GM-CSF基因修饰肺癌细胞疫苗的抗肿瘤活性及其与化疗的协同作用

目的评价粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因修饰肿瘤疫苗的抗肿瘤活性,探讨其与化疗联合应用的抗肿瘤效果。方法以小鼠肺癌Lewis细胞系接种C57BL/6小鼠建立动物模型。利用含GM-CSF基因的重组质粒GM-CSF-pIRES2-EGFP,转染小鼠肺癌细胞系Lewis和LA795细胞,分别制备自体(Lewis细胞)和同种异体(LA795细胞)肿瘤疫苗。小鼠皮下接种Lewis细胞(1×107个)后5 d,采用GM-CSF分泌性肿瘤疫苗接种3次,同时设PBS组和单纯疫苗组作为对照,检测疫苗治疗前后脾细胞对Lewis细胞杀伤活性的变化,以及血清白介素4(IL-4)和γ干扰素(IFN-γ)的水平。观察GM-CSF分泌性肿瘤疫苗接种及联合化疗对小鼠生存期的影响。结果GM- CSF分泌性自体或同种异体肿瘤疫苗,均可诱导小鼠脾细胞对Lewis细胞杀伤活性增高,第3次接种后分别为(42.0±2.5)%和(39.6±7.3)%;同时血清中Th1类细胞因子IFN-γ的水平升高,而Th2类因子IL-4的水平无显著变化。GM-CSF分泌性肿瘤疫苗治疗组小鼠生存期与对照组比较,差异无统计学意义(P>0.05);而化疗联合疫苗组小鼠生存期较单独治疗组及对照组明显延长(P<0.05)。结论GM-CSF基因修饰的肿瘤疫苗可刺激机体产生特异性免疫反应;化疗的联合应用有助于提高肿瘤疫苗的治疗效果。     

小鼠肺癌B7疫苗细胞FLB2C诱导的抗肿瘤免疫应答

目的：研究小鼠肺癌细胞与活化B淋巴细胞融合的疫苗细胞FLB2c诱导细胞免疫反应情况。方法：用母本细胞LA795作对照，采用体外混合淋巴细胞培养，观察FLB2c细胞刺激T细胞增殖情况，通过CTLL细胞MTT法测定FLB2c刺激T细胞产生分泌IL－2的作用，用FLB2c免疫小鼠，观察疫苗体内诱导小鼠CTL活性的能力。结果：FLB2c细胞在体外刺激T细胞增殖的作用明显比LA795强；FLB2c能刺激T细胞分泌IL－2，而LA795则不能；FLB2c细胞在体内能诱导CTL活性，其诱导CTL在体外对FLB2c细胞和LA795的杀伤率分别为34％－25．3％，而LA795诱导的CTL对FLB2c和LA795杀伤率分别为10．5％和12．25％，两者的杀伤率有显著性差异。结论：FLB2c细胞在体内外均能刺激T细胞免疫应答，其以能力明显强于未融合的LA795小鼠肺癌细胞，将其作为疫苗细胞将有可能通过提高机体免疫应答而达到治疗肺癌的效果。本研究为进一步探讨该疫苗的应用价值奠定了免疫学基础。     

Dendritic cells fused with allogeneic breast cancer cell line induce tumor antigen-specific CTL responses against autologous breast cancer cells

Dendritic cell (DC)/tumor cell fusion vaccine has been revealed as a promising tool for the antitumor immunotherapy. Previous research has shown that fusion hybrids of human DCs and autologous tumor cells can induce cytotoxic T lymphocyte (CTL) responses against autologous tumor cells in animal models and human clinical trials. However, a major restriction factor for the clinical use is the difficulty for preparation of sufficient amount of autologous tumor cells especially for the patients with metastasis cancer whose primary tumor lesion is not clear or has been resected. In this study, allogeneic breast cancer cell line MCF-7 cells were electrofused to autologous DCs from patient with breast cancer as a strategy to deliver shared breast cancer antigens to DCs. Fusion cells generated by autologous DCs and allogeneic MCF-7 were able to induce autologous T lymphocytes proliferation, high levels of IFN-gamma production and CTL responses. CTLs induced by DCs/allogeneic MCF-7 fusion cells were able to kill autologous breast cancer cells in an antigen specific and HLA restriction manner. Our study may provide the experiment basis for the use of allogeneic breast cancer cell line in the DC/tumor cell fusion cell vaccination strategy.     

