is collected, and the radioactivity is measured with a gamma counter. The cytotoxicity can be determined in the same way as in the ADCC activity.
Cells of which proliferation is inhibited by the anti-ROBO1 antibody are not particularly limited as long as they express ROBO1, and are preferably cancer cells, and more preferably hepatic cancer cells, lung cancer cells, breast cancer cells, uterine cancer cells, gastric cancer cells, brain tumor cells and large intestine cancer cells. The anti-ROBO1 antibody can be used for the purpose of treating and preventing diseases attributable to cell proliferation, for instance, hepatocellular cancer, lung cancer, breast cancer, uterine cancer, gastric cancer, brain tumor, large intestine cancer and the like.
The cell growth inhibitor and anticancer agent of the present invention may be administered orally or parenterally. Preferably it is administered parenterally in the form of, for example, injectable formulation, nasal administration formulation, pulmonary administration formulation, percutaneous administration formulation. An injectable formulation may be administered systemically or locally, for instance, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like. The administration route may be suitably selected according to the age of the patient and the symptoms. The dose may be selected within the range of 0.0001 mg to 1000 mg per kg body weight per administration. Alternatively, the dose may be selected for instance within the range of 0.001 to 100000 mg/body per patient. However, the therapeutic agent of the present invention is not limited to these doses.
The cell growth inhibitor and anticancer agent of the present invention can be formulated according to conventional methods (for instance, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A), and may also contain pharmaceutically acceptable carriers and additives, for example, but not limited to, surfactant, diluent, colorant, perfume, preservative, stabilizer, buffer, suspending agent, isotonization agent, bonded, disintegrant, lubricant, fluidity promoting agent, flavoring agent and the like. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatine, medium chain fatty acid triglyceride, polyoxyethylene hardened castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salt, and the like.
In addition, the present invention provides a method for inducing damages in a
ROBO1 expressing cell and a method for inhibiting cell growth by contacting a ROBO1 expressing cell with an antibody that binds to ROBO1. The antibody that binds to ROBO1 to be contained in the cell growth inhibitor of the present invention is as described above. The cell that is bound by the anti-ROBO1 antibody is not particularly limited as long as the cell is expressing ROBO1, preferably a cancer cell, more preferably a hepatic cancer cell, a lung cancer cell, a breast cancer cell, a uterine cancer cell, a gastric cancer cell, a brain tumor cell and a large intestine cancer cell.
All the contents of the patent references and other references explicitly referred to in this description are incorporated herein by reference. In addition, the entire contents of the specification and the drawings of Japanese Patent Application Nos. 2004-102862, 2004-227899 and 2005-004024, which are the basic applications for the priority of the present application, are incorporated herein by reference.
EXAMPLES
The present invention will be described in further detail with the following examples; however these examples are not to limit the scope of the present invention.
Example 1
mRNA Expression Analysis of ROBO1 in Various Types of Cancer
1-1. ROBO1 Gene Expression Analysis Using Gene Chip
To search for genes for which the expression is enhanced in cancer cells, various RNAs as well as total RNAs shown in Table 1 were prepared from various extracted tissues by conventional method using ISOGEN (manufactured by Nippon Gene) and analyzed.







TABLE 1







Tissues and cell lines used for the ROBO1 gene expression analysis





















Number of



Serum


Tissue
Origin
Lot#
Tissue
Origin
Cases
Tumor
Cell line
Medium
(%)



















whole brain
Clontech 64020-1
101041
glioblastoma
clinical sample
3
brain tumor
U251
DMEM
10


tonsil
Clontech 6574-1
1030830
lung cancer(adenocarcinoma)
clinical sample
12
breast cancer
MCF7
RPMI1640
10


callosal body
Clontech 6577-1
1010486
hepatic cancer (moderately differentiated)
clinical sample
3
esophageal cancer
TE2
RPMI1640
10


caudate nucleus
Clontech 6575-1
120289
hepatic cancer(well differentiated)
clinical sample
3
stomach cancer
AGS
RPMI1640
10


thalamus
Clontech 6582-1
1070147
stomach cancer
clinical sample
3

GT3
DMEM
10


hippocampus
Clontech 6578-1
1050638
lung cancer(small cell cancer)
clinical sample
1

Kato[1]
PMI1640:DMEM = 1:1
10


pituitary gland
Clontech 8584-1
2010981
lung cancer(small cell cancer)
clinical sample
1

MKN45
RPMI1640
10


cerebellum
Clontech 64035-1
1010033
lung cancer(small cell cancer)
clinical sample
1

MKN74
RPMI1640
10


thyroid gland
Stratagone 735040
510225
lung cancer(small cell cancer)
clinical sample
1

2M
DMEM
10


salivary gland
Clontech 64026-1
1011322
lung cancer(small cell cancer)
clinical sample
1

2MD3
DMEM
10


lung
clinical sample
14887
lung cancer(small cell cancer)
clinical sample
1
colorectal cancer
CAC02
DMEM
20


trachea
Clontech 64091-1
1010201
lung cancer(small cell cancer)
clinical sample
1

DLD1
RPMI1640
10


skeletal muscle
Ambion 7982
091P0101C
lung cancer(small cell cancer)
clinical sample
1

hCT116
McCoy5A
10


heart
Ambion 7966
110P43B
lung cancer(small cell cancer)
clinical sample
1

LOVO
HamF12:DMEM = 1:1
10


kidney
Ambion 7976
071P04B
lung cancer(small cell cancer)
clinical sample
1

SW480
RPMI1640
10


adrenal gland
Clontech64096-1
2020671
lung cancer(squamous cancer)
clinical sample
1
hepatic cancer
Alexander
DMEM
10


liver
clinical sample (Surgery)
N4
lung cancer(squamous cancer)
clinical sample
1

HepG2
DMEM
10


pancreas
Ambion 7954
091P0104A
lung cancer(squamous cancer)
clinical sample
1

HLE
DMEM
10


spleen
Ambion 7970
061P18A
lung cancer(squamous cancer)
clinical sample
1

HuH6
DMEM
10


stomach
clinical sample
MN15
lung cancer(squamous cancer)
clinical sample
1

HuH7
DMEM
10



(Surgery)










small intestine
Ambion 7984
091P0201A
kidney cancer
clinical sample
1
pancreas cancer
Capan1
DMEM
20


large intestine
Ambion 7986
071P10B
kidney cancer
clinical sample
1

KLM1
RPMI1640
10


bladder
Ambion 7990
81P0101A
colorectal cancer
clinical sample
1

Pane1
RPMI1640
10


bone marrow
Clontech64106-1
1110932
colorectal cancer
clinical sample
1

Paca2
RPMI1640
10


peripheral
clinical sample
—
colorectal cancer
clinical sample
1
kidney cancer
Caki2
RPMI1640
10


mononuclear











blood cell











testis
Clontech64027-1
6120257
colorectal cancer
clinical sample
1
lung cancer
A549
DMEM
10


prostate
Ambion 7938
081P0103A
colorectal cancer
clinical sample
1

Lu130
RPMI1640
10


ovary
Ambion 7974
051P42A
colorectal cancer
clinical sample
1

H1359
RPMI1640
10


uterus
Stratagone 735042
1100640
colorectal cancer-liver metastasis
clinical sample
1

H157
RPMI1640
10


placenta
Ambion 7950
061P33B
colorectal cancer-liver metastasis
clinical sample
1

H1648
HamF12:DMEM = 1:1
10


hepatic cirrhosis
clinical sample
—
colorectal cancer-liver metastasis
clinical sample
1

H2009
HamF12:DMEM = 1:1
10


embryo brain
Clontech64094-1
2020902
colorectal cancer-liver metastasis
clinical sample
1

H23
RPMI1640
10


embryo liver
CHEMICON356
21060678
colorectal cancer-liver metastasis
clinical sample
1

