Radiopharmaceutical Chemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Eur J Nucl Med Mol Imaging. 2010 Jul;37(7):1397-407. doi: 10.1007/s00259-010-1392-6. Epub 2010 Feb 16.
Recombinant antibodies isolated from human antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of recombinant antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope (68)Ga.
In order to combine the promising tumour targeting properties of antibodies with (68)Ga, four antibody variants with the same specificity and origin only differing in molecular weight were constructed for comparison. Therefore, the binding domains of a single-chain fragment variable (scFv) isolated from a human naïve antibody library were modified genetically to construct the respective full-length IgG, the tria- and diabody variants. These molecules were conjugated with the bifunctional chelating agent N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) to enable (68)Ga labelling at ambient temperature and compared in biodistribution and immuno-PET imaging experiments.
The antibody variants with identical specificity proved to have the correct molecular weight, high binding affinity and specificity to their antigen, EpCAM. Radiometal complexation was efficiently performed at room temperature leading to (68)Ga-labelled antibodies with unchanged binding properties compared to the original antibody variants. The best targeting properties were obtained with the scFv and especially with the diabody. The triabody showed higher absolute tumour uptake but only moderate clearance from circulation.
The antibody variants differed considerably in normal organ uptake, clearance from circulation and tumour accumulation. The data demonstrate the feasibility of imaging solid tumours with the (68)Ga-labelled diabody format. This type of recombinant protein might be a promising carrier even for the short-lived radiometal (68)Ga to support e.g. the management of immunotherapy which may provide important information regarding receptor expression of solid tumours.
从人源抗体文库中分离得到的重组抗体具有极好的亲和力和高靶特异性。由于全长 IgG 从循环中清除速度不够慢，本研究旨在确定哪种重组抗体片段最适合用短寿命放射性同位素（68）Ga 对上皮细胞黏附分子（EpCAM）表达的肿瘤进行成像。
为了将抗体的有前途的肿瘤靶向特性与（68）Ga 结合，构建了 4 种具有相同特异性和来源但分子量不同的抗体变体进行比较。为此，对从人源天然抗体文库中分离的单链片段可变区（scFv）的结合域进行遗传修饰，构建相应的全长 IgG、三聚体和二聚体变体。这些分子与双功能螯合剂 N，N'-双[2-羟基-5-（羧乙基）苄基]乙二胺-N，N'-二乙酸（HBED-CC）缀合，以实现室温下（68）Ga 标记，并在生物分布和免疫 PET 成像实验中进行比较。
具有相同特异性的抗体变体证明具有正确的分子量、对其抗原 EpCAM 的高结合亲和力和特异性。在室温下高效地进行放射性金属络合，导致与原始抗体变体相比，（68）Ga 标记的抗体保持不变的结合特性。最佳的靶向特性是通过 scFv 获得的，特别是通过二聚体获得的。三聚体显示出更高的绝对肿瘤摄取量，但从循环中清除速度较慢。
抗体变体在正常器官摄取、从循环中清除和肿瘤积累方面差异很大。这些数据证明了用（68）Ga 标记的二聚体形式对实体瘤进行成像的可行性。这种类型的重组蛋白即使对于短寿命放射性金属（68）Ga 也可能是一种有前途的载体，甚至可以支持免疫治疗的管理，这可能为实体瘤的受体表达提供重要信息。