Combination of Fasl and GM-CSF confers synergistic antitumor immunity in an in vivo model of the murine Lewis lung carcinoma

Gene transfer of Fas ligand (FasL) to tumor cells has been demonstrated to inhibit tumor growth in vivo, and neutrophils are primarily responsible for this immunoprotection. The granulocyte-macrophage colony stimulating factor (GM-CSF) secreted by tumor vaccine can recruit dendritic cells (DCs) for efficient antigen presentation to T cells that generate the tumor-specific response. To investigate whether the combination of FasL and GM-CSF can efficiently suppress tumor growth, we have established Lewis lung carcinoma (LLC-1) cells that are transduced with GM-CSF (LLC/GM-CSF), FasL (LLC/FasL) or both genes (LLC/FasL/GM-CSF) to test their tumorigenic potential in vivo. Mice inoculated with LLC/GM-CSF display high survival rates along with reduction of tumor growth. In contrast, none of the mice injected with LLC/FasL or LLC/FasL/GM-CSF develop tumors. Specific memory immune response and delayed LLC-1 tumor growth are found in mice immunized with LLC-1/FasL or LLC-1/FasL/GM-CSF. Furthermore, therapeutic effects are observed only when LLC-1/FasL/GM-CSF tumor vaccine is employed to retard growth of preexisting LLC-1 tumors. Tumor growth is also completely suppressed in mice injected with a mixture of LLC-1 and LLC-1/FasL/GM-CSF. In addition, IL-12 production, cytotoxic T-cell activity and IgG against LLC-1 are manifested in mice injected with LLC/FasL/GM-CSF. Our data show that FasL-induced pathway triggers expression of proinflammatory cytokines, including IL-1 beta, IL-6, MIP-2 and MCP-1, while GM-CSF-dependent pathway promotes functional maturation and activation of DCs. Taken together, the results indicate that dual gene-based delivery with FasL and GM-CSF may serve as a more effective tumor vaccine to suppress lung cancer cell growth in vivo.     

ANTITUMOR EFFECTS OF HUMAN IL-15 GENE MODIFIED LUNG CANCER CELL LINE

ＡＮＴＩＴＵＭＯＲＥＦＦＥＣＴＳＯＦＨＵＭＡＮＩＬ－１５ＧＥＮＥＭＯＤＩＦＩＥＤＬＵＮＧＣＡＮＣＥＲＣＥＬＬＬＩＮＥＳｈｅｎＹｏｎｇｑｕａｎ１沈永泉ＣｕｉＬｉａｎｘｉａｎ２崔莲仙ＨｅＷｅｉ１何维ＸｕｅＬｉ１薛莉ＢａＤｅｎｉａｎ１巴德年１Ｉｎｓｔ...     

A novel DNA vaccine containing four mimicry epitopes for gastric cancer

Gastric cancer is one of the most common malignant tumors in China. This paper focuses on the development of a DNA vaccine containing four mimotopes of MG7Ag for gastric cancer (multi-epitope vaccine). By inoculating BALB/c mice, the vaccine was characterized and compared with a similar vaccine containing only one mimotope (mono-epitope vaccine) and other controls. Cellular ELISA indicated that serum titer of antibody against MG7Ag was significantly higher in mice immunized with the multi-epitope vaccine than that in the group immunized with the mono-epitope vaccine (0.8627 vs 0.6754, P < 0.05). And ELISPOT assay showed that the number of INF-gamma spots induced by multi-epitope vaccine was significantly larger than that of the group immunized with mono-epitope vaccine(93.3 vs 70.7, P < 0.05). Two weeks after tumor challenge, the weight of tumor in each mouse was evaluated, and the tumor masses formed in the mice immunized with multi-epitope vaccine were markedly smaller than those formed in the mice immunized with mono-epitope vaccine. These studies demonstrated that both humoral and cellular response were induced by the two vaccines and the efficiency of multi-epitope vaccine is stronger than that of the mono-epitope vaccine.     

