【作者】 施桂兰；

【导师】 张叔人；

【作者基本信息】 北京协和医学院 ， 免疫学， 2011， 博士

【摘要】 研究背景生物治疗已经成为继手术、放疗、化疗后的第4种抗癌疗法。其中细胞过继免疫治疗是肿瘤的生物治疗的主要手段之一。治疗所采用的细胞通常来源于患者自体细胞。由于肿瘤患者一般处于免疫低功状态,有些患者很难诱导出较强的和足量的抗肿瘤免疫活性细胞。若采用MHC完全匹配的供者免疫细胞作过继免疫治疗比较理想,但供者难求。如采用MHC不相合异体免疫细胞可能会产生移植物抗宿主反应,也无法诱导针对宿主肿瘤特异性的细胞毒性T细胞(CTL)。而父母和子女之间半相合的供者较易寻找。供者免疫细胞有可能通过与患者MHC相合部分诱导产生肿瘤抗原特异性CTL；而不相合部分产生免疫佐剂效应。但应用半相合CTL进行过继免疫治疗尚缺乏深入研究。目的研究MHC半相合肿瘤特异性CTL过继免疫治疗肿瘤的效应和机制。探索细胞过继免疫治疗肿瘤的新途径。方法应用TC-1肿瘤细胞裂解物冲击C57BL/6与Balb/C的杂交一代(F1)小鼠骨髓来源的树突细胞(TP-DC),再与小鼠脾细胞混合培养,诱导肿瘤特异性CTL(TP-DC-TC); C57BL/6 (C57)荷瘤鼠以TC-1肿瘤细胞的皮下成瘤为动物治疗模型,当肿瘤直径约为5毫米时,经尾静脉回输丝裂霉素C灭活TP-DC-TC予以治疗。或用丝裂霉素C灭活的C57小鼠来源的TC-1肿瘤细胞直接免疫F1小鼠获得肿瘤特异性CTL。进而将CTL经丝裂霉素C灭活后经尾静脉输入C57小鼠TC-1肿瘤细胞人工肺转移模型。两个动物模型分别治疗3次,每次间隔5-7天,观察肿瘤大小和生存期。人工肺转移模型的小鼠末次治疗后5天,眼眶采血,用ELISA检测血浆IL-2、IL-10、IFN-γ,TGF-β水平；用流式细胞术检测脾细胞和外周血细胞中CD3+CD4+T、CD3+CD8+T、Treg、MDSC、NK等免疫细胞亚群的变化和宿主免疫效应细胞的H-2表型。组织切片检测半相合CTL治疗对心、肝、肾、脾的影响。结果ELISPOT结果显示体外或体内均可诱导半相合肿瘤特异性CTL。应用该细胞治疗皮下荷TC-1肿瘤的C57小鼠,其肿瘤生长速度减缓,生存延长。在人工肺转移C57小鼠模型中与同系小鼠CTL过继免疫治疗相比,灭活半相合CTL免疫治疗后小鼠肺表面肿瘤结节数量明显减少,小鼠的生存期显著延长。半相合CTL治疗显著上调了Thl细胞因子IL-2和IFN-γ的分泌,并下调免疫抑制因子TGF-β和IL-10产生。流式仪检测证明：半相合CTL治疗后CD4+T, CD8+T和NK细胞在外周血和脾细胞中的比例明显增高；并且诱导了宿主自身肿瘤特异性CTL和NK细胞。而Treg、MDSC细胞亚群的比例显著下降；流式检测宿主免疫细胞的H-2表型结果发现,上述免疫细胞均源于宿主自身。HE染色证实,接受半相合CTL治疗小鼠的心、肝、肾、脾的无明显的病理变化。结论灭活半相合肿瘤特异性CTL过继免疫治疗,既可特异性杀伤肿瘤,又对宿主抗肿瘤免疫起到佐剂效应。该疗法不失为一种简单、安全有效的抗肿瘤疗法。研究背景溶瘤性单纯疱疹病毒(OncolyticHSV,简称为oHSV)具有强效、特异、广谱、安全及易于生产制备等特点。oHSVl已进入临床Ⅰ-Ⅲ期试验阶段。虽有文献报道oHSV2对肿瘤细胞的杀伤活性比oHSVl强,在治疗动物膀胱癌以及卵巢癌腹腔转移时,疗效明显好于oHSVl,但对oHSV2的研究较少。另外,oHSV作为载体还可以携带各种治疗性基因(如GM-CSF, TNF-α、药物前体转化酶、IL-12、FGFR2-IIIb, TRP1等),因此,我们已构建成有自主知识产权的新型oHSV2hGM-CSF,以改进病毒的安全性(优于现有的oHSV2)及增强病毒的溶瘤活性(优于现有的oHSVl),同时具有佐剂效应,增强机体抗肿瘤免疫。目的构建新的含有表达人GM-CSF的溶瘤2型疱疹病毒(oHSV2hGM-CSF),初步验证其体外溶瘤效果及其对小鼠B16R黑色素瘤模型的体内疗效。方法采用PCR、分子克隆及同源重组技术,从HG52野生病毒株的基因组中剔除ICP34.5及ICP47基因并插入hGM-CSF表达序列,构建hGM-CSF溶瘤活性体外实验,将HeLa, Eca-109和PG等16种肿瘤细胞分别接种到24孔板,分别用oHSV1hGM-CSF和oHSV2hGM-CSF (MOI=1)感染24h、48h后,显微镜观察细胞病变。动物实验,以表达HSV感染受体的C57BL/6小鼠黑色素瘤细胞B16R为模型。当肿瘤生长至直径约7-8mm3时,瘤内分别注射2.3 X106 PFU oHSV1hGM-CSF和oHSV2hGM-CSF,共3次,每次间隔3天,同时测量肿瘤大小,并观察生存期。结果PCR扩增及DNA序列分析证实各目的基因被剔除及hGM-CSF表达盒插入到ICP34.5的基因位点。oHSV1hGM-CSF和oHSV2hGM-CSF MOI-1感染肿瘤细胞后,24h即能观察到细胞病变,且溶瘤谱较广,oHSV2hGM-CSF感染的肿瘤细胞合胞体现象比较多。从感染48h的图像来分析,oHSV2hGM-CSF对HeLa, HepG2、SK-Mel-28 B16R U87-MG,的溶瘤效果优于oHSV1hGM-CSF。B16R细胞的黑色素瘤的动物实验发现,接受oHSV2hGM-CSF治疗后,能明显延长治疗组荷瘤鼠的生存期(P<0.01)；减缓肿瘤的生长(oHSV1hGM-CSF组, P<0.05; oHSV2hGM-CSF组, P<0.01)。结论oHSV2hGM-CSF对人肿瘤细胞溶瘤谱较广,对荷B16R黑色素瘤瘤内注射有较好的抗肿瘤效应,具有较好的开发前景,有望成为临床治疗肿瘤的制剂。更多还原

【Abstract】 Background Biotherapy of malignant diseases has become the fourth treatment modality besides surgery, chemo-and radiotherapy and the adoptive immune competent cells (AICs) infusion is one of the important mean for biotherapy. AICs are often derived from the cancer patient autologous immune cells which can do little in combating tumor cells due to the defected immune function of the cancer patient. A healthy donor with identical MHC would be ideal but it is very difficult to find such a donor. The allologous AICs has been demonstrated to be able to induce not tumor-specific CTLs but strong graft anti-host reaction (GAHR). On the other hand, it has been proposed that the semi-allogeneic AICs may use their allogeneic part to function as a powerful adjuvant to break the tumor immune tolerance or