Antibodies to troponin i and methods of use thereof

Abstract

The subject invention relates to antibodies to troponin I as well as methods of use thereof. In particular, such antibodies may be used to detect Troponin I in a patient and may also be used in the diagnosis of, for example, a myocardial infarction or acute coronary syndrome.

1 . A Chinese Hamster Ovary (CHO) cell line, referred to as TnI 19C7 AM1 hG1 CHO 204, designated by American Type Culture Collection (ATCC) deposit number PTA-9816. 
     
     
         2 . A recombinant antibody produced by said CHO cell line of  claim 1 . 
     
     
         3 . An isolated binding protein comprising an antigen-binding domain which binds to Troponin I, said antigen-binding domain comprising at least one complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of: GYTFTDYNLH (SEQ ID NO:52), YIYPYNGITGYNQKFKS (SEQ ID NO:53), DAYDYDYLTD (SEQ ID NO:54), RTSKNVGTNIH (SEQ ID NO:55), YASERLP (SEQ ID NO:56) and QQSNNWPYT (SEQ ID NO:57). 
     
     
         4 . The binding protein of  claim 3 , wherein said binding protein comprises at least 3 CDRs. 
     
     
         5 . The binding protein according to  claim 4 , wherein said binding protein comprises at least 6 CDRs. 
     
     
         6 . The binding protein according to  claim 3  further comprising a human acceptor framework. 
     
     
         7 . An isolated nucleic acid molecule encoding a binding protein, wherein the amino acid sequence of the variable heavy chain of said binding protein has at least 70% identity to SEQ ID NO.:25. 
     
     
         8 . The isolated nucleic acid molecule of  claim 7 , wherein the amino acid sequence of the variable light chain of said binding protein has at least 70% identity to SEQ ID NO.:28. 
     
     
         9 . An isolated nucleic acid molecule encoding a binding protein, wherein the amino acid sequence of the variable heavy chain of said binding protein is SEQ ID NO.:25. 
     
     
         10 . An isolated nucleic acid molecule encoding a binding protein, wherein the amino acid sequence of the variable light chain of said binding protein is SEQ ID NO.:28. 
     
     
         11 . The isolated nucleic acid molecule of  claim 10 , wherein said binding protein further comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO.:25. 
     
     
         12 . An isolated binding protein comprising the amino acid sequence of SEQ ID NO.:25. 
     
     
         13 . An isolated binding protein comprising the amino acid sequence of SEQ ID NO.:28. 
     
     
         14 . A vector comprising said isolated nucleic acid molecule of  claim 9  or  claim 10 . 
     
     
         15 . An isolated host cell comprising said vector of  claim 14 . 
     
     
         16 . A method of producing a binding protein capable of binding to Troponin I, comprising culturing said host cell of  claim 15  for a time and under conditions sufficient to produce said binding protein. 
     
     
         17 . An isolated protein produced according to the method of  claim 16 . 
     
     
         18 . A pharmaceutical composition comprising the binding protein of  claim 3  and a pharmaceutically acceptable carrier. 
     
     
         19 . A method of detecting Troponin I antigen in a test sample comprising the steps of:
 a) contacting said test sample with an antibody which binds to Troponin I and comprises the amino acid sequence of SEQ ID NO:25 for a time and under conditions sufficient for the formation of antibody/antigen complexes; and   b) detecting presence of said complexes, presence of said complexes indicating presence of Troponin I antigen in said test sample.   
     
     
         20 . The method of  claim 19  wherein said antibody further comprises the amino acid sequence of SEQ ID NO:28. 
     
     
         21 . The method of  claim 20  wherein said antibody is produced by CHO cell line TnI 19C7 AM1 hG1 CHO 204 having ATCC deposit designation PTA-9816 
     
     
         22 . A method of detecting Troponin I antigen in a test sample comprising the steps of:
 a) contacting said test sample with a first antibody which binds to Troponin I and comprises SEQ ID NO:25 for a time and under conditions sufficient for the formation of first antibody/antigen complexes;   b) adding a conjugate to said first antibody/antigen complexes, wherein said conjugate comprises a second antibody attached to a signal generating compound capable of generating a detectable signal, for a time and under conditions sufficient to form first antibody/antigen/second antibody complexes; and   c) detecting presence of a signal generating by said signal generating compound, presence of said signal indicating presence of Troponin I antigen in said test sample.   
     
     
         23 . The method of  claim 22 , wherein said first antibody further comprises SEQ ID NO:28. 
     
     
         24 . The method of  claim 23 , wherein said first antibody is produced by CHO cell line TnI 19C7 AM1 hG1 CHO 204 having ATCC deposit designation PTA-9816. 
     
     
         25 . A method of detecting Troponin I antigen in a test sample comprising the steps of:
 a) contacting Troponin I antigen with an antibody to Troponin I for a time and under conditions sufficient to form Troponin I antigen/antibody complexes, wherein said antibody comprises the amino acid sequence of SEQ ID NO:25 and is labeled with a signal-generating compound capable of generating a detectable signal;   b) adding said test sample to said Troponin I antigen/antibody complexes for a time and under conditions sufficient to form Troponin I antigen/antibody/Troponin I test sample antigen complexes; and   c) detecting presence of a signal generating by said signal generating compound, presence of said signal indicating presence of Troponin I antigen in said test sample.   
     
     
         26 . The method of  claim 25  wherein said antibody further comprises the amino acid sequence of SEQ ID NO:28. 
     
     
         27 . The method of  claim 24  wherein said antibody is produced by CHO cell line TnI 19C7 AM1 hG1 CHO 204 cell line having ATCC deposit designation PTA-9816. 
     
     
         28 . A method of detecting Troponin I antigen in a test sample comprising the steps of:
 a) contacting said test sample with 1) a Troponin I reference antigen, wherein said antigen is attached to a signal generating compound capable of generating a detectable signal and 2) an antibody to Troponin I antigen wherein said antibody comprises the amino acid sequence of SEQ ID NO:25, for a time and under conditions sufficient to form Troponin I reference antigen/antibody complexes; and   b) detecting a signal generated by said signal generating compound, wherein the amount of Troponin I antigen detected in said test sample is inversely proportional to the amount of Troponin I reference antigen bound to said antibody.   
     
     
         29 . The method of  claim 28  wherein said antibody further comprises the amino acid sequence of SEQ ID NO:28. 
     
     
         30 . The method of  claim 29  wherein said antibody is produced by CHO cell line TnI 19C7 AM1 hG1 CHO 204 having ATCC deposit designation PTA-9816. 
     
     
         31 . A method of diagnosing acute coronary syndrome or myocardial infarction in a patient suspected of having one of these conditions comprising the steps of:
 a) isolating a biological sample from said patient;   b) contacting said biological sample with an antibody which binds to Troponin I and comprises the amino acid sequence of SEQ ID NO:25, for a time and under conditions sufficient for formation of Troponin I antigen/antibody complexes; and   c) detecting presence of said Troponin I antigen/antibody complexes;   d) dissociating said Troponin I antigen present in said complexes from said antibody present in said complexes; and   e) measuring the amount of dissociated Troponin I antigen, wherein an amount of Troponin I antigen greater than approximately 1-5 times the Troponin I value of the 99 th  percentile of a normal population indicates a diagnosis of acute coronary syndrome or myocardial infarction in said patient.   
     
     
         32 . A method of diagnosing acute coronary syndrome or myocardial infarction in a patient suspected of having one of these conditions comprising the steps of:
 a) isolating a biological sample from said patient;   b) contacting said biological sample with a first antibody which binds to Troponin I and comprises the amino acid sequence of SEQ ID NO:25 for a time and under conditions sufficient for the formation of Troponin I antigen/antibody complexes;   c) adding a conjugate to the resulting Troponin I antigen/antibody complexes for a time and under conditions sufficient to allow said conjugate to bind to the bound Troponin I antigen, wherein said conjugate comprises a second antibody attached to a signal generating compound capable of generating a detectable signal;   d) detecting the presence of Troponin I antigen which may be present in said biological sample by detecting a signal generated by said signal generating compound; and   e) measuring the amount of Troponin I antigen present in said test sample by measuring the intensity of said signal, an amount of Troponin I antigen greater than approximately 1-5 times the value of the 99 th  percentile of a normal population indicating a diagnosis of acute coronary syndrome or myocardial infarction in said patient.   
     
     
         33 . A kit comprising a container containing said recombinant antibody of  claim 2  or said binding protein of  claim 3 . 
     