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。     

肺癌肿瘤全溶物脉冲自体树突状细胞体外诱生T细胞抗肿瘤反应的研究

目的　探讨非小细胞肺癌 (NSCLC)患者全瘤溶解物 (WTL)冲击的自体树突状细胞 (DCs)瘤苗体外诱生T细胞介导的抗肿瘤反应。方法　在含重组人粒细胞 巨噬细胞 集落刺激因子 (rhGM CSF)和重组人白细胞介素 4 (rhIL 4 )的培养条件下 ,以 10例患者的贴壁外周血单核细胞 (PBMCs)衍生不成熟的树突状细胞 (imDCs) ,分别添加肿瘤坏死因子 (TNFα)、自体WTL或WTL TNFα诱导成熟 ,即分别为DCs/TNF、DCs/WTL和 DCs/WTL ,用流式细胞仪和混合淋巴细胞反应 (MLR) ,检测细胞表面分子的表达变化及其刺激异基因或同基因的T细胞增殖能力。将 DCs/WTL和自体T细胞共培养1～ 2周 ,以诱导细胞毒性T淋巴细胞 (CTL) ,用计数γ 干扰素 (IFN γ)释放的酶联免疫斑点 (ELISPOT)试验和乳酸脱氢酶 (LDH)释放的细胞毒试验 ,分别检测该CTL中识别自体肿瘤细胞释放特异性IFN γ的T细胞数和溶瘤活性。结果　以 30 μg/ml蛋白的WTL体外冲击自体 10 6imDCs,可导致上调DC表型 ,包括CD1a、CD83和CD86以及HLA DR的分子表达 ,与常规TNFα诱导成熟的DCs表型相似。虽然两者均能显著刺激异基因T细胞的增殖 ,但其刺激自体T淋巴细胞的增殖能力 ,DCs/WTL却显著高于DCs/TNF(P <0 .0 5 )。将体外与 DCs/WTL共培养的自体T细胞作为反应或效应细胞 ,再次     

Myelopoiesis-associated immune suppressor cells in mice bearing metastatic Lewis lung carcinoma tumors: gamma interferon plus tumor necrosis factor alpha synergistically reduces immune suppressor and tumor growth-promoting activities of bone marrow cells and diminishes tumor recurrence and metastasis.

Metastatic Lewis lung carcinoma (LLC) tumors stimulate myelopoiesis and, consequently, induce bone marrow cells to become immune suppressive to T cell blastogenesis and macrophage activation for tumor necrosis factor alpha (TNF-alpha) secretion. The suppressor cells phenotypically resembled granulocytic-monocytic progenitor cells. In order to diminish the presence of these immune suppressor cells, LLC-bearing mice were treated with low doses of gamma interferon (IFN-gamma) (100 units/mouse) plus TNF-alpha (10 units/mouse). Treatment of LLC-bearing mice with these low doses of IFN-gamma plus TNF-alpha diminished the suppressive activity of their bone marrow cells, as measured by the effect on normal macrophage activation to secrete TNF-alpha. In in vivo adoptive transfer studies, bone marrow from placebo-treated LLC-bearers stimulated tumor establishment and metastasis, while the bone marrow of IFN-gamma-plus TNF-alpha-treated tumor-bearers diminished LLC establishment and metastasis. The effect of the low dose treatments with IFN-gamma and/or TNF-alpha on the recurrence of excised s.c. tumors was also assessed. Treatment of mice following tumor excision with either IFN-gamma, TNF-alpha, or the combination of IFN-gamma plus TNF-alpha reduced recurrence. However, in the animals with recurring tumors only the combined IFN-gamma plus TNF-alpha treatment effectively diminished the development of lung metastases. These results demonstrate that low dose IFN-gamma plus TNF-alpha treatment diminishes the presence of suppressor and tumor growth-promoting activities of bone marrow and reduces tumor recurrence and metastasis.     

