目的：约 65-90% 的非小细胞肺癌 (NSCLC) 表达上皮生长因子受体 (EGFR) 作为跨膜蛋白，该蛋白通过特定配体（包括表皮生长因子和转化生长因子 α (TGFα)）的结合而被激活。将 EGFR 确定为癌基因导致了针对 EGFR 的抗癌疗法的开发，包括全长人 IgG2 单克隆抗体帕尼单抗。本研究的主要目标是通过免疫 PET 研究 64Cu 标记的帕尼单抗在皮下和转移性 EGFR 阳性 NSCLC 异种移植物中的作用。方法：将双功能螯合剂 2-S-(4-异硫氰酸苄基)-1,4,7-三氮杂环壬烷-1,4,7-三乙酸 (NOTA-NCS) 连接至帕尼单抗。使用基质辅助激光解吸/电离 (MALDI) 质谱法测定每种帕尼单抗的螯合剂数量。使用放射TLC测量64Cu掺入NOTA-帕尼单抗的效率。表达 EGFR 的上皮样 H1299-luc NSCLC 细胞用于体外和体内实验。在帕尼单抗存在和不存在的情况下测量[64Cu]Cu-NOTA-帕尼单抗的细胞摄取。在雄性 NSG 小鼠中培养皮下和转移性 H1299-luc 肿瘤模型。通过[18F]FLT PET 分析肺部和转移部位肿瘤的存在。在两种肿瘤模型中注射后 2、24 和 48 小时，使用 [64Cu]Cu-NOTA-帕尼单抗进行免疫 PET 成像，作为静态 PET 成像。通过帕尼单抗的阻断实验证实了靶点证明。在两种动物肿瘤模型中进行了详细的离体生物分布实验，以确认通过免疫 PET 成像获得的生物分布曲线。结果：MALDI 分析证实每个抗体附着约 1.5 个 NOTA。 [64Cu]CuCl2 的放射性标记效率为 93.8 ± 5.7%，摩尔活性为 0.65 MBq/μg。 H1299 细胞中 [64Cu]Cu-NOTA-帕尼单抗的细胞摄取研究表明，摄取随着时间的推移而增加，达到 29.1 ± 2.9% 放射性 (Bq)/mg 蛋白质 (n = 3)，并在 45 分钟时达到稳定水平。添加 25 μg 帕尼单抗可将放射性配体摄取降低至 1.22 ± 0.06% 放射性/毫克蛋白质 (n = 3)。 PET 成像显示皮下肿瘤中 [64Cu]Cu-NOTA-帕尼单抗的高摄取：注射后 24 小时和 48 小时后标准化摄取值 (SUV) 平均值分别达到 4.70 ± 0.42 和 5.37 ± 0.40 (n = 5)。施用 1 mg 帕尼单抗可将肿瘤摄取显着降低至 1.94 ± 0.22 和 1.66 ± 0.08（n = 4；p < 0.001）。在转移模型中，从肝脏和肺部病变中分析了以下 SUV 平均值：注射后 24 小时和 48 小时后分别为 5.55 ± 0.34 和 6.28 ± 0.46（n = 6 只小鼠的 23 个病变），也显着降低至 2.53 ± 0.39 和注射 1 mg 帕尼单抗后，值为 2.31 ± 0.15（n = 4 只小鼠的 16 个病变；p < 0.001）。详细的离体生物分布证实了两种模型中的免疫 PET 分析。帕尼单抗将皮下肿瘤的放射性摄取从 11.01 ± 0.72 (n = 4) 降低至 3.67 ± 0.33% ID/g (n = 5; p < 0.001)，并将转移性肝病灶的放射性摄取从 29.44 ± 8.14 (n = 4) 降低至 8.35分别为 ± 1.30% ID/g（n = 5；p < 0.001）。结论：[64Cu]Cu-NOTA-帕尼单抗成功用于表达 EGFR 的皮下和转移性 NSCLC 肿瘤的免疫 PET 成像。这一结果为开发针对癌症中的 EGFR 的放射治疗以及选择正确的患者在正确的时间进行正确的治疗奠定了基础。

Objective: About 65-90% of nonsmall cell lung cancer (NSCLC) express the epithelial growth factor receptor (EGFR) as a transmembrane protein that is activated by binding of specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). Identifying EGFR as an oncogene has led to the development of anticancer therapeutics directed against EGFR, including the full-length human IgG2 monoclonal antibody panitumumab. The main goal of the present study was to investigate 64Cu-labeled panitumumab with immuno-PET in subcutaneous and metastatic EGFR-positive NSCLC xenografts. Methods: Bifunctional chelating agent 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclo-nonane-1,4,7-triacetic acid (NOTA-NCS) was attached to panitumumab. The number of chelators per panitumumab was determined using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy. The incorporation efficiency of 64Cu into NOTA-panitumumab was measured by using radio-TLC. EGFR-expressing