利用64Cu-NOTA帕尼单抗的免疫PET成像在皮下及转移性非小细胞肺癌异种移植模型中的应用

目的：大约65-90%的非小细胞肺癌（NSCLC）表达上皮生长因子受体（EGFR），作为一种跨膜蛋白被激活，激活方式包括结合特定配体如表皮生长因子和转化生长因子α（TGFα）。确认EGFR作为癌基因推动了抗癌药物的研发，包括全长人源IgG2单克隆抗体帕尼单抗。本研究的主要目标是探讨标记为64Cu的帕尼单抗结合免疫PET在皮下和转移性EGFR阳性NSCLC异种移植模型中的成像效果。方法：将双功能螯合剂2-硫代氰基苯甲基-1,4,7-三唑环非烷-1,4,7-三乙酸（NOTA-NCS）连接到帕尼单抗上。利用基质辅助激光解吸/离子化（MALDI）质谱法测定每个抗体上的螯合剂数。通过放射性薄层色谱（radio-TLC）测定64Cu在NOTA-帕尼单抗中的结合效率。采用表达EGFR的上皮样H1299-luc+非小细胞肺癌细胞进行体内外实验。在有无帕尼单抗存在的情况下，测定[64Cu]Cu-NOTA-帕尼单抗在细胞中的摄取。采用雄性NSG小鼠建立皮下和转移性H1299-luc肿瘤模型，利用[18F]FLT PET分析肺部和转移部位的肿瘤。将[64Cu]Cu-NOTA-帕尼单抗在注射后2、24、48小时进行静态PET成像，验证靶向性通过阻断实验。在动物模型中进行详细的离体生物分布实验，以确认免疫PET成像获得的生物分布特征。结果：MALDI分析确认每个抗体连接约1.5个NOTA。用[64Cu]CuCl2的放射标记效率为93.8±5.7%，摩尔活性为0.65 MBq/μg。在H1299细胞中，[64Cu]Cu-NOTA-帕尼单抗的细胞摄取随时间增加，达到29.1±2.9%（Bq）/毫克蛋白（n=3），在45分钟时达到平台期。加入25 μg帕尼单抗后，放射配体的摄取降低至1.22±0.06%（Bq）/毫克蛋白（n=3）。PET成像显示在皮下肿瘤中[64Cu]Cu-NOTA-帕尼单抗的摄取率较高：24和48小时的标准化摄取值（SUV）平均值分别为4.70±0.42和5.37±0.40（n=5）。给药1 mg帕尼单抗显著降低肿瘤摄取率，分别为1.94±0.22和1.66±0.08（n=4；p<0.001）。在转移模型中，肝脏和肺损伤的SUV平均值在注射后24和48小时分别为5.55±0.34和6.28±0.46（均为6只小鼠的23个病灶），在注射1 mg帕尼单抗后显著降低至2.53±0.39和2.31±0.15（均为4只小鼠的16个病灶；p<0.001）。详细的离体生物分布分析验证了免疫PET的成像结果：帕尼单抗显著降低了皮下肿瘤的放射性摄取，从11.01±0.72% ID/g（n=4）降至3.67±0.33% ID/g（n=5；p<0.001），在转移性肝损伤中从29.44±8.14%ID/g（n=4）降至8.35±1.30%ID/g（n=5；p<0.001）。结论：[64Cu]Cu-NOTA-帕尼单抗成功应用于EGFR表达的皮下和转移性NSCLC肿瘤的免疫PET成像，为开发靶向EGFR的放射性治疗和患者筛选提供基础。

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.