Immunotherapy of tumors with protein vaccine based on chicken homologous Tie-2.

PURPOSE: Tie-2 is an endothelium-specific receptor tyrosine kinase known to play a key role in tumor angiogenesis. The present study explores the feasibility of immunotherapy of tumors by using a protein vaccine based on chicken Tie-2 as a model antigen to break the immune tolerance against Tie-2 in a cross-reaction between the xenogeneic homologous and self-Tie-2. EXPERIMENTAL DESIGN AND RESULTS: In this study, a chicken homologous Tie-2 protein vaccine (chTie-2) and a corresponding mouse Tie-2 vaccine as a control were prepared and the antitumor effect of these vaccines was tested in two tumor models (murine B16F10 melanoma and murine H22 hepatoma). Immunotherapy with chTie-2 was found effective in two tumor models. Autoantibodies against mouse Tie-2 were detected in sera of mice immunized with chTie-2 through Western blot analysis and ELISA assay. Anti-Tie-2 antibody-producing B cells were detectable by ELISPOT. Histologic examination revealed that autoantibodies were deposited on the endothelial cells of tumor tissues. Purified immunoglobulins from chTie-2-immunized mice could induce the apoptosis of human umbilical vein endothelial cells in vitro. Importantly, adoptive transfer of purified immunoglobulins led to antitumor effect in vivo; apparently, angiogenesis was significantly inhibited in these tumors. Furthermore, the antitumor activity and production of autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. CONCLUSIONS: Our findings may provide a vaccine strategy for cancer therapy and show the potential utilization of interference with Tie-2 pathway.     

肺一丸对小鼠移植瘤VEGF的表达及肺转移相关性研究

目的 :探讨血管生成因子的表达对荷瘤小鼠肿瘤生长及肺转移的影响 ,以及肺一丸对其干预作用。方法 :采用LA795肺腺癌皮下接种T73 9小鼠模型。灌胃及腹腔给药法 ,观察中药肺一丸对肿瘤血管生成、VEGF的表达及肺转移的抑制作用。结果 :肺一丸能够抑制荷瘤小鼠肿瘤生长 ,高剂量抑瘤率为 49 6% ;降低血管密度 ,肺转移抑制率为 47 62 %。结论 :肺一丸能够通过调控肿瘤VEGF的表达 ,抑制肿瘤血管生成 ,起到抑制肿瘤生长和抗转移作用。     

Immunotherapy of murine colon cancer using receptor tyrosine kinase EphA2-derived peptide-pulsed dendritic cell vaccines

BACKGROUND: Further optimization of dendritic cell (DC)-based vaccines is required clinically against advanced stage cancer. Given the broad range of expression levels observed in the recently defined tumor antigen EphA2 in a diverse types of cancers, especially in advanced stage or metastatic cancers, the authors evaluated the effectiveness of vaccination using DCs pulsed with EphA2-derived peptides (Eph-DCs) in a murine colon cancer model. METHODS: EphA2 protein expression levels were evaluated in advanced colorectal carcinoma tissues from 10 patients by Western blot analysis. C57BL/6 mice were immunized with Eph-DCs twice weekly. Interferon gamma (IFN-gamma) ELISPOT assays were used for the analysis of CD8-positive T cells that were specific for EphA2-derived peptide. Immunized mice were challenged subcutaneously with EphA2-positive murine colorectal adenocarcinoma (MC38) mouse colon tumors or with EphA2-negative BL6 melanoma tumors. In some experiments, mice were injected with anti-CD8, anti-CD4, or antiasialo GM1 antibody to deplete corresponding lymphocyte subsets. RESULTS: Among 10 samples of advanced colorectal carcinoma, 6 samples (60%) overexpressed EphA2. IFN-gamma ELISPOT assays revealed that EphA2-derived peptide-specific CD8-positive T cells were generated by immunization with Eph-DCs. Immunization with Eph-DCs inhibited MC38 tumor growth compared with immunization using unpulsed DCs or phosphate-buffered saline. In contrast, Eph-DC vaccination had no effect on BL6 growth. Antibody depletion studies revealed that both CD8-positive T cells and CD4-positive T cells, but not natural killer cells, played critical roles in the efficacy observed for immunizations with Eph-DCs. Eph-DC vaccines resulted in long-term antitumor immunity against a rechallenge with MC38 tumor cells. CONCLUSIONS: The current results demonstrated that Eph-DC vaccines may represent a promising preventative/therapeutic modality in the cancer setting.     

