Direct radiolabeling of antibodies and other proteins with technetium or rhenium

Abstract

Proteins containing one or more disulfide bonds are radiolabeled with radionuclides of technetium or rhenium for use in diagnosis and treatment of a variety of pathologic conditions. Radiolabeling is accomplished by partial reduction of the disulfide bonds of the protein using Sn (II), or using other reducing agents followed by the addition of Sn (II), removal of excess reducing agent and reduction by-products, and addition of a specified amount of pertechnetate or perrhenate reducing agent, such as stannous tartrate, with the addition accomplished in such manner that further reduction of the protein is limited. The resulting product may be stored frozen or lyophilized, with radiolabeling accomplished by the addition of pertechnetate or perrhenate solution.

What is claimed is: 
     
       1. A method of radiolabeling a protein containing one or more monosulfide or disulfide bonds with a radionuclide to obtain stable labeling, comprising the steps of: a) incubating the protein containing monosulfide or disulfide bonds with a first Sn (II) agent, the period of incubation being sufficient to allow formation of Sn (II)-containing and sulfur-containing complexes and formation of Sn (IV) reaction by-products, while preventing excessive fragmentation of the protein;   b) purifying the reduced protein to substantially remove uncomplexed Sn agents and other impurities;   c) adding a second Sn (II) agent to the purified protein with Sn (II)-containing and sulfur-containing complexes in a sufficient mount to reduce the radionuclide yet not generate significant radiochemical impurities, the radionuclide to be added in a subsequent step; and   d) radiolabeling the purified protein with the Sn (II)-containing and sulfur-containing complexes by adding the radionuclide, whereby the Sn (II) agents reduce the radionuclide and the reduced radionuclide forms radionuclide-containing and sulfur-containing complexes.   
     
     
       2. The method of claim 1 wherein the source of the first Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0. 
     
     
       3. The method of claim 1 wherein the source of the second Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0. 
     
     
       4. The method of claim 1 wherein the source of the second Sn (II) agent comprises at least one member selected from the group consisting of stannous glucoheptonate, stannous gluconate and stannous phosphonate. 
     
     
       5. The method of claim 1 wherein the protein comprises a member selected from the group consisting of monoclonal antibodies, monoclonal antibody fragments and polyclonal antibodies. 
     
     
       6. The method of claim 1 wherein following step c), and prior to step d), the purified protein with Sn (II)-containing and sulfur-containing complexes and second Sn (II) agent are frozen in a vial, whereby the frozen purified protein with Sn (II)-containing and sulfur-containing complexes and second Sn (II) agent can be maintained for an indefinite period before radiolabeling in step d) by the addition of the radionuclide to the vial. 
     
     
       7. The method of claim 1 wherein following step c), and prior to step d), the purified protein with Sn (II)-containing and sulfur-containing complexes and second Sn (II) agent are lyophilized in a vial, whereby the lyophilized purified protein with Sn (II)-containing and sulfur-containing complexes and second Sn (II) agent can be maintained for an indefinite period before radiolabeling in step d) by the addition of the radionuclide to the vial. 
     
     
       8. The method of claim 1 wherein 85 percent or more of the radionuclide is strongly bonded to the protein. 
     
     
       9. The method of claim 1 wherein the radionuclide comprises a member selected from the group consisting of technetium and rhenium. 
     
     
       10. The method of claim 9 wherein the radionuclide is technetium-99m in the form of sodium pertechnetate. 
     
     
       11. A method of radiolabeling a protein containing one or more monosulfide or disulfide bonds with a radionuclide to obtain stable labeling, comprising the steps of: a) incubating the protein containing monosulfide or disulfide bonds with a Sn (II) agent, the period of incubation being sufficient to allow formation of Sn (II)-containing and sulfur-containing complexes and formation of Sn (IV) reaction by-products, while preventing excessive fragmentation of the protein;   b) purifying the reduced protein to remove uncomplexed Sn agents and other impurities yet retaining Sn (II) in a sufficient mount to reduce the radionuclide and not generate significant radiochemical impurities, the radionuclide to be added in a subsequent step; and   c) radiolabeling the purified protein with the Sn (II)-containing and sulfur-containing complexes by adding the radionuclide, whereby the Sn (II) agent reduces the radionuclide and the reduced radionuclide forms radionuclide-containing and sulfur-containing complexes.   
     