     
         35 . An isolated binding protein comprising an antigen-binding domain, wherein said antigen-binding domain comprises at least one CDR comprising an amino acid sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   CDR-VH1. 
                 
                 
                 
                 
               
                     
                   X 1 -X 2 -X 3 -X 4 -X 5- X 6- X 7- X 8- X 9- X 10 , 
                   (SEQ ID NO: 63) 
                 
             
                
               
            
             
                
               
            
           
         
       
       wherein:
 X 1  is G; 
 X 2  is Y; 
 X 3  is T or S; 
 X 4  is F; 
 X 5  is T; 
 X 6  is D; 
 X 7  is Y; 
 X 8  is N; 
 X 9  is I or L; and 
 X 10  is H. 
 CDR-VH2. X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 X 13 -X 14 -X 15 -X 16 -X 17  (SEQ ID 
 
       
         
           
                 
                 
               
                     
                   CDR-VH2. 
                 
                     
                   (SEQ ID NO: 64) 
                 
                     
                   X 1 -X 2 -X 3 -X 4 -X 5 -X 6- X 7- X 8- X 9- X 10- X 11- X 12- X 13- X 14-   
                 
                     
                     
                 
                     
                   X 15- X 16- X 17 , 
                 
             
                
                
                
                
                
               
            
           
         
       
       wherein:
 X 1  is Y; 
 X 2  is I; 
 X 3  is Y; 
 X 4  is P; 
 X 5  is Y; 
 X 6  is N; 
 X 7  is G; 
 X 8  is I; 
 X 9  is T; 
 X 10  is G; 
 X 11  is Y; 
 X 12  is N; 
 X 13  is Q; 
 X 14  is K; 
 X 15  is F; 
 X 16  is K; and 
 X 17  is S. 
 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 66) 
                 
                 
                 
                 
               
                     
                   CDR-VH3. 
                   X 1 -X 2 -X 3 -X 4 -X 5 -X 6- X 7- X 8- X 9- X 10- X 11 , 
                 
             
                
               
            
             
                
               
            
           
         
       
       wherein:
 X 1  is D; 
 X 2  is A or F; 
 X 3  is Y; 
 X 4  is D; 
 X 5  is Y or S; 
 X 6  is D; 
 X 7  is W, Y or A; 
 X 8  is L; 
 X 9  is A or T; and 
 X 10  is Y or D. 
 
       
         
           
                 
                 
               
                     
                   CDR-VL1. 
                 
                 
                 
                 
               
                     
                   X 1 -X 2 -X 3 -X 4 -X 5 -X 6- X 7 , 
                   (SEQ ID NO: 66) 
                 
             
                
               
            
             
                
               
            
           
         
       
       wherein:
 X 1  is R; 
 X 2  is A or T; 
 X 3  is S; 
 X 4  is Q or K; 
 X 5  is S or N; 
 X 6  is I or V; 
 X 7  is G; 
 X 8  is T; 
 X 9  is N; 
 X 10  is I; and 
 X 11  is Y or H. 
 
       
         
           
                 
                 
               
                     
                   CDR-VL2. 
                 
                 
                 
                 
               
                     
                   X 1 -X 2 -X 3 -X 4 -X 5 -X 6- X 7- X 8- X 9- X 10 , 
                   (SEQ ID NO: 67) 
                 
             
                
               
            
             
                
               
            
           
         
       
       wherein:
 X 1  is Y; 
 X 2  is A or G; 
 X 3  is S or T; 
 X 4  is E; 
 X 5  is S or R; 
 X 6  is I, L or V; and 
 X 7  is S, P or F. 
 
       and 
       
         
           
                 
                 
               
                     
                   CDR-VL3. 
                 
                 
                 
                 
               
                     
                   X 1 -X 2 -X 3 -X 4 -X 5 -X 6- X 7- X 8- X 9 , 
                   (SEQ ID NO: 68) 
                 
             
                
               
            
             
                
               
            
           
         
       
       wherein:
 X 1  is Q; 
 x 2  is Q; 
 X 3  is S; 
 X 4  is N; 
 X 5  is N; 
 X 6  is W; 
 X 7  is P; 
 X 8  is Y; and 
 X 9  is T. 
 
     
     
         36 . The binding protein of  claim 3 , wherein said binding protein is selected from the group consisting of: an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, and a functionally active epitope-binding fragment thereof.