H2347
RPMI1640
10





colorectal cancer-liver metastasis
clinical sample
1

H522
RPMI1640
10





colorectal cancer-liver metastasis
clinical sample
1
cervix cancer
Hela
DMEM
10





colorectal cancer
clinical sample
1









colorectal cancer
clinical sample
1









pancreas cancer
clinical sample
1









pancreas cancer
clinical sample
1









pancreas cancer
clinical sample
1









pancreas cancer
clinical sample
1









Gene expression analysis was carried out using 10 ng each of total RNAs, subjecting to GeneChip U133 (manufactured by Affymetrix) according to the Expression Analysis Technique Manual (manufactured by Affymetrix). Genes overexpressed in cancer cells was searched based on the mean value of the expression score set at 100 for total gene, it was apparent that expression was notably enhanced for ROBO1 mRNA (Probe ID: 213194_at HG-U133A), by 26-fold in moderately-differentiated hepatocellular cancer (236.4), and by 62-fold in poorly-differentiated hepatocellular cancer (563), compared to healthy liver (9.1). In addition, the expression of ROBO1 mRNA was also enhanced in large intestine cancer, with a two-fold enhancement or more in five out of eight cases in primary large intestine cancer, compared to healthy large intestine (21.4). In addition, enhancement was also marked in three out of seven cases in metastasizing hepatic cancer of large intestine cancer, compared to healthy liver and large intestine. One case where ROBO1 was enhanced two-fold or more compared to healthy lung was also present in pulmonary parvicellular cancer (FIG. 1a,b).
In the analyses in cancer cell lines, expression of ROBO1 showed a score of 100 in U251, which is a cell line derived from human brain tumor, Alexander, HLE, HuH6, HuH7 and HepG2, which are derived from a human hepatic cancer cell line, Lu1320 and H522, which are derived from a human lung cancer cell line, and Hela, which is derived from human uterine cervical cancer, and the like, suggesting that expression of ROBO1 is stimulated not only in hepatocellular cancer, large intestine cancer and lung cancer shown above, but also in a wide range of cancers such as brain tumor and uterine cervical cancer (FIG. 1c).
In addition, analyses were carried out in the same way as described above in well-differentiated, moderately-differentiated (attributed to HBV or HCV viral infection) and poorly-differentiated hepatocellular cancer for hepatocellular cancers, as well as for hepatitis site, hepatic cirrhosis site, which are non-cancer sites, and healthy liver, using GeneChipTM HG-U95B Target (manufactured by Affymetryx). Total RNA was prepared from extracted tissues each from three cases, and 5 μg each of total RNA from three cases were mixed and subjected to the GeneChip analysis. The values are shown with the mean value of the total chip score normalized to 100.
As a result, the ROBO1 gene (Probe ID: 55461_at_HG-U95B) was expressed in an extremely low amount in healthy liver and non-cancer sites, while a remarkable increase in expression was observed in from well-differentiated hepatocellular cancer to poorly-differentiated hepatocellular cancer. It clearly shows that the expression was enhanced in hepatocellular cancer (FIG. 2).
Example 2
1-2. ROBO1 mRNA Expression Analysis by Quantitative PCR
Quantitative PCR was carried out using RNA prepared from healthy liver, hepatitis site, and hepatic cirrhosis site, as well as from tissue extirpated from hepatic cancer and from non-cancer site from the same tissue. PCR reaction was carried out with the iCycleriQ real time PCR analysis system (manufactured by BIO-RAD) using as the template DNA a single-stranded cDNA synthesized using the reverse transcriptase Superscript II (manufactured by GIBCO BRL) from the total RNA prepared from each tissue, to quantify the amount of expression of mRNA expression. The primer for ROBO1 was designed according to GenBank ID (NM_133631). Each 25 μL PCR reaction solution was prepared to contain 500 mM KCl, 100 mM Tris-HCl (pH8.3), 20 mM MgCl2, 0.1% gelatine, 1.25 mM dNTPs (dATP, dCTP, dGTP, dTTP) each, 1 μL of each single-stranded cDNA, 5 pmol each of ROBO1 sense primer (SEQ ID NO: 1) and ROBO1 antisense primer (SEQ ID NO: 2), 0.75 μL of SYBR Green I (1000-fold diluted solution, manufactured by Takara Shuzo), 0.25 μL of recombinant Taq polymerase Mix (FG Pluthero, Rapid purification of high-activity Taq DNA polymerase, Nucl. Acids. Res. 1993 21: 4850-4851). The reaction comprised of a primary denaturation for 3 minutes at 94° C., and 40 cycles of 15 seconds at 94° C., 15 seconds at 63° C., and 30 seconds at 72° C. The amount expressed in each authentic preparation was calculated using the software associated with the iCycler iQ real time analysis system. In addition, the amount of human β-actin gene expressed in individual RNA was also analyzed in the same way as described above using a sense primer (SEQ ID NO: 3) and an antisense primer (SEQ ID NO: 4) specific to human β-actin. The analysis result of ROBO1 was corrected with the analysis result of human β-actin (ROBO1/β-actin×100) and served as the amount of ROBO1 mRNA expressed.
As a result, similarly to the result from the GeneChip analysis, expression of ROBO1 mRNA was almost not observed in healthy liver and hepatitis site, as well as hepatic cirrhosis site; in contrast, the expression of ROBO1 mRNA was observed to be enhanced in a number of hepatocellular cancer sites. In particular, in comparisons of cancer site and non-cancer site within the same tissue, the expression was enhanced two-fold or more in 8 cases out of the 9 cases analyzed (Table 2).







TABLE 2







Rate of enhancement of ROBO1 gene expression by quantitative PCR


analysis


Quantitative PCR analysis (versus beta-actin (%))













Non-cancer

Expression


Patient No.
Stage
site
Cancer Site
increase rate














#1
moderately-
16
712
44.5



differentiated


#12
moderately-
62
488
7.9



differentiated


#15
moderately-
19
40
2.1



differentiated


#21
moderately-
19
64
3.4



differentiated


#30
moderately-
46
118
2.6



differentiated


#22
poorly-
16
304
19.0



differentiated


#104
poorly-
N/A
826



differentiated


#108
poorly-
15
32
2.1



differentiated


#111
poorly-
161
43
0.3



differentiated


#115
poorly-
154
685
4.4



differentiated










Pair with a difference of
8 cases



two-fold or more:










