Antibody-metal ion complexes

• US 4,741,900 A
• Filed: 08/31/1984
• Issued: 05/03/1988
• Est. Priority Date: 11/16/1982
• Status: Expired due to Term

• Current Assignee: Cytogen Corporation
• Original Assignee: Cytogen Corporation
• Inventors: Siegel, Richard C., McKearn, Thomas J., Alvarez, Vernon L., Lee, Chyi, Rodwell, John D., Goers, John W. F.
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/646,328
• Time in Patent Office: 1,341 Days
• Field of Search: 260/112 R, 260/112 B, 424/85, 514/2, 514/6, 435/7, 435/29, 530/335, 530/387-391
• US Class Current: 424/1.53
• CPC Class Codes: A61K 2121/00 Preparations for use in therapy
• CPC Class Codes: A61K 2123/00 Preparations for testing in vivo
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 49/085 conjugated systems
• CPC Class Codes: A61K 49/16 Antibodies; Immunoglobulins; Fragments thereof
• CPC Class Codes: A61K 51/1093 conjugates with carriers being antibodies
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/534 with radioactive label
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/577 involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens; G01N33/53 - G01N33/576 take precedence
• CPC Class Codes: Y10S 435/972 Modified antibody, e.g. hybrid, bifunctional

Abstract

This invention relates to antibody-metal ion complexes having a metal ion coordinately bound to a compatible chelator covalently bound to an antibody or antibody fragment. Also described are methods for intermediates in the preparation of antibody-metal ion complexes. Therapeutic and in vitro and in vivo diagnostic methods utilizing such antibody-metal ion complexes are described.

Claims

1. A method for preparing an antibody-chelator conjugate, comprising:

(a) reacting an antibody or antibody fragment with an oxidizing agent to form an aldehyde group in the carbohydrate moiety of the antibody or antibody fragment, in which the carbohydrate moiety is not part of nor directly involved with the antigen binding site of the antibody or antibody fragment; and

(b) reacting the aldehyde group of the resultant oxidized antibody or antibody fragment with a compatible chelator containing an amine group selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups to form a water-soluble antibody-chelator conjugate being characterized by (i) substantially the same immunospecificity as the unconjugated antibody or antibody fragment; and

(ii) aqueous solubility such that the antibody-chelator conjugate, when reacted with a metal ion, is suitable for in vivo administration.

2. The method according to claim 1, wherein the oxidizing agent is an enzyme.

3. The method according to claim 1, wherein the oxidizing agent is an oxygen acid.

4. The method according to claim 1, wherein the antibody fragment is selected from the group consisting of Fab fragments, (Fab'"'"')₂ fragments and half antibody molecules.

5. The method according to claim 1, wherein the antibody or antibody fragment is a monoclonal antibody or a monoclonal antibody fragment.

6. The method according to claim 1, wherein the compatible chelator is an amine-containing derivative of diethylenetriaminepentaacetic acid.

7. The method according to claim 1, wherein the compatible chelator is selected from the group consisting of p-aminoaniline-diethylenetriaminepentaacetic acid, hydrazide-diethylenetriaminepentaacetic acid, phenylhydrazide-diethylenetriaminepentaacetic acid, hydroxylamine-diethylenetriaminepentaacetic acid, semicarbazide-diethylenetriaminepentaacetic acid, thiosemicarbazide-diethylenetriaminepentaacetic acid, polyethyleneimine-diethylenetriaminepentaacetic acid, p-phenylenediamine-diethylenetriaminepentaacetic acid, α

-N-diethylenetriaminepentaacetic acid-L-lysine, glycyl-tyrosyl-lysine-diethylenetriaminepentaacetic acid, diethylenetriaminepentaacetic acid mono[(4-aminophenyl)methyl ] amide and L-lysine benzyl ester-diethylenetriaminepentaacetic acid.

8. The method according to claim 1, wherein the compatible chelator is an amine-containing derivative of a chelator selected from the group consisting of ethylenediaminetetraacetic acid, dimercaptosuccinic acid, 2,3-dimercaptopropanesulfonic acid and metallothioein.

9. The method according to claim 1, wherein thee compatible chelator is an amine-containing derivative of a cryptate.

10. The method according to claim 1, wherein the antibody-chelator conjugate is stabilized by exposure to an effective amount of a reducing agent.

15. The method according to claim 2, wherein the radioisotope emits alpha particles.

16. The method according to claim 15, wherein the radioisotope is selected from the group consisting of ²¹¹ Bismuth, ²¹² Bismuth, ²¹³ Bismuth and ²¹⁴ Bismuth.

