Toward in Vivo Imaging of Heart Disease Using a Radio labeled Single-Chain Fv Fragment Targeting Tenascin-C
Toward in Vivo Imaging of Heart Disease Using a Radio labeled Single-Chain Fv Fragment Targeting Tenascin-C
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DOI：
10.1021/ac202159p
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发表时间：
2011-12-01
影响因子：
7.4
通讯作者：
Irie, Toshiald
中科院分区：
文献类型：
--
作者：
Kobayashi, Norihiro;Odaka, Kenichi;Irie, Toshiald
针对特定靶分子的抗体可用作分析试剂，不仅可用于体外免疫分析，还可用于非侵入性体内成像，例如免疫核素成像。在后一种情况下，重要的是减小抗体分子的大小，以实现合适的活体“诊断动力学”并生成更高分辨率的图像。对于这些目的，单链抗体片段(scFvs；M(R)<；30 kDa)比完整的免疫球蛋白(类似于150 kDa)或Fab(或Fab‘)片段(类似于50 kDa)具有更大的潜力。我们最近观察到心肌梗死后心脏修复部位tenascin-C(TNC)表达增强，促使我们开发了针对TNC的放射性标记单链抗体，用于心脏病的活体成像。我们克隆了小鼠抗TNC单抗4F10的重链可变区和轻链可变区的基因，并将它们组合在一起创建了单基因。将获得的ScFv-4F10基因在大肠杆菌中表达，产生可溶性ScFv蛋白。ScFv-4F10对TNC具有亲和力(K(A)=3.5×10(7)M(-1))，与抗体4F10的Fab片段(K(A)=1.3×10(7)M(-1)相似)，具有较高的实用价值。然后将半胱氨酸残基添加到C-末端，通过螯合基团实现位点特异性WIN标记。将得到的(111)In标记单链抗体应用于急性心肌梗死大鼠模型。生物分布和定量放射自显影研究表明，梗死区心肌的放射性摄取高于非梗死区。单光子发射计算机断层扫描(SPECT)提供了与体外观察相一致的活体心脏图像。我们的结果将促进心脏病诊断策略的进步。
Antibodies specific to a particular target molecule can be used as analytical reagents, not only for in vitro immunoassays but also for noninvasive in vivo imaging, e.g., immunoscintigraphies. In the latter case, it is important to reduce the size of antibody molecules in order to achieve suitable in vivo "diagnostic kinetics" and generate higher-resolution images. For these purposes, single-chain Fv fragments (scFvs; M(r) < 30 kDa) have greater potential than intact immunoglobulins (similar to 150 kDa) or Fab (or Fab') fragments (similar to 50 kDa). Our recent observation of enhanced tenascin-C (Tnc) expression at sites of cardiac repair after myocardial infarction prompted us to develop a radiolabeled scFv against Tnc for in vivo imaging of heart disease. We cloned the genes encoding the heavy and light chain variable domains of the mouse anti-Tnc monoclonal antibody 4F10, and combined them to create a single gene. The resulting scFv-4F10 gene was expressed in E. coli cells to produce soluble scFv proteins. scFv-4F10 has an affinity for Tnc (K(a) = 3.5 x 10(7) M(-1)), similar to the Fab fragment of antibody 4F10 (K(a) = 1.3 x 10(7) M(-1)) and high enough to be of practical use. A cysteine residue was then added to the C-terminus to achieve site-specific win labeling via a chelating group. The resulting (111)In-labeled scFv was administered to a rat model of acute myocardial infarction. Biodistribution and quantitative autoradiographic studies indicated higher uptake of the radioactivity at the infarcted myocardium than the noninfarcted one. Single photon emission computed tomography (SPECT) provided in vivo cardiac images that coincided with the ex vivo observations. Our results will promote advances in diagnostic strategies for heart disease.