Example 3
Preparation of Anti-ROBO1 Antibody
In order to test the possibility of detecting cancer using an anti-ROBO1 antibody, an anti-ROBO1 antibody was generated.
3-1. Preparation of Antigen
3-1-1. Isolation of ROBO1 cDNA
In order to carry out expression of ROBO1, a ROBO1 cDNA was first isolated as follows. A single stranded cDNA was prepared from Hep3B cell following the method described above, and used as a template in amplification by the PCR method using primer RBV2F-TA (SEQ ID NO: 5) and RBR-TA (SEQ ID NO: 6). Primer RBV2F-TA was designed to hybridize with the 5′-end of the ROBO1 gene (GenBank: NM_133631), and RBR-TA was designed to hybridize to the 3′-end. The PCR method was carried out by preparing the reaction solution according to protocols of the LA-PCR kit (TAKARA manufactured by), and comprised of a primary denaturation for two minutes at 95° C., and 30 cycles of 15 seconds at 94° C., 15 seconds at 63° C., and 5 minutes at 72° C., and then the last elongation reaction under conditions comprising 10 minutes at 72° C. As a result, a band near approximately 5 kbp corresponding to the predicted ROBO1 sequence was successfully detected. The specifically amplified fragment obtained by the PCR method was inserted into pcDNA3.1/V5-H is TOPO (manufactured by Invitrogen) by the TA cloning method. The base sequence was examined by an established method to confirm that the isolated cDNA corresponded to ROBO1.
3-1-2. Preparation of Recombinant Baculovirus Expressing the N-Terminal Site of ROBO1
A region containing from the N-terminus to the first immuno globulin region (Ig1) of ROBO1 was expressed as a fusion protein with the membrane protein gp64 of the baculovirus with the ROBO1 cDNA isolated above as a template, the gene coding for the region containing from the N-terminus to the first immuno globulin region (Ig1) of ROBO1 was amplified by the PCR method using the RB_BVF primer (SEQ ID NO: 7) and the RB_BVR primer (SEQ ID NO: 8), and inserted into the pGEM-Te vector (manufactured by Promega). After verifying the base sequence by an established method, a gene fragment was digested with the restriction endonuclease KpnI and inserted into the pBucSurf vector (manufactured by Novagen) to construct a transfer vector ROBO1N/pBS. Then, 4 μg of ROBO1N/pBS was cut using the restriction endonuclease BplI (manufactured by Fermentas) and linearized and introduced together with Bac-N-Blue DNA into Sf9 cell according to the instructions of Invitrogen to prepare a recombinant baculovirus expressing a fusion protein of ROBO1-Ig1 and gp64.
The recombinant virus prepared as above was added to infect Sf9 cells (2×106 cells/mL) at the MOI of 5, which were then cultured at 27° C. for 3 days. Budding baculoviruses (BV) expressing the fusion protein of ROBO1-Ig1 and gp64 were recovered from the culture supernatant after 3 days culture. The culture solution was centrifuged at 800×g for 15 minutes to remove cells and cell debris, then the recovered culture supernatant was centrifuged at 45,000×g for 30 minutes. The precipitate was resuspended in PBS, and cell components were removed by further centrifuging at 800×g. The supernatant was centrifuged again at 45,000×g, and the obtained precipitate was suspended with PBS to serve as the BV fraction to be used as antigen for immunization.
3-2. Preparation of Anti-ROBO1 Monoclonal Antibody
An anti-ROBO1 monoclonal antibody was generated using as antigen the ROBO1-Ig1 expressing BV prepared by the above method. ROBO1-Ig1 expressing BV corresponding to a protein amount of 1 mg suspended in PBS were mixed with 200 ng of pertussis toxin, and subcutaneously injected into a gp64 transgenic mouse (WO03/104453) as an initial immunization. In a subsequent immunization, only ROBO1-Ig1 expressing BV corresponding to a protein amount of 500 μg was injected subcutaneously. As a final immunization, 250 μg of ROBO1-Ig1 expressing BV was administered intravascularly. After 3 days, spleen cells were isolated from the mouse, and fused with mouse P3U1 cells by a conventional method to establish a hybridoma cell. A hybridoma cell producing anti-ROBO1 antibody was selected by ELISA, in which the antigen ROBO1-Ig1 expressing BV used for immunization was immobilized on a solid phase. For the ELISA method, ROBO1-Ig1 expressing BV was left for one day and night in a 96-well flat-bottomed plate (manufactured by Falcon) at 4° C. at a final concentration of 10 μg/ml, then blocked with TBS buffer solution containing 40% Block Ace reagent (manufactured by Dainippon Pharmaceutical Co., Ltd.). A hybridoma culture supernatant was added, and the reaction was let to take place at room temperature for one hour. Next, HRP labeled antimouse IgG antibody (manufactured by Jackson) was added at room temperature for 1 hour, washed 4 times, then 3,3′,5,5′-tetramethylbenzin (TMB) reagent (manufactured by Sigma) was added at room temperature for one hour. The reaction was stopped with 0.5N sulfuric acid, and the optical density at 492 nm was measured with the microplate reader Multickan JX (manufactured by Labsystems).
As a result, hybridoma cells A7241A and A7225A were successfully established which produce a monoclonal antibody that binds to ROBO1. Each monoclonal antibody was prepared from the culture supernatant of the hybridoma cells by ammonium sulfate precipitation.
Example 4
Detection of ROBO1 Protein Molecule Using Anti-ROBO1 Antibody
In order to test the reactivity of the anti-ROBO1 antibody prepared by the above description, ROBO1 was detected using cell lysates from cell lines overexpressing ROBO1 and from various cancer cell lines. First, reactivity of the anti-ROBO1 antibody A7241A was verified by Western analysis using HEK293 cells forced to express ROBO1. The full length ROBO1 gene expression vector (ROBO1/pcDNA3.1) containing cDNA coding for ROBO1 inserted into pcDNA3.1/V5-H is TOPO (manufactured by Invitrogen) was used as an animal cell expression vector. Next, 1 μg of ROBO1/psDNA3.1 or pcDNA3.1 (Mock) as a negative control was introduced into 5×104 COST cells or 2×105 HEK293 cells using the FuGene6 reagent (manufactured by Roche Diagnostics), to express ROBO1 transiently. Cells were recovered three days after introduction of the expression vector, and the cultured cells were solubilized in the RIPA buffer solution (150 mM sodium chloride, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM tris-hydroxymethyl-aminomethane hydroxy aminomethane hydrochloride (pH 8.0)) to prepare a cell lysate.
An amount corresponding to 3 μg of protein of each lysate was subjected to SDS-polyacrylamide gel. The proteins were separated by SDS-PAGE and transferred to Hybond-P (manufactured by Amersham Bioscience). Then, the protein was detected with ECL plus (manufactured by Amersham Bioscience) using an anti-His antibody (manufactured by Sigma) or the A7241A antibody (1 μg/mL) as primary antibody, and using HRP labeled antimouse IgG antibody (manufactured by Jackson) as secondary antibody. A band of approximately 260 kD molecular weight reacting specifically with the anti-His antibody was detected. This band was considered as the full length ROBO1. A band with the same molecular weight was also observed in the cell lysate using the anti-ROBO1 antibody A7241A. From the above results, the anti-ROBO1 antibody A7241A was shown to be capable of detecting a full length ROBO1 protein specifically. A few bands with small molecular weights were also detected for A7241A, but not detected with the anti-His antibody, suggesting that they are degradation products lacking the C-terminus. Indeed, a band of approximately 120 kD molecular weight was detected in the culture supernatant (FIG. 3). Although the estimated molecular weight of full length ROBO1 is 190 kD, it was detected at a higher position (approximately 260 kD) than 250 kD of the prestained molecular weight markers (manufactured by Bio-Rad), probably due to modification of glycosyl chain. In addition, a band thought to be ROBO1 was also detected in the Mock test using HEK293 cell and pcDNA3.1. ROBO1 is thought to be expressed in HKE293 cells, which were derived from human embryo kidney, since ROBO1 is expressed during the fetal stage.
Next, Western analysis was carried out for cell lysates from various cancer cell lines. As a result, a band of approximately 260 kD molecular weight thought to be the full length ROBO1 was detected successfully only in the cell lines with a high mRNA expression score, which is consistent with the GeneChip U133 analysis results (FIG. 4). In addition, a plurality of bands with small molecular weights were detected in the same way, suggesting that, depending on the cell line, the number of links in a glycosyl chain is different, or ROBO1 with different molecular weights are present due to degradation or splicing.
It was examined whether soluble ROBO1 fragment can be detected in the culture supernatant of cancer cell lines expressing ROBO1. In the culture supernatant of a hepatic cancer cell line highly expressing ROBO1, a band with the same molecular weight as the culture supernatant of the overexpressing cells was also detected by the anti-ROBO1 antibody (FIG. 3).
From the above result, it was clear that the ROBO1 monoclonal antibody A7241A can specifically detect ROBO1, and that the degree of mRNA expression by the GeneChip analysis was consistent with the degree of ROBO1 protein expression. In addition, the examination using anti-ROBO1 antibody clearly showed that soluble ROBO1 fragment was present in the culture supernatant of ROBO1 expressing cell, strongly suggesting that the presence or the absence of cancer cell can be determined by detecting soluble ROBO1.
Example 5
Immunohistological Staining of Hepatocellular Cancer Tissue
Immunohistological staining analysis of clinical sample of hepatocellular cancer was carried out using anti-ROBO1 monoclonal antibody.
A section from a fixed paraffin embedded preparation of a hepatocellular cancer extirpated tissue sliced to 4 μm was mounted on a slide glass, and left at 37° C. for about 16 hours to dry extensively. The slide glass was deparaffinized by soaking three times in 100% xylene for 5 minutes each, then hydrophylized by soaking three times in 100% alcohol for 5 minutes and in 70% ethanol for 5 minutes. Then, after washing three times in 50 mM TBS buffer solution (50 mM Tris, pH 7.4, 150 mM NaCl) for 5 minutes, the antigen was activated by reacting in a citrate buffer (10 mM, pH 7.0) at 120° C. for 10 minutes. Following the activation of the antigen, the slide glass washed with a TBS buffer solution three times for 5 minutes each. Then, A7241A antibody diluted to 10 μg/mL was reacted at room temperature for one hour. Next, the endogenous peroxidase was inactivated with 0.3% hydrogen peroxide for 15 minutes at room temperature. After washing three more times with a TBS buffer solution, ENVISION+ kit/HRP (manufactured by DAKO) was added as the secondary antibody for one hour. After washing three times with TBS buffer solution for 5 minutes each, DAB (3,3′-diaminobenzidine tetrahydrochloride) was added for color development. Hematoxylin was used for contrast staining of the nucleus.
