以EGFR和IGF-ⅠR为靶点的双特异性融合蛋白的构建及其抗非小细胞肺癌活性研究

Epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor Ⅰ both over-expressed on non-small cell lung cancer (NSCLC) and there are complicated crosstalk between the two receptors, making them promising targets for cancer therapy. Lidamycin (LDM) is an enediyne antibiotic with highly potent antitumor activity. The conjugates or fusion proteins which composed of antibody/ligand and lidamycin exhibited strong antitumor efficacy. This study was to construct a novel bispecific fusion protein EGF-LDP-IGF-AE consisting of EGFR and IGF-ⅠR specific ligands (EGF and IGF) and lidamycin, and investigate its antitumor efficacy. The bispecific fusion protein EGF-LDP-IGF-AE was constructed by two steps. First, the fusion gene egf-ldp-igf which contains the genes of EGF, apoprotein of lidamycin (LDP) and IGF was cloned and inserted into pET30a expression vector, then the fusion protein EGF-LDP-IGF was expressed in E.Coli and purified by affinity chromatography. Second, the energized fusion protein EGF-LDP-IGF-AE was prepared by integrating the active enediyne chromophore of lidamycin (AE) into the EGF-LDP-IGF protein. The binding affinity and in vitro cytotoxicity of EGF-LDP-IGF-AE protein to NSCLC cells was measured in the study. Moreover, the binding affinity and in vitro cytotoxicity of the corresponding monospecific fusion proteins EGF-LDP-AE and LDP-IGF-AE was also analyzed for comparison. The in vivo efficacy of growth inhibition by energized fusion protein EGF-LDP-IGF-AE was evaluated with human NSCLC xenografts model. In addition, compared with lidamycin and corresponding monospecific fusion proteins, whether the bispecific fusion protein EGF-LDP-IGF-AE showed decreased toxicity to normal tissues and increased growth inhibitory effects on tumors is another focus of this study, and this will provide evidences for exploring the importance of dual-inhibiting EGFR/IGF-ⅠR in NSCLC treatment. The highly potent antitumor activity, the smaller molecular weight and dual-targeting property suggested that EGF-LDP-IGF-AE protein would be a promising candidate for NSCLC-targeting therapy.

表皮生长因子受体（EGFR）和胰岛素样生长因子受体Ⅰ（IGF-ⅠR）在人非小细胞肺癌中呈过表达状态，且二者之间可发生多种形式的相互作用，是非小细胞肺癌治疗的理想靶点；力达霉素(LDM)是一种强效的抗肿瘤抗生素，在细胞实验及动物模型中均显示了良好的抗肿瘤活性。本项目以EGFR和IGF-ⅠR的天然配体EGF和IGF作为导向载体，以LDM作为"弹头"药物，制备一种兼具双靶向、小型化和高效化的新型融合蛋白EGF-LDP-IGF-AE。检测该融合蛋白与EGFR和IGF-ⅠR受体的亲和活性、体外细胞毒效应以及与相应的单靶点融合蛋白相比，其毒副作用是否减小。采用动物模型评价EGF-LDP-IGF-AE对EGFR/IGF-ⅠR高表达的非小细胞肺癌移植瘤的生长抑制作用，并与相应单靶点融合蛋白的抑瘤作用进行比较，探讨有效杀伤EGFR/IGF-ⅠR双阳性细胞是否对人非小细胞肺癌的治疗具有更积极的意义。

表皮生长因子受体（EGFR）和胰岛素样生长因子受体1（IGF-1R）在人非小细胞肺癌（Non-small cell lung cancer, NSCLC）中呈高表达状态，且二者之间可发生多种形式的相互作用，是非小细胞肺癌治疗的理想靶点；力达霉素(Lidamycin, LDM)是一种具有烯二炔结构的强效抗肿瘤抗生素，它与抗体或配体的偶联物显示了良好的抗肿瘤疗效。本项目首先将EGFR和IGF-IR的天然配体EGF和IGF-1与LDM的辅基蛋白LDP进行融合表达，获得融合蛋白EGF-IGF-LDP。之后再将EGF-IGF-LDP蛋白与LDM的发色团分子AE在体外进行分子组装，制备了一种烯二炔强化融合蛋白EGF-IGF-LDP-AE，并检测了该融合蛋白对NSCLC的体内外抗肿瘤活性。MTT结果显示EGF-IGF-LDP-AE对NSCLC细胞系A549、H520、H460和H1299均具有非常强烈的杀伤活性，其IC50值均小于0.01nmol/L，而且其杀伤活性显著高于LDM和两种相应的单靶向性强化融合蛋白EGF-LDP-AE和LDP-IGF-AE。细胞周期和细胞凋亡检测结果显示，EGF-IGF-LDP-AE可导致细胞发生显著的G2/M期阻滞，并以浓度依赖性的方式诱导NSCLC细胞发生凋亡。在人NSCLC细胞A549裸鼠移植瘤模型中，EGF-IGF-LDP-AE同样具有显著的抑制肿瘤生长的作用，在0.2mg/kg和0.4mg/kg 的剂量时抑瘤率分别达到了69.7%和和80.5%，抑瘤效果显著高于LDM最大耐受剂量（0.05mg/kg）处理组。且EGF-IGF-LDP-AE对肿瘤的生长抑制作用也显著高于相同剂量的EGF-LDP-AE和LDP-IGF-AE处理组。EGF-IGF-LDP-AE处理可抑制由EGF和IGF-1诱导的EGFR和IGF-1R的激活，并进而抑制了两个主要的下游信号分子p42/44 MAPK及AKT的活化，这可能是EGF-IGF-LDP-AE发挥强效抗肿瘤效应的分子机制之一。本项目以EGF和IGF-1作为导向载体，以LDM作为“弹头”药物构建的融合蛋白EGF-IGF-LDP-AE具有双靶向和小型化（分子量仅27kDa）的特点，并对NSCLC表现出了高效的体内外抗肿瘤活性，具有开发成为新型NSCLC靶向治疗药物的潜能。