epithelial-like H1299-luc+ NSCLC cells were used for in vitro and in vivo experiments. Cell uptake of [64Cu]Cu-NOTA-panitumumab was measured in the presence and absence of panitumumab. Subcutaneous and metastatic H1299-luc tumor models were grown in male NSG mice. The presence of tumors at lung and metastatic sites was analyzed by [18F]FLT PET. Immuno-PET with [64Cu]Cu-NOTA-panitumumab was performed as static PET imaging at 2, 24, and 48 h postinjection in both tumor models. Proof-of-target was confirmed by blocking experiments with panitumumab. Detailed ex vivo biodistribution experiments were performed in both animal tumor models to confirm biodistribution profiles obtained by immuno-PET imaging. Results: MALDI analysis confirmed the attachment of ∼1.5 NOTA per antibody. Radiolabeling efficiency with [64Cu]CuCl2 was 93.8 ± 5.7% and a molar activity of 0.65 MBq/μg. Cellular uptake studies with [64Cu]Cu-NOTA-panitumumab in H1299 cells demonstrated increasing uptake over time, reaching 29.1 ± 2.9% radioactivity(Bq)/mg protein (n = 3) and plateauing at 45 min. Addition of 25 μg of panitumumab reduced radioligand uptake to 1.22 ± 0.06% radioactivity/mg protein (n = 3). PET imaging revealed high uptake of [64Cu]Cu-NOTA-panitumumab in subcutaneous tumors: Standardized uptake values (SUV)mean reached 4.70 ± 0.42 and 5.37 ± 0.40 (n = 5) after 24 and 48 h postinjection, respectively. Administration of 1 mg panitumumab reduced tumor uptake significantly to 1.94 ± 0.22 and 1.66 ± 0.08 (n = 4; p < 0.001). In the metastatic model, the following SUVmean were analyzed from liver and lung lesions: 5.55 ± 0.34 and 6.28 ± 0.46 (both n = 23 lesions from 6 mice) after 24 and 48 h postinjection, which was also significantly reduced to 2.53 ± 0.39 and 2.31 ± 0.15 (both n = 16 lesions from 4 mice; p < 0.001) after injection of 1 mg panitumumab. Detailed ex vivo biodistribution confirmed immuno-PET analysis in both models. Panitumumab reduced radioactivity uptake into subcutaneous tumors from 11.01 ± 0.72 (n = 4) to 3.67 ± 0.33% ID/g (n = 5; p < 0.001), and in metastatic liver lesions from 29.44 ± 8.14 (n = 4) to 8.35 ± 1.30% ID/g (n = 5; p < 0.001), respectively. Conclusions: [64Cu]Cu-NOTA-panitumumab was successfully used for immuno-PET imaging of EGFR-expressing subcutaneous and metastatic NSCLC tumors. This result represents the basis for developing radiotheranostics for targeting EGFR in cancers and for selecting the right patients for the right treatment at the right time.