11. A method for preparing an antibody-metal ion complex, comprising:

(a) reacting an antibody or antibody fragment with an oxidizing agent to form an aldehyde group in the carbohydrate moiety of the antibody or antibody fragment, in which the carbohydrate moiety is not part of nor directly involved with the antigen binding site of the antibody or antibody fragment;

(b) reacting the aldehyde group of the resultant oxidized antibody or antibody fragment with a compatible chelator containing an amine group selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups to form a water-soluble antibody-chelator conjugate; and

(c) combining the antibody-chelator conjugate with a metal ion under conditions which allow chelation of the metal ion to the antibody-chelator conjugate to form a water-soluble antibody-metal ion complex being characterized by (i) substantially the same immunospecificity as the unconjugated antibody or antibody fragment and (ii) an aqueous solubility such that the antibody metal ion complex is suitable for in vivo administration.

12. The method according to claim 11, wherein the metal ion is a radioisotope.

13. The method according to claim 12, wherein the radioisotope emits data particles.

14. The method according to claim 13, wherein the radioisotope is selected from the group consisting of ⁴⁶ Scandium, ⁴⁷ Scandium, ⁴⁸ Scandium, ⁷² Gallium and ⁷³ Gallium.

17. The method according to claim 12, wherein the radioisotope emits positron particles.

18. The method according to claim 17, wherein the radioisotope is selected from the group consisting of ⁴³ Scandium, ⁴⁴ Scandium, ⁵² Iron, ⁵⁵ Cobalt and ⁶⁸ Gallium.

19. The method according to claim 11, wherein the metal ion is paramagnetic.

20. The method according to claim 19, wherein the metal ion is selected from the group consisting of ⁵⁴ Iron, ⁵⁶ Iron, ⁵⁷ Iron, ⁵⁸ Iron, ¹⁵⁷ Gadolinium and ⁵⁵ Manganese.

21. The method according to claim 11, wherein the oxidizing agent is an enzyme.

22. The method according to claim 11, wherein the oxidizing agent is an oxygen acid.

23. The method according to claim 11, wherein the antibody fragment is selected from the group consisting of Fab fragments, (Fab'"'"')₂ fragments and half antibody molecules.

24. The method according to claim 11, wherein the antibody or antibody fragment is a monoclonal antibody or monoclonal antibody fragment.

25. The method according to claim 11, wherein the compatible chelator is an amine-containing derivative of diethylenetriaminepentaacetic acid.

26. The method according to claim 11, wherein the compatible chelator is selected from the group consisting of p-aminoaniline-diethylenetriaminepentaacetic acid, hydrazide-diethylenetriaminepentaacetic acid, phenylhydrazide-diethylenetriaminepentaacetic acid, hydroxylamine-diethylenetriaminepentaacetic acid, semicarbazide-diethylenetriaminepentaacetic acid, thiosemicarbazide-diethylenetriaminepentaacetic acid, polyethyleneimine-diethylenetriaminepentaacetic acid, p-phenylenediamine-diethylenetriaminepentaacetic acid, α

-N-diethylentriminepentacetic acid L-lysine, glycyl-tyrosyl-lysinediethylentriaminepentaacetic acid, diethylenetriaminepentaacetic acid mono [(4-aminophenyl)methyl ]amide and L-lysine benzyl ester-diethylenetriaminepentaacetic acid.

27. The method according to claim 11, wherein the compatible chelator is an amine-containing derivative of a chelator selected from the group consisting of ethylenediaminetetraacetic acid, dimercaptosuccinic acid, 2,3-dimercaptopropanesulfonic acid and metallothioein.

28. The method according to claim 11, wherein the compatible chelator is an amine-containing derivative of a cryptate.

29. The method according to claim 11, wherein the antibody-metal ion complex is stabilized by exposure to an effective amount of a reducing agent.

30. The method according to claim 11, further comprising the step of:

(d) separating the antibody-metal ion complexes from non-chelated metal ions to obtain antibody-metal ion complexes substantially free of non-chelated metal ions.

31. The method according to claim 30, wherein the separation is accomplished by high performance gel permeation liquid chromatography.

