Determination of AM-binding proteins and the association of adrenomedullin (AM) therewith

Abstract

The present invention provides methods for the isolation, identification, and purification of adrenomedullin (AM)-binding proteins. Also, provided are methods for utilizing the purified AM-binding proteins, or functional portions thereof, to diagnose, treat, and monitor AM-related diseases, for example, diseases or disorders associated with abnormally elevated AM levels. In addition, the present invention provides a newly identified complex between AM and a specific AM-binding protein 1 (AMBP-1); which has been isolated and identified herein as factor H (fH). The invention also provides AM/AMBP complexes, particularly AM/FH complexes, and antibodies specifically reactive with this complexes. Further provided are methods for identifying and purifying complexes of AM and an AM binding protein using anti-AM/fH antibodies, and methods for treating conditions such as cancer or diabetes utilizing compositions comprising these antibodies. The present invention additionally provides methods for identifying antagonists agents that inhibit the function of AM, factor H, or the AM/factor H complex. The invention also provides methods for treating conditions such as cancer or diabetes using these antagonist agents.

1 . A method for measuring adrenomedullin levels in a sample, comprising: 
 i) incubating the sample with a chaotropic agent to dissociate adrenomedullin and factor H;    ii) fractionating the sample to obtain a peptide fraction; and    iii) quantifying adrenomedullin levels in the peptide fraction of the sample.    
     
     
         2 . The method according to  claim 1 , wherein the sample is selected from the group consisting of plasma, serum, cell lysate, and tissue extract samples.  
     
     
         3 . The method according to  claim 1 , wherein the chaotropic agent is sodium thiocyanate.  
     
     
         4 . The method according to  claim 1 , wherein the sample is fractionated by a C18 matrix.  
     
     
         5 . The method according to  claim 1 , wherein the adrenomedullin levels are quantified in step (iii) by radioimmunoassay analysis.  
     
     
         6 . A method for monitoring adrenomedullin concentrations in a subject having a condition associated with elevated levels of adrenomedullin, wherein the condition selected from the group consisting of heart disease, pulmonary disease, liver cirrhosis, cancer, diabetes, sepsis, and inflammation, comprising: 
 i) incubating a sample from the subject to be analyzed with isolated factor H, or an adrenomedullin-bindable peptide fragment thereof, under conditions allowing binding of factor H or the factor H peptide to adrenomedullin in the sample    ii) quantifying the amount of adrenomedullin bound to factor H or the factor H peptide; and    iii) comparing the levels of adrenomedullin in the subject with levels of adrenomedullin in normal individuals.    
     
     
         7 . The method according to  claim 6 , wherein the monitoring of adrenomedullin concentration assists in diagnosing a condition, determining the severity of a condition, or following the recovery course of a condition.  
     
     
         8 . The method according to  claim 6 , wherein the sample is selected from the group consisting of plasma, serum, cell lysate, and tissue extract samples.  
     
     
         9 . The method according to  claim 6 , wherein the cancer is selected from the group consisting of urinary bladder, urethral, renal, rectal, colon, small intestine, gastric, esophageal, salivary gland, gallbladder, liver, breast, vaginal, endometrial, ovarian, cervical, prostate, skin, lung, and brain cancers.  
     
     
         10 . The method according to  claim 6 , wherein the factor H or the factor H peptide comprises a label.  
     
     
         11 . The method according to  claim 10 , wherein the label is non-radioactive.  
     
     
         12 . The method according to  claim 10 , wherein the label is an enzyme.  
     
     
         13 . The method according to  claim 6 , wherein prior to step (i), the sample is fractionated to obtain a peptide fraction.  
     
     
         14 . The method according to  claim 13 , wherein the sample is fractionated using a C18 matrix.  
     
     
         15 . The method according to  claim 13  or  14 , wherein prior to fractionation, the sample is incubated with a chaotropic agent.  
     
     
         16 . The method according to  claim 15 , wherein the chaotropic agent is sodium thiocyanate.  
     
     
         17 . A kit for detecting adrenomedullin concentrations in a sample comprising: 
 i) factor H or an adrenomedullin-binding peptide fragment thereof; and    ii) at least one component to facilitate or detect AM-binding.    
     
     
         18 . The kit according to  claim 17 , wherein the peptide comprises a label.  
     
     
         19 . The kit according to  claim 17 , wherein the label is non-radioactive.  
     
     
         20 . The kit according to  claim 17 , wherein the label comprises an enzyme.  
     
     
         21 . A method of identifying an antagonist agent, comprising: 
 i) contacting adrenomedullin with a test compound under conditions that allow the adrenomedullin to bind to the test compound, and thereby form a stable association;    ii) detecting the adrenomedullin-test compound association of step (i); and    iii) contacting the adrenomedullin-test compound association with factor H under conditions that allow the test compound to inhibit the complexation of adrenomedullin and factor H, wherein inhibition indicates antagonist activity.    
     
     
         22 . A method of identifying an antagonist agent, comprising: 
 i) contacting factor H with a test compound under conditions to allow the factor to bind to the test compound, and thereby form a stable association;    ii) detecting the factor H-test compound association of step (i);    iii) contacting the factor H-test compound association with adrenomedullin under conditions that allow the test compound to inhibit the complexation of adrenomedullin and factor H, wherein inhibition indicates antagonist activity.    
     
     
         23 . A pharmaceutical composition comprising an antagonist agent identified according to the method of  claim 21  or  22 , and a physiologically acceptable carrier, excipient, or diluent.  
     
