Basic study of polynucleotide vaccine for tumor immunotherapy

【目的】肌内注射质粒型表达载体等裸DNA，导致编码蛋白在肌肉中表达，当编码蛋白为非自身样病毒蛋白时，引发宿主对其的免疫反应。本研究将探讨这种新方法是否适用于肿瘤免疫治疗。[方法]首先将几种CAT表达载体肌内注射，用肌肉提取物进行CAT检测，发现CAG启动子和CE启动子在肌肉中最有效。编码一种在小鼠黑色素瘤细胞系B16上表达的黑色素瘤抗原的cDNA，编号为H52。插入CAG启动子（pCAGH52）的下游。另一个编码小鼠纤维肉瘤细胞系Meth-A中表达的p53突变蛋白的cDNA被插入CE启动子（pCEp53）的下游。C57BL或BALB/c小鼠肌内注射100微克pCAGH52或pCEp53，每隔一周注射3次。最后一次注射一周后，分别皮下注射10万个B16细胞和10万个甲基a。[结果]治疗组与对照组肿瘤发生率无差异，注射DNA未延长生存期。[结论]单纯注射DNA不足以引起宿主对自体肿瘤的免疫反应。由于注射DNA后产生的蛋白质数量很少，因此需要一些佐剂才能被抗原提呈细胞识别。

[PURPOSE] Intramuscular injection of naked DNA such as plasmid-type expression vector leads to the expression of coded protein in the muscle, and host immune response against the protein is elicited when it is non-self like viral protein. In present study we investigate whether this new method is applicable to cancer immunotherapy or not. [METHOD] At first several kinds of CAT expression vectors were intramusculary injected to perform CAT assay with muscular extract and CAG promoter and CE promoter were found to be most efficient in muscle. cDNA designated as H52, which codes one of melanoma antigens expressed on murine melanoma cell line B16. was inserted downstreams of CAG promoter (pCAGH52). Another cDNA, which codes mutant p53 protein expressed in murine fibrosarcoma cell line Meth-A, was inserted downstreams of CE promoter (pCEp53). C57BL or BALB/c mice were intramusculary injected thrice at one week interval with 100 microgram of pCAGH52 or pCEp53 respectively. One week after the last injection those mice were injected subcutaneously with 100000 cells of B16 or Meth-A respectively. [RESULT] There was no difference in tumor incidence between treated group and control group and survival was not prolonged by DNA injection. [CONCLUSION] Simple DNA injection is not sufficient to elicit host immune response against autologous tumor. Becausae the amount of protein produced after DNA injection is very small, some adjuvant would be necessary to be recognized by antigen presenting cells.