Isolation and characterization of tumor-specific antigen toward cancer gene therapy

采用PCR方法从黑色素瘤患者cDNA文库中分离黑色素瘤特异性抗原cDNA。MAGE-1和mage - 3通过hvj脂质体注入小鼠肌肉，可诱导对肿瘤抗原产生抗体反应。然后，我们在荷瘤小鼠身上测试了癌症基因疫苗的预防作用。op100具有免疫原性，在人体内可诱导抗体和细胞毒性T细胞（CTL）反应。在小鼠黑色素瘤模型中使用gplOO DNA疫苗来评估C57BL/6小鼠对B16黑色素瘤的免疫力。肌内注射DNA-HVJ-AVE脂质体可诱导抗gp100抗体和CTL反应。gp100 DNA-HVJ-AVE脂质体免疫延迟B16黑色素瘤细胞攻击小鼠的肿瘤发展。用gplOO DNA-HVJ-AVE脂质体免疫的小鼠比单独用HVJ-AVE脂质体免疫的对照组小鼠存活时间更长。接下来，研究了一种RNA黑色素瘤疫苗在小鼠黑色素瘤模型中通过不同途径给药时诱导保护性免疫。利用pSFV3表达载体体外合成人黑色素瘤相关抗原gplOO mRNA，包封于HVJ-脂质体中。肌内或皮内注射mrna - hvj脂质体对诱导抗B16黑色素瘤攻击的保护性免疫无效。然而，通过将gplOO mRNA hvj脂质体直接注射到小鼠脾脏免疫，可诱导抗gp 100抗体和抗黑色素瘤CTL反应。与对照组相比，将gplOO mRNA注入脾脏免疫可延缓肿瘤生长并显著延长存活时间。这些临床前研究表明，RNA肿瘤抗原疫苗策略在人类癌症治疗和预防方面具有潜在的应用前景。

We isolated melanoma specific antigen cDNA from melanoma patient cDNA library by PCR.MAGE-1 and 3 were introduced into mouse muscle by HVJ-liposomes and could induce antibody rtesponse tu tumor antigen. Then, we tested preventive effects of cancer gene vaccination in tumorbearing mice. Op 100 is immunogenic and shown to induce both antibody and cytotoxic T cell (CTL) responses in humans. The gplOO DNA vaccine was used in a mouse melanoma model to assess immunity against the B16 melanoma of C57BL/6 mice. Intrasmuscular injection of the DNA-HVJ-AVE liposomes induced both anti-gp100 anyibody and CTL responses. Gp 100 DNA-HVJ-AVE liposome immunization delayed tumor development in mice challenged with B16 melanoma cells. Mice limmunized with gplOO DNA-HVJ-AVE liposomes survived longer compared with control mice immunized with HVJ-AVE liposome alone. Next, an RNA melanoma vaccine was investigated to induce protective immunity in a mouse-melanoma model when administered by various routes. The human melanoma-associated antigen gplOO mRNA was in vitro synthesized by pSFV3 expression vector and encapsulated in HVJ- liposomes. Immunization by either intramuscular or intradermal injection of the mRNA-HVJ-liposomes was ineffective for the induction of protective immunity against B16 melanoma challenge. However, immunization by direct injection of the gplOO mRNA HVJ-liposomes into mouse spleen induced both anti-gp 100 Ab and CTL responses against melanoma. Immunizations with gplOO mRNA into the spleen delayed tumor growth and significantly prolonged survival compared to control treated mice. These pre-clinical studies demonstrate that an RNA tumor antigen vaccine strategy has potential application for human cancer treatment and prevention.