应用细胞因子缓释微球的肿瘤疫苗治疗肝癌的研究

目的 探讨采用细胞因子缓释微球的肿瘤疫苗预防和治疗肝癌的疗效及抗癌机制。方法 我们研制开发了一种肿瘤疫苗,其组成是固定的肿瘤细胞或组织碎片、细胞因子缓释微球和免疫辅助药。采用多聚甲醛固定的小鼠Hepal-6细胞或肿瘤碎片、微球包装的GM—CSF和／IL—2和合成TiterMax Gold等不同成份的瘤苗皮内接种C57BL／6J小鼠,随后肝内接种活体Hepal-6细胞。结果 对照组15只小鼠全部发展成肝肿瘤;含有固定Hepal-6细胞和IL-2及GM—CSF微球的肿瘤疫苗,80％小鼠获得保护。再加入免疫辅助剂TiterMax Gold的肿瘤疫苗,则87％小鼠获得保护。将Hepal-6细胞接种于左躯干皮下。肿瘤长至直径5mm时,皮内接种肿瘤疫苗2次。结果显示,对照组肿瘤继续生长。疫苗组在第2次接种后7—10天,10只小鼠中9只肿瘤生长受到抑制,随后明显缩小。60％小鼠的肿瘤完全消散。细胞毒性实验结果显示,未接种疫苗的小鼠脾细胞不能杀灭Hepal-6细胞和其他肿瘤细胞;而接种疫苗的小鼠脾细胞对Hepal-6细胞杀瘤活性达41％,但对B16—Fl,Lewis肺癌细胞(LLC),肾癌细胞(Renca),膀胱癌细胞(MBT-2)则无效。疫苗的Ⅰ期临床实验结果显示肝癌疫苗能有效地预防肝癌术后复发,诱导DTH反应。结论 肝癌疫苗能有效预防和治疗原发性肝癌,其抗瘤机制是诱导内源性抗原特异性CTL反应,其杀瘤特性是由典型的：MHC—Ⅰ限制的CD8^ T细胞所介导的。

Fixed-tumor vaccine: A practical formulation with cytokine-microspheres for protective and therapeutic antitumor immunity

Objective: To study the protective and therapeutic antitumor immunity against hepatocellular carcinoma (HCC) with the fixed-tumor vaccine.Methods: A tumor vaccine consisting of fixed tumor cells or fixed tumor fragments combined with sustained-releasers of cytokines and a non-toxic adjuvant was developed. C57BL/6J mice were immunized intra-dermally with the vaccine on day 0 and 7, followed by intrahepatic challenge with live Hepa 1–6 cells.Results: All of 15 nonimmunized control mice developed the hepatoma. Protection of mice immunized with fixed Hepa 1–6 cells and both of IL-2/GM-CSF microspheres or further mixed with TiterMax Gold reached 80% and 87%, respectively. Mass growth of the established tumors, vaccinated twice at 5 mm in diameter, the tumor of control animals continued to grow. However, 7–10 days after the second injection of the tumor vaccine, the tumor growth was suppressed in 9 of 10 mice and then markedly reduced. Complete tumor regression was observed in 60% (6/10) of mice. Splenocytes from the control mice were not able to lyse target Hepa 1–6 cells and other tumor cells. In contrast splenocytes from the vaccinated mice exhibited a 41% lytic activity against the Hepa 1–6 cells tested at an effector/target (E/T) ratio of 5, whereas they did not exhibited such activity against the melanoma cells (B16-F1), Lewis lung carcinoma cells (LLC), renal carcinoma cells (Renca), and bladder carcinoma cells (MBT-2). The cytotoxic activity was inhibited by the treatment with anti-CD3, anti-CD8, and anti-MHC-class I monoclonal antibodies but not with anti-CD4 and anti-MHC-class II antibodies. In the Phase-I clinical trial, vaccination of HCC patients with the autologous vaccine is a well-tolerated treatment and induces fixed tumor fragment-specific immunity.Conclusion: Fixed HCC vaccination elicited protective and therapeutic antitumor immunity against HCC. The tumor vaccine elicited antigen specific CTL response lysis of the target HCC was mediated by the typical MHC-class I restricted CD8⁺ T cells.