32. A method for preparing an antibody-metal ion complex, comprising:

(a) reacting an antibody or antibody fragment with an oxidizing agent to form an aldehyde group in the carbohydrate moiety of the antibody or antibody fragment, in which the carbohydrate moiety is not part of nor directly involved with the antigen binding site of the antibody or antibody fragment; and

(b) reacting the aldehyde group of the resultant oxidized antibody or antibody fragment with a chelator-metal ion complex, said chelator-metal ion complex comprising a compatible chelator-metal ion complex comprising a compatible chelator containing an amine group selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups coordinately bound to a metal ion, to form a water-soluble antibody-metal ion complex being characterized by (i) substantially the same immunospecificity as the unconjugated antibody or antibody fragment, and (ii) aqueous solubility such that the antibody-chelator metal ion complex is suitable for in vivo administration.

33. The method according to claim 32, wherein the metal ion is a radioisotope.

34. The method according to claim 33, wherein the radioisotope emits beta particles.

35. The method according to claim 34, wherein the radioisotope is selected from the group consisting of ⁴⁶ Scandium, ⁴⁷ Scandium, ⁴⁸ Scandium, ⁷² Gallium and ⁷³ Gallium.

36. The method ccording to claim 33, wherein the radioisotope emits alpha particles.

37. The method according claim 36, wherein the radioisotope is selected from the group consisting of ²¹¹ Bismuth, ²¹² Bismuth, ²¹³ Bismuth and ²¹⁴ Bismuth.

38. The method according to claim 33, wherein the radioisotope emits positron particles.

39. The method according to claim 38, wherein the radioisotope is selected from the group consisting of ⁴³ Scandium, ⁴⁴ Scandium, ⁵² Iron, ⁵⁵ Cobalt and ⁶⁸ Gallium.

40. The method according to claim 32, wherein the metal ion is paramagnetic.

41. The method according to claim 40, wherein the metal ion is selected from the group consisting of ⁵⁴ Iron, ⁵⁶ Iron, ⁵⁷ Iron, ⁵⁸ Iron, ¹⁵⁷ Gadolinium and ⁵⁵ Manganese.

42. The method according to claim 32, wherein the oxidizing agent is an enzyme.

43. The method according to claim 32, wherein the oxidizing agent is an oxygen acid.

44. The method according to claim 32, wherein the antibody fragment is selected from the group consisting of Fab fragments, (Fab'"'"')₂ fragments and half antibody molecules.

45. The method according to claim 32, wherein the antibody or antibody fragment is a monoclonal antibody or monoclonal antibody fragment.

46. The method according to claim 32, wherein the compatible chelator is an amine-containing derivative of diethylenetriaminepentaacetic acid.

47. The method according to claim 32, wherein the compatible chelator is selected from the group consisting of p-aminoaniline-diethylenetriaminepentaacetic acid, hydrazide-diethylenetriaminepentaacetic acid, phenylhydrazide-diethylenetriaminepentaacetic acid, hydroxylamine-diethylenetriaminepentaacetic acid, semicarbazide-diethylenetriaminepentaacetic acid, thiosemicarbazide-diethylenetriaminepentaacetic acid, polyethyleneimine-diethylenetriaminepentaacetic acid, p-phenylenediamine-diethylenetriaminepentaacetic acid, α

-N-diethylenetriaminepentaacetic acid-L-lysine, glycyl-tyrosyl-lysine-diethylenetriaminepentaacetic acid, diethylenetriaminepentaacetic acid mono[(4-aminophenyl)methyl]amide and L-lysine benzyl ester-diethylenetriaminepentaacetic acid.

48. The method according to claim 32, wherein the compatible chelator is an amine-containing derivative of a chelator selected from the group consisting of ethylenediaminetetraacetic acid, dimercaptosuccinic acid, 2,3-dimercaptopropanesulfonic acid and metallothioein.

49. The method according to claim 32, wherein the compatible chelator is an amine-containing derivative of a cryptate.

50. The method according to claim 32, werein the antibody-metal ion complex is stabilized by exposure to an effective amount of a reducing agent.

51. The method according to claim 32, further comprising the step of:

(c) separating the antibody-metal ion complexes from non-chelated metal ions to obtain antibody-metal ion complexes substantially free of non-chelated metal ions.

52. The method according to claim 51, wherein the separation is accomplished by high performance gel permeation liquid chromatography.

53. An antibody-chelator conjugate, comprising:

a compatible chelator attached through a covalent bond to a carbohydrate moiety of an oxidized antibody or antibody fragment, in which the carbohydrate moiety is not part or nor directly involved with the antigen binding site of the antibody or antibody fragment, said water-soluble antibody-chelator conjugate being characterized (i) substantially the same immunospecificity as the unconjugated antibody or antibody fragment, and (ii) aqueous solubility such that the antibody-chelator conjugate, when reacted with a metal ion, is suitable for in vivo administration.