ignorance to some degree and use their autologous part to induce the MHC-restricted tumor-specific CTLs. So far, only semi-allogeneic CTL adoptive immunotherapy for the treatment of hematopoietic malignancies has been reported. Therefore, the semi-allogeneic CTL adoptive immunotherapy with less toxicity and more specific tumor targeting and adjuvant effects are to be studied.Objective This study was aimed at investigating the anti-tumor efficacy of the transfused tumor specific T cells with the surface semi-allogeneic antigens and to explore a new way on adoptive immunotherapy of cancer.Methods In order to produce the tumor-antigen specific semi-allogeneic lymphocytes, the lst-generation hybrid mice named F1 cross (H-2b/d) were bred by cross-mating C57BL/6 and Balb/c mice. The Fl tumor lysate-pulsed dendritic cells (TP-DC) and antigen specific CTLs (TP-DC-TCs) were prepared in vitro, and then the TP-DC-TCs were inactivated by MMC and transferred into the TC-1 bearing mice (C57BL/6). For evaluating therapeutic TP-DC-TCs infusion,1X1O5TC-1 tumor cells were injected s.c. into the right flank, and after the mice developed palpable tumor (approximately 5 mm diameter),3X107 antigen specific TP-DC-TCs were transferred into the recipients via the tail vein.In order to rapidly produce the tumor-antigen specific semi-allogeneic splenocytes (SPs), the Fl cross (H-2b/d) mice were then immunized with the mitomycin C (MMC) inactivated TC-1 tumor cells. These SPs were used as donor cells to transfuse into TC-1 bearing C57BL/6 mice (an artificial pulmonary metastases ainimal model was established by the i.v. injection of lxlO5 TC-1 cells) via intravenous injections three times at 7-d intervals in 3X107. On day 5 after the third treatment, the heparinized whole obital blood was collected and the plasma IL-2, IL-10, IFN-y, TGF-βwere examed by ELISA. To measure cell preparations of CD3+CD4+, CD3+CD8+, Treg, MDSC and NK mice were sacrificed on the 5th day after the third SPs administion, and their spleen cells were prepared and analysed by flow cytometry. The pathological characteristics of heart, spleen, liver and kidney were by HE staining. The phenotype of H-2 from recipients leucocyte was analysed by flow cytometry also.Results our results illuminated that the treatment of C57BL/6 tumor-bearing recipients receiving TP-DC-TCs from Fl mice resulted in a slower tumor growth and survived longer term. It was found in our study that both specific and nonspecific immune responses were triggered after SPs intra-vein transfusion (3x107/100ul, tail vein) with no obvious side effects. Intravenous transfusion of MMC inactivated semi-allogeneic SPs resulted in the increased IL-2 and IFN-y release, the decreased TGF-P excretion, the proliferation of specific CD4+and CD8+and the generation of nonspecific immune response (NK cells). In addition, the semi-allogeneic SP transfusion has shown to be able to delay TC-1 tumor growth and prolong survival in the established TC-1 tumor model. Our data indicate that the transfusion of tumor specific allogeneic SPs may induce anti-tumor therapeutic effects resulted from both the specific and nonspecific immune responses. There were less pathological characteristics