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To directly radiolabel an anti-hepatoma mAb fragment HAb18 F(ab’)2 with 99mTc …

Biology Articles » Biophysics » Medical Biophysics » Direct technetium-99m labeling of anti-hepatoma monoclonal antibody fragment: a radioimmunoconjugate for hepatocellular carcinoma imaging » Discussion

- Direct technetium-99m labeling of anti-hepatoma monoclonal antibody fragment: a radioimmunoconjugate for hepatocellular carcinoma imaging

Great efforts have been made to develop a method that can be used for the direct labeling of mAbs with 99mTc[16]. Earlier studies involved the incubation of mAbs with stannous phthalate tartrate solution for up to 21h at room temperature, which was named “pretinning” method. Clinical success with this method has been claimed by the author[23].
One aim of our study was to further evaluate the role of stannous as a reducing agent in the direct labeling of mAb F(ab’) 2 with 99mTc. The difference between the “pretinning” method and this method is that we use GH instead of phthalate-tartrate as transfer ligand and stabilizer to avoid Sn or Tc-c olloid formation. To do this, we investigated the effect of the quantity of Sn/ GH on the labeling time and efficiency. When the molar ratio of Sn/GH to mA b F(ab’)2 was constant, we found that there was no obvious difference on the number of-SH between the reduction time of 20min and 30min or even longer[ 24]. The whole labeling process can be accomplished within 1.5h. Hnatowich et al. reported that labeling efficiency in the case of the stannous ion-reduced a ntibodies was generally in excess of 70%[12], however, in our method mol ar ratio of Sn/GH to mAb was an important parameter to obtain good labeling re sults, and molar ratio of 401 or higher were needed to get labeling effici ency of more than 80% (Table 1). The low percentage of free 99mTcO4 and radiocolloid in each sample implied that pH 5.3 and GH are the opti mal pH value and transfer ligand. Under this condition, the labeled mAb HAb18 F( ab’)2 keeps its immunoreactivity. Autoradiography of SDS-PAGE had only one m igration of component identical to that of native HAb18 F(ab’)2 determination by staini ng with Coomassie brilliant blue R250 (Figure 2), which demonstrated that S n/GH reduction is mild and does not destroy interchain bridges in mAbs. Labeli ng efficiency of 2% in control experiments using unreduced HAb18 F(ab’)2 indicated that there was no exchange with the low affinity sites and also demonstrated tha t reduction of disulfides is a necessary initial step in 99mc direct labeling of antibodies. The bond between SH and Tc is stronger than that of N -Tc or O-Tc which was verified by the challenge assay of 99mTc-HAb18 F(ab’)2 in the presence of EDTA. We found that EDTA even at a molar rat io of 100001 failed to remove a significant amount of 99mTc, this is in agreement with the results of Rhodes et al[8]. But L-cys at 6251 remove one-tenth of the label (Figure 5). Despite such insta bility of the label, there was no in vivo evidence of release of pertechneta te due to no thyroid imaging observed in the whole imaging process (Figure 7) . Tumor localization of 99mTc-HAb18 F(ab’)2 was successfully dem onstrated in a human tumor/nude mouse xenograft model. Biodistribution and ima ging results showed the highest tumor uptake at 24h post-injection. Where as kidney levels were foun d to be higher in the whole process. Accumulation of radioactivity in the kidney may be the result of retention of this metallic radionuclide by the kidney pro ximal tubule[25], the possible release of 99mTc-labeled cysteine and gluta thione[26]stemming from the radioimmunoconjugate catabolism, and the r elative amount of 99mTc-GH. A technique has been used in patients to block renal tubule uptake of 99mTc-anti-CEA Fab’ fragments by amin o acid infusion[27].
In conclusion, a radioimmunoimaging conjugate for hepatoma detection was prepare d by direct labeling mAb HAb18 F(ab’)2-with 99mTc using stannous/g lucoheptonate as reducing agent. Although the labeling efficiency is not satisf actory to some degree, it has several advantapes: simple, easy and quick, besid es, the labeled mAb fragment retains its immunoreactivity. Biodistribution and imaging studies reveal that this conjugate is useful for the detection of hepato ma.

ACKNOWLEGDGMENTS The authors are grateful to Dr. Wang Jing for mice imaging and the Department of Nuclear Medicine of Shaanxi People’s Hospital for their support of this project.

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