54. The antibody-chelator conjugate according to claim 53, wherein the covalent bond is an enamine, hydrazone, oxime, phenylhydrazone, semicarbazone, thiosemicarbazone, or a reduced form thereof.

55. The antibody-chelator conjugate according to claim 53, wherein the antibody fragment is selected from the group consisting of Fab fragments, (Fab'"'"')₂ fragments and half antibody molecules.

56. The antibody-chelator conjugate according to claim 53, wherein the antibody or antibody fragment is a monoclonal antibody or monoclonal antibody fragment.

57. The antibody-chelator conjugate according to claim 53, wherein the compatible chelator is an amine-containing derivative of diethylenetriaminepentaacetic acid.

58. The antibody-chelator conjugate according to claim 57, wherein the compatible chelator is selected from the group consisting of p-aminoaniline-diethylenetriaminepentaacetic acid, hydrazide-diethylenetriaminepentaacetic acid, phenylhydrazide-diethylenetriaminepentaacetic acid, hydroxylamine-diethylenetriaminepentaacetic acid, semicarbazide-diethylenetriaminepentaacetic acid, thiosemicarbazide-diethylenetriaminepentaacetic acid, polyethyleneimine-diethylenetriaminetetraacetic acid p-phenylenediamine-diethylenetriaminepentaacetic acid, α

-N-diethylenetriaminepentaacetic acid-L-lysine, glycyl-tyrosyl-lysine-diethylenetriaminepentaacetic acid, diethylenetriaminepentaacetic acid mono[(4-aminophenyl)methyl]amide and L-lysine benzyl ester-diethylenetriamine-pentaacetic acid.

59. The antibody-chelator conjugate according to claim 53, wherein the compatible chelator is an amine-containing derivative of a chelator selected from the group consisting of ethylenediaminetetraacetic acid, dimercaptosuccinic acid, 2,3-dimercaptopropanesulfonic acid and metallothioein.

60. The antibody-chelator conjugate according to claim 53, wherein the compatible chelator is an amine-containing derivative of a cryptate.

61. An antibody-metal ion complex, comprising:

an antibody-chelator conjugate comprising a compatible chelator attached through a covalent bond to a carbohydrate moiety of an oxidized antibody or antibody fragment, in which the carbohydrate moiety is not part of nor directly involved with the antigen binding site of the antibody or antibody fragment;

said water-soluble antibody-chelator conjugate coordinately bound through said compatible chelator to a metal ion to form a water-soluble antibody-metal ion complex being characterized by (i) substantially the same immunospecificity as the unconjugated antibody or antibody fragment, and (ii) aqueous solubility such that the antibody-chelator metal ion complex is suitable for in vivo administration.

62. The antibody-metal ion complex according to claim 61, wherein the metal ion is a radioisotope.

63. The antibody-metal ion complex according to claim 62, wherein the radioisotope emits beta particles.

64. The antibody-metal ion complex according to claim 63, wherein the radioisotope is selected from the group consisting of ⁴⁶ Scandium, ⁴⁷ Scandium, ⁴⁸ Scandium, ⁷² Gallium and ⁷³ Gallium.

65. The antibody-metal ion complex according to claim 62, wherein the radioisotope emits alpha particles.

66. The antibody-metal ion complex according to claim 65, wherein the radioisotope is selected from the group consisting of ²¹¹ Bismuth, ²¹² Bismuth, ²¹³ Bismuth ²¹⁴ Bismuth.

67. The antibody-metal ion complex according to claim 62, wherein the radioisotope emits positron particles.

68. The antibody-metal ion complex according to claim 67, wherein the radioisotope is selected from the group consisting of ⁴³ Scandium, ⁴⁴ Scandium, ⁵² Iron, ⁵⁵ Cobalt and ⁶⁸ Gallium.

69. The antibody-metal ion complex according to claim 61, wherein the metal ion is paramagnetic.

70. The antibody-metal ion complex according to claim 69, wherein the metal ion is selected from the group consisting of ⁵⁴ Iron, ⁵⁶ Iron, ⁵⁷ Iron, ⁵⁸ Iron, ¹⁵⁷ Gadolinium and ⁵⁵ Manganese.

71. The antibody-metal ion complex according to claim 61, wherein the covalent bond is an imine, hydrazone, oxime, phenylhydrazone, semicarbazone, thiosemicarbazone or a reduced form thereof.

