Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
Laboratory of Molecular Pathology and Cancer Genomics, Research Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul, Republic of Korea.
Nucl Med Biol. 2022 Nov-Dec;114-115:135-142. doi: 10.1016/j.nucmedbio.2022.04.003. Epub 2022 Apr 8.
Claudin-3 (CLDN3), a tight junction protein, regulates cell-to-cell interactions in epithelial or endothelial cell sheets. During tumorigenesis, epithelial cells are transformed, and tumor cells proliferate through out-of-plane division, resulting in external exposure of CLDN3. Since alterations of CLDN3 expression are associated with cancer progression and higher CLDN3 expression is observed in most ovarian cancers, we tested the feasibility of using a CLDN3-specific antibody as a novel imaging tracer.
After reducing the CLDN3-specific antibodies to expose the -SH groups, click chemistry was used to conjugate the radioactive isotope In or the fluorescent protein FNR648. Human ovarian cancer OVCAR-3 and glioblastoma U87MG cells were used as CLDN3-positive and -negative cells. Flow cytometry was used to determine the CLDN3 IgG1 monoclonal antibody binding to both cell lines. OVCAR-3 cells were injected subcutaneously into mice to establish a xenograft model. In-labeled CLDN3 antibodies (370 kBq/50 μL) were administered intravenously into mice. After 24 h, organs, including tumors, were excised and measured with a γ-counter. Images were acquired with the IVIS optical imaging system and SPECT/CT.
The labeling efficiency of NOTA-In and antibody-NOTA-In was 98.52% and 100%, respectively. FNR648-labeled CLDN3 antibody bound to the cell surface of OVCAR-3 and U87MG with 83.4% and 5.7% specificity, respectively. In OVCAR-3 tumor xenografted mice, CLDN3 IgG1 antibody showed a 2.5-fold higher tumor uptake (20.4 ± 7.4% ID/g) than human IgG1 (8.8 ± 2.6% ID/g) at 24 h post injection. The CLDN3 antibody fluorescence signal in the tumor peaked at 24 h post injection.
We have successfully conjugated a radioisotope and a fluorescent protein with CLDN3-specific antibodies and verified the specific binding of labeled antibodies to OVCAR-3 tumors in a mouse model. Our data suggested that CLDN3-specific human monoclonal antibodies could be used as a useful theranostic tracer.
紧密连接蛋白 3（CLDN3）调节上皮细胞或内皮细胞层的细胞间相互作用。在肿瘤发生过程中，上皮细胞发生转化，肿瘤细胞通过平面外分裂增殖，导致 CLDN3 暴露于细胞外。由于 CLDN3 表达的改变与癌症进展有关，并且大多数卵巢癌中观察到 CLDN3 表达升高，因此我们测试了使用 CLDN3 特异性抗体作为新型成像示踪剂的可行性。
将 CLDN3 特异性抗体还原以暴露 -SH 基团后，使用点击化学将放射性同位素 In 或荧光蛋白 FNR648 缀合到抗体上。用人卵巢癌细胞系 OVCAR-3 和胶质母细胞瘤 U87MG 细胞作为 CLDN3 阳性和阴性细胞。使用流式细胞术确定 CLDN3 IgG1 单克隆抗体与这两种细胞系的结合。将 OVCAR-3 细胞皮下注射到小鼠中以建立异种移植模型。将放射性标记的 CLDN3 抗体（370kBq/50μL）静脉内注射到小鼠体内。24 小时后，取出包括肿瘤在内的器官，并使用γ计数器进行测量。使用 IVIS 光学成像系统和 SPECT/CT 采集图像。
NOTA-In 和抗体-NOTA-In 的标记效率分别为 98.52%和 100%。FNR648 标记的 CLDN3 抗体分别与 OVCAR-3 和 U87MG 细胞表面的结合特异性为 83.4%和 5.7%。在 OVCAR-3 肿瘤异种移植小鼠中，CLDN3 IgG1 抗体在注射后 24 小时的肿瘤摄取率（20.4±7.4%ID/g）是人类 IgG1 的 2.5 倍（8.8±2.6%ID/g）。注射后 24 小时，CLDN3 抗体的荧光信号在肿瘤中达到峰值。
我们已成功将放射性同位素和荧光蛋白与 CLDN3 特异性抗体缀合，并在小鼠模型中验证了标记抗体与 OVCAR-3 肿瘤的特异性结合。我们的数据表明，CLDN3 特异性人源单克隆抗体可用作有用的治疗性示踪剂。
