Monoclonal antibodies against human colon carcinoma-associated antigens and uses therefor

Abstract

Monoclonal antibodies, in particular 33.28 and 31.1, and chimeric antibodies, in particular mouse/humans chimeric Chi #1 specific for glycoprotein antigens of colon carcinoma-associated antigens which are immunogenic in humans, are disclosed. Such antibodies, and fragments and derivatives thereof, are useful in immunodiagnosis and immunotherapy of human colon, breast, and ovarian cancer, and for purification of antigens which can serve as immunotherapeutic agents. Methods of detecting the colon carcinoma-associated antigen in a sample, and methods for treating subjects having colon, breast, and ovarian carcinomas are disclosed.

1. A monoclonal antibody specific for a purified human colon carcinoma-associated protein antigen, wherein said antigen has the following characteristics:
 (a) said antigen is purified to the extent that the membrane fractions are free of HL-A antigen and are substantially free from non-immunogenic glycoprotein fractions;  
 (b) said antigen is not detectable on normal colon cancer free human tissues;  
 (c) said antigen is not detectable on human carcinoma cells other than colon carcinoma cells;  
 (d) said antigen is specifically immunogenic in humans; and  
 (e) said antigen induces an immune response in humans having colon carcinoma which is expressed as cell mediated immunity  which is murine monoclonal antibody  33 . 28  as produced by hybridoma PCA  33 . 28 , deposited with the American Type Culture Collection and assigned accession number PTA- 5413 . 
 
     
     
       2. An antibody according to  claim 1  which is mouse monoclonal antibody  33 . 28  (ATCC HB- 12315 ) or an antibody which binds specifically to a colon carcinoma-associated epitope that specifically binds to monoclonal antibody  3328 . 
     
     
       3. An antibody according to claim  2    1  wherein said colon carcinoma-associated antigen is a protein having a molecular weight of about 61.1 kilodaltons as measured by gradient polyacrylamide gel electrophoresis. 
     
     
       4. An antibody according  claim 1   A monoclonal antibody specific for a purified human colon carcinoma-associated protein antigen, which is mouse monoclonal antibody  31 . 1 , as produced by hybridoma PCA  31.1 (ATCC HB-12314) , deposited with the American Type Culture Collection and assigned accession number PTA-2497 or an antibody which binds specifically to a colon carcinoma-associated epitope that specifically binds to monoclonal antibody  31 . 1  . 
     
     
       5. An  A monoclonal antibody according to  claim 4  wherein said colon carcinoma-associated antigen is a protein having a molecular weight of about  72  kilodaltons  which comprises an antigen-binding region obtained from the H chain of a murine monoclonal antibody  33 . 28  as produced by hybridoma PCA  33 . 28 , deposited with the American Type Culture collection and assigned action number PTA- 5413 . 
     
     
       6. An antibody according to claim  2    1  wherein said colon carcinoma-associated antigen is a glycoprotien, the protein component having a molecular weight of 61.1 kilodaltons as measured by gradient polyacrylamide gel electrophoresis. 
     
     
       7. An antibody according to  claim 1 ,  4  or  5  immobilized on a solid phase. 
     
     
       8. An antibody according to  claim 1 ,  4  or  5  which is detectably labeled. 
     
     
       9. An antibody according to  claim 8  wherein said detectable label is a radiolabel. 
     
     
       10. An antibody according to  claim 1 ,  4  or  5  conjugated to a cytotoxic radionuclide. 
     
     
       11. An antibody according to  claim 1 ,  4  or  5  conjugated to a cytotoxic drug. 
     
     
       12. An antibody according to  claim 1 ,  4  or  5  conjugated to a cytotoxic protein. 
     
     
       13. A composition comprising an antibody according to  claim 10  in combination with a pharmaceutically acceptable carrier. 
     
     
       14. A composition comprising an antibody according to  claim 11  in combination with a pharmaceutically acceptable carrier. 
     
     
       15. A composition comprising an antibody according to  claim 12  in combination with a pharmaceutically acceptable carrier. 
     
     
       16. A monoclonal antibody against the monoclonal antibody of  claim 1 . 
     
     
       17. A monoclonal antibody against the monoclonal antibody of  claim 2 . 
     
     
       18. A monoclonal antibody against the monoclonal antibody of  claim 3 . 
     
     
       19. A monoclonal antibody against the monoclonal antibody of  claim 4 . 
     
     
       20. A monoclonal antibody against the monoclonal antibody of  claim 5 . 
     
     
       21. A monoclonal antibody against the monoclonal antibody of  claim 6 . 
     
     
       22. An immunoassay for detecting a colon carcinoma-associated antigen which binds to mouse monoclonal antibody  33.28 (ATCC HB-12315)  as produced by hybridoma PCA 33.28, deposited with the American Type Culture Collection and assigned accession number PTA-5413,  in a sample comprising:
 (a) contacting said sample with an effective binding amount of the antibody according to  claim 1 ; and  
 (b) detecting said antigen by detecting the binding of the antibody to the purified  colon carcinoma-associated protein antigen.  
 