As a result, as shown in FIG. 5, hepatocellular cancer tissue was stained specifically by the anti-ROBO1 antibody, indicating that the expression of ROBO1 protein was specifically stimulated in hepatocellular cancer. In addition, the anti-ROBO1 antibody was shown to be useful for the immunohistochemical diagnosis of cancers highly expressing ROBO1, such as hepatocellular cancer.
Example 6
Detection of Soluble ROBO1 Protein (sROBO1) in the Serum of Hepatocellular Cancer Patient
The results of Example 4 showed that a fragment of ROBO1 protein was released and existed as soluble ROBO1. From this fact, a soluble ROBO1 protein (sROBO1) is considered to be present in the serum of a patient with cancer expressing ROBO1 at a elevated level, such as a hepatocellular cancer, and is useful as a diagnosis marker. The presence of soluble ROBO1 in each serum from 24 hepatocellular cancer patients, 6 hepatic cirrhosis patients and 6 hepatitis patients was examined by a Western analysis using the A7241A antibody. The SDS-PAGE and the Western analysis were carried out as described above with 5 μl of each patient serum. The culture supernatant of the hepatic cancer cell line Alexander (ALX) was used as a positive control. As a result, as shown in FIG. 6, sROBO1 was detected in the serum of a hepatocellular cancer patient in 23 out of 24 cases, while it was not detected in the sera of hepatic cirrhosis and hepatitis patients. From these results, sROBO1 was shown to be present also in the serum of a patient with ROBO1-expressing cancer, and the detection of sROBO1 was shown to be extremely efficient as a serodiagnosis marker of ROBO1-expressing cancer, such as hepatocellular cancer.
Example 7
Preparation of Soluble ROBO1 (sROBO1-His)
A soluble ROBO1 with a His-tag added to the C-terminus of the extracellular region of ROBO1 (sROBO1-His) was prepared as described below.
The gene coding for the extracellular region was amplified by the PCR method with ROBO1 cDNA as a template and using the primer RBV2F-TA (SEQ ID NO: 13) and the primer RB_SH_TA (SEQ ID NO: 14). The PCR product was directly inserted into the pBlueBack4.5-TOPO vector and the sequence was analyzed to generate a transfer vector sROBO1/pBB having the correct base sequence. A recombinant baculovirus was prepared using 4 μg of sROBO1/pBB by a similar method to Example 3.
Next, sROBO1-His was prepared as described below. 2×106/mL of Sf9 cells were infected with the sROBO1-His expressing recombinant baculovirus at an MOI of 5, cultured for 3 days at 27° C., and the culture supernatant was recovered. sROBO1-His contained in the culture supernatant was purified using Ni-NTA superflow (QIAGEN) according to the enclosed protocol. The purification product was concentrated using Centircon-10 (manufactured by Amicon), and the buffer was exchanged to PBS to prepare sROBO1-His.
Example 8
Evaluation of Serum from sROBO1-Immunized Rabbit
8-1. Detection of ROBO1 Protein Molecule by Western Analysis
The purified sROBO1 antigen (100 μg/0.5 mL/animal) suspended in PBS was mixed with 0.5 mL of Freund complete adjuvant (manufactured by DIFCO) to form an emulsion, and administered by subcutaneous injection to New Zealand White rabbits (10 weeks old females, manufactured by Clea Japan) as an initial immunization. Subsequently, with an interval of 2 weeks, a total of four immunizations were carried out by subcutaneous injections of an emulsion containing 100 μg/0.5 mL of purified sROBO1 antigen suspended in PBS mixed with 0.5 mL of Freund complete adjuvant. Blood was collected prior to immunization and after the third and the fourth immunization, and the increase in the levels of the antibody against sROBO1 was determined by the ELISA method. sROBO1 was immobilized on a solid phase onto an ELISA polystyrene plate, and a series of dilution of rabbit antiserum was added to the plate. The plate was reacted with horseradish peroxidase labeled antirabbit IgG antibody (manufactured by Cappel) and colored with 3,3′,5,5′-tetramethylbenzidine reagent (TMB reagent; manufactured by Cytech) to assay the antibody titer. After the increase in the antibody level was observed, whole blood was collected to obtain anti-ROBO1 rabbit antiserum.
The full length ROBO1 molecule was detected by Western analysis using the serum from sROBO1 immunized rabbit. Cell lysate was prepared from HEK293 cell overexpressing a full length ROBO1 using RIPA buffer solution as described above, and an amount corresponding to 10 μg of protein was subjected to SDS-polyacrylamide gel. The proteins were separated and transferred to Hybond-P (manufactured by Amersham Bioscience). Then, the proteins were detected with ECL plus (manufactured by Amersham Bioscience) using a 100-fold diluted serum from sROBO1 immunized rabbit as primary antibody, and using HRP labeled antirabbit IgG antibody (manufactured by Amersham Bioscience) as secondary antibody. A band of approximately 260 kD molecular weight thought to be the full length ROBO1 molecule was detected, which was similarly to the positive control A7241A antibody (FIG. 7). From the above results, the serum from an sROBO1 immunized rabbit was shown to be capable of detecting the full length ROBO1 molecule by the Western analysis.
8-2. Detection of ROBO1 Protein by FACS Analysis
In order to evaluate whether serum from sROBO1 immunized rabbit could detect ROBO1 on the cell surface, FACS analysis was carried out using HEK293 cells forced to express ROBO1. HEK293 cells forced to express ROBO1 or negative control HEK293 cells were suspended in a FACS solution (PBS containing 1% albumin and 0.1% NaN3). Serum from sROBO1 immunized rabbit was added to the cell suspension and let to react at 4° C. for 60 minutes. After washing twice with a FACS solution, FITC labeled antirabbit F(ab)2 antibody (manufactured by DAKO) was added and let to react at 4° C. for 30 minutes. Then, the cells were washed twice with a FACS solution and analyzed by FACS with FACScalibur (manufactured by Beckton Dickinson) following the user manual. The anti-V5-tag antibody (manufactured by Invitrogen) was used as the positive control for the experiment, and the FITC labeled antimouse IgG antibody (manufactured by Jackson) was used as the secondary antibody. Since V5-tag was conjugated to the C terminus of the intracellular region of ROBO1, a FACS solution containing 0.1% saponin was used when the primary antibody was used, so that the antibody can detect the intracellular V5-tag.
The result is shown in FIG. 8. Similarly to the peak shift of the positive control anti-V5 antibody, a specific peak shift was also detected in the blood of sROBO1-immunized rabbit only in HEK293 cell forced to express ROBO1.
In addition, FACS analysis was carried out according to the same method as described above in hepatic cancer cell line HepG2 cells, which inherently express ROBO1, as shown in FIG. 9. A specific peak shift was observed in the serum of sROBO1 immunized rabbit, showing that ROBO1 molecule having the original structure on the cell surface could also be detected.
Example 9
Purification of Rabbit Anti-Human ROBO1 Polyclonal Antibody
Anti-human ROBO1 rabbit polyclonal antibody prepared from serum of sROBO1 immunized rabbit was purified by affinity chromatography with solid-phased ROBO1. A sROBO1-His affinity column was prepared using CNBr-Activated Sepharose 4B (Amersham Pharmacia Biotech #17-0430-02) according to the enclosed text, and immobilizing 0.7 mg of purified sROBO1-His antigen per 1 mL of gel, according to the enclosed documentation. Then, anti-ROBO1 rabbit polyclonal antibody was purified according to conventional methods from the rabbit serum obtained in Example 8.
Example 10
Establishment of ROBO1 Measurement System and Detection of ROBO1 in Blood by ELISA
An ELISA detection system was constructed using the anti-ROBO1 rabbit polyclonal antibody obtained in Example 9. Anti-ROBO1 rabbit polyclonal antibody was diluted in PBS at a concentration of 5 μg/mL, and was dispensed into a 96-well immuno-plate, 50 μL per well each. After the plate was let to stand overnight at 2 to 8° C., the plate washed three times with PBS containing 0.05% Tween 20, and coated with 150 μL Immunoassay Stabilizer (ABI #10-601-001) per well for one hour. After the solution was discarded, the plate was dried at 37° C. for two hours to obtain a solid-phased anti-ROBO1 antibody plate. For the biotinylated anti-ROBO1 antibody used for detection, anti-ROBO1 rabbit polyclonal antibody and Sulfo-NHS-LC-Biotin (Pierce #21335) were prepared at 0.12 mg/mL and 46 μg/mL, respectively, using a 50 mM carbonate buffer solution at pH 8.5, and let to stand at room temperature for two hours. Unreacted biotin reagent was removed using PD-10 (Pharmacia #17-0851-01), and biotinylated anti-ROBO1 antibody was diluted to 1 μg/mL with PBS containing 30% calf serum. Biotinylated anti-ROBO1 antibody was detected using streptavidin labeled peroxidase (Vector #SA-5004) diluted at 3 μg/mL in tris-hydroxymethyl-aminomethane buffered physiological saline containing 30% calf serum. TMB reagent (Cytech #TM490041) was used as the substrate for peroxidase and TMB stop agent (Cytech #TSB999) was used as stop reagent for the substrate reaction.
ROBO1 concentration was measured in the sera from 72 cases of healthy subjects, 79 cases of hepatic cancer patients, 67 cases of hepatic cirrhosis/chronic hepatitis patients and 22 cases of other cancer patients. Purified sROBO1-His was used as a standard for measuring ROBO1 concentrations. Serum or sROBO1-His was diluted to 1/90 fold with tris-hydroxymethyl-aminomethane buffer solution containing 20% rabbit serum and 1% BSA, respectively, and 100 μL each was dispensed into each well of the antibody solid phased plate. After incubating at room temperature for two hours, 25 μL of biotinylated anti-ROBO1 antibody was dispensed into each well. After incubating at room temperature for two hours, the reaction solution was removed from the well, 100 μL each of streptavidin labeled peroxidase reagent was dispensed. After incubating at room temperature for 30 minutes, the plate was washed 5 times with PBS containing 0.05% Tween 20. 100 μL each of TMB reagent was added to each well, and incubated at room temperature for 30 minutes. 100 μL of TMB stop reagent was added, and the absorption at a wavelength of 450 nm was measured with an EIA plate reader (Corona Electric Co., #MTP-120) with a reference wavelength of 630 nm.
As a result, a concentration-dependent increase of absorption was observed with sROBO1-His. The concentration in each serum was determined by regression to the standard absorption curve (FIG. 10). The results are shown in Tables 3, 4, 5 and 6.







