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

通过聚合酶链式反应从黑色素瘤患者的c DNA文库中分离出黑色素瘤特异性抗原c DNA，将MAGE-1和MAGE-3通过脂质体导入小鼠肌肉中，并能诱导产生抗体rtesponse tu抗原。然后，我们检测了肿瘤基因疫苗对荷瘤小鼠的预防作用。OP 100具有免疫原性，可在人体内诱导抗体和细胞毒性T细胞(CTL)反应。将gplOO DNA疫苗应用于小鼠黑色素瘤模型，评价C57BL/6小鼠对B16黑色素瘤的免疫力。肌肉注射DNA-HVJ-AVE脂质体可诱导抗gp100抗体和CTL反应。GP-100DNA-HVJ-AVE脂质体免疫延缓了B16黑色素瘤细胞攻击小鼠的肿瘤生长。免疫gplOO DNA-HVJ-AVE脂质体的小鼠比单独免疫HVJ-AVE脂质体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，当通过不同途径给药时，可以在小鼠黑色素瘤模型中诱导保护性免疫。用pSFV3表达载体体外合成了人黑色素瘤相关抗原gplOO mRNA，并将其包裹在脂质体中。无论是肌肉注射还是皮内注射，均不能有效地诱导小鼠对B16黑色素瘤的保护性免疫。然而，将gplOO mR-hvJ-脂质体直接注射到小鼠脾内可诱导抗gp-100抗体和抗黑色素瘤的CTL反应。与对照组相比，脾内注射gplOO mRNAs可延缓肿瘤生长，显著延长小鼠的生存期。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中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.