表达CD40L/Dm-dNK瘤裂解腺病毒乳腺癌的靶向免疫及分子治疗

Breast cancer is a major health problem worldwide. Small targeted molecules in an adjuvant setting have shown to extend relapse-free survival of patients with early-stage disease; however, therapeutic options for advanced, metastatic malignancies remain incurable. Cancer-selective biotherapy or gene therapy approaches have been considered to be the next horizon toward developing a cure for breast cancer. The immunosuppressive environment of breast cancer is a primary obstacle to the efficacy of immunostimulatory and vaccine approaches. The capacity of the adenoviral early gene E1A to down-regulate Her-2/neu activity has led to phase I clinical trial for breast cancers in this regard. CD40 ligand (CD40L) plays a central role in immunoregulation and also directly modulates epithelial cell growth and differentiation; We previously showed that the oncolytic adenovirus armed with Deoxyribonucleoside Kinase of Drosophila Melanogaster （Dm-dNK) produced a synergistic inhibitive effect of adenovirus replication when combining with anti-virus nucleoside analogs, while maintaining specific cancer cell killing activity, and the CD40 receptor is commonly expressed in primary breast cancer tissues. Here we evaluate a novel approach to arm an oncolytic adenovirus with CD40L and Dm-dNK to stimulate beneficial immunological responses in vitro and in vivo. A double targeted chimeric adenovirus controlled by the hTERT promoter and expressing CD40L-dNK was constructed, and the breast cancer growth regulatory activities of an oncolytic adenoviral construct carrying the CD40L/Dm-dNK transgene (AdCD40L-dNK) will be examined. Dm-dNK will serve as a analog of thymidine kinase (TK) to promote the virus replicate; Dm-dNK/prodrug could also be the last attack to tumor cell and the safety-control of the system. In vitro and in vivo evaluations will be carried out on AdCD40L-dNK to validate selective viral replication and CD40L transgene activity in hypoxia inducing factor and estrogen receptor expressing human breast cancer cells. In this project, we will characterize the antitumor properties of a novel, conditional replicative,oncolytic adenoviral construct (AdCD40L-dNK) carrying the multifunctional molecule CD40L and Dm-dNK in the form of an integrated transgene. This novel strategy together with the previously known immune-activating features of CD40L and molecular chemotherapy of Dm-dNK/prodrug, will support the potential applicability of AdCD40L-dNK for preclinical treatment of human breast cancer.

乳腺癌是世界范围的主要健康难题。早期肿瘤辅助治疗的小分子靶向治疗可以减少复发延长生存，而晚期及复发者尚不能治愈。肿瘤选择性生物及基因治疗为乳腺癌带来了新曙光。免疫抑制环境的产生是肿瘤免疫治疗的主要障碍。腺病毒E1A基因可以下调Her-2基因并已进入I期临床试验。CD40配体(CD40L)用于免疫调节及协调上皮细胞生长分化。以前的研究显示瘤裂解腺病毒装配黑腹果蝇脱氧核苷酸激酶(Dm-dNK)保持肿瘤杀伤活性，但加药可抑制腺病毒的复制。该项目将评估瘤裂解腺病毒装载CD40L 及Dm-dNK新的体系。Dm-dNK将可能作为TK类似物促进病毒复制，而又可作为安全控制机制及分子化疗的抗肿瘤杀伤机制。本项目将证实AdCD40L-dNK体系的多重抗肿瘤特性；这一携带多功能免疫激活因子CD40L和Dm-dNK/癌前药物的分子化疗特性新的治疗体系AdCD40L-dNK可能成为乳腺癌临床前试验的潜在治疗新策略

肿瘤的形成和发展是一个多种因素影响的复杂的进程，单种基因疗法无法达到理想的肿瘤抑制效果，多基因治疗能显著增强抗肿瘤效应。之前的一系列研究证明了Dm-dNK /癌前药物（比如BVDU）系统在乳腺癌细胞体外及体内的有效杀伤作用，在此不予赘述。CD40 配体（CD40L）主要能增强免疫效应，促进生成CD8(+)中心记忆T细胞，通过促进生成B细胞，激活树突状细胞增强机体对肿瘤上皮细胞的免疫抑制作用。同时CD40-CD40L 相互作用对多种实体肿瘤细胞如卵巢癌、胰腺癌、直肠癌、乳腺癌等的直接肿瘤抑制作用已得到证实。.本项研究旨在探索Dm-dNK/prodrug 自杀系统和外源性CD40L 联合基因治疗在乳腺癌细胞体内和体外的杀伤作用。 应用选择复制性腺病毒携带能够同时表达Dm-dNK 和CD40L 的P74-dNK-CD40L直接进入乳腺癌细胞系验证其杀伤作用。研究表明联合基因治疗在MDA-MB-231(CD40+阳性乳腺癌细胞)较Dm-dNK/prodrug 和CD40L 单基因治疗组发挥显著的肿瘤抑制作用（P＜0.05）.而在MCF7（CD40-乳腺癌细胞系）和MRC5（人正常细胞系）细胞系中未发现如此差异。同时将MDA-MB-231 乳腺癌细胞系注入BALB/C 鼠脂肪垫构建体内动物模型，瘤内注射P74，P74-dNK, P74-CD40L, P74-dNK-CD40L。P74-dNK和P74-dNK-CD40L组在病毒注射后第2-8天5mg/kg日二次瘤内注射，验证体内的肿瘤杀伤作用。P74-dNK-CD40L较其他各组肿瘤体积明显减小（P＜0.05），具有统计学差异。本研究证实了Dm-dNK/prodrug 和CD40L联合分子基因治疗在乳腺癌细胞的显著杀伤作用，为临床治疗乳腺癌提供了一条新的道路。