Nucleolin-mediated cancer diagnostics and therapy

Abstract

The present invention provides for diagnostic kits for identifying cancer patients who are more susceptible to cancer therapies employing endostatin and other angiogenesis inhibitors, based upon the discovery that Nucleolin is a specific receptor for Endostatin. In particular, the diagnostic kits include antibody molecules against Nucleolin, DNA or RNA molecules that specifically bind to nucleic acid molecules encoding Nucleolin. The present invention also discloses methods of screening for angiogenesis inhibitors which specifically interact with Nucleolin, and act as angiogenesis inhibitors in an analogous manner as Endostatin. In addition, the present invention discloses methods of inhibiting the proliferation of endothelial cells or angiogenesis of tumor by administering an anti-nucleolin antibody linked to a cytotoxic agent such as tumor necrosis factor alpha to the endothelial cells.

1 . A kit for determining the susceptibility of a subject to endostatin cancer therapy, comprising: 
 a. A label that labels nucleolin; and    b. A usage instruction for performing a screening of a sample of said subject with said label such as that an amount of nucleolin present in the sample is determined.    
     
     
         2 . The kit of  claim 1 , wherein the subject is a mammal.  
     
     
         3 . The kit of  claim 1 , wherein the subject is a human.  
     
     
         4 . The kit of  claim 1 , wherein the nucleolin being labeled is cell surface nucleolin.  
     
     
         5 . The kit of  claim 1 , wherein the nucleolin being labeled is systemic nucleolin.  
     
     
         6 . The kit of  claim 1 , wherein the label comprises an antibody that specifically binds to nucleolin.  
     
     
         7 . The kit of  claim 6 , wherein the antibody is a monoclonal antibody.  
     
     
         8 . The kit of  claim 6 , wherein the antibody is a polyclonal antibody.  
     
     
         9 . The kit of  claim 1 , wherein the label comprises a nucleic acid molecule.  
     
     
         10 . The kit of  claim 9 , wherein the nucleic acid molecule is a DNA molecule.  
     
     
         11 . The kit of  claim 9 , wherein the nucleic acid molecule is an RNA molecule.  
     
     
         12 . A method of determining the likelihood of success of endostatin cancer therapy in a subject, comprising: 
 a. Screening a sample from said subject for the level of expression of nucleolin; and    b. Determining if said subject is susceptible to endostatin cancer therapy based on the amount of nucleolin expression.    
     
     
         13 . A method of producing a nucleolin-specific angiogenesis inhibitor effective in inhibiting angiogenesis, comprising: 
 a. Applying an appropriate binding assay to a pool of candidate molecules, thereby obtaining a plurality of nucleolin-specific molecules;    b. Testing each of the plurality of nucleolin-specific molecules for its effectiveness of inhibiting angiogenesis using an anti-angiogenesis assay; and    c. Selecting the resulting nucleolin-specific molecule which is effective in inhibiting angiogenesis as demonstrated by the anti-angiogenesis assay.    
     
     
         14 . The method of  claim 13 , wherein the nucleolin-specific angiogenesis inhibitor is a protein or peptide.  
     
     
         15 . The method of  claim 13 , wherein the nucleolin-specific angiogenesis inhibitor is a small molecule.  
     
     
         16 . The method of  claim 13 , wherein the nucleolin-specific angiogenesis inhibitor is used in curing an angiogenesis-dependent disease.  
     
     
         17 . The method of  claim 16 , wherein the angiogenesis-dependent disease is a cancer.  
     
     
         18 . The method of  claim 16 , wherein the angiogenesis-dependent disease is an endothelial cell disease.  
     
     
         19 . A method of selecting an angiogenesis inhibitor having the ability to inhibit endothelial proliferation and/or migration when added to proliferating endothelial cells in vitro, comprising the steps of: 
 a. Using a pharmaceutically acceptable method to discover molecules that specifically interact with nucleolin as the target molecule;    b. Testing the molecules thus derived from step (a) for their effectiveness in inhibiting endothelial cell proliferation and/or migration; and    c. Determining the molecule thus derived which are effective in inhibition of endothelial cell proliferation and/or migration, wherein the effectiveness of the anti-angiogenesis function of said molecule is compared to that of endostatin.    
     
     
         20 . The method of  claim 19 , wherein the nucleolin-specific molecule is a protein or polypeptide.  
     
     
         21 . The method of  claim 19 , wherein the nucleolin-specific molecule is a small molecule.  
     
     
         22 . A method of increasing the receptiveness of a target cell to an angiogenesis inhibitor, comprising: 
 a. Introducing an exogenous nucleolin into the target cells, thereby obtaining a plurality of modified target cells expressing the exogenous nucleolin; and    b. Measuring the killing rate of the modified target cells by endostatin.    
     
