在皮下和转移性非小细胞肺癌异种移植物中，具有64cu-nota panitumumumab对EGFR的免疫PET成像

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）是一种通过特定配体的结合而激活的跨膜蛋白，包括表皮生长因子和转化生长因子α（TGFα）。将EGFR识别为癌基因，导致针对EGFR的抗癌治疗剂的发展，包括全长的人IgG2单克隆抗体panitiTumumab。本研究的主要目的是研究64CU标记的panitumumumab，并在皮下和转移性EGFR阳性NSCLC异种移植物中使用免疫-ET-PET。方法：双功能螯合剂2-S-（4-异硫氰苯二苯基）-1,4,7- triazacyclo-nonane-1,4,7-毛酸（nota-ncs）连接到panitumumumab。使用基质辅助激光解吸/电离（MALDI）质谱法确定每个持纤维单抗的螯合剂数量。使用Radio-TLC测量了64CU在NOTA-PANITUMUMUMAB中的掺入效率。表达EGFR的上皮样H1299-LUC+ NSCLC细胞用于体外和体内实验。在存在和不存在panitumumab的情况下测量了[64cu] Cu-Nota-Panitumumab的细胞摄取。雄性NSG小鼠生长皮下和转移性H1299-LUC肿瘤模型。通过[18F] FLT PET分析了肺部和转移性部位的肿瘤的存在。在两个肿瘤模型中，用[64cu] Cu-Nota-Panitumumab在2、24和48 h的静态PET成像中进行免疫PET作为静态PET成像。通过阻止panitumumab的实验来证实目标证明。在两个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分析证实了每种抗体约1.5 NOTA的附着。 [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细胞中使用[64CU] Cu-Nota-Panitumumab进行的细胞摄取研究表明，随着时间的推移摄取量增加，达到29.1±2.9％的放射性（BQ）/mg蛋白（n = 3）（n = 3），在45分钟内达到了高原。添加25μg的Panitumumab降低了放射性吸收量为1.22±0.06％放射性/mg蛋白（n = 3）。 PET成像显示在皮下肿瘤中[64cu] Cu-Nota-Panitumumab的摄取高：分别在注射后24和48 h后，标准化的吸收值（SUV）平均达到4.70±0.42和5.37±0.40（n = 5）。 1 mg panitumumab的施用可显着降低肿瘤的摄取至1.94±0.22和1.66±0.08（n = 4; p <0.001）。在转移模型中，在注射后24和48 h后，从肝脏和肺部病变中分析了以下suvmean：5.55±0.34和6.28±0.46（均来自6只小鼠的n = 23个病变），这也显着降低至2.53±0.39和2.31±0.39和2.31±0.15（n = 16 l = 16 l = 16 l = 16 lesions; Panitumumab。详细的离体生物分布确认了这两个模型中的免疫PET分析。 Panitumumab从11.01±0.72（n = 4）降低到3.67±0.33％ID/G（n = 5; P <0.001），在29.44±8.14（n = 4）（n = 4）到8.35±1.30％/g（n = 0.00;结论：[64cu] Cu-Nota-Panitumumab成功地用于表达EGFR的皮下和转移性NSCLC肿瘤的免疫-PET成像。该结果代表了开发用于靶向癌症EGFR的放射性抑制剂的基础，并在正确的时间选择合适的患者进行正确的治疗。

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.