     
     
       23. An immunoassay for detecting a colon carcinoma-associated antigen which binds to mouse monoclonal antibody  31.1 (ATCC HB-12314) , as produced by hybridoma PCA 31.1, deposited with the American Type Culture Collection and assigned accession number PTA-2497,  in a sample comprising:
 (a) contacting said sample with an effective binding amount of the antibody according to claim  1    4  or  claim 5 ; and  
 (b) detecting said antigen by detecting the binding of the antibody to the purified  colon carcinoma-associated protein antigen.  
 
     
     
       24. A method for diagnosing colon cancer in humans comprising:
 (a) removing a histological specimen from a patient suspected of having a colon cancer;  
 (b) contacting the specimen with monoclonal antibody  33.28 (ATCC HB-12315) , as produced by hybridoma PCA 33.28, deposited with the American Type Culture Collection and assigned accession number PTA-5413 ;  
 (c) staining the specimen with an immunohistochemical stain; and  
 (d) detecting the presence of the antigen-antibody complex by the stain.  
 
     
     
       25. A method for diagnosing colon cancer in humans comprising:
 (a) removing a histological specimen from a patient suspected of having colon-  carcinoma;  
 (b) contacting the specimen with mouse monoclonal antibody  31.1 (ATCC HB-12314) , as produced by hybridoma PCA 31.1, deposited with the American Type Culture Collection and assigned accession number PTA-2497) ;  
 (c) staining the specimen with an immunohistochemical stain; and  
 (d) detecting the presence of the antigen-antibody complex.  
 
     
     
       26. A method according to  claim 24  wherein the stain is an avidin-biotin immunoperoxidase stain. 
     
     
       27. A method according to  claim 25  wherein the stain is an avidin-biotin immunoperoxidase stain. 
     
     
       28. A kit for the immunohistochemical detection of colon carcinoma comprising:
 (a) mouse monoclonal antibody  31.1 (ATCC HB-12314) , as produced by hybridoma PCA 31.1, deposited with the American Type Culture Collection and assigned accession number PTA-2497 ;  
 (b) reagents for immunoperoxidase and secondary antibody;  
 (c) immunoperoxidase; and  
 (d) colorizing reagents.  
 
     
     
       29. A kit for the immunohistochemical detection of colon carcinoma comprising:
 (a) mouse monoclonal antibody  33.28 (ATCC HB-12315 , as produced by hybridoma PCA 33.28, deposited with the American Type Culture Collection and assigned accession number PTA-5413 ;  
 (b) reagents for immunoperoxidase and secondary antibody;  
 (c) immunoperoxidase; and  
 (d) colorizing reagents.  
 
     
     
       30. A compartmentalized kit for the detection of a human colon carcinoma-associated antigen, wherein the antigen has the following characteristics:
 (a) said antigen is purified to the extent that the membrane fractions are free of HL-A antigen and are substantially free from non-immunogenic glycoprotein fractions;    (b) said antigen is not detectable on normal colon cancer free human tissues;    (c) said antigen is not detectable on human carcinoma cells other than colon carcinoma cells;    (d) said antigen is specifically immunogenic in humans; and    (e) said antigen induces an immune response in humans having colon carcinoma which is expressed as cell mediated immunity,    
       said kit comprising a first container adapted to contain an antibody to said antigen or an active component thereof, and a second container adapted to contain a second antibody to said antigen or an active component thereof, said second antibody being labeled with a reporter molecule capable of giving a detectable signal. 
     
     
       31. A kit according to  claim 30  wherein the reporter molecule is a radioisotope, an enzyme, a fluorescent molecule, a chemiluminescent molecule or a bioluminescent molecule. 
     
     
       32. A kit according to  claim 30  wherein the reporter molecule is an enzyme. 
     
     
       33. A kit according to  claim 30  wherein the kit further comprises a third container adapted to contain a substrate for the enzyme. 
     