烟曲霉醇联合环磷酰胺对小鼠LA795肺腺癌转移的抑制作用

背景与目的:血管生成抑制剂联合化疗药物治疗肿瘤成为目前研究热点之一。本研究旨在观察烟曲霉醇(fumagillol,TNP-470)联合环磷酰胺(cyclophosphamide,CTX)对肺腺癌小鼠异体移植转移的协同抑制作用,并初步探讨TNP-470抑制肿瘤转移的相关机制。方法:将40只接种高转移性LA795肺腺癌细胞的T739裸小鼠随机分成5组:对照组、溶剂组、TNP-470组(30mg/kg)、CTX组(40mg/kg)、联合组(TNP-47030mg/kg CTX40mg/kg)。实验3周后,处死全部小鼠。剥离皮下肿瘤称瘤重并计算抑瘤率;取出双肺观察表面肿瘤转移情况,计算肿瘤肺转移发生率,计数各组小鼠肺表面转移结节数及计算出肺表面结节转移抑制率。免疫组化和图像分析系统检测皮下移植瘤中微血管密度(microvesseldensity,MVD)、P-选择素表达并定量分析。结果:联合组抑瘤率(81.5%)明显高于其他各组(P<0.01),Q值等于1.21,说明两药合用具有协同作用。与对照组(12.13±4.02)相比,联合组(1.75±1.71)、TNP-470组(4.75±3.34)、CTX组(8.50±2.67)肺表面转移结节数明显下降;同时TNP-470组和联合用药组皮下肿瘤内MVD、P-选择素表达与对照组相比均下降,差异有显著性(P<0.01),而CTX组对此则无明显影响。结论:TNP-470与CTX对LA795肺腺癌的肺结节转移具有协同抑制作用;TNP-470抑制LA795肺腺癌转移与其抑制肿瘤内P-选择素表达有关。     

Antimetastatic activity of a preventive cancer vaccine

The development of prophylactic cancer vaccines that protect healthy hosts from tumor development leaves open the question whether such vaccines are also effective against established tumors and metastases. We tested the therapeutic activity of a proven prophylactic anti-HER-2/neu vaccine against successive stages of mammary carcinoma progression in HER-2/neu transgenic mice. The vaccine consisted of transgenic mammary carcinoma cells expressing HER-2/neu and two adjuvants: allogeneic class I histocompatibility antigens and interleukin (IL)-12. Vaccination of mice bearing lung micrometastases resulted in a 90% inhibition of metastasis development, whereas vaccination of mice with incipient local tumors was ineffective. The antimetastatic response was hampered by immune tolerance, as the protection of transgenic mice was lower than that of wild-type congenics not tolerant to HER-2/neu. A significant gain in immunotherapeutic activity in transgenic mice was obtained through the coadministration of anti-CD25 monoclonal antibody targeting regulatory T cells, which resulted in a >99% inhibition of metastasis. The immune responses elicited in transgenic mice comprised the activation of lung granulocytes and macrophages and of systemic adaptive responses based on helper T cells and their cytokines (IFN-gamma and IL-4) and anti-HER-2/neu antibodies. Dissection of relevant antimetastatic mechanisms by means of knockout mice and of depleting antibodies revealed a major difference between tumor prevention, which was completely dependent on anti-HER-2/neu antibodies, and metastasis therapy, which was antibody independent. In conclusion, a vaccine successfully developed for cancer immunoprevention showed a strong therapeutic activity against lung metastases mediated by protective immune mechanisms distinct from those preventing the onset of primary mammary carcinoma.     