72. The antibody-metal ion complex according to claim 61, wherein the antibody fragment is selected from the group consisting of Fab fragments, (Fab'"'"')₂ fragments and half antibody molecules.

73. The antibody-metal ion complex according to claim 61, wherein the antibody or antibody fragment is a monoclonal antibody or monoclonal antibody fragment.

74. The antibody-metal ion complex according to claim 61, wherein the compatible chelator is an amine-containing derivative of diethylenetriaminepentaacetic acid.

75. The antibody-metal ion complex according to claim 61, wherein the compatible chelator is selected from the group consisting of p-aminoaniline-diethylenetriaminepentaacetic acid, hydrazide-diethylenetriaminepentaacetic acid, phenylhydrazide-diethylenetriaminepentaacetic acid, hydroxylamine-diethylenetriaminepentaacetic acid, semicarbazide-diethylenetriaminepentaacetic acid, thiosemicarbazide-diethylenetriaminepentaacetic acid, polyethyleneimine-diethylenetriaminepentaacetic acid, p-phenylenediamine-diethylenetriaminepentaacetic acid, α

-N-diethylenetriaminepentaacetic acid-L-lysine, glycyl-tyrosyl-lysine-diethylenetriaminepentaacetic acid, diethylenetriaminepentaacetic acid mono[(4-aminophenyl)methyl]amide and L-lysine benzyl ester-diethylenetriaminepentaacetic acid.

76. The antibody-metal ion complex according to claim 61, wherein the compatible chelator is an amine-containing derivative of a chelator selected from the group consisting of ethylenediaminetetraacetic acid, dimercaptosuccinic acid, 2,3-dimercaptopropanesulfonic acid and metallothioein.

77. The antibody-metal ion complex according to claim 61, wherein the compatible chelator is an amine-containing derivative of a cryptate.

78. The antibody-metal ion complex according to claim 61, substantially free of non-chelated metal ions.

79. A method for in vivo imaging a specific tissue, comprising:

(a) administering to an animal or a human an effective amount of a water-soluble antibody metal ion complex which

(i) is immunoreactive with and immunospecific for an antigenic determinant of the specific tissue and substantially non-immunoreactive with and non-immunospecific for non-specific tissue and the antigenic determinant is not found in substantial amounts in non-specific tissue; and

(ii) comprises an antibody or antibody fragment, a compatible chelator and metal ion in which the compatible chelator is covalently bound to the antibody or antibody fragment at a carbohydrate moiety located outside the antigen binding region of the antibody or antibody fragment, and coordinately bound to the metal ion so that the antibody-metal ion complex is characterized by substantially the same immunospecificity as the antibody or antibody fragment and an aqueous solubility such that the antibody-metal ion complex is suitable for administration in vivo; and

(b) detecting whether the antibody-metal ion complex localized at the specific tissue.

80. A method for therapeutic treatment of a cellular disorder, comprising:

administering to an animal or a human a therapeutically effective amount of a water-soluble antibody metal-ion complex which

(a) is immunoreactive with and immunospecific for a target site associated with the cellular disorder and substantially non-immunoreactive with and non-immunospecific for tissue not associated with the cellular disorder; and

(b) which comprises an antibody or antibody fragment, a compatible chelator and a metal ion which emits cytotoxic beta or alpha particles, in which the compatible chelator is covalently bound to the antibody or antibody fragment at a carbohydrate moiety located outside the antigen binding region of the antibody or antibody fragment, and coordinately bound to the metal ion so that the antibody-metal ion complex is characterized by substantially the same immunospecificity as the antibody or antibody fragment and an aqueous solubility such that the antibody-metal ion complex is suitable for administration in vivo.

81. A method for testing for an antigen, comprising:

(a) mixing a sample suspected of containing the antigen with a water-soluble antibody metal ion complex which

(i) is immunoreactive with and immunospecific for the antigen; and

(ii) which comprises an antibody or antibody fragment, a compatible chelator and metal ion in which the compatible chelator is covalently bound to the antibody or antibody fragment at a carbohydrate moiety located outside the antigen binding region of the antibody or antibody fragment, and coordinately bound to the metal ion so that the antibody-metal ion complex is characterized by substantially the same immunospecificity as the antibody or antibody fragment and an aqueous solubility such that the antibody-metal ion complex is suitable for administration in vivo; and

(b) detecting any interaction of the antibody-metal ion complex with the sample.