of heart, spleen, liver and kidney were found by HE staining.Conclusion Adoptive transfer of semi-allogeneic tumor specific CTL provides a promising approach to cancer immunotherapy. Background Oncolytic virus (OV) therapy is based on the concept of using live viruses to selectively infect and replicate in cancer cells, with minimal destruction of non-neoplastic tissue. And the evidences have recorded their specificity, wild-oncolytic effecty, good safety, fast replication and facilitated process. oHSVl has enterⅠ-Ⅲclinical pilot. However, there a few studies demonstrated that oHSV2 is more oncolytic effective than oHSVl in the treatment of bladder cancer and peritoneal metastasis of ovarian cancer in mice models. Further more, HSV2 is an enveloped double-stranded DNA virus containing a large, well characterized, fully sequenced genome of about 152kb of DNA. While the large size makes genetic manipulations cumbersome, it also provides ample opportunities to remove genes that are not essential for replication and insert therapeutic transgenes within the viral backbone, just as a vector carrying DNA encoding GM-CSF, TNF-a, IL-12, TRPland prodrug-converting enzyme. Therefore, our lab has constructed a newly oHSV2hGM-CSF aiming at enhancing the safety and the activity of oncolytic. oHSV2hGMCSF may promote the anti-tumor immunity as adjuvant.Objective This study was to construct a new oHSV2hGM-CSF and evaluate the oncolytic activity in vitro and in vivo in parallel with oHSVlhGM-CSF.Methods oHSV2hGM-CSF is a replication-competent, attenuated HSV type 2 based on the HG52 virus (an HSV2 strain). It has been engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and inserted the gene encoding hGM-CSF. To measure the in vitro killing effect of the viruses,15 human tumor cell lines (HeLa, Eca-109 and PG etc.) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at 1 pfu/cell (Multiplicity of infection, MOI=1), or left uninfected. Cells were harvested 24 and 48 hours post infection, and observed at microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3x106 pfu for three times when tumor volume reached 7-8mm3. And the tumor volume was measured at 3 day interval and animal survival was monitored after treatment.Results Both oHSV2hGM-CSFand oHSVlhGM-CSF induce widespread cytopathic effects after 24h infection. oHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSVlhGM-CSF. In the in vitro killing experiments for the cell lines of HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSVlhGM-CSF under the same conditions. For the animal experiments, it is concluded that the oHSV2hGM-CSF significantly inhibited tumor growth. The long term therapitic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days post tumor cells inoculation whereas mice in the PBS group all were dead by day 22 (p<0.01). The anti-tumor mechanism of newly constructed oHSV2hGM-CSF against B16R cell carcinoma appeared to include the directly oncolytic activity and the induction of antitumor immunity in some degree.Conclusion These data demonstrate that the newly constructed oHSV2hGM"CSF have potent anti-tumor activity in vitro for many tumor cell lines and in vivo for the established B16R tumor models.更多还原