     
       34. A compartmentalized kit for the detection of a human colon carcinoma-associated antigen, wherein the antigen has the following characteristics:
 (a) said antigen is purified to the extent that the membrane fractions are free of HL-A antigen and are substantially free from non-immunogenic glycoprotein fractions;    (b) said antigen is not detectable on normal colon cancer free human tissues;    (c) said antigen is not detectable on human carcinoma cells other than colon carcinoma cells;    (d) said antigen is specifically immunogenic in humans; and    (e) said antigen induces an immune response in humans having colon carcinoma which is expressed as cell mediated immunity,    
       said kit comprising a first container adapted to contain monoclonal antibody  31 . 1  (ATCC HB- 12314 ) to said antigen and a second container adapted to contain a second antibody to said antigen or an active component thereof, said second antibody being labeled with a reporter molecule capable of giving a detectable signal. 
     
     
       35. A kit according to  claim 34  wherein the reporter molecule is a radioisotope, an enzyme, a fluorescent molecule, a chemiluminescent molecule or a bioluminescent molecule. 
     
     
       36. A kit according to  claim 32  wherein the reporter molecule is an enzyme. 
     
     
       37. A kit according to  claim 33  wherein the kit further comprises a third container adapted to contain a substrate for the enzyme. 
     
     
       38. A compartmentalized kit for the detection of a human colon carcinoma-associated antigen, wherein the antigen has the following characteristics:
 (a) said antigen is purified to the extent that the membrane fractions are free of HL-A antigen and are substantially free from non-immunogenic glycoprotein fractions;    (b) said antigen is not detectable on normal colon cancer free human tissues;    (c) said antigen is not detectable on human carcinoma cells other than colon carcinoma cells;    (d) said antigen is specifically immunogenic in humans; and    (e) said antigen induces an immune response in humans having colon carcinoma which is expressed as cell mediated immunity,    
       said kit comprising a first container adapted to contain monoclonal antibody  33 . 28  (ATCC HB- 12315 ) to said antigen and a second container adapted to contain a second antibody to said antigen or an active component thereof, said second antibody being labeled with a reporter molecule capable of giving a detectable signal. 
     
     
       39. A kit according to  claim 38  wherein the reporter molecule is a radioisotope, an enzyme, a fluorescent molecule, a chemiluminescent molecule or a bioluminescent molecule. 
     
     
       40. A kit according to  claim 38  wherein the reporter molecule is an enzyme. 
     
     
       41. A kit according to  claim 38  wherein the kit further comprises a third container adapted to contain a substrate for the enzyme. 
     
     
       42. The monoclonal antibody of  claim 1  which is a chimeric antibody. 
     
     
       43. The  A chimeric antibody according to  claim 42   which is a chimeric mouse/human antibody Chi  #1 as produced by the cell line deposited with the American Type Culture Collection and assigned accession number  (ATCC  CRL- 12316) .    
     
     
       44. The chimeric antibody according to  claim 42  wherein said colon carcinoma-associated antigen is a protein having a molecular weight of  72  kilodalton. 
     
     
       45. A composition comprising the chimeric antibody according to claim  42    43  in combination with a pharmaceutically acceptable carrier. 
     
     
       46. A monoclonal antibody against the chimeric antibody of  claim 42 . 
     
     
       47. An immunoassay for detecting a colon carcinoma-associated antigen which binds to the mouse/human chimeric antibody Chi  #1 as produced by the cell line deposited with the American Type Culture Collection and assigned accession number  (ATCC  CRL- 12316) of    claim 42     in a sample comprising:  
 (a) contacting said sample with the Chi # 1  antibody according to  claim 42  ; and  
 (b) detecting said antigen by detecting the binding of said antibody to the purified  colon carcinoma-associated protein antigen.  
 
     
     
       48. A method for diagnosing colon cancer in humans comprising:
 (a) removing a histological specimen from a patient suspected of having a colon carcinoma;  
 (b) contacting the specimen with a chimeric antibody which binds to an antigen  according to claim  1    43 ;  
 (c) staining the specimen with an immunohistochemical stain; and  
 (d) detecting the presence of the antigen-antibody complex by the stain.  
 
     
     
       49. A method for diagnosing colon cancer in humans comprising:
 (a) removing a histological specimen from a patient suspected of having a colon carcinoma;  
 (b) contacting the specimen with mouse/human chimeric antibody which binds to an antigen which binds to mouse/human chimeric antibody Chi  #1 (ATCC  as produced by the cell line deposited with the American Type Culture Collection and assigned accession number  CRL- 12316) ;    
 (c) staining the specimen with an immunohistochemcial stain; and  
 (d) detecting the presence of the antigen-antibody complex by the stain.  
 
     
     
       50. A kit for the immunohistochemical detection of colon carcinoma comprising:
 (a) mouse/human chimeric antibody Chi #1 (ATCC CRL-12316);  
 (b) reagents for immunoperoxidase and secondary antibody;  
 (c) immunoperoxidase; and  
 (d) colorizing reagents.