A combination of flk1-based DNA vaccine and an immunomodulatory gene (IL-12) in the treatment of murine cancer

AIM: The aim of this study was to investigate the antivasculature effects and the antitumor effects of combining attenuated Salmonella typhimurium vaccine strain encoding murine vascular endothelial growth factor (VEGF) receptor-2 (flk1) with plasmid DNA vector encoding the murine IL-12 (mIL-12) gene. METHODS: Mouse models of Gl261 glioblastoma were treated with combining orally given attenuated Salmonella typhimurium vaccine strain encoding flk1 with direct intratumoral injection of a nonviral plasmid DNA vector encoding the murine IL-12 (mIL-12) gene. The volumes of tumors were observed. Cytolytic T lymphocyte (CTL) response was measured by a 4-hour 51Cr release assay, vessle density and tumor cell proliferation were observed by immunostaining, and tumor apoptosis was determined by TUNEL staining. RESULTS: Compared to mice receiving single agent therapy, received either oral immunization flk1-based vaccine only or the therapeutic gene-IL-12 plasmid DNA only or those in the control group, the combination therapy groups developed a strong CTL response and showed more significantly inhibited tumor growth, apoptosis of tumor cells, and reduced neovascularization and cell proliferation in these mice. CONCLUSIONS: The therapy of attenuated Salmonella typhimurium vaccine strain encoding flk1 combined with the interleukin-12 gene has significant synergistic effect against tumors.     

胃癌MG7-Ag模拟表位口服DNA疫苗的研制

目的　利用减毒鼠伤寒沙门氏菌研制胃癌MG7 Ag模拟表位的口服DNA疫苗 ,并观察其对小鼠的免疫效能及保护作用。方法　构建MG7 Ag模拟表位和通用性辅助性T细胞表位融合基因的真核表达载体。将真核表达载体转入减毒鼠伤寒沙门氏菌得到模拟表位的口服DNA疫苗。以 1× 10 8cfu疫苗菌口服免疫C5 7BL/6J小鼠 ,以携带空载体的沙门氏菌和PBS口服作为对照。以ELISA法检测小鼠血清中抗MG7 Ag抗体的滴度 ,以H TDR掺入法检测小鼠脾淋巴细胞对人工合成的MG7 Ag抗原肽刺激的增殖能力。同时 ,用表达MG7 Ag的小鼠艾氏腹水瘤细胞进行肿瘤攻击 ,观察疫苗对小鼠的保护作用。结果　口服疫苗可诱导小鼠产生MG7抗体 ,但各组小鼠脾淋巴细胞体外刺激增殖实验差异无显著性。肿瘤攻击 2周后 ,疫苗免疫组 7只小鼠中有 2只未见肿瘤形成 ,而对照组 4只小鼠则全部成瘤。结论　胃癌MG7 Ag模拟表位的口服DNA疫苗具有免疫原性 ,可以诱导小鼠产生抗肿瘤免疫 ,并具有一定保护作用     

携带flk1的重组减毒沙门氏菌的构建及其抗肿瘤血管生成的研究

目的:观察携带鼠血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,flk1)的重组减毒鼠伤寒沙门氏菌诱导的flk1特异性免疫应答及抗肿瘤血管生成作用.方法:构建真核表达载体pcDNA3.1-flk1,并将其转化到减毒鼠伤寒沙门氏菌SL7207中,体外感染小鼠巨噬细胞,用Western印迹对表达产物进行鉴定,将重组菌口服免疫小鼠,分析免疫后小鼠体内的特异性CTL应答.采用藻酸盐微囊实验观察其对体内肿瘤血管生成的影响.结果:免疫印迹试验表明携带pcD-NA3.1-flk1表达载体的重组菌感染的小鼠巨噬细胞能表达flk1蛋白,免疫小鼠脾淋巴细胞产生针对flk1的特异性CTL活性.重组疫苗菌的免疫能够明显抑制小鼠体内肿瘤血管的形成.结论:携带flk1的重组减毒沙门氏菌经口服免疫,诱导小鼠产生抗flk1的特异性免疫反应,且能明显产生抗肿瘤血管生成作用.     

Oral administration of attenuated S. typhimurium carrying shRNA-expressing vectors as a cancