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Use of bifunctional chelating agents for radiolabeling antibodies
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Radiolabeled MonoclonalAntibodies USE OF BIPUNCTIONAL CHELATING AGl3W'SFOR RADIOLABELING ANTIBODIES. F. Meares. University of California, Davis, CA, USA. -___
Claude
The applications of monoclonal antibodies (MAbs) may be greatly expanded by tagging them with metal ions chosen for their desirable physical properties. This is done with "bifunctional" chelating agents (BCA) that incorporate a metal-chelating group and a chemically reactive or biologically active group. Several factors have to be considered in the design of a chelator with the most useful properties, for example the complex simultaneous equilibria and rates of reaction of the particular metal ion in biological media. A suitable molecular structure then may be chosen for a BCA, and a chemical synthesis designed. General procedures to attach the BCA to the antibody must be devised. The MAb-BCA conjugate must be isolated, and characterized as to extent of conjugation and immunoreactivity. Finally, buffer and pH conditions must be found that allow rapid, efficient addition of short-lived radionuclides to the protein-bound chelating groups without affecting the biological properties of the antibody, and should prevent the binding of metal ions to sites on the protein other than the attached chelating groups. For example, Cu-67 is a promising radionuclide for radioimmunotherapy. When free Cu(Z+)-67 is added to human serum, it is readily bound to serum albumin (HSA). The apparent equilibrium constant (1ogK CUHSA) for the binding of Cu (II) to HSA at physiologic pH is 16.2. This number refers to the state of protonation of HSA at pH 7.4, and would be different at another pH. The log K for Cu (II)-EDTA at pH 7.4 (15.9) may be calculated using thermodynamic Cu (II)-EDTA equilibrium constant (18.7) and the known protonbinding constants for EDTA. A comparison indicates that at equilibrium, under physiological conditions, practically all the Cu injected as Cu-EDTA will be HSA-bound. The presence of a backbone substituent on EDTA, for example a benzyl group generally increases the stability of a metal-EDTA chelate by l-2 log units, with little change in the proton-binding constants. However, for 10-5M benzyl-EDTA, the position of equilibrium still strongly favors the binding of Cu to HSA. For the macrocycle TETA, 1ogK CuTETA after correction for protonation is 12.6. Thus under physiologic conditions, CuTETA is thermodynamically less stable than CuEDTA. The substitution of benzyl groups on TETA does not change this situation. However, in contrast to Cu-benzyl-EDTA, Cu-benzyl-TETA has been shown to remain intact for several days in serum. It has been found that the loss of Cu from DTPA to albumin also occurs readily. This chelate (1ogK CuDTPA = 17 at pH 7.4) is thermodynamically more stable than Cu-benzyl-TETA as well. These studies demonstrate that the loss of Cu from these chelates is controlled by kinetic factors which depend on each chelate's structure. Macrocyclic structures such as that of Cu-TETA are notable for their slow kinetics.
CONF'ARISON OF TC-99M AND IN-111 IABELING OF DTPA CONJUCATED ANTIBODIES. Chang H. Paik, W. C. Eckelman and R. C. Reba. George Washington University Medical Center, Washington, DC, USA. An acylation reaction using cyclic DTPA dianhydride (CDTPAA) is chosen to discuss important parameters for optimum radiolabeling of antibodies (Ab). Indium-ill labeling of antibody-DTPA conjugates (Ab-DTPA) can be successfully carried out in a pH 4-6 medium. Denaturation of Ab occurs below pH 4 and In-111 hydroxide formation is appreciable above pH 6. Another important information for successful radiolabeling of Ab-DTPA is relative reactivity for In-111 among immunoreactive Ab-DTPA (sAb-DTPA), nonimmunoreactive DTPA (nAb-DTPA), and free DTPA. Antihuman serum albumin antibody IgG isotype (Ab, 8.7 mgfml) was reacted with cDTPAA at various cDTPAA/Ab molar ratios between 1 and 40 to answer the questions on relative reactivity. These reactions produced between 0.1 and 11 DTPA molecules conjugated per Ab. The number of DTPA molecules per sAb and nAb from the same reaction was similar when titrated with In ion, indicating that antibody deactivation is not solely determined by how many DTPA molecules are conjugated. The sAb-DTPA remaining after the above reactions was between 88 and 66% according to UV peak intensities of nAb and sAb. The percent In-111-sAb-DTPA from In-111 labeling of Ab-DTPA was similar to that determined by the UV analysis. This indicates that the reactivfties of sAb-DTPA and nAb-DTPA for In-111 are similar. Comparison of DTPA conjugation yields both by In titration method and by no-carrier-addedIn-111 labeling indicates that Ab-DTPA with >l DTPA per Ab is as reactive as free DTPA toward In-111, but more reactive than that containing