     
       12. The method of claim 11 wherein the source of the first Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0. 
     
     
       13. The method of claim 11 wherein the protein comprises a member selected from the group consisting of monoclonal antibodies, monoclonal antibody fragments and polyclonal antibodies. 
     
     
       14. The method of claim 11 wherein following step b), and prior to step c), the purified protein with Sn (II)-containing and sulfur-containing complexes is frozen in a vial, whereby the frozen purified protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step c) by the addition of the radionuclide to the vial. 
     
     
       15. The method of claim 11 wherein following step b), and prior to step c), the purified protein with Sn (II)-containing and sulfur-containing complexes is lyophilized in a vial, whereby the lyophilized purified protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step c) by the addition of the radionuclide to the vial. 
     
     
       16. The method of claim 11 wherein 85 percent or more of the radionuclide is strongly bonded to the protein. 
     
     
       17. The method of claim 11 wherein the radionuclide comprises a member selected from the group consisting of technetium and rhenium. 
     
     
       18. The method of claim 17 wherein the radionuclide is technetium-99m in the form of sodium pertechnetate. .[.19. A method of radiolabeling a protein containing one or more monosulfide or disulfide bonds with a radionuclide to obtain stable labeling, comprising the steps of: a) incubating the protein containing monosulfide or disuifide bonds with a first Sn (II) agent, the period of incubation being sufficient to allow formation of Sn (II)-containing and sulfur-containing complexes, while preventing excessive fragmentation of the protein;   b) complexing the free Sn agents with a complexing agent so as not to further reduce the protein, while retaining complexed Sn agents for reducing the radionuclide, the radionuclide to be added in a subsequent step; and   c) radiolabeling the protein with the Sn (II)-containing and sulfur-containing complexes by adding the radionuclide, whereby the completed Sn (II) agents reduce the radionuclide and the reduced radionuclide forms radionuclide-containing and sulfur-containing   
     
     
        complexes..]..[.20.  The method of claim 19 wherein the complexing agent is polyaminocarboxylic acid..]..[.21. The method of claim 19 wherein the polyaminocarboxylic acid comprises at least one member selected from the group consisting of EDTA and DTPA..]..[.22. The method of claim 19 wherein the source of the first Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0..]..[.23. The method of claim 19 wherein the protein comprises a member selected from the group consisting of monoclonal antibodies, monoclonal antibody fragments and polyclonal antibodies..]..[.24. The method of claim 19 wherein following step b), and prior to step c), the free Sn complexes and protein with Sn (II)-containing and sulfur-containing complexes are frozen in a vial, whereby the frozen free Sn complexes and protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step d) by the addition of the radionuclide to the vial..]..[.25. The method of claim 19 wherein following step b), and prior to step c), the free Sn complexes and protein with Sn (II)-containing and sulfur-containing complexes are lyophilized in a vial, whereby the lyophilized free Sn complexes and protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step d) by addition of the radionuclide to the vial..]..[.26. The method of claim 19 wherein 85 percent or more of the radionuclide is strongly bonded to the protein..]..[.27. The method of claim 19 wherein the radionuclide comprises a member selected from the group consisting of technetium and rhenium..]..[.28. The method of claim 27 wherein the radionuclide is technetium-99m in the form of sodium pertechnetate..]..[.29. The method of claim 19 wherein following step b) a second Sn (II) agent is added to the combination of the completing agent and protein with Sn (II)-containing and sulfur-containing complexes in a sufficient mount to completely reduce the radionuclide, whereby the reduced radionuclide forms radionuclide-containing and sulfur-containing complexes..]..[.30. The method of claim 29 wherein the source of the second Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0..]..[.31. The method of claim 29 wherein the source of the second Sn (II) agent comprises a member selected from the group consisting of stannous glucoheptonate, stannous gluconate and stannous phosphonate..]..[.32. The method of claim 29 wherein following the addition of the second Sn (II) agent and prior to radiolabeling, the free Sn complexes, second Sn (II) agent and protein with Sn (II)-containing and sulfur-containing complexes are frozen in a vial, whereby the frozen free Sn complexes, second Sn (II) agent and protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling by the 
     
