Development of Radiochemically Pure Antibodies1

There are presently three approaches to radiolabeling antibodies: direct radiolabeling; radiolabeling of a chelating agent conjugated to an antibody; and radiolabeling of a chelating agent before conjugation to an antibody. Using either the direct radiolabeling or radiolabeling of a chelating agent conjugated to an antibody has not led to a radiochemically pure ""Tc-labeled protein. High radiochemical purity is obtained using a prelabeled ligand; therefore this method is preferred. One of the major efforts since the first Conference on Radioimmunodetection and Radioimmunotherapy of Cancer has been the development of antibodies that are radiochemically pure in vitro and remain so in vivo. Since radioiodine has been one of the first radionuclides used to label antibodies, many studies have centered around the preparation of radiochemically pure antibodies either by direct iodination or by indirect labeling through a reagent such as the Bolton-Hunter ligand. The cur rent research emphasis is on in vivo stability using various iodinated species less susceptible to enzymatic deiodination (1, 2). There have been two approaches to radiolabeling antibodies with metallic radionuclides using bifunctional chelates; either the chelating agent is first attached to the antibody followed by radiolabeling or the chelating agent is first radiolabeled and the resulting chelate is bound to the antibody. Very few experiments have shown that first attaching the chelating agent to the antibody results in an "instant" kit in which all the radiolabel is bound via the chelating agent. For 99mTc,the indirect labeling methods have mostly involved the use of DTPAbound to the protein. DTPA is considered a bifunctional chelate in that it binds to the protein and to the "Tc. Other chelating agents, derivatives of DADS (3), derivatives of bis-A'-methylthiosemicarbazone (4), and metallothionein (5), have been conjugated to antibodies for "Tc labeling. Although 99mTclabeling con ditions and biodistribution studies of the labeled products were reported, a systematic investigation of the effectiveness of this latter chelator for complexing "Tc in competition with anti body has been reported only in abstract form (6). Hnatowich's group (7, 8) has carried out various experiments to determine the expected ratio of "Tc binding to the chelating agent in the bifunctional chelate compared to direct binding to the antibody. Using 50 Mg/ml DTPA as the molar equivalent to 20 mg/ml IgG, (0.13 ^mol/ml) various binding conditions were tested. Without protein present, DTPA complexed re duced "Tc efficiently, but not quantitatively (Table 1). In a subsequent paper, they determined the appropriate tin:DTPA ratio. As has been observed earlier, too little stannous ion resulted in a high percentage of pertechnetate and too much stannous ion resulted in tin colloid. The labeling of conjugated protein via the bifunctional chelate is about 80%. The distri bution in normal mice at l h is similar for '"In-DTPA-IgG and "Tc-DTPA-IgG. Three N2S2 (diaminodithio) ligands were studied by Liang et ' Presented at the "Second Conference on Radioimmunodetection and Ra dioimmunotherapy of Cancer." September 8-10. 1988, Princeton, NJ. 2The abbreviations used are: DTPA. diethylenetriaminepentaacetic acid; DADS. bis(mercaptoacetyl)ethylenediamine; DADT. diaminodithiol. al. (9) to determine if they had advantages over DTPA as bifunctional chelates of antibodies (Table 2). Optimal concen trations of tin and chelator were determined to be 2.5 to 5 /ig/ ml SnCl2-2H2O and 50 Mg/ml of chelator. The optimal pH range was 5 to 8. Labeling in the presence of 5 mg/ml of IgG was used to determine the percentage expected to be labeled via the N2S2 chelating agent and the percentage expected to be directly bound to the antibody (Table 3). The DADT-3C-2OH in which the ethylene backbone is re placed with a propylene backbone has the highest percentage binding. This appears to be the only example of a chelating agent that can compete quantitatively with an equimolar con centration of IgG. If the source of 99mTcis 99mTc-EDTA at 100 Mg/ml, then the requirement for chelator concentration is less (Table 4). Using 150,000 as the molecular weight of IgG there are 33 nmol/ml of IgG. If the molecular weight of the free amine is used (236), then 3 ng of DADT are 13 nmol/ml and 8 Mgof DADT are 34 nmol/ml. These molar values do not agree with the molar ratios stated by Liang et al. (9). However, it appears that the chelation is more effective when 9VrnTc-EDTA is used than when the chelator is labeled directly. Yokoyama's group (10) has developed the neutral 99mTc chelate ketobisthiosemicarbazone for use in bifunctional che lates. Various derivatives have been prepared including the pcarbonylethylphenylglycol derivative, which has been conju gated to antibody by activating the acid group using the phosphorylazide method. Stannous ascorbate was used as the reduc ing agent and 99mTc-ascorbate as the transfer agent. As in most of these studies the optimal pH is between 4.5 and 6.2. The labeling experiments were carried out to determine what per centage of the 99mTc binds to the chelating agent and what percentage binds directly to the antibody. At ratios of chelating agent to antibody that preserve the immunological activity (i.e., 1:1 molar ratio), about 15-20% of the "Tc is directly bound to the protein. In vitro and in vivo stability of the derivatized antibody is high. Although direct binding of 99mTcto antibodies has been reported (11-14) the stability and inertness of the bond are in question. There appear to be two binding sites on the antibody, one with high affinity and one with lower affinity. Steigman et al. (15) were the first to suggest that sulfhydryl groups were responsible for the direct binding of 99mTc.Paik et al. (16, 17) substantiated this hypothesis by carrying out a number of competitive experiments and by titrating the sulfhy dryl groups using Ellman's reagent. The available sulfhydryl groups were determined after 30 min incubation of stannous chloride with antibody. The stannous chloride was present in 10-fold excess over the protein. The results obtained are re ported in Table 5. The presence of both high affinity sites and low affinity sites on the protein was defined by competition studies using DTPA. The high affinity sites were determined as those which bound 99nTc even at large molar excesses of DTPA. The percentage bound to protein was determined at various ratios of DTPA to protein. These results are obtained only under conditions of direct competition between DTPA and antibody. If the protein is labeled first, then DTPA is effective only at high pH. One 780s on May 26, 2017. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from RADIOCHEMICALLV PURE ANTIBODIES Table l DTPA binding ofstannous reduced """Tc" Table 6 Percentage of loss of radioactivity from n"Tc-labeled antibodies"