Antigen-specific cancer immunotherapy using a GM-CSF secreting allogeneic tumor cell-based vaccine

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cell-based vaccines are currently one of the major forms of cancer vaccines. However, the preparation of GM-CSF-transduced autologous tumor vaccines is time-consuming and technically challenging. In addition, the host antigen presenting cells, rather than the tumor vaccine cells themselves, present tumor-specific antigens and prime the host T cells. Therefore, we tested the efficacy of antigen-specific allogeneic tumor vaccines. We used human papillomavirus 16 (HPV-16) E7 protein as a model tumor antigen, which is associated with the development of most cervical carcinoma. B16, a C57BL/6 (H-2(b)) derived melanoma cell line, was genetically engineered to produce GM-CSF alone (B16GM), HPV-16 E7 alone (B16E7), or both (B16GME7). These vaccine cells were injected into BALB/c (H-2(d)) mice (10(6) cells/mouse). Two weeks later, mice were challenged with 10(5) live HPV-16 E7(+) BL-1 (H-2(d)) tumor cells and monitored for tumor progression twice weekly. To determine the effective cell population in the antitumor immunity elicited by B16GME7, we carried out in vivo antibody depletion experiments using CD4 and CD8 specific antibodies. In addition, as a measure of the immune responses produced by B16GME7, we performed an in vitro cytotoxic T lymphocyte assay using a standard chromium release method. We found that all of the mice vaccinated with B16GME7 remained tumor free 49 days post-BL-1 challenge. In contrast, mice vaccinated with B16GM and B16E7 did not show any tumor protection against a similar dose of BL-1 cells. Furthermore, the antitumor immunity produced by B16GME7 was dependent on both CD4 and CD8 T cells. In addition, E7-specific cytotoxic T lymphocyte activity could be readily demonstrated in mice immunized with B16GME7. These results suggest that allogeneic tumor cells transduced with GM-CSF and the tumor antigen, HPV-16 E7, cannot only generate an E7-specific cytotoxic T lymphocytes response in vitro, but can also elicit a potent antitumor immune response against an E7 expressing tumor in vivo.     

Combination effect of vaccination with IL2 and IL4 cdna transfected cells on the induction of a therapeutic immune response against lewis lung carcinoma cells

In order to develop a more effective method of immunotherapy we have transfected mouse interleukin-2 (IL2) or mouse interleukin-4 (IL4) cDNA into a spontaneous non-immunogenic murine lung cancer, Lewis lung carcinoma (LLC). IL2 cDNA transfection more strongly decreases tumorigenicity of LLC than IL4 cDNA transfection. Recombinant-human-IL2 treatment of mice that were transplanted with untransfected LLC could not prolong their survival. In contrast, vaccination with IL2-cDNA-transfected LLC (LLC-IL2) and LLC-ILZ mixed with IL4-cDNA-transfected LLC (LLC-114) could significantly suppress tumor growth of LLC in a tumor-specific manner. The vaccination with LLC-IL2 mixed with the same number of LLC-IL4 cells was more suppressive to the growth of LLC than that with LLC-ILZ cells alone, while LLC-IL4 vaccination alone was ineffective. Nude, severe-combined-immune-deficient (SCID) and beige mice were unable to reject LLC-IL2 cells. However, immunodeficient mice responded to LLC-IL2, but not to LLC, since their survival times after transplantation with LLC-IL2 cells were significantly longer than the survival time of normal or immunodeficient mice transplanted with untransfected LLC cells. We conclude that vaccination with IL2-producing tumors and, with more pronounced effect, in combination with IL4-producing tumors, is able to induce an immune response to this normally non-immunogenic tumor. Tumor rejection appears to be achieved by the combined activity of CTL and NK cells. This strategy has potential for new immunotherapeutic interventions in cancer patients.     

小剂量5-FU提高携带VEGF siRNA的腺病毒载体对肿瘤细胞的转导效率和基因治疗效果的研究

Background and Objective:Adenovirus vectors were widely used in gene therapy for tumors.We used adenovirus vector to transfer small interfering RNA(siRNA) against vascular epithelium growth factor A(VEGF-A) molecules to mouse lung adenoma LA795 cells and used low dose of chemotherapeutic drugs to further elevate the infection efficiency of adenovirus vector in and therapeutic effect of RNAi on tumor cells.Met hods:LA795 cells were infected by Ad/EGFP and treated with different dosages of gemcitabin,epirubic...     

