King's College London , Division of Imaging Sciences and Biomedical Engineering, 4th Floor Lambeth Wing, St. Thomas' Hospital, SE1 7EH, London, United Kingdom.
Bioconjug Chem. 2012 May 16;23(5):1029-39. doi: 10.1021/bc300037w. Epub 2012 Apr 13.
High radiolabeling efficiency, preferably to high specific activity, and good stability of the radioimmunoconjugate are essential features for a successful immunoconjugate for imaging or therapy. In this study, the radiolabeling efficiency, in vitro stability, and biodistribution of immunoconjugates with eight different bifunctional chelators labeled with (64)Cu were compared. The anti-CD20 antibody, rituximab, was conjugated to four macrocyclic bifunctional chelators (p-SCN-Bn-DOTA, p-SCN-Bn-Oxo-DO3A, p-SCN-NOTA, and p-SCN-PCTA), three DTPA derivatives (p-SCN-Bn-DTPA, p-SCN-CHX-A″-DTPA, and ITC-2B3M-DTPA), and a macrobicyclic hexamine (sarcophagine) chelator (sar-CO2H) = (1-NH2-8-NHCO(CH2)3CO2H)sar where sar = sarcophagine = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane). Radiolabeling efficiency under various conditions, in vitro stability in serum at 37 °C, and in vivo biodistribution and imaging in normal mice over 48 h were studied. All chelators except sar-CO2H were conjugated to rituximab by thiourea bond formation with an average of 4.9 ± 0.9 chelators per antibody molecule. Sar-CO2H was conjugated to rituximab by amide bond formation with 0.5 chelators per antibody molecule. Efficiencies of (64)Cu radiolabeling were dependent on the concentration of immunoconjugate. Notably, the (64)Cu-NOTA-rituximab conjugate demonstrated the highest radiochemical yield (95%) under very dilute conditions (31 nM NOTA-rituximab conjugate). Similarly, sar-CO-rituximab, containing 1/10th the number of chelators per antibody compared to that of other conjugates, retained high labeling efficiency (98%) at an antibody concentration of 250 nM. In contrast to the radioimmunoconjugates containing DTPA derivatives, which demonstrated poor serum stability, all macrocyclic radioimmunoconjugates were very stable in serum with <6% dissociation of (64)Cu over 48 h. In vivo biodistribution profiles in normal female Balb/C mice were similar for all the macrocyclic radioimmunoconjugates with most of the activity remaining in the blood pool up to 48 h. While all the macrocyclic bifunctional chelators are suitable for molecular imaging using (64)Cu-labeled antibody conjugates, NOTA and sar-CO2H show significant advantages over the others in that they can be radiolabeled rapidly at room temperature, under dilute conditions, resulting in high specific activity.
高放射性标记效率，最好是高比活度，以及放射性免疫偶联物的良好稳定性，是成像或治疗用成功免疫偶联物的必备特征。在这项研究中，比较了用八种不同双功能螯合剂标记的（64）Cu 标记的免疫偶联物的放射性标记效率、体外稳定性和生物分布。抗 CD20 抗体利妥昔单抗与四个大环双功能螯合剂（p-SCN-Bn-DOTA、p-SCN-Bn-Oxo-DO3A、p-SCN-NOTA 和 p-SCN-PCTA）、三个 DTPA 衍生物（p-SCN-Bn-DTPA、p-SCN-CHX-A″-DTPA 和 ITC-2B3M-DTPA）和一个大环己二胺（sarcophagine）螯合剂（sar-CO2H =（1-NH2-8-NHCO（CH2）3CO2H）sarsar = sarcophagine = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane））偶联。在不同条件下进行放射性标记效率研究，在 37°C 下的血清中体外稳定性，以及在正常小鼠体内的 48 小时内的生物分布和成像。除了 sar-CO2H 之外，所有螯合剂都通过硫脲键形成与利妥昔单抗偶联，每个抗体分子平均偶联 4.9±0.9 个螯合剂。sar-CO2H 通过酰胺键与利妥昔单抗偶联，每个抗体分子偶联 0.5 个螯合剂。（64）Cu 放射性标记效率取决于免疫偶联物的浓度。值得注意的是，（64）Cu-NOTA-rituximab 缀合物在非常稀释的条件下（31 nM NOTA-rituximab 缀合物）表现出最高的放射化学产率（95%）。同样，与其他缀合物相比，sar-CO-rituximab 每个抗体分子中含有 1/10 的螯合剂，但保留了高标记效率（98%），抗体浓度为 250 nM。与含有 DTPA 衍生物的放射性免疫偶联物相比，后者的血清稳定性较差，所有大环放射性免疫偶联物在 48 小时内均非常稳定，血清中（64）Cu 的解离率<6%。在正常雌性 Balb/C 小鼠体内的生物分布曲线相似，所有大环放射性免疫偶联物的大部分活性在 48 小时内仍留在血池中。虽然所有大环双功能螯合剂都适合使用（64）Cu 标记的抗体缀合物进行分子成像，但 NOTA 和 sar-CO2H 比其他螯合剂具有显著优势，因为它们可以在室温下、在稀释条件下快速标记，从而获得高比活度。
