一种含有细菌白蛋白结合结构域（Zag）的（67）Ga标记抗TNF VHH单域抗体的生物分布
Biodistribution of a (67)Ga-labeled anti-TNF VHH single-domain antibody containing a bacterial albumin-binding domain (Zag).
作者信息
Morais Maurício, Cantante Cátia, Gano Lurdes, Santos Isabel, Lourenço Sara, Santos Catarina, Fontes Carlos, Aires da Silva Frederico, Gonçalves João, Correia João D G
机构信息
Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10, ao km 139.7, 2695-066 Bobadela LRS, Portugal.
CPM-URIA Fac. Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; IMM, Fac. Med., Universidade de Lisboa, Av. Prof. Egas Moniz 1649-028 Lisboa, Portugal.
出版信息
Nucl Med Biol. 2014 May;41 Suppl:e44-8. doi: 10.1016/j.nucmedbio.2014.01.009. Epub 2014 Jan 17.
INTRODUCTION
Small domain antibodies (sdAbs) present high potential for both molecular in vivo imaging and therapy. Owing to the low molecular weight they are rapidly cleared from blood circulation, and new strategies to extend their half-lifes are needed for therapeutic applications. We have selected a bacterial albumin-binding domain (ABD) from protein Zag to be fused to an anti-tumor necrosis factor (TNF) single variable-domain heavy-chain region antibody (VHH) to delay blood clearance, and evaluated the biodistribution profile of the fusion protein.
METHODS
The anti-TNF VHH and the fusion protein VHH-Zag were conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA). The anti-TNF and albumin-binding properties of the conjugates NOTA-VHH and NOTA-VHH-Zag were assessed by enzyme-linked immunosorbent assay (ELISA). The radioconjugates (67)Ga-NOTA-VHH and (67)Ga-NOTA-VHH-Zag were obtained by reaction of (67)GaCl3 with the corresponding conjugates at room temperature. Biodistribution studies were performed in healthy female CD-1 mice.
RESULTS
The immunoreactivity of the VHH-based proteins is preserved upon conjugation to NOTA as well as after radiometallation. The radiochemical purity of the radioconjugates was higher than 95% as determined by ITLC-SG after purification by gel filtration. The biodistribution studies showed that the Zag domain affected the pharmacokinetic properties of VHH, with impressive differences in blood clearance (0.028 ± 0.004 vs 1.7 ± 0.8 % I.A./g) and total excretion (97.8 ± 0.6 vs 25.5 ± 2.1 % I.A.) for (67)Ga-NOTA-VHH and (67)Ga-NOTA-VHH-Zag, respectively, at 24h p.i.
CONCLUSION
The Zag domain prolonged the circulation time of VHH by reducing the blood clearance of the labeled fusion protein (67)Ga-NOTA-VHH-Zag. In this way, the anti-TNF VHH in fusion with the Zag ABD presents a higher therapeutic potential than the unmodified VHH.
引言
小分子结构域抗体（sdAbs）在分子体内成像和治疗方面具有很高的潜力。由于其分子量低，它们会迅速从血液循环中清除，因此治疗应用需要新的策略来延长其半衰期。我们从蛋白质Zag中选择了一个细菌白蛋白结合结构域（ABD），将其与抗肿瘤坏死因子（TNF）单可变结构域重链区域抗体（VHH）融合以延迟血液清除，并评估了融合蛋白的生物分布情况。
方法
将抗TNF VHH和融合蛋白VHH-Zag与S-2-(4-异硫氰酸苄基)-1,4,7-三氮杂环壬烷-1,4,7-三乙酸（p-SCN-Bn-NOTA）偶联。通过酶联免疫吸附测定（ELISA）评估偶联物NOTA-VHH和NOTA-VHH-Zag的抗TNF和白蛋白结合特性。通过在室温下使氯化镓（67）GaCl3与相应的偶联物反应获得放射性偶联物（67）Ga-NOTA-VHH和（67）Ga-NOTA-VHH-Zag。在健康雌性CD-1小鼠中进行生物分布研究。
结果
与NOTA偶联后以及放射性金属化后，基于VHH的蛋白质的免疫反应性得以保留。通过凝胶过滤纯化后，用ITLC-SG测定，放射性偶联物的放射化学纯度高于95%。生物分布研究表明，Zag结构域影响了VHH的药代动力学特性，注射后24小时，（67）Ga-NOTA-VHH和（67）Ga-NOTA-VHH-Zag的血液清除率（分别为0.028±0.004与1.7±0.8%注射剂量/克）和总排泄率（分别为97.8±0.6与25.5±2.1%注射剂量）存在显著差异。
结论
Zag结构域通过降低标记融合蛋白（67）Ga-NOTA-VHH-Zag的血液清除率延长了VHH的循环时间。通过这种方式，与Zag ABD融合的抗TNF VHH比未修饰的VHH具有更高的治疗潜力。
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