启动自身免疫防御的双重靶向叶酸-半抗原免疫制剂的抗肺癌作用

Nowadays lung cancer remains as strong menace to human health. Chemothreapy depending solely upon anticancer drugs to produce anticancer function shows severe side-effects and low therapeutic efficacy, and recidivation and metastasis often occur. In order to improve the therapeutic efficay against lung cancer，prevent its metastasis and recidivation, and recover immune recognition of the organism to cancer cells and activate the immune defence, this project presents an immune preparation of doubly targeting folate(FA)-heptan with the aid of simultaneous specific mediation of ligand-receptor and antigen-antibody. We use the anticancer drug adriamycin (ADM) to take the place of usually used heptan. Our new vaccine consists of a conjugate of ADM-foreign protein, enabling the organism to produce antibody against ADM, and FA-ADM, which acts as (1) an molecular bridge of double functions, forming a conjugate of folate receptor (on the cancer cell surface) -FA-ADM-antibody against ADM, thus the conjugate starts immune cells etc. to recognize the cancer cells and kill and eliminate them, and (2) an anticancer drug on the cell surface, thus chemotherapy is achieved at the same time as the folate receptor (over expressed on the cancer cell surface) mediated FA-ADM actively targets to the cancer cell. So our vaccine serves as a stone to hit two birds, which not only takes a temporary solution, but effects a permanent cure against lung cancer. Therefore, our studies on the new preparation with doubly actively targeting functions will allow for accurately killing the cancer cells and avoiding the damage of normal cells, thus can improve the therapeutic efficacy against lung cancer and reduce side-effects with the help of activation of immune defence and recovery of immune recognition. The project provides new research ideas and innovation, and hopefully a solid experimental foundation for the therapy of lung cancer.

目前肺癌威胁巨大，依靠化疗药的抗癌治疗副作用大、疗效差，易复发和转移。为了治疗肺癌，恢复机体对癌细胞的免疫识别，启动免疫防御，本项目拟同时利用配体-受体、抗原-抗体的特异性为导向，构建双重靶向叶酸-半抗原免疫制剂。改用抗癌药阿霉素为半抗原，以阿霉素-异体蛋白偶联物作抗原发挥类似疫苗的作用，促使机体产生抗阿霉素抗体，而叶酸-阿霉素修饰物既是免疫治疗发挥靶向引导作用的双功能分子桥，可形成癌细胞表面的叶酸受体-叶酸-阿霉素-抗阿霉素抗体的复合物，并由该抗体复合物启动免疫细胞等对癌细胞的杀灭和清除功能；又可通过癌细胞表面过度表达的叶酸受体介导，发挥叶酸-阿霉素的主动靶向化疗作用，达到对肺癌标本兼治的目的。因此通过研究这种兼具两种主动靶向优势的新型制剂，可体现准确杀灭癌细胞，避免损害正常细胞，从而提高疗效，减少毒副反应，并恢复机体免疫识别，启动免疫防御的特点，为治疗肺癌提供新的研究思路和实验基础。

按照计划完成了叶酸-阿霉素（FA-ADM）、阿霉素-牛血清白蛋白（ADM-BSA）的制备及鉴定；以ADM-BSA为免疫原，分别免疫健康新西兰兔、健康Balb/c鼠，在2种动物体内均获得了特异性多抗；在体外细胞试验研究中发现，FA-ADM对MTT法考察的抑制癌细胞生长效果显著，证实了FA-ADM具有显著的主动靶向抗癌作用；经荧光显微镜观察证实FA-ADM的抗癌作用强与其易于被癌细胞吞噬有关；ELISA法考察发现，在癌细胞表面形成了“叶酸受体FR—FA-ADM—抗ADM抗体”复合物，证明FA-ADM具有引导抗体与癌细胞结合的连接桥作用，从而有利于启动机体免疫系统发挥抗癌作用。并且，在原计划之外，以所得ADM-BSA对荷瘤鼠进行免疫也获得了特异性多抗，又进一步对体内产生多抗的荷瘤鼠用FA-ADM进行治疗，试验组为：用ADM-BSA免疫荷瘤鼠使其体内产生抗ADM抗体后，注射FA-ADM，发挥分子桥促进免疫治疗与主动靶向化疗的双重作用；阳性对照组1为：荷瘤鼠体内无抗体，注射FA-ADM进行主动靶向化疗，结果肿瘤相对体积为试验组的3.3倍；阳性对照组2：用ADM-BSA免疫荷瘤鼠使其体内产生抗ADM抗体后，注射ADM进行普通化疗，结果肿瘤相对体积为试验组的4.2倍；阴性对照组：用ADM-BSA免疫荷瘤鼠使其体内产生抗体，仅注射生理盐水，不治疗，结果肿瘤相对体积为试验组的5.9倍。由于试验组动物体内的癌块相对生长速度显著低于各对照组，从而证实了本项目设计的启动自身免疫防御的双重靶向免疫制剂的抗癌作用疗效显著。因此，本项目取得了满意的实验结果，达到并超额完成项目预定的研究目标。.完成论文5篇：已发表SCI论文 2篇，平均影响因子5.551；核心期刊1篇，全国会议大会交流1篇，另有1篇论文已i投稿SCI期刊。申请中国发明专利1项。培养了硕士生1名。超额完成本项目考核指标。