Targeted therapy of gastrointestinal cancer using human anti-CEA antibody

我们构建了一个纤维修饰的Ad5载体（Adv-FZ33），该载体将葡萄球菌蛋白a衍生的igg结合域（Z33）插入到knob蛋白的HI环中。我们在体外和体内水平上评估了使用这种修饰载体的抗cea抗体复合物对cea阳性胃癌的重靶向程度和治疗效果。与抗cea单抗结合的Adv-FZ33载体感染后，LacZ基因的体外表达量比与对照单抗结合的载体高约20倍。ax3cap - fz33是一种Adv-FZ33衍生载体，表达一种治疗性基因，即大肠杆菌尿嘧啶磷酸核糖基转移酶（UPRT），可将5FU直接转化为5-氟吡啶单磷酸。当ax3cap - fz33与抗cea单抗结合时，5FU对胃癌细胞的细胞毒性增强（IC_<50>值的19.7倍）。接下来，我们研究了Ax3U…More P-FZ33结合抗cea mAb + 5FU对腹膜播散性胃癌小鼠的生存影响。ax3cap - fz33 /anti-CEA mAb/5FU组的中位生存时间显著长于PBS和5FU治疗组（p<0.01，与PBS和5FU治疗组相比）。此外，我们还检测了哪一种IgG亚型对Adv-FZ33和抗cea单抗介导的基因传递有效。BIACORE分析显示，人IgG1和IgG4对蛋白A的亲和常数（Ka）比小鼠IgG1高1000倍，而仅比小鼠IgG2a或IgG3高10倍。在表达cea的细胞中，Adv-FZ33与任何亚型的抗cea人IgG的转基因表达量明显高于任何类型的病毒-抗cea小鼠IgG的转基因表达量。特别是，人IgG4介导的转导效率比小鼠IgG1提高了200倍。我们发现Ab和蛋白A的反应性在Adv-FZ33传递Ab依赖基因的过程中起着关键作用。少

We constructed a fiber modified Ad5 vector (Adv-FZ33) in which an IgG-binding domain (Z33) derived from staphylococcal protein A was inserted into the HI loop of knob protein. We evaluated on both in vitro and in vivo levels the extent of retargeting towards and therapeutic effectiveness against CEA-positive gastric cancers when using the anti-CEA antibody complex with this modified vector. In vitro LacZ gene expression after infection with Adv-FZ33 vectors conjugated to anti-CEA mAb was approximately 20 times higher than that obtained with vector conjugated to the control mAb. We generated Ax3CAUP-FZ33, which is an Adv-FZ33 derivative vector expressing a therapeutic gene, namely E. coli uracil phosphoribosyltransferase (UPRT) which converts 5FU directly to 5-fluorouridine monophosphate. When conjugated with the anti-CEA mAb, Ax3CAUP-FZ33 enhanced the cytotoxicity of 5FU (19.7-fold in terms of IC_<50> values) against gastric cancer cells. Nextl, we examined the survival effects of Ax3U … More P-FZ33 conjugated with anti-CEA mAb plus 5FU in mice with peritoneal disseminated gastric cancer. The median survival time of the Ax3CAUP-FZ33/anti-CEA mAb/5FU group was significantly longer than those of the PBS and 5FU only treatment groups (p<0.01, versus PBS and 5FU only groups).Furthermore, we examined which subtype of IgG was effective on gene delivery mediated by Adv-FZ33 and anti-CEA mAb. BIACORE analysis showed that human IgG1 and IgG4 had 1,000-fold higher affinity constants (Ka) for protein A compared with mouse IgG1, whereas only 10-fold higher than mouse IgG2a or IgG3. In CEA-expressing cells, the transgene expression using Adv-FZ33 and any subtype of anti-CEA human IgG was significantly increased compared with combinations of any type of virus-anti-CEA mouse IgG. Especially, human IgG4 mediated transduction efficiency showed 200-fold enhancements compared with mouse IgG1. We found that the reactivity of Ab and protein A played a key role in the Ab dependent gene delivery by Adv-FZ33. Less