     
        addition of the radionuclide to the vial..]..[.33.  The method of claim 29 wherein following the addition of the second Sn (II) agent and prior to radiolabeling, the free Sn complexes, second Sn (II) agent and protein with Sn (II)-containing and sulfur-containing complexes are lyophilized in a vial, whereby the lyophilized free Sn complexes, second Sn (II) agent and protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling by the addition of the radionuclide to the vial..]..[.34. The method of claim 29 wherein 85 percent or more of the radionuclide is strongly bonded to the 
     
     
        protein..].35. A method of radiolabeling a protein containing monosulfide or disulfide bonds with a radionuclide to obtain stable labeling, comprising the steps of: a) incubating the protein containing monosulfide or disulfide bonds with a first reducing agent, the period of incubation being sufficient to reduce available disulfide bonds to reactive sulfide groups while preventing excessive fragmentation of the protein;   b) adding a source of first Sn (II) agent to the reduced protein to allow formation of Sn (II)-containing and sulfur-containing complexes;   c) purifying the reduced protein with Sn (II)-containing and sulfur-containing complexes to substantially remove the first reducing agent and impurities; and   d) radiolabeling the purified reduced protein with Sn (II)-containing and sulfur-containing complexes by adding the radionuclide, whereby the complexed Sn(II) agent reduces the radionuclide and the reduced radionuclide and reduced protein form radionuclide-containing and   
     
     
        sulfur-containing complexes. 36. The method of claim 35 wherein the radionuclide comprises a member selected from the group consisting of 
     
     
        technetium and rhenium. 37. The method of claim 35, wherein the 
     
     
        radionuclide is technetium-99m in the form of sodium pertechnetate. 38. The method of claim 35. wherein the first reducing agent comprises at least one member selected from the group consisting of 2-mercaptoethanol; 1,4-dithiothreitol, 2,3-dihydroxybutane-1,4-dithiol, 2-aminoethanethiol 
     
     
        HCl; 2-mercaptoethylamine; thioglycolate; cyanide; and cysteine. 39. The method of claim 35, wherein the source of the first Sn (II) agent of step b) is present in a solution comprising alkali metal tartrate having a pH 
     
     
        of between approximately 5.0 and 6.0. 40. The method of claim 35 wherein the protein comprises a member selected from the group consisting of monoclonal antibodies, monoclonal antibody fragments and polyclonal 
     
     
        antibodies. 41. The method of claim 35 wherein following step c), and prior to step d), the purified reduced protein with Sn (II)-containing and sulfur-containing complexes are frozen in a vial, whereby the purified reduced protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step d) 
     
     
        by the addition of the radionuclide to the vial. 42. The method of claim 35 wherein following step c), and prior to step d), the purified reduced protein with Sn (II)-containing and sulfur-containing complexes are lyophilized in a vial, whereby the lyophilized purified reduced protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step d) by the addition 
     
     
        of the radionuclide to the vial. 43. The method of claim 35 wherein 85 percent or more of the radionuclide is strongly bonded to the protein. 
     
     
            The method of claim 35 wherein following step c) a second reducing agent is added to the purified reduced protein with Sn (II)-containing and sulfur-containing complexes in a sufficient mount to completely reduce the radionuclide, whereby the reduced radionuclide forms 
     
     
        radionuclide-containing and sulfur-containing complexes. 45. The method of claim 44 wherein the second reducing agent comprises a source of the Sn (II) agent present in a solution comprising alkali metal tartrate having a 
     
     
        pH of between approximately 5.0 and 6.0. 46. The method of claim 44 wherein the second reducing agent comprises a member selected from the group consisting of stannous glucoheptonate, stannous gluconate and 
     
     
        stannous phosphonate. 47. The method of claim 44 wherein following the addition of the second reducing agent and prior to radiolabeling, the second reducing agent and purified reduced protein with Sn (II)-containing and sulfur-containing complexes are frozen in a vial, whereby the second reducing agent and purified reduced protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period 
     
     
        before radiolabeling by the addition of the radionuclide to the vial. 48. The method of claim 44 wherein following the addition of the second reducing agent and prior to radiolabeling, the second reducing agent and purified reduced protein with Sn (II)-containing and sulfur-containing complexes are lyophilized in a vial, whereby the lyophilized second Sn reducing agent and purified reduced protein with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period 
     
     
        before radiolabeling by the addition of the radionuclide to the vial. 49. The method of claim 44 wherein 85 percent or more of the radionuclide 
     
     
        is strongly bonded to the protein. .Iadd.50.  A method of radiolabeling a whole antibody or F(ab') 2  antibody fragment containing one or more disulfide bonds with a radionuclide to obtain stable labeling, comprising the steps of: a) incubating the antibody or fragment with a first Sn (II) agent, the period of incubation being sufficient to reduce a portion of the disulfide bonds and to allow formation of Sn (II)-containing and sulfur-containing complexes, while preventing excessive fragmentation of the antibody or fragment;   b) thereafter complexinh the free Sn agents with a subsequently added polyaminocarboxylic acid complexing agent so as not to further reduce the antibody or fragment, while retaining complexed Sn agents for reducing radionuclide, the radionuclide to be added in a subsequent step; and   c) radiolabeling the antibody or fragment with the Sn (II)-containing and sulfur-containing complexes by adding the radionuclide, whereby the complexed Sn (II) agents reduce the radionuclide and the reduced radionuclide forms radionuclide-containing and sulfur-containing complexes. .Iaddend..Iadd.51. The method of claim 50 wherein the polyaminocarboxylic acid comprises at least one member selected from the group consisting of EDTA and DTPA. .Iaddend..Iadd.52. The method of claim 50 wherein the source of the first Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0   
     
     
        and 6.0. .Iaddend..Iadd.53.  The method of claim 50 wherein the antibody or fragment is a member selected from the group consisting of monoclonal antibodies, monoclonal F(ab') 2  fragments and polyclonal antibodies. .Iaddend..Iadd.54. The method of claim 50 wherein following step b), and prior to step c), the free Sn complexes and antibody or fragment with Sn(II)-containing and sulfur-containing complexes are frozen in a vial, whereby the frozen free Sn complexes and antibody or fragment with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step d) by the addition of the radionuclide to the vial. .Iaddend..Iadd.55. The method of claim 50 wherein following step b), and prior to step c), the free Sn complexes and antibody or fragment with Sn (II)-containing and sulfur-containing complexes are lyophilized in a vial, whereby the lyophilized free Sn complexes and antibody or fragment with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step d) by addition of the radionuclide to the vial. .Iaddend..Iadd.56. The method of claim 50 wherein 85 percent or more of the radionuclide is strongly bonded to the antibody or fragment. .Iaddend..Iadd.57. The method of claim 50 wherein the radionuclide comprises a member selected from the group consisting of technetium and rhenium. .Iaddend..Iadd.58. The method of claim 50 wherein the radionuclide is technetium-99m in the form of sodium pertechnetate. 
     
     
        .Iaddend..Iadd.59.  The method of claim 50 wherein following step b) a second Sn (II) agent is added to the combination of the complexing agent and antibody or fragment with Sn (II)-containing and sulfur-containing complexes in a sufficient amount to completely reduce the radionuclide, whereby the reduced radionuclide forms radionuclide-containing and sulfur-containing complexes. .Iaddend..Iadd.60. The method of claim 59 wherein the source of the second Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0. .Iaddend..Iadd.61. The method of claim 59 wherein the source of the second Sn (II) agent comprises a member selected from the group consisting of stannous glucoheptonate, stannous gluconate and stannous phosphonate. .Iaddend..Iadd.62. The method of claim 59 wherein following the addition of the second Sn (II) agent and prior to radiolabeling, the free Sn complexes, second Sn (II) agent and antibody or fragment with Sn (II)-containing and sulfur-containing complexes are frozen in a vial, whereby the frozen free Sn complexes, second Sn (II) agent and antibody or fragment with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling by the addition 
     
     
        of the radionuclide to the vial. .Iaddend..Iadd.63.  The method of claim 59 wherein following the addition of the second Sn (II) agent and prior to radiolabeling, the free Sn complexes, second Sn (II) agent and antibody or fragment with Sn (II)-containing and sulfur-containing complexes are lyophilized in a vial whereby the lyophilized free Sn complexes, second Sn (II) agent and antibody or fragment with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling by the addition of the radionuclide to the vial. .Iaddend..Iadd.64. The method of claim 59 wherein 85 percent or more the radionuclide is strongly bonded to the antibody or fragment. .Iaddend.