     
         23 . The method of  claim 22 , wherein the target cell is a cancer cell.  
     
     
         24 . The method of  claim 22 , wherein the target cell is an endothelial cell.  
     
     
         25 . The method of  claim 22 , wherein said angiogenesis inhibitor is endostatin.  
     
     
         26 . The method of  claim 22 , wherein the exogenous nucleolin is introduced into the target cell via a viral vector.  
     
     
         27 . A method of enhancing the anti-angiogenesis effect of an angiogenesis inhibitor on a target endothelial cell, comprising 
 a. Introducing into said target cell a pharmaceutically effective amount of exogenous nucleolin molecule, said nucleolin molecule being able to express in said target cell; and    b. Incubating said target cell with said angiogenesis inhibitor, thereby causing the inhibition of the growth of said target cell.    
     
     
         28 . The method of  claim 27 , wherein said angiogenesis inhibitor is endostatin.  
     
     
         29 . The method of  claim 27 , wherein said target endothelial cell is a cancer cell.  
     
     
         30 . A diagnostic kit for assaying the individual sensitivity of target cells towards angiogenesis inhibitors, comprising: 
 a. A molecule that specifically bind to an nucleolin molecule; and    b. A pharmaceutically acceptable carrier.    
     
     
         31 . The diagnostic kit of  claim 30 , wherein the angiogenesis inhibitor is endostatin.  
     
     
         32 . The diagnostic kit of  claim 30 , wherein the target cell is a cancer cell.  
     
     
         33 . A diagnostic kit for determining the target cancer cells susceptible to anti-angiogenesis inhibitor treatment, comprising: 
 a. An antibody against nucleolin; and    b. A pharmaceutically acceptable carrier.    
     
     
         34 . The diagnostic kit of  claim 33 , wherein the antibody is a polyclonal antibody.  
     
     
         35 . The diagnostic kit of  claim 33 , wherein the antibody is a monoclonal antibody.  
     
     
         36 . A method of determining a patient's sensitivity to endostatin therapy, comprising contacting a sample of a patient with an antibody against nucleolin, and detecting the formation of a complex between the antibody and nucleolin, a higher level of presence of the complex indicating a higher likelihood of success of endostatin therapy.  
     
     
         37 . A method of increasing the efficacy of angiogenesis inhibitors on controlling the growth of a cancer in a patient having such cancer, comprising: 
 a. Identifying the presence of the level of endogenous nucleolin molecules in a sample of the cancer of said patient; and    b. Determining the likelihood of efficacy of angiogenesis inhibitor on said cancer patient using the level of expression of nucleolin in said patient, a higher level of nucleolin indicating a higher degree of success of such angiogenesis inhibitor treatment.    
     
     
         38 . The method of  claim 37 , wherein said angiogenesis inhibitor is endostatin.  
     
     
         39 . The method of  claim 37 , wherein the detection of nucleolin level in such sample of said patient is performed by immunoprecipitation of anti-nucleolin antibody and nucleolin in said sample.  
     
     
         40 . A method of identifying target cancer cells which are susceptible to an anti-angiogenesis inhibitor treatment, comprising: 
 a. Generating an anti-nucleolin antibody;    b. Contacting a sample from a subject with said anti-nucleolin antibody; and    c. Identifying target cancer cells that are susceptible to anti-angiogenesis inhibitor treatment as indicated by the level of nucleolin present in the sample, a higher level indicating a higher susceptibility.    
     
     
         41 . The method of  claim 40 , wherein the anti-nucleolin antibody is polyclonal antibody.  
     
     
         42 . The method of  claim 40 , wherein the anti-nucleolin antibody is monoclonal antibody.  
     
     
         43 . The method of  claim 40 , wherein the anti-angiogenesis inhibitor is endostatin.  
     
     
         44 . A method of inhibiting the proliferation and/or migration of a plurality of endothelial cells in a cell sample, comprising: 
 a. Linking an anti-nucleolin antibody with a cytotoxic agent, thereby forming an anti-nucleolin toxic antibody; and    b. Administering said anti-nucleolin toxic antibody to said cell sample, thereby inhibiting the proliferation and/or growth of said plurality of endothelia cells.    
     
     
         45 . The method of  claim 44 , wherein the cytotoxic agent is a cytokine.  
     
     
         46 . The method of  claim 44 , wherein the cytotoxic agent is a tumor necrosis factor.  
     
     
         47 . The method of  claim 44 , wherein the cell sample is derived from cancer patients.