微环DNA载体介导BiTE治疗卵巢癌的应用基础研究

In spite of recent advances in diagnostic and therapeutic techniques, ovarian cancer remains the principal cause of gynaecological cancer-associated death over the past three decades. In our previous study, the progression of intraperitoneal human ovarian cancer xenograft was significantly inhibited by bispecific T cell engager (BiTE) specific for CD3 and epithelial cell adhesion molecule expressed using minicircle DNA (MC). However, these tumors were not completely eradicated by the monotherapy. Herein, we drafted a multi-target therapy based on MC-BiTE system for the treatment of ovarian cancer. In this project, the expression pattern of tumor-associated antigen in ovarian cancer was investigated by gene expression profile and tissue microarray, which was employed to validate optimized combination covering diverse clones. A group of BiTEs targeted the combination of antigens will be generated using MC in vivo to harness polyclonal cytotoxic T-cells and cause highly efficient lysis of ovarian cancer. The enhanced local concentration of BiTEs in peritumoral region will alleviate the immunosuppressive action of ovarian cancer microenvironment and reduce their systemic toxicity. This study will provide further perspective and method for multi-targeted strategy for clinical ovarian cancer gene therapy.

卵巢癌是死亡率最高的女性生殖系统恶性肿瘤，缺乏有效的治疗方法和措施。本研究前期发现，靶向上皮细胞粘附分子和CD3的双特异性T细胞衔接器（Bispecific T cell engager, BiTE）可以显著抑制卵巢癌移植瘤生长，但无法彻底清除肿瘤。申请人拟通过基因表达谱和组织微阵列技术分析卵巢癌组织中相关抗原表达模式，优化抗原靶点组合，覆盖不同的肿瘤细胞亚群，构建一组针对上述靶点组合的BiTE，研究其体外协同T细胞激活作用和抗肿瘤活性；采用微环DNA载体在移植瘤小鼠腹腔高效表达BiTE，提高肿瘤局部区域药物浓度，克服卵巢癌微环境的免疫抑制作用，形成持久和多克隆的抗肿瘤T细胞免疫反应。该研究将阐明基于微环DNA-BiTE多靶点疗法的抗卵巢癌活性和作用机制，为实现安全、有效的卵巢癌基因疗法提供理论基础和实验依据，并为其临床推广打下坚实基础。

卵巢癌是死亡率最高的女性生殖系统恶性肿瘤，缺乏有效的治疗方法和措施。随着肿瘤基因组学、蛋白质组学、表观遗传学等研究进展，发现卵巢癌具有高度的异质性。卵巢癌的发生、发展是一个多靶点多环节调控的结果，不同组织病理学类型的卵巢癌具有完全迥异的遗传突变，提示单靶点指导卵巢癌诊疗的局限性。本项目执行期间，通过对转录组、蛋白质组和组织微阵列组化检测结果分析分析筛选卵巢癌靶点，构建了候选靶点库及微环DNA-Bispecific T cell engager表达载体。开发腹腔靶向投递系统，将MC-BiTE靶向投递至体内提高肿瘤局部区域药物浓度，实现持久和多克隆的抗肿瘤T 细胞免疫反应，抑制卵巢癌移植瘤的生长。.基于以上策略，本研究获得以下成果：.1. 筛选了卵巢癌相关抗原表达模式，选择EpCAM、HER2、MUC1、MUC16、Mesothelin作为候选靶点，构建了靶向EpCAM、HER2的微环DNA-BiTE分子（MC-BiTEs）。.2. 开发了基于磷酸钙纳米材料（CaPO）的非病毒基因递送系统，通过腹腔注射可以实现MC-BiTE在小鼠体内高效和持续表达。.3. 腹腔注射CaPO/MC-BiTE联合过继T细胞疗法可以抑制人卵巢癌腹腔异种移植瘤的生长，显著延长荷瘤小鼠生存时间。.上述研究阐明了MC-BiTE多靶点基因疗法的抗卵巢癌活性和作用机制，为构建安全、有效、可负担的卵巢癌多靶点基因疗法提供了理论基础和实验依据。