Cafferata Eduardo G, Macció Daniela R, Lopez Maria V, Viale Diego L, Carbone Cecilia, Mazzolini Guillermo, Podhajcer Osvaldo L
Laboratorio de Terapia Molecular y Celular, Instituto Leloir and Instituto de Investigaciones Bioquimicas de Buenos Aires, Argentina.
Clin Cancer Res. 2009 May 1;15(9):3037-49. doi: 10.1158/1078-0432.CCR-08-1161. Epub 2009 Mar 31.
A33 antigen is a membrane-bound protein expressed in intestinal epithelium that is overexpressed in 95% of primary and metastatic colorectal carcinomas but is absent in most epithelial tissues and tumor types. We hypothesized that A33 promoter might be useful in the design of a conditionally replicative adenovirus for the treatment of colorectal cancer (CRC).
We cloned an A33 promoter fragment (A33Pr) that extends from -105 to +307 bp. Using luciferase activity as a reporter gene, we showed that A33Pr was active in CRC cell lines. We next constructed a conditionally replicative adenovirus named AV22EL where E1A was placed under the control of A33Pr. The tumor-specific oncolytic effect of AV22EL was investigated both in vitro and in vivo.
AV22EL induced specific in vitro lysis of human CRC cell lines that expressed A33 and have negligible lytic capacity on cells that lacked or had minimal A33 expression, including normal human colonic cells. In vivo, a marked reduction of tumor growth and increased long-term survival rates were observed in nude mice xenografted with s.c. CRC tumors. Combination with 5-fluorouracil induced an additive effect in vitro with no toxic effects in vivo. Remarkably, AV22EL completely eliminated established hepatic metastases in >90% of mice and restored hepatic function according to biochemical parameters. Its systemic administration induced E1A expression only in the hepatic metastasis but not in normal organs.
These data show that AV22EL is a stringently regulated and potent oncolytic agent for the treatment of CRC.
A33抗原是一种在肠上皮中表达的膜结合蛋白，在95%的原发性和转移性结直肠癌中过表达，但在大多数上皮组织和肿瘤类型中不存在。我们推测A33启动子可能有助于设计一种用于治疗结直肠癌（CRC）的条件性复制腺病毒。
我们克隆了一个从-105至+307 bp的A33启动子片段（A33Pr）。以荧光素酶活性作为报告基因，我们证明A33Pr在结直肠癌细胞系中具有活性。接下来，我们构建了一种名为AV22EL的条件性复制腺病毒，其中E1A置于A33Pr的控制之下。在体外和体内研究了AV22EL的肿瘤特异性溶瘤作用。
AV22EL诱导了表达A33的人结直肠癌细胞系的特异性体外裂解，而对缺乏或仅有极少A33表达的细胞（包括正常人结肠细胞）的裂解能力可忽略不计。在体内，皮下接种结直肠癌肿瘤的裸鼠中观察到肿瘤生长显著减少，长期生存率提高。与5-氟尿嘧啶联合使用在体外产生了相加效应，在体内无毒性作用。值得注意的是，AV22EL在>90%的小鼠中完全消除了已形成的肝转移，并根据生化参数恢复了肝功能。其全身给药仅在肝转移灶中诱导E1A表达，而在正常器官中不诱导。
这些数据表明AV22EL是一种严格调控且有效的用于治疗结直肠癌的溶瘤剂。
