In Vitro Analysis and In Vivo Tumor Targeting of a Humanized, Grafted Nanobody in Mice Using Pinhole SPECT/Micro-CT
In Vitro Analysis and In Vivo Tumor Targeting of a Humanized, Grafted Nanobody in Mice Using Pinhole SPECT/Micro-CT
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DOI：
10.2967/jnumed.109.069823
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发表时间：
2010-07-01
影响因子：
9.3
通讯作者：
Devoogdt, Nick
中科院分区：
文献类型：
--
作者：
Vaneycken, Ilse;Govaert, Jochen;Devoogdt, Nick
纳米抗体是一种新型的免疫球蛋白样抗原结合蛋白，具有有益的药理学和药代动力学特性，非常适合针对细胞抗原进行分子成像或治疗目的。然而，由于其骆驼科动物的非人类来源，纳米抗体在临床使用时可能的免疫原性令人担忧。在这里，我们提出了一种快速生成用于分子成像目的的人源化纳米抗体的新策略。方法：我们将 NbCEA5（一种对结肠癌标志物癌胚抗原 (CEA) 具有特异性的纳米抗体）的抗原结合环基因移植到人源化纳米抗体支架的框架上。该支架之前在我们的实验室中被描述为稳定的纳米抗体，可以作为来自供体纳米抗体的抗原结合环的通用环受体，并且在大约10个关键的表面暴露位点进行了额外突变，以类似于人类可变免疫球蛋白结构域的序列。 3 种重组纳米抗体（NbCEA5、人源化支架和人源化 CEA5 移植物）在细菌中产生并纯化。未标记和 Tc-99m 标记的纳米抗体在体外进行了生化表征，并作为异种移植肿瘤的 SPECT/CT 探针进行了测试。结果：通过比较这些纳米抗体在酶联免疫吸附测定中识别可溶性 CEA 蛋白的能力和通过表面等离振子共振的能力，以及在流式细胞术中与 CEA 阳性 LS174T 结肠癌细胞和 CEA 转染但未转染的中国仓鼠卵巢细胞结合的能力，证实了环移植的成功。结合的特异性通过竞争研究得到证实。所有纳米抗体都是热稳定的，可以用 Tc-99m 有效标记，并在结合研究中识别可溶性和膜结合的 CEA 蛋白。最后，使用针孔 SPECT/micro-CT 在 LS174T 荷瘤小鼠中静脉注射 Tc-99m 标记的纳米抗体进行生物分布实验。这些体内实验揭示了所有纳米抗体的肿瘤靶向特异性和快速肾脏清除率，除肾脏外的所有器官中信号均较低。结论：本研究显示了抗原结合环移植有效生成人源化纳米抗体的效力，这些纳米抗体保留了肿瘤无创体内成像的靶向能力。
Nanobodies are a novel type of immunoglobulinlike, antigen-binding protein with beneficial pharmacologic and pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. However, because of their camelid, nonhuman origin, the possible immunogenicity of Nanobodies when used in the clinic is a concern. Here we present a new strategy to quickly generate humanized Nanobodies for molecular imaging purposes. Methods: We genetically grafted the antigen-binding loops of NbCEA5, a Nanobody with specificity for the colon carcinoma marker carcinoembryonic antigen (CEA), onto the framework of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory as a stable Nanobody that can serve as a universal loop acceptor for antigen-binding loops from donor Nanobodies and has been additionally mutated at about 10 crucial surface-exposed sites to resemble the sequence of human variable immunoglobulin domains. The 3 recombinant Nanobodies (NbCEA5, humanized scaffold, and humanized CEA5 graft) were produced in bacteria and purified. Unlabeled and Tc-99m-labeled Nanobodies were biochemically characterized in vitro and tested as probes for SPECT/CT of xenografted tumors. Results: The success of loop-grafting was confirmed by comparing these Nanobodies for their capacity to recognize soluble CEA protein in enzyme-linked immunosorbent assay and by surface plasmon resonance and to bind to CEA-positive LS174T colon carcinoma cells and CEA-transfected but not untransfected Chinese hamster ovary cells in flow cytometry. Specificity of binding was confirmed by competition studies. All Nanobodies were heat-stable, could be efficiently labeled with Tc-99m, and recognized both soluble and membrane-bound CEA protein in binding studies. Finally, biodistribution experiments were performed with intravenously injected Tc-99m-labeled Nanobodies in LS174T tumor-bearing mice using pinhole SPECT/micro-CT. These in vivo experiments revealed specificity of tumor targeting and rapid renal clearance for all Nanobodies, with low signals in all organs besides the kidneys. Conclusion: This study shows the potency of antigen-binding loop-grafting to efficiently generate humanized Nanobodies that retain their targeting capacities for noninvasive in vivo imaging of tumors.