Radiolabeled MonoclonalAntibodies USE OF BIPUNCTIONAL CHELATING AGl3W'SFOR RADIOLABELING ANTIBODIES. F. Meares. University of California, Davis, CA, USA. -___
Claude
The applications of monoclonal antibodies (MAbs) may be greatly expanded by tagging them with metal ions chosen for their desirable physical properties. This is done with "bifunctional" chelating agents (BCA) that incorporate a metal-chelating group and a chemically reactive or biologically active group. Several factors have to be considered in the design of a chelator with the most useful properties, for example the complex simultaneous equilibria and rates of reaction of the particular metal ion in biological media. A suitable molecular structure then may be chosen for a BCA, and a chemical synthesis designed. General procedures to attach the BCA to the antibody must be devised. The MAb-BCA conjugate must be isolated, and characterized as to extent of conjugation and immunoreactivity. Finally, buffer and pH conditions must be found that allow rapid, efficient addition of short-lived radionuclides to the protein-bound chelating groups without affecting the biological properties of the antibody, and should prevent the binding of metal ions to sites on the protein other than the attached chelating groups. For example, Cu-67 is a promising radionuclide for radioimmunotherapy. When free Cu(Z+)-67 is added to human serum, it is readily bound to serum albumin (HSA). The apparent equilibrium constant (1ogK CUHSA) for the binding of Cu (II) to HSA at physiologic pH is 16.2. This number refers to the state of protonation of HSA at pH 7.4, and would be different at another pH. The log K for Cu (II)-EDTA at pH 7.4 (15.9) may be calculated using thermodynamic Cu (II)-EDTA equilibrium constant (18.7) and the known protonbinding constants for EDTA. A comparison indicates that at equilibrium, under physiological conditions, practically all the Cu injected as Cu-EDTA will be HSA-bound. The presence of a backbone substituent on EDTA, for example a benzyl group generally increases the stability of a metal-EDTA chelate by l-2 log units, with little change in the proton-binding constants. However, for 10-5M benzyl-EDTA, the position of equilibrium still strongly favors the binding of Cu to HSA. For the macrocycle TETA, 1ogK CuTETA after correction for protonation is 12.6. Thus under physiologic conditions, CuTETA is thermodynamically less stable than CuEDTA. The substitution of benzyl groups on TETA does not change this situation. However, in contrast to Cu-benzyl-EDTA, Cu-benzyl-TETA has been shown to remain intact for several days in serum. It has been found that the loss of Cu from DTPA to albumin also occurs readily. This chelate (1ogK CuDTPA = 17 at pH 7.4) is thermodynamically more stable than Cu-benzyl-TETA as well. These studies demonstrate that the loss of Cu from these chelates is controlled by kinetic factors which depend on each chelate's structure. Macrocyclic structures such as that of Cu-TETA are notable for their slow kinetics.
CONF'ARISON OF TC-99M AND IN-111 IABELING OF DTPA CONJUCATED ANTIBODIES. Chang H. Paik, W. C. Eckelman and R. C. Reba. George Washington University Medical Center, Washington, DC, USA. An acylation reaction using cyclic DTPA dianhydride (CDTPAA) is chosen to discuss important parameters for optimum radiolabeling of antibodies (Ab). Indium-ill labeling of antibody-DTPA conjugates (Ab-DTPA) can be successfully carried out in a pH 4-6 medium. Denaturation of Ab occurs below pH 4 and In-111 hydroxide formation is appreciable above pH 6. Another important information for successful radiolabeling of Ab-DTPA is relative reactivity for In-111 among immunoreactive Ab-DTPA (sAb-DTPA), nonimmunoreactive DTPA (nAb-DTPA), and free DTPA. Antihuman serum albumin antibody IgG isotype (Ab, 8.7 mgfml) was reacted with cDTPAA at various cDTPAA/Ab molar ratios between 1 and 40 to answer the questions on relative reactivity. These reactions produced between 0.1 and 11 DTPA molecules conjugated per Ab. The number of DTPA molecules per sAb and nAb from the same reaction was similar when titrated with In ion, indicating that antibody deactivation is not solely determined by how many DTPA molecules are conjugated. The sAb-DTPA remaining after the above reactions was between 88 and 66% according to UV peak intensities of nAb and sAb. The percent In-111-sAb-DTPA from In-111 labeling of Ab-DTPA was similar to that determined by the UV analysis. This indicates that the reactivfties of sAb-DTPA and nAb-DTPA for In-111 are similar. Comparison of DTPA conjugation yields both by In titration method and by no-carrier-addedIn-111 labeling indicates that Ab-DTPA with >l DTPA per Ab is as reactive as free DTPA toward In-111, but more reactive than that containing