Active specific immunotherapy of renal cell carcinoma: cellular and humoral immune responses

We investigated the effect of vaccinating renal cell carcinoma (RCC) patients with irradiated autologous or allogeneic tumor cells and Newcastle disease virus (NDV) as adjuvant on cellular and humoral antitumor immunity. By Western blot analysis, we found that vaccination induced antibody formation in 33 of 34 patients against NDV proteins but not against tumor cell related antigens. NDV proteins detected had molecular weights of 53 kDa, 55-56 kDa, and 66 kDa. ADCC by patients' isolated PBMC and patients' sera against autologous or allogeneic tumor cells was not enhanced after vaccine treatment in a nonradioactive cytotoxicity assay. Target cells infected with NDV were lysed more effectively (p < 0.05) in ADCC after vaccination than noninfected targets. Natural cellular cytotoxicity of patients' isolated PBMC was not altered during vaccine treatment. Specific lysis rates against autologous and allogeneic RCC cells not exceeded 10% (effector:target ratio 50:1). Specific lysis of K-562 cells was > 20%; a slight decrease in lysis during vaccination was not significant. Numbers of lymphocyte subsets from patients' peripheral blood analyzed by FACS revealed significant expression of CD20+ (p < 0.02) and CD39+ (p < 0.03) cell numbers by vaccine therapy. Cytokine detection in patients' sera by ELISA showed significant increases (p < 0.05) for IFN-gamma and TNF-alpha but not for IFN-alpha four h post vaccination. Thus, immunomodulation with autologous or allogeneic RCC tumor cell vaccines is mainly due to cytokine induction, whereas tumor specific humoral or cellular responses are not detectable in patients' peripheral blood.     

Enhanced immune response to gastric cancer specific antigen Peptide by coencapsulation with CpG oligodeoxynucleotides in nanoemulsion

CpG oligodeoxynucleotides (CpG ODN) have been shown to have potent adjuvant activity for a wide range of antigens. Of particular interest is their improved activity when closely associated with the antigen. The purpose of this study is to construct a nanovaccine coencapsulated with a gastric cancer specific antigen MG7 mimotope peptide and adjuvant CpG ODN 1645 using new nanotechnology as nanoemulsion and evaluate its immunocompetence. Nanoemulsion vaccine was prepared using magnetic ultrasound methods. BALB/c mice were immunized and the in vivo effectiveness was evaluated using tumor challenge assay. It was shown that the tumor masses formed in the mice immunized with coencapsulated nanovaccine (0.0825 g) markedly smaller (P < 0.01) than those formed in the mice immunized with nanovaccine encapsulated with antigen peptide alone (0.4465 g). A tumor inhibiting rate as high as 82.5% of the coencapsulated nanovaccine was obtained, while nanovaccine encapsulated with peptide only could not achieve the same effect (28.5%) (P < 0.01). Enzyme-linked immunospot assay (ELISPOT) showed that immunization using MG7 mimotope peptide coencapsulated with CpG ODN within the same nanoemulsion enhanced the frequency of splenocytes secreting IFN-gamma significantly (P < 0.01) when compared with immunization using MG7 peptide encapsulated in nanoemulsion alone (197spots/1 x 10(6) vs. 73 spots/1 x 10(6)). Cellular ELISA indicated that serum titer of antibody against MG7-Ag was significantly higher (P < 0.01) in mice immunized with coencapsulation form nanovaccine (0.7884) than that in the group immunized with nanovaccine encapsulated with MG7 peptide alone (0.3616). Using intracellular flow cytometric analysis, it was found that the IFN-gamma response was contributed by CD4+ T-cells. Our experiments suggest that a vaccinal approach using nano-delivery system carrying in tumoral epitope and CpG ODN as adjuvant may have important implications for cancer therapy.     

Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector

Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors – B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials.