Background

To develop the extracellular matrix metalloproteinase inducer (CD147)-targeting therapeutic strategies, accurate detection of CD147 expression in tumors is crucial. Owing to their relatively low molecular weights and high affinities, nanobodies (Nbs) may be powerful candidates for cancer diagnosis and therapy. In this study, we developed a novel CD147-targeted nanobody radiotracer, [¹²⁴I]I-NB147, which provides guidance for the noninvasive detection of CD147-overexpressing cancers.

Methods

CD147 expression in human cancers was detected via immunohistochemistry (IHC) on tissue microarrays (TMAs). Western blot (WB) and flow cytometry were used to screen CD147-positive malignant melanoma (MM), triple-negative breast cancer (TNBC), and pancreatic cancer (PCA) cell lines. The CD147 nanobody (NB147) was labeled with [¹²⁴I]INa using Iodogen as the oxidizing agent and was purified by the PD-10 column. The physicochemical properties, affinity, metabolic characteristics, biodistribution, and immunoPET imaging of [¹²⁴I]I-NB147 were evaluated Moreover, [¹⁸F]F-FDG was used as a control. Finally, CD147 expression analysis was performed via multiplex immunofluorescence (MxIF) and autoradiography on human cancer specimens and tumor-bearing mice tissues.

Results

TMAs results revealed that CD147 is highly expressed in MM, TNBC, and PCA. A CD147-specific nanobody, NB147, was successfully generated with excellent in vitro binding characteristics. [¹²⁴I]I-NB147 was obtained with high radiochemical yield and purity, and was stable for at least 4 h in vitro. WB and FCM revealed that CD147 was positive in A375, MDA-MB-435 and ASPC1 cells, whereas SK-MEL-28, 4T1 and BXPC3 cells presented low expression levels. The radio-ELISA results indicated that [¹²⁴I]I-NB147 had a high binding affinity to CD147. The uptake of [¹²⁴I]I-NB147 was significantly different between CD147 high-expression cells and CD147 low-expression cells (P < 0.001). The biological half-life of the distribution and clearance phases were 0.05 h and 1.58 h, respectively. In CD147-positive tumor models, the [¹²⁴I]I-NB147 accumulated in A375, MDA-MB-435, and ASPC1 tumors, and the uptake value was significantly higher than that of [¹⁸F]F-FDG. Uptake in SK-MEL-28, BXPC3, and 4T1 tumors was not clearly observed. Finally, through autoradiography and histological studies, the correlation analysis between tumor uptake and CD147 expression level was determined.

Conclusions

The high expression of CD147 in MM, TNBC, and PCA tissuesand in tumor cells was verified. The CD147 nanobody, NB147 was produced and radiolabeled to obtain the immunoPET probe, [¹²⁴I]I-NB147, which showed high affinity to CD147 and precise visualization for accurate diagnosis of CD147-expressing lesions in different cancers. These results provide insight into the imaging and binding properties of nanobody NB147 over extended periods of time, reinforcing its potential in developing radionuclide therapies for CD147-positive cancer patients.

Graphical abstract

中文翻译：

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放射性碘纳米抗体免疫 PET 探针用于泛癌中 CD147 的体内检测

 背景

为了开发细胞外基质金属蛋白酶诱导剂 （CD147） 靶向治疗策略，准确检测肿瘤中 CD147 的表达至关重要。由于其相对较低的分子量和高亲和力，纳米抗体 （Nbs） 可能是癌症诊断和治疗的有力候选者。在这项研究中，我们开发了一种新型 CD147 靶向纳米抗体放射性示踪剂 [¹²⁴I]I-NB147，为无创检测 CD147 过表达癌症提供了指导。

 方法

通过组织微阵列 （TMA） 上的免疫组织化学 （IHC） 检测 CD147 在人癌症中的表达。Western blot （WB） 和流式细胞术筛选 CD147 阳性恶性黑色素瘤 （MM） 、三阴性乳腺癌 （TNBC） 和胰腺癌 （PCA） 细胞系。CD147 纳米抗体 （NB147） 以碘原为氧化剂，用 [¹²⁴I]INa 标记，并通过 PD-10 柱纯化。评价了 [¹²⁴I]I-NB147 的理化性质、亲和力、代谢特性、生物分布和免疫 PET 成像此外，以 [¹⁸F]F-FDG 作为对照。最后，通过多重免疫荧光 （MxIF） 和放射自显影对人癌症标本和荷瘤小鼠组织进行 CD147 表达分析。

 结果

TMAs 结果显示 CD147 在 MM、TNBC 和 PCA 中高表达。成功生成了具有优异体外结合特性的 CD147 特异性纳米抗体 NB147。[¹²⁴我]I-NB147 具有高放射化学产率和纯度，并且在体外稳定至少 4 小时。WB 和 FCM 显示 CD147 在 A375 、 MDA-MB-435 和 ASPC1 细胞中呈阳性，而 SK-MEL-28 、 4T1 和 BXPC3 细胞呈低表达水平。放射ELISA结果显示，[¹²⁴I]I-NB147与CD147具有较高的结合亲和力。CD147 高表达细胞和 CD147 低表达细胞对 [¹²⁴I] I-NB147 的摄取差异显著 （P < 0.001）。分布期和清除期的生物半衰期分别为 0.05 h 和 1.58 h。在 CD147 阳性肿瘤模型中，[¹²⁴I]I-NB147 在 A375 、 MDA-MB-435 和 ASPC1 肿瘤中积累，摄取值显著高于 [¹⁸F]F-FDG。未明确观察到 SK-MEL-28 、 BXPC3 和 4T1 肿瘤的摄取。最后，通过放射自显影和组织学研究，确定肿瘤摄取与 CD147 表达水平的相关性分析。

 结论

验证了 CD147 在肿瘤细胞 MM 、 TNBC 和 PCA 组织沙中的高表达。产生 CD147 纳米抗体 NB147 并进行放射性标记以获得免疫 PET 探针 [¹²⁴I]I-NB147，该探针对 CD147 具有高亲和力和精确可视化，可准确诊断不同癌症中表达 CD147 的病变。这些结果为纳米抗体 NB147 在较长时间内的成像和结合特性提供了见解，增强了其为 CD147 阳性癌症患者开发放射性核素疗法的潜力。

 图形摘要