Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules
Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules
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DOI：
10.1021/bc700307y
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发表时间：
2008-01-01
影响因子：
4.7
通讯作者：
Tolmachev, Vladimir
中科院分区：
文献类型：
--
作者：
Ahlgren, Sara;Orlova, Anna;Tolmachev, Vladimir
亲和体分子是一类新的小（7 kDa）支架亲和蛋白，其表现出作为体内放射性核素靶向试剂的有前途的性质。亲和体支架不含半胱氨酸，因此不依赖于二硫键。因此，可以通过引入一个半胱氨酸将单个巯基工程化到蛋白质中。巯基反应性双功能螯合剂的偶联可以实现重组产生的亲和体分子的位点特异性标记。在本研究中，评价了1，4，7，10-四氮杂环十二烷-1，4，7-三-乙酸-10-马来酰亚胺乙基乙酰胺（MMA-DOTA）用于In-111标记抗HER 2亲和体分子HiS（6）-Z（HER 2：342）-Cys和Z（HER 2：2395）-Cys的用途。半胱氨酸残基的引入不影响蛋白质的亲和力，其对于HiS（6）-Z（HER 2：342）-Cys为29 pM，对于Z（HER 2：2395）-Cys为27 pM，与亲本Z（HER 2：342）的22 pM相当。使用螯合剂与蛋白质的1：1摩尔比将MMA-DOTA缀合至DTT还原的亲和体分子，偶联效率为93%。将缀合物用标记。In-111的比放射性高达7 GBq/mmol，保留了与靶HER 2的结合。在体内，非His-标记的变体In-111-[MMA-DOTA-CyS 61]-Z（HER 2：2395）-Cys表现出比其含His-标记的对应物明显更低的肝脏摄取。在携带表达HER 2的LS 174 T异种移植物的小鼠中，In-111-[MMA-DOTA-Cys（61）]- Z（HER 2：2395）-CYS显示特异性和快速的肿瘤定位，以及从血液和非特异性区室的快速清除，导致肿瘤-血液比为18 +/- 8，已经在注射后1 h。四小时的注射，肿瘤与血液的比例为138 +/- 8。异种移植物在注射后1 h已清晰可见。
Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for In-111-labeling of anti-HER2 Affibody molecules HiS(6)-Z(HER2:342)-Cys and Z(HER2:2395)-CYS has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for HiS(6)-Z(HER2:342)-Cys and 27 pM for Z(HER2:2395)-CYS, comparable with 22 pM for the parental Z(HER2:342). MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with. In-111 to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non His-tagged variant In-111-[MMA-DOTA-CyS61]-Z(HER2:2395)-Cys demonstrated appreciably lower liver uptake than its His-tagcontaining counterpart. In mice bearing HER2-expressing LS174T xenografts, In-111-[MMA-DOTA-Cys(61)]- Z(HER2:2395)-CYS showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.