TABLE 3







Concentration of ROBO1 in blood of healthy subjects










ID
ROBO1



NO.
(ng/ml)












1
1210
44


2
1211
44


3
1212
37


4
1213
39


5
1214
56


6
1215
39


7
1216
41


8
1217
38


9
1218
33


10
1219
38


11
1220
42


12
1221
41


13
1222
33


14
1223
39


15
1224
30


16
1225
38


17
1226
30


18
1227
44


19
1228
36


20
1229
37


21
1230
43


22
1231
44


23
1232
38


24
1233
40


25
1234
22


26
1235
26


27
1236
35


28
1237
40


29
1238
41


30
1239
42


31
1240
36


32
1241
45


33
1242
19


34
1243
35


35
1244
39


36
1245
36


37
1246
36


38
1247
39


39
1248
42


40
1249
27


41
1250
34


42
1251
34


43
1252
29


44
1253
30


45
1254
33


46
1255
23


47
1256
15


48
1257
28


49
1258
39


50
1259
41


51
1260
40


52
1261
43


53
1262
35


54
1263
34


55
1264
36


56
1265
34


57
1266
22


58
1267
21


59
1268
35


60
1269
31


61
1270
29


62
1271
27


63
1272
33


64
1273
28


65
1274
29


66
1275
36


67
1276
47


68
1277
41


69
1278
33


70
1279
38


71
1280
33


72
1281
56








Mean
36


SD
8


Mean + 3SD
58


Mean + 6SD
81
















TABLE 4







Concentration of ROBO1 in blood of hepatic cancer patients
















AFP-




ID
AFP
PIVKA
L3
ROBO1



NO.
(ng/ml)
(U/ml)
(%)
(ng/ml)

















 1
428
2647
1292

75



 2
442
1976

31.8
163



 3
275
1670
130
79.4
152



 4
538
168


226



 5
229
146

30.6
196



 6
381
125
17
11.6
170



 7
159
101
13

143



 8
168
82.7

2.4
112



 9
545
73.3

17.9
176



10
585
60.7
50
49.4
58



11
42
56.3
10
12.6
83



12
512
48.5
41
47.1
70



13
360
47.9
10
3.9
83



14
356
39
1565

76



15
520
36.9
10
7.2
81



16
66
34
89

139



17
337
32.5

1.7
79



18
510
30.6

1.5
92



19
322
30
1824

83



20
225
29
19

80



21
231
28


160



22
314
28
—
2,1
103



23
350
26
18

98



24
402
25
2117

80



25
289
22
10
7.8
107



26
549
21
893

75



27
155
21
26
5.8
80



28
527
19
14

47



29
122
19
167

60



30
151
17
559

72



31
5
16.4

8.8
100



32
481
14.2
315
2
129



33
330
14
91

147



34
359
13
14

37



35
196
12
10
2
152



36
425
11.9
892
0.5
157



37
28
10.7
31
2.4
92



38
271
10.1
13.8

63



39
108
9
33

57



40
453
8
12
0
105



41
346
7
10

93



42
221
7
16
0
51



43
333
6.9
12
0
50



44
584
6.2
19
0
51



45
95
6
42
6.7
91



46
82
5.7
15
0
51



47
340
5.6
16
0
42



48
264
5
13

83



49
4
5
26
0
52



50
130
4.8
15
0
58



51
23
4
14

45



52
256
4
25
—
44



53
341
3


73



54
483
3
17

41



55
466
3
15

43



56
228
2
15

36



57
36
1
14

47



58
426



56



59
251



53



60
141



53



61
230



31



62
366



27



63
452



115



64
328



111



65
429

47

111



66
507



108



67
239

15

77



68
41



70



69
203

14

67



70
50



65



71
457



64



72
266



63



73
320

21

60



74
279



50



75
255



49



76
534



47



77
395



44



78
487
—
—
—
80



79
175
—
—
—
56



positive
n
27
16
6
36




%
46
30
18
46





Note:


Blanks in the table indicate “unmeasured”.













TABLE 5







Concentration of ROBO1 in blood of hepatic cirrhosis and chronic


hepatitis patients


















AFP-




ID

AFP
PIVKA
L3
ROBO1



NO.
diagnosis
(ng/ml)
(U/ml)
(%)
(ng/ml)
















1
791
LC
59.8

9.8
112


2
515
LC
41
—
—
129


3
740
LC
36
16
2.1
95


4
71
LC
33


125


5
187
LC
24.4
12
5.6
107


6
443
LC
22
—
—
106


7
20
LC
20


94


8
777
LC
20


92


9
675
LC
17


95


10
601
LC
16
10
—
87


11
445
LC
13
—
—
88


12
378
LC
13
23
—
87


13
940
LC
9


75


14
628
LC
8


57


15
260
LC
6.3
10
0  
61


16
245
LC
6
19
—
82


17
67
LC
6
18

79


18
776
LC
6


61


19
932
LC
6
35

58


20
137
LC
6


60


21
574
LC
6
—
—
50


22
220
LC
5.7

0  
87


23
533
LC
5


105


24
1165
LC
5


57


25
59
LC
5
11

59


26
62
LC
4
11

110


27
1149
LC
4
10

101


28
80
LC
4
—
—
77


29
375
LC
4
—
—
48


30
878
LC
4


46


31
1128
LC
4


47


32
69
LC
3
10

76


33
138
LC
3


74


34
513
LC
3
—
—
66


35
105
LC
3
12

54


36
570
LC
3


50


37
561
LC
2
13
—
40


38
1030
LC
2
14

28


39
671
LC
1


87


40
1198
LC
1
26

81


41
479
LC
1
—
—
59


42
172
LC
1
21
—
50


43
49
LC
1
—
—
47


44
409
LC
1
—
—
45


45
374
LC
—
12
—
105


46
222
LC
—
—
—
102


47
1143
LC

11

91


48
565
LC



87


49
870
LC

15

83


50
380
LC



80


51
193
LC



76


52
169
LC



66


53
921
LC

10

62


54
922
LC

14

50


55
691
LC



48


56
981
LC



30


57
116
LC



66


58
1035
LC

10

53


59
192
CH
17


43


60
458
CH
15


43


61
379
CH
6
14

32


62
772
CH
4
14

28


63
421
CH
3


32


64
568
CH
—
—
—
39


65
1090
CH

13

33


66
302
CH

17

59


67
285
CH



45




n
8
 0
0  
15




%
15
 0
0  
22





Note:


Blanks in the table indicate “unmeasured”.













TABLE 6







Concentration of ROBO1 in blood of cancer patients other than hepatic


cancer













ROBO1



ID number
diagnosis
(ng/ml)














1
43
leukemia
27


2
58
leukemia
61


3
84
leukemia
56


4
182
leukemia
64


5
204
leukemia
52


6
159
lung cancer
128


7
161
lung cancer
57


8
76
brain tumor
15


9
29
gallbladder cancer
41


10
175
cholangiocarcinoma
53


11
168
colorectal cancer
50


12
68
pancreas cancer
54


13
71
esophageal carcinoma
10


14
106
myeloma
31


15
49
stomach cancer
1


16
50
stomach cancer
80


17
69
stomach cancer
43


18
70
stomach cancer
67


19
105
stomach cancer
18


20
113
stomach cancer
39


21
147
stomach cancer
128


22
48
submandibular gland cancer
30



positive
n
2




%
9.1









From the fact that the mean value and standard deviation 36 ng/mL and 8 ng/mL in the measurements of ROBO1 concentration in 72 cases of healthy subject sera, 81 ng/mL, that is mean value+(6× standard deviation), was taken as the cutoff value. As a result, all the cases of healthy subject serum samples were below this cutoff value; in contrast, 46% (36 cases out of 79 cases) of hepatic cancer patient samples, 22% (15 cases out of 67 cases) of hepatic cirrhosis/chronic hepatitis patient samples and 9% (2 cases out of 22 cases) of other cancer patient samples showed not less than the cutoff value, indicating ROBO1 positive. The positive rate of AFP, which is generally used as a diagnostic marker for hepatic cancer, was 46% (27 cases out of 59 cases) and 15% (8 cases out of 52 cases) in hepatic cancer patient samples and hepatic cirrhosis/chronic hepatitis patient samples, respectively. thus the sensitivity and specificity in hepatic cancer diagnosis were approximately equivalent between AFP and ROBO1. Meanwhile, 10 out of 32 cases of AFP negative hepatic cancer patient samples showed ROBO1 positive, and PIVKA was also negative in 5 cases of these 10 cases. It suggests that hepatic cancer can be diagnosed with ROBO1 even in the cases that could not be diagnosed as hepatic cancer by existing diagnostic methods. In fact, the positive rate was 46% by AFP alone, while it was 63% by a combination with ROBO1.
The above results showed that ROBO1 measurement system has an approximately equivalent capability to the existing AFP measurement system in the diagnosis of hepatic cancer, and it is possible to establish a cancer diagnostic method with high sensitivity and specificity by combining ROBO1 with existing cancer markers.
The values shown in Tables 3, 4, 5 and 6 are plotted by disease and shown in FIG. 11. The mean ROBO1 concentration in sera of healthy subjects (NHS), chronic hepatitis (CH), hepatic cirrhosis (LC) and hepatic cancer (HCC), were 34.8, 39.3, 74.0 and 84.4 ng/mL, respectively. This result clearly shows that the ROBO1 concentration in the sera from each group of healthy subject, chronic hepatitis, hepatic cirrhosis and hepatic cancer patients increased proportionally to the progression of liver disease.
Next, in order to determine whether the ROBO1 concentration varies also in time-course measurements for each individual, ROBO1 concentration in blood samples collected before onset of hepatic cancer was measured. ROBO1 concentration in blood was observed to be increased proportionally to progression in 2 out of 3 cases tested (FIG. 12). No increase proportional to progression was observed in one case, probably because the maximum value had been reached, which is higher than the healthy subject (30 to 40 ng/mL).
These results show that not only the diagnosis of hepatic cancer but also the severity of liver disease in the patient can be managed by measuring or monitoring the time-course of the ROBO1 concentration in serum from the patients with liver disease.
Example 11
Measurement of Complement-Dependent Cytotoxicity (CDC Activity)
Creation of Human Albumin Veronal Buffer (HAVB)
A solution was prepared by dissolving 12.75 g of NaCl (Special Grade, Wako Pure Chemical Industries, Ltd.), 0.5625 g of Na-barbital (Special Grade, Wako Pure Chemical Industries, Ltd.), and 0.8625 g of barbital (Special Grade, Wako Pure Chemical Industries, Ltd.) in Milli Q water and filled to 200 mL, and autoclaveed (121° C., 20 minutes). A volume of 100 mL of autoclaved hot Milli Q water was added and found to be pH 7.43 (recommended pH: 7.5), which served as 5× veronal buffer. An amount of 0.2205 g of CaCl2.2H2O (Special Grade, Junsei Chemical Co., Ltd.) was dissolved in 50 mL Milli Q water to obtain 0.03 mol/L, which served as a CaCl2 solution. An amount of 1.0165 g of MgCl2.6H2O (Special Grade, Junsei Chemical Co., Ltd.) was dissolved in 50 mL Milli Q water to obtain 0.1 mol/L, which served as a MgCl2 solution. A volume of 100 mL of 5× veronal buffer, 4 mL of human serum albumin (25% Buminate®, 250 mg/mL human serum albumin concentration, Baxter), 2.5 mL of CaCl2 solution, 2.5 mL of MgCl2 solution, 0.1 g of KCl (Special Grade, Junsei Chemical Co., Ltd.), and 0.5 g of glucose (D(+)-glucose, glucose anhydrous, Special Grade, Wako Pure Chemical Industries, Ltd.) were dissolved in Milli Q water, and brought to 500 mL. This served as HAVB. After sterilization by filtration, the solution was stored at a set temperature of 5° C.
Preparation of Target Cell
HEK293 cell forced to express ROBO1 was cultured in a DMEM culture medium (SIGMA) supplemented with 10% FBS (Thermo Trace) and 0.5 mg/mL Geneticin (GIBCO), released from the dish using cell detachment buffer solution (GIBCO), dispensed at 1×104 cells/well in each well of a 96-well U bottomed plate (BECTON DICKINSON), and cultured overnight. To the culture 5.55 MBq of chromium-51 was added and incubated in a 5% carbon dioxide incubator at 37° C. for one hour. The cells were washed twice with HAVB, and 50 μL of HAVB was added to prepare a target cell.
Preparation of Baby Rabbit Complement
For a complement solution, baby rabbit complement (BABY RABBIT COMPLEMENT, CEDARLANE), which was prepared immediately before use, was dissolved in 1 mL per vial of injectable distilled water (Fuso Pharmaceutical Industries, Ltd.) at the time of examination.
Chromium Release Test (CDC Activity)
Anti-ROBO1 antiserum (anti-ROBO1 rabbit polyclonal antibody) was diluted with HAVB to obtain 1/50 and 1/500 fold antibody solutions. A volume of 50 μL each of antibody solution was added to target cells, and let to stand on ice for 15 minutes. Next, 100 μg/mL each of complement solution was added to each well (with final antibody dilution ratios of 1/200 and 1/2000), and was let to stand in a 5% carbon dioxide incubator at 37° C. for 90 minutes. After centrifugation of the plate, 100 μL each of supernatant was collected from each well, and the radioactivity was measured with a gamma counter. The specific chromium release rate was determined according to the following equation:

specific chromium release rate (%)=(A−C)/(B−C)×100

where A represents the radioactivity (cpm) in a well, B represents the mean value of radioactivity (cpm) in a well where 100 μL of 2% NP-40 aqueous solution (Nonidet P-40, Nacalai Tesque Co, Ltd.) and 50 μL of HAVB were added to the target cells, and C represents the mean value of radioactivity (cpm) in a well where 150 μL of HAVB was added to the target cells. The test was carried out in triplicate, and mean value and standard deviation were calculated for the CDC activity (%).
The results are shown in FIG. 13. It was apparent that the anti-ROBO1 antiserum, that is the anti-ROBO1 polyclonal antibody, showed dose-dependent CDC activity against ROBO1 expressing HEK293 cells. No CDC activity was detected with rabbit preserum or without addition of antibody.
Example 12
Preparation of Monoclonal Antibody that Binds to Extracellular Region of ROBO1
12-1. Preparation of Recombinant Baculovirus Expressing the N-Terminal Region of ROBO1
The fibronectin III region (FnIII) present in the extracellular region of ROBO1 was expressed as a fusion protein with the baculovial membrane protein gp64. A gene coding for the third fibronectin region of ROBO1 was amplified by the PCR method with the ROBO1 cDNA as a template, using the gp4F primer (SEQ ID NO: 15) and the gp4R primer (SEQ ID NO: 16), and inserted into the pGEM-Te vector (manufactured by Promega). After the nucleotide sequence was confirmed by an established method, a gene fragment cleaved by restriction endonuclease KpnI was inserted into the pBucSurf vector (manufactured by Novagen), to construct the transfer vector ROBO1 gp4/pBS. Then, 4 μg of ROBO1gp4/pBS was cut and linearized with the restriction endonuclease BO (manufactured by Fermentas), and introduced together with a Bac-N-Blue DNA into Sf9 cell according to the instructions from Invitrogen, to prepare a recombinant baculovirus that expresses a fusion protein of FnIII from ROBO1 and gp64.









SEQ ID NO: 15:



GGTACCCGCACCCAGTGCCCCACCCCAAGG



 



SEQ ID NO: 16:



GGTACCGCATCTGAAATCTGCTGAGCGAGG






The recombinant virus prepared as described above was added to infect SD cells (2×106 cells/mL) at an MOI of 5, which were cultured at 27° C. for 3 days. Budding baculoviruses (BV) expressing the fusion protein of ROBO1-FnIII and gp64 were recovered from the culture supernatant after 3 days culture. The culture solution was centrifuged at 800×g for 15 minutes to remove cells and cell debris, and the culture supernatant was centrifuged at 45,000×g for 30 minutes. The precipitate was suspended in PBS and further centrifuged at 800.times.g to remove cell constituents. The supernatant was centrifuged again at 45,000×g and the precipitate was suspended in PBS to serve as BV fraction for use in immunization.
12-2. Preparation of Anti-ROBO1 Monoclonal Antibody
The ROBO1-FnIII expressing BV prepared by the above method was used as an antigen to generate an anti-ROBO1 monoclonal antibody. ROBO1-FnIII expressing BV corresponding to a protein amount of 1 mg was suspended in PBS and mixed with 200 ng of pertussis, and then subcutaneously injected into a gp64 transgenic mouse (WO03/104453) for an initial immunization. In a subsequent immunization, only ROBO1-FnIII expressing BV corresponding to a protein amount of 500 μg was injected subcutaneously. As a final immunization, 250 μg of ROBO1-FnIII expressing BV was administered intravascularly. After 3 days, spleen cells were isolated from the mouse and fused with mouse NS-1 cells according to conventional methods to establish a hybridoma cell. Hybridoma cell producing anti-ROBO1 antibody was selected by ELISA using the solid-phased ROBO1-FnIII expressing BV, the antigen used for immunization. In the ELISA method, ROBO1-FnIII expressing BV was left for one day and night in a 96-well flat-bottomed plate (manufactured by Falcon) at 4° C. at final concentration of 10 μg/ml, and blocked with a TBS buffer solution containing 40% Block Ace reagent (manufactured by Dainippon Pharmaceutical Co., Ltd.). A hybridoma culture supernatant was added, and the reaction was let to take place at room temperature for one hour. Next, HRP labeled antimouse IgG antibody (manufactured by Jackson) was added at room temperature for one hour, washed 4 times, and 3,3′,5,5′-tetramethylbenzin (TMB) reagent (manufactured by Sigma) was added at room temperature for one hour. The reaction was stopped with 0.5N sulfuric acid, and the optical density at 492 nm was measured with the microplate reader Multickan JX (manufactured by Labsystems).
A hybridoma cell B2318C producing a monoclonal antibody that binds to ROBO1 was established successfully. The monoclonal antibody was prepared from the culture supernatant of the hybridoma cells by the ammonium sulfate precipitation method.
Example 13
Measurement of Complement-Dependent Cytotoxicity (CDC Activity)
Preparation of human albumin•veronal•buffer (HAVB), target cells, and baby rabbit complement were carried out similarly to Example 11.
B2318C antibody (anti-ROBO1 monoclonal antibody) was diluted with HAVB, 50 μL each was added to target cells, and let to stand on ice for 15 minutes. Next, 100 μL/mL each of complement solution was added to each well (prepared with final concentrations of antibody of 1 μg/mL and 10 μg/mL), and incubated in a 5% carbon dioxide incubator at 37° C. for 90 minutes. After centrifugation of the plate, 100 μL each of supernatant was collected from each well, and the radioactivity was measured with a gamma counter. The specific chromium release rate was determined according to the following equation:

