Lin Xinfeng, Zhu Hua, Luo Zheng, Hong Ye, Zhang Hong, Liu Xijuan, Ding Huirong, Tian Huifang, Yang Zhi
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, 52 Fucheng Road, Haidian District, 100142, Beijing, China.
Mol Imaging Biol. 2014 Dec;16(6):877-87. doi: 10.1007/s11307-014-0742-3.
Obinutuzumab is the first fully humanized and glycoengineered monoclonal antibody (mAb) directly targeting CD20 antigen, which is expressed on B cell lymphocytes and the majority of non-Hodgkin's lymphoma (NHL). This study aims to design a diagnostic molecular probe, Cy7-Obinutuzumab (Cy7-Obi), in which Cy7 is a near-infrared fluorescent dye. This probe is used to noninvasively image CD20 antigen expressed in NHL cells.
Cy7-Obi probe was synthesized through nucleophilic substitution reaction between NHS-Cy7 and obinutuzumab. After purification, the conjugate was fully characterized by a series of methods. The immunoreactivity and molecular specificity of the probe were confirmed using flow cytometry and in vitro microscopy on Raji (CD20-positive) cells. For in vivo imaging, Cy7-Obi probe (1 nmol) was injected intravenously in severe combined immunodeficiency (SCID) mice bearing Raji tumors which overexpress CD20 (n = 3) and was imaged with near-infrared fluorescence (NIRF) at 6, 9, 12, 24, 60, and 96 h post-probe injection. For pre-block, obinutuzumab (3.25 mg) was injected intravenously in tumor-bearing mice 6 h before the administration of Cy7-Obi probe.
The synthesized Cy7-Obi probe in this paper mimics obinutuzumab in both structure and function. Flow cytometry analysis of the probe and obinutuzumab on Raji cells showed minor difference in binding affinity/specificity with CD20. The probe showed significant fluorescence signal when it was examined on Raji cells using in vitro microscopy. The fluorescence signal can be blocked by pretreatment with obinutuzumab. The probe Cy7-Obi also showed high tumor uptake when it was examined by in vivo optical imaging on Raji tumor-bearing mice. The tumor uptake can be blocked by pretreatment with obinutuzumab (n = 3, p < 0.05). The in vivo imaging results were also confirmed by ex vivo imaging of dissected organs. Finally, the probe Cy7-Obi has shown excellent tumor targeting and specificity through immunofluorescence analysis.
We have shown that humanized Cy7-Obi probe can be used for NIRF imaging successfully. The probe may be an effective and noninvasive diagnostic molecular probe capable of tracking CD20 overexpression in NHL.
奥滨尤妥珠单抗是首个直接靶向CD20抗原的完全人源化且经糖基工程改造的单克隆抗体（mAb），CD20抗原在B淋巴细胞和大多数非霍奇金淋巴瘤（NHL）中表达。本研究旨在设计一种诊断性分子探针，即Cy7-奥滨尤妥珠单抗（Cy7-Obi），其中Cy7是一种近红外荧光染料。该探针用于对NHL细胞中表达的CD20抗原进行无创成像。
通过NHS-Cy7与奥滨尤妥珠单抗之间的亲核取代反应合成Cy7-Obi探针。纯化后，通过一系列方法对缀合物进行全面表征。使用流式细胞术和对Raji（CD20阳性）细胞进行体外显微镜检查，确认了探针的免疫反应性和分子特异性。对于体内成像，将Cy7-Obi探针（1 nmol）静脉注射到携带过表达CD20的Raji肿瘤的重症联合免疫缺陷（SCID）小鼠（n = 3）体内，并在注射探针后6、9、12、24、60和96小时用近红外荧光（NIRF）成像。对于预阻断，在给予Cy7-Obi探针前6小时，将奥滨尤妥珠单抗（3.25 mg）静脉注射到荷瘤小鼠体内。
本文合成的Cy7-Obi探针在结构和功能上均模拟奥滨尤妥珠单抗。对Raji细胞上的探针和奥滨尤妥珠单抗进行流式细胞术分析，结果显示与CD20的结合亲和力/特异性存在细微差异。当使用体外显微镜在Raji细胞上检测时，该探针显示出显著的荧光信号。该荧光信号可通过奥滨尤妥珠单抗预处理来阻断。在携带Raji肿瘤的小鼠上进行体内光学成像时，探针Cy7-Obi也显示出高肿瘤摄取。肿瘤摄取可通过奥滨尤妥珠单抗预处理来阻断（n = 3，p < 0.05）。解剖器官的离体成像也证实了体内成像结果。最后，通过免疫荧光分析表明探针Cy7-Obi具有出色的肿瘤靶向性和特异性。
我们已证明人源化的Cy7-Obi探针可成功用于NIRF成像。该探针可能是一种能够追踪NHL中CD20过表达的有效且无创的诊断性分子探针。
