Use of p97 and iron binding proteins as diagnostic and therapeutic agents

Abstract

The invention related to a GPI-anchored p97 and a soluble form of p97 and derivatives thereof and methods for preparing the same. Methods of using p97 in modulating iron transport, in the delivery of therapeutic agents, and in the treatment of conditions involving disturbances in iron metabolism are described. The treatment and diagnosis of Alzheimer's Disease in view of the finding that p97 and transferrin receptor are markers for microglial cells associated with senile plaques are also described.

We claim:  
     
         1 . Isolated p97 having a glycosyl-phosphatidylinositol anchor.  
     
     
         2 . A method of preparing a cleaved form of the isolated p97 having a glycosyl-phosphatidylinositol anchor as claimed in  claim 1 , comprising incubating a cell which expresses the isolated p97 on its surface with an enzyme that cleaves glycosyl-phosphatidylinositol (GPI) anchors, to produce the cleaved form of the isolated p97, and isolating the cleaved form.  
     
     
         3 . An isolated soluble p97 which is hydrophilic.  
     
     
         4 . The isolated soluble p97 as claimed in  claim 3  labelled with a label selected from the group consisting of fluorescent molecules, luminescent molecules, enzymes, substances having therapeutic activity, toxins, and radionuclides.  
     
     
         5 . A method for treating conditions involving disturbances in iron metabolism comprising administering an iron modulating amount of p97 or a stimulant, agonist or antagonist of p97.  
     
     
         6 . A method for treating conditions involving disturbances in iron metabolism comprising administering an iron modulating amount of p97, or a stimulant, agonist or antagonist thereof.  
     
     
         7 . A method for identifying a stimulant, agonist or antagonist of p97-mediated iron uptake comprising: incubating a cell expressing p97 on its surface and a substance suspected of being a stimulant, agonist or antagonist of p97 in the presence of iron and in the absence of transferrin, measuring the amount of iron uptake into the cell and identifying a stimulant, agonist or antagonist of p97-mediated iron uptake by comparing the amount of iron uptake in the cell with the amount of iron uptake in a cell from a control incubation in the absence of the substance.  
     
     
         8 . A method as claimed in  claim 7  wherein the cell is incubated in the presence of labelled iron and wherein the amount of iron uptake in the cell is determined by measuring the amount of labelled iron in the cell.  
     
     
         9 . A method as claimed in  claim 8  wherein the label is radioactive.  
     
     
         10 . A method as claimed in  claim 7  wherein the substance is an antibody specific for p97.  
     
     
         11 . A composition for delivering an agent across the blood brain barrier comprising p97 conjugated to the agent, a substance which is capable of specifically binding to p97 conjugated to the agent, or a p97 fusion protein containing p97 or the N-terminal or C-terminal portion of p97 sequence fused to the agent, and a pharmaceutically acceptable carrier or diluent.  
     
     
         12 . A method of delivering an agent across the blood brain barrier comprising peripherally administering a composition as claimed in  claim 11 .  
     
     
         13 . A method for diagnosing or monitoring Alzheimer's Disease in a patient, comprising determining the concentration of p97 in a sample from the patient and comparing the determined concentration to the level of p97 in other samples from the patient, control subjects and/or Alzheimer Disease patients.  
     
     
         14 . A method for diagnosing or monitoring Alzheimer's Disease, as claimed in  claim 13  which is a radioimmunoassay, immunofluorescent assay, competitive assay, or enzyme linked immunosorbant assay.  
     
     
         15 . A method for diagnosing or monitoring Alzheimer's Disease as claimed in  claim 13  wherein the sample from the patient is a serum sample.  
     
     
         16 . A method for diagnosing or monitoring Alzheimer's Disease as claimed in  claim 13  wherein the sample from the patient is a cerebrospinal fluid sample.  
     
     
         17 . A method for diagnosing or monitoring Alzheimer's disease as claimed in  claim 13  wherein the sample is from a patient being monitored to assess the efficacy of a therapeutic treatment for Alzheimer's Disease.  
     
     
         18 . A method for diagnosing or monitoring Alzheimer's disease as claimed in  claim 13  wherein the therapeutic treatment is the administration of a pharmaceutical composition suspected of affecting Alzheimer's Disease.  
     
     
         19 . A method for diagnosing or monitoring Alzheimer's disease as claimed in  claim 13  wherein the patient is diagnosed as having Alzheimer's disease by a serum concentration of p97 of at least 20 ng/ml.  
     
     
         20 . A method for monitoring Alzheimer's Disease, comprising detecting the presence of transferrin receptors, detecting the presence of p97 on microglial cells associated with amyloid plaques in a patient or detecting the presence of p97 in serum or cerebrospinal fluid.  
     
     
         21 . A method for treating Alzheimer's Disease in a patient comprising administering p97, transferrin, transferrin receptor, antibody specific to p97 or transferrin receptor, or an iron chelator to deplete iron in the brain of the patient.  
     
     
         22 . A method for treating Alzheimer's Disease comprising the step of administering to a patient p97, transferrin receptor, or a substance which is capable of binding to p97 or labelled transferrin receptor which are labelled with a substance having therapeutic activity or a toxin.