specific chromium release rate (%)=(A−C)/(B−C)×100

where A represents the radioactivity (cpm) in a well, B represents the mean value of radioactivity (cpm) in a well where 100 μL of 2% NP-40 aqueous solution (Nonidet P-40, Nacalai Tesque Co, Ltd.) and 50 μL of HAVB were added to the target cells, and C represents the mean value of radioactivity (cpm) in a well where 150 μL of HAVB was added to the target cells. The test was carried out in triplicate, and mean value and standard deviation were calculated for the CDC activity (%).
The results are shown in FIG. 14. The anti-ROBO1 monoclonal antibody B2318C demonstrated dose-dependent CDC activity against ROBO1 expressing HEK293 cells.
The anti-ROBO1 monoclonal antibody B2318C demonstrated dose-dependent CDC activity on Alexander (PLC/PRF/5) cells, also a liver cancer cell line, which is similar to the effect against ROBO1 expressing HEK293 cell (FIG. 15).
Example 14
Measurement of ADCC Activity Using Mouse Bone Marrow-Derived Effector Cells
14-1. Preparation of Mouse Bone Marrow-Derived Effector Cell Solution
Bone marrow cells were collected from the femoral bone of a SCID mouse (10 weeks old male, Clea Japan), and suspended at 5×105 cells/mL in a 10% FBS/RPMI 1640 culture medium. Mouse GM-CSF (PeproTech) and human IL-2 (PeproTech) were added at 10 ng/mL and 50 ng/mL, respectively, and the cells were cultured in a 5% carbon dioxide incubator at 37° C. for 5 days. After the culture, the cells were peeled with a scraper, washed once with culture medium, suspended at 5×106/cells/mL in 10% FBS/RPMI 1640 culture medium to prepare a mouse bone marrow-derived effector cell solution.
14-2. Preparation of Target Cell
HEK293 cells overexpressing ROBO1 were maintained in a DMEM culture medium (manufactured by Sigma) containing 10% FBS (manufactured by ThermoTrace) and 500 ng/mL Geneticine (Invitrogen), and removed from the dish using the Cell Dissociation Buffer (Invitrogen). The cells were dispensed at 1×104 cells/well in each well of a 96-well U bottomed plate (Falcon), and cultured overnight. After the culture, 5.55 MBq of chromium-51 was added and incubated in a 5% carbon dioxide incubator at 37° C. for four hours. The cells were washed three times with culture medium, and 50 μL of 10% FBS/RPM11640 culture medium was added to prepare a target cell.
14-3. Chromium Release Test (ADCC Activity)
A volume of 50 μL of B2318C antibody (anti-ROBO1 monoclonal antibody) solution was added to target cells, and let to stand on ice for 15 minutes. Then, 100 μL of mouse bone marrow-derived effector cell solution (5×105 cells/well) was added, and incubated in a 5% carbon dioxide incubator at 37° C. for 4 hours (prepared at final antibody concentrations of 1 μg/mL and 10 μg/mL). The plate was centrifuged and the radioactivity in 100 μL of culture supernatant was measured with a gamma counter. The specific chromium release rate was determined according to the following equation:

specific chromium release rate (%)=(A−C)×100/(B−C)

where A represents the mean value of radioactivity (cpm) in a well, B represents the mean value of radioactivity (cpm) in a well where 100 μL of 2% NP-40 aqueous solution (Nonidet P-40, Code No. 252-23, Nacalai Tesque Co, Ltd.) and 50 μL of 10% FBS/RPMI culture medium were added to the target cells, and C represents the mean value of radioactivity (cpm) in a well where 150 μL of 10% FBS/RPMI culture medium was added to the target cells. The test was carried out in triplicate, and mean value and standard deviation were calculated for the ADCC activity (%).
The results are shown in FIG. 16. The anti-ROBO1 monoclonal antibody B2318C demonstrated dose-dependent ADCC activity against ROBO1 expressing HEK293 cells.
These results demonstrated that anti-ROBO1 monoclonal antibody would be effective in the treatment of ROBO1-expressing cancer.
The present invention is useful in the diagnosis and treatment of cancers, as well as in monitoring of progression of hepatitis.

## Claims

1. A method for treating ROBO1 expressing cancer comprising administrating to a patient in need of such a treatment a pharmaceutical composition comprising an antibody that binds to the third fibronectin region of ROBO1 as an active ingredient, wherein the antibody has ADCC or CDC activity.

2. The method according to claim 1, wherein the cancer is hepatocellular cancer.

3. A method for inducing cell damages in a ROBO1 expressing cell comprising bringing a ROBO1 expressing cell into contact with an antibody that binds to the third fibronectin region of ROBO1, wherein the antibody has ADCC or CDC activity.

4. A method for inhibiting the growth of a ROBO1 expressing cell comprising bringing a ROBO1 expressing cell into contact with an antibody that binds to the third fibronectin region of ROBO1, wherein the antibody has ADCC or CDC activity.

5. The method according to claim 4, wherein the ROBO1 expressing cell is a cancer cell.

6. The method according to claim 5, wherein the cancer cell is a hepatocellular cancer cell.

7. The method according to claim 3, wherein the ROBO1 expressing cell is a cancer cell.

8. The method according to claim 7, wherein the cancer cell is a hepatocellular cancer cell.

9. The method according to claim 1, wherein the third fibronectin region of ROBO1 is an amino acid sequence encoded by the KPN1-KPN1 restriction fragment of a nucleic acid amplified by the PCR method with a nucleotide sequence set forth in SEQ ID NO: 12 as a template, using a forward primer consisting of set forth in SEQ ID NO: 15 and the reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 16.

10. A method for treating ROBO1 expressing cancer comprising administrating to a patient in need of such a treatment a pharmaceutical composition comprising an antibody having ADCC or CDC activity as an active ingredient, wherein the antibody is obtained by immunization of a mammal with the third fibronectin region of ROBO1, wherein the third fibronectin region of ROBO1 is an amino acid sequence encoded by the KPN1-KPN1 restriction fragment of a nucleic acid amplified by the PCR method with the nucleotide sequence set forth in SEQ ID NO: 12 as a template, using a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and the reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 16.

11. The method according to claim 3, wherein the third fibronectin region of ROBO1 is an amino acid sequence encoded by the KPN1-KPN1 restriction fragment of a nucleic acid amplified by the PCR method with the nucleotide sequence set forth in SEQ ID NO: 12 as a template, using a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and the reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 16.

12. A method for inducing cell damages in a ROBO1 expressing cell comprising bringing a ROBO1 expressing cell into contact with an antibody having ADCC or CDC activity, wherein the antibody is obtained by immunization of a mammal with the third fibronectin region of ROBO1, wherein the third fibronectin region of ROBO1 is an amino acid sequence encoded by the KPN1-KPN1 restriction fragment of a nucleic acid amplified by the PCR method with the nucleotide sequence set forth in SEQ ID NO: 12 as a template, using a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO 15 and the reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 16.

13. The method according to claim 4, wherein the third fibronectin region of ROBO1 is an amino acid sequence encoded by the KPN1-KPN1 restriction fragment of a nucleic acid amplified by the PCR method with the nucleotide sequence set forth in SEQ ID NO: 12 as a template, using a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and the reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 16.

14. A method for inhibiting the growth of a ROBO1 expressing cell comprising bringing a ROBO1 expressing cell into contact with an antibody having ADCC or CDC activity, wherein the antibody is obtained by immunization of a mammal with the third fibronectin region of ROBO1, wherein the third fibronectin region of ROBO1 is an amino acid sequence encoded by the KPN1-KPN1 restriction fragment of a nucleic acid amplified by the PCR method with the nucleotide sequence set forth in SEQ ID NO: 12 as a template, using a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and the reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 16.