     
         24 . A method of inhibiting the growth or proliferation of cancer or tumor cells, comprising: 
 i) administering to cancer or tumor cells an antagonist agent identified according to the method of  claim 21  or  22  in an amount effective to inhibit complexation of adrenomedullin and factor H, and a physiologically acceptable carrier, excipient, or diluent; and    ii) inhibiting the growth or proliferation of the cancer or tumor cells.    
     
     
         25 . The method according to  claim 24 , wherein the cells are selected from the group consisting of urinary bladder, urethral, renal, rectal, colon, small intestine, gastric, esophageal, salivary gland, gallbladder, liver, breast, vaginal, endometrial, ovarian, cervical, prostate, skin, lung, and brain cancer or tumor cells.  
     
     
         26 . An isolated and purified protein complex comprising adrenomedullin and factor H.  
     
     
         27 . An antibody specifically reactive with an epitope formed by the complexation of adrenomedullin and factor H, or a complex-specific fragment thereof.  
     
     
         28 . The antibody according to  claim 27 , wherein the antibody is a monoclonal antibody.  
     
     
         29 . A pharmaceutical composition comprising the antibody according to  claim 28 , and a physiologically acceptable carrier, excipient, or diluent.  
     
     
         30 . A method of isolating adrenomedullin/factor H protein complexes from a biological sample, comprising: 
 i) providing a solid support having bound thereto the antibody of  claim 27  or  28 ;    ii) contacting said support with the sample; and    iii) eluting the adrenomedullin/factor H complex that binds to the antibody bound to the support.    
     
     
         31 . The method according to  claim 30 , wherein the biological sample is selected from the group consisting of plasma, serum, cell lysate, and tissue extract samples.  
     
     
         32 . A method for detecting adrenomedullin/factor H protein complexes in a biological sample, comprising: 
 i) incubating the sample with the antibody of  claim 27  or  28  under conditions allowing binding of the antibodies to the adrenomedullin/factor H complex in the sample; and    ii) determining binding of the antibodies to the adrenomedullin/factor H complex.    
     
     
         33 . The method according to  claim 32 , wherein the sample is selected from the group consisting of plasma, serum, cell lysate, and tissue extract samples.  
     
     
         34 . The method according to  claim 32 , wherein the antibodies are labeled.  
     
     
         35 . The method according to  claim 32 , wherein the antibodies are non-radioactively labeled.  
     
     
         36 . The method according to  claim 32 , wherein the antibodies are labeled with an enzyme.  
     
     
         37 . A kit for detecting adrenomedullin/factor H complex concentrations in a sample comprising: 
 i) an antibody specifically reactive with an epitope formed by the complexation of adrenomedullin and factor H, or a complex-specific fragment thereof; and    ii) at least one component to facilitate or detect adrenomedullin/factor H complex-binding.    
     
     
         38 . The kit according to  claim 37 , wherein the antibody comprises a label.  
     
     
         39 . The kit according to  claim 38 , wherein the label is non-radioactive.  
     
     
         40 . The kit according to  claim 38 , wherein the label comprises an enzyme.  
     
     
         41 . A method of inhibiting the growth or proliferation of cancer or tumor cells, comprising: 
 i) administering to cancer or tumor cells an antibody specifically reactive with an epitope formed by the complexation of adrenomedullin and factor H or a complex-specific fragment thereof, in an amount effective to inhibit activity of the adrenomedullin/factor H complex, and a physiologically acceptable carrier, excipient, or diluent; and    ii) inhibiting the growth or proliferation of the cancer or tumor cells.    
     
     
         42 . The method according to  claim 41 , wherein the cells are selected from the group consisting of urinary bladder, urethral, renal, rectal, colon, small intestine, gastric, esophageal, salivary gland, gallbladder, liver, breast, vaginal, endometrial, ovarian, cervical, prostate, skin, lung, and brain cancer or tumor cells.  
     
     
         43 . The method according to  claim 41 , wherein the antibody is humanized.  
     
     
         44 . The method according to  claim 41 , wherein the antibody is recombinant.  
     
     
         45 . A method of treating a condition associated with decreased secretion of insulin by pancreatic cells, comprising: 
 i) administering to the pancreatic cells an antagonist agent identified according to the method of  claim 21  or  22  in an amount effective to inhibit complexation of adrenomedullin and factor H, and a physiologically acceptable carrier, excipient, or diluent; and    ii) increasing the secretion of insulin by the pancreatic cells.    
     
     
         46 . The method according to  claim 45 , wherein the condition is selected from the group consisting of diabetes and pheochromocytoma.  
     
     
         47 . The method according to  claim 45 , wherein the condition is type 2 diabetes.  
     
     
         48 . A method of treating a condition associated with decreased secretion of insulin by pancreatic cells, comprising: 
 i) administering to the pancreatic cells an antibody specifically reactive with an epitope formed by the complexation of adrenomedullin and factor H or a complex-specific fragment thereof, in an amount effective to inhibit activity of the adrenomedullin/factor H complex, and a physiologically acceptable carrier, excipient, or diluent; and    ii) increasing the secretion of insulin by the pancreatic cells.    
     
     
         49 . The method according to  claim 48 , wherein the condition is selected from the group consisting of diabetes and pheochromocytoma.  
     
     
         50 . The method according to  claim 48 , wherein the condition is type 2 diabetes.  
     
     
         51 . A method of treating a condition associated with an autoimmune response to pancreatic cells, comprising: 
 i) administering factor H to the pancreatic cells in an amount effective to inhibit complement-mediated cell lysis, and a physiologically acceptable carrier, excipient, or diluent; and    ii) decreasing the autoimmune response to the pancreatic cells.    
     
     
         52 . The method according to  claim 51 , wherein the condition is type 1 diabetes.