基于适配子的分子探针进行肺癌EGFR突变体的多模式分子显像和靶向治疗的实验研究

Aptamers are nucleotide acids that can bind to their targets with high specificity and high affinity. They have characeristics of low molecular weight, target specificity, high permeability and so on, and represents an ideal probe for targeted molecular imaging and therapy. EGFR antibodies did not show good results in molecular imaging owing to their high molecular weight and low tissue uptaking. The T790M mutation of EGFR (EGFR-T790M) is highly expressed lung cancer and accounts for 50% in acquired resistance to EGFR-TKI drugs . We have obtained and identified aptamers to the type III mutation of EGFR (EGFRvIII) which is specific target in glioma and detected its disgnois and therapy in vitro and in vivo by aptamers labeling with 188Re. In this project, we are going to screen and identify the aptamers with high specificity to EGFR-T790M protein which is specifically for lung cancer. We will produce the aptamers labeled with 68Ga and quantum dots, and detect the EGFR mutations in animal models of lung cancer using dual-modality imaging, and to evaluate the availability and practicability of the radiolabelling aptamers in imaging the EGFR mutations in lung cancer, it combined the quantum dots of Higher tissue penetration, higher signal to noise ratio with PET/CT providing higher quality tomographic images. And we will also detect the inhibition effects on animal model by the 188Re radiolabelled aptamers in vivo. It can provide a new multi-modality molecular probe for lung cancer with EGFR-T790M in early diagnosis, individualized treatment, resistance detection and curative effect detection, and also provide a radiopharmaceutical drug in targeted therapy of lung cancer.

适配子是一种高特异性和高亲和力结合靶标的核酸分子，有分子量小、靶向性高、组织渗透性高等特点，有望成为理想的分子成像探针和靶向治疗分子。EGFR抗体显像由于分子大、组织摄取低等原因而效果不好。EGFR的T790M突变体在肺癌中高表达，与EGFR-TKI的继发耐药相关（约占50%）。我们曾利用SELEX技术筛选EGFRvIII突变体的适配子，用188Re标记适配子进行了胶质瘤诊疗的研究。本研究拟对肺癌特异的EGFR-T790M突变蛋白进行SELEX筛选得到适配子，行68Ga和量子点双标记，结合了量子点组织穿透力更强、信噪比更高和PET/CT可提供更高质量图像的特点，在肺癌的动物模型上进行双模式分子显像，对其进行定性、定位和定量分析；另外还尝试研究188Re标记适配子的体内抑瘤作用。为肺癌早期诊断、个体化治疗、耐药检测、靶向治疗和疗效检测提供一种全新的多模式分子影像技术和核素靶向治疗药物。

EGFRT790M突变是肺癌早期诊断、靶向治疗药物、耐药检测等相关研究的热点。适配体（Aptamer），又称适配子，是通过SELEX筛选得到的与靶蛋白高亲和力和高特异性结合的寡核苷酸，可作为理想的分子探针和核酸类治疗分子，应用于肿瘤等疾病的诊断和靶向治疗。本项目通过SELEX技术筛选出特异识别EGFR T790M突变片段的适配体，主要取得以下成果：（1）构建EGFR T790M突变多肽，通过磁珠SELSEX技术得到高亲和力的适配体，并进行高通量测序和二级结构预测；（2）流式细胞术检测适配体与靶标的结合常数Kd值在nM水平，具有高特异性和高亲和力；（3）细胞功能实验验证EGFR T790M特异的适配体能抑制肺癌细胞的增殖、迁移和侵袭，同时肺癌动物模型瘤内给药显示有体内抑瘤效果；（4）适配体给药联合放射实验验证EGFR T790M特异的适配体有放射增敏作用；（5）通过免疫印迹实验验证适配体作用的关键信号通路为抑制EGFR和AKT的磷酸化，同时适配体给药联合X线照射后可通过抑制DNA损伤修复信号通路起放射增敏效果。（6）将适配体进行荧光素标记后，尾静脉给药于肺癌荷瘤裸鼠模型，通过活体成像显示适配体有体内靶向性。以上结果表明成功筛选得到针对EGFR T790M突变的新型核酸药物适配体，为研发肺癌的早期诊断、靶向治疗、在体肿瘤组织EGFR表达水平检测和分子显像提供新的技术平台和研究工具。