Eder Matthias, Wängler Björn, Knackmuss Stefan, LeGall Fabrice, Little Melvyn, Haberkorn Uwe, Mier Walter, Eisenhut Michael
Radiopharmaceutical Chemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Eur J Nucl Med Mol Imaging. 2008 Oct;35(10):1878-86. doi: 10.1007/s00259-008-0816-z. Epub 2008 May 29.
The success of (68)Ga-labeled peptides for positron emission tomography of neuroendocrine tumors is mainly depending on the complex chemistry of this radioisotope. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the chelator of choice has however limitations if its application is expanded to heat-sensitive proteins. Recombinant antibodies like single chain Fv or diabodies belong to this class of proteins. They are suited to provide imaging contrast despite the short-lived (68)Ga because of their rapid blood clearances and nanomolar affinities. The heterobifunctional agent N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) was chosen as an alternative ligand because this agent is complexing [(68)Ga]Ga(3+) much faster than DOTA at ambient temperatures.
A versatile technology for HBED-CC conjugation of proteins and (68)Ga-labeling has been developed. This included HBED-CC-tetrafluorophenol (TFP) ester synthesis, coupling to the antibody at various pH and complexation reactions performed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer under different conditions.
The synthesis of the monoreactive 2,3,5,6-tetrafluorophenolate of HBED-CC at a carboxyl group not participating in complex formation used Fe(HBED-CC) for ester formation. The removal of Fe(3+) from purified (HBED-CC)TFP ester was achieved with RP(18) cartridge technology. The conjugation chemistry was performed with mAb425 which binds to the epidermal growth factor receptor (EGFR). This protein was used for optimizing purposes only. The influence of complexation parameters like temperature, pH, reaction time, and HBED-CC/antibody ratio on the biological activity of this model antibody was investigated. Furthermore, the outcome of this labeling procedure on the biological activity of a recombinant diabody (50 kDa) was studied.
It is known that small HBED-CC/antibody ratios are prerequisites for minimal interference of labels with antigen-binding domains. Here, the coupling of about one HBED-CC per antibody proved to be sufficient for efficient (68)Ga labeling, pointing to the successful application of (68)Ga for molecular imaging with small recombinant proteins.
（68）Ga标记的肽用于神经内分泌肿瘤正电子发射断层扫描的成功主要取决于这种放射性同位素的复杂化学性质。然而，如果将1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸（DOTA）（首选螯合剂）的应用扩展到热敏蛋白，则存在局限性。重组抗体如单链Fv或双抗体属于这类蛋白。由于它们的快速血液清除率和纳摩尔亲和力，尽管（68）Ga半衰期短，但它们仍适合提供成像对比度。选择异双功能试剂N,N'-双[2-羟基-5-（羧乙基）苄基]乙二胺-N,N'-二乙酸（HBED-CC）作为替代配体，因为该试剂在环境温度下比DOTA更快地络合[（68）Ga]Ga（3+）。
已开发出一种用于蛋白质与HBED-CC缀合及（68）Ga标记的通用技术。这包括HBED-CC-四氟苯酚（TFP）酯的合成、在不同pH下与抗体偶联以及在4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）缓冲液中在不同条件下进行的络合反应。
在不参与络合形成的羧基处合成HBED-CC的单反应性2,3,5,6-四氟苯酚盐时，使用[Fe（HBED-CC）]（-）进行酯形成。用RP（18）柱技术从纯化的（HBED-CC）TFP酯中去除Fe（3+）。偶联化学是用与表皮生长因子受体（EGFR）结合的单克隆抗体425进行的。该蛋白仅用于优化目的。研究了络合参数如温度、pH、反应时间和HBED-CC/抗体比例对该模型抗体生物活性的影响。此外，还研究了这种标记程序对重组双抗体（50 kDa）生物活性的影响。
已知小的HBED-CC/抗体比例是标记对抗原结合域干扰最小的前提条件。在此，每个抗体偶联约一个HBED-CC被证明足以进行有效的（68）Ga标记，这表明（
