肝癌部位药物“工厂”的建立及其抗肝癌活性研究

Chemotherapy is one of the proven effective anti-cancer means because of the difficulties in specific tumor targeter identified. The prodrug designation improved the chemotherapy targeting and reduce the side effects with the low transfer efficiency and poor security however. We found that human umbilical cord derived mesenchymal stem cells (HUMSCs) can homogenize to hepatocellular microenvironment and differentiate into hepatocyte-like cells with AFP promoter actived. In this study, we proposed a new strategy of "PAG-acetylated prodrug" carried by HUMSC and regulated by AFP promoter.We want to establish the "prodrug factory" in the cancer in situ and metastases. The Pgp was further expressed on the HUMSC membrane so that the chemotherapy drugs in it will discharge timely which will protect HUMSC itself and the "high concentration and multi-drug combined chemotherapy" can be formed in the tumor microenvironment.“Pgp modified HUMSC-AFPPromotor-PGA-DOXP” strategy combined the advantages of HUMSC, prodrug and Pgp so that it will achieved the cancer targeted high concentration chemotherapy. It is an innovative strategy for cancer treatment with research value and clinical application value.

“特异肿瘤靶点”存在的不确定性和发现困难，使得化疗仍是目前已证实的最有效抗癌手段之一。“前药”设计能显著提高化疗靶向性，但现有抗体、基因等导向手段效率低、安全性差。前期我们发现人脐带来源间充质干细胞（HUMSC）能向肝癌微环境归巢、向肝样细胞分化伴随AFP特异性启动。在此基础上，本课题提出HUMSC运载、AFP调控的“二次靶向”“前药”策略，在肝癌原位和转移灶内成功建立“青霉素G酰化酶PAG-乙酰化前药加工厂”，并创新性的利用Pgp外排泵功能，将HUMSC打造成能抵抗化疗恶劣微环境的前药加工与化疗药释放工厂，最终实现肿瘤局部“高浓度的多药联合化疗”目标，很好的解决了肿瘤异质性高、易耐药的难题。“Pgp修饰的HUMSC-AFPPromotor-PGA-DOXP肝癌局部药厂”理念巧妙整合了HUMSC靶向、前药放大及Pgp外排三大系统优势，开创式的提出一条极具研究和临床实用价值的肿瘤治疗新策略。

本课题成功构建了pAd-hTERT-CD3-HAC、pAd-hTERT-CD3scFv、pAd-hTERT-HAC、pcDNA3.1(+)-CD3-HAC、pcDNA3.1(+)-CD3scFv及pcDNA3.1(+)-HAC载体，证明双特异性融合蛋白CD3-HAC可乳腺癌细胞中表达分泌，并成功结合于细胞表面。活细胞工作站监测结果表明了PBMCs与AdCD3-HAC感染的MDA-MB-231乳腺癌细胞之间的相互作用。LDH释放实验结果表明CD3-HAC能够特异性介导T细胞对PD-L1阳性细胞的杀伤。HAC功能蛋白可以阻断PD-1与PD-L1相互作用。联合低剂量5-FU可增敏腺病毒对靶细胞的感染，进而促进PBMCs的细胞毒作用。腺病毒AdTrack及慢病毒LentiR.E1A共感染MSCs后，腺病毒可在MSCs中复制及包装，最终裂解MSCs并释放具有感染能力的完整腺病毒颗粒。成功建立MDA-MB-231肺转移小鼠模型，经基因修饰的MSCs在尾静脉注射24小时后即可归巢至肿瘤局部，然后能够产生新的病毒并感染肿瘤细胞，实现体内CD3-HAC融合蛋白介导T细胞对肿瘤细胞的杀伤。体内抑瘤实验证明，MSC.AdCD3-HAC.E1A + PBMC单独或与5-FU联用能明显延长小鼠的生存期。本课题建立的由基因修饰的MSCs运载CD3-HAC双特异性融合蛋白的局部靶向治疗系统，在增强免疫应答的同时，避免了PD-1/PD-L1免疫检查点阻断剂的副作用，为恶性转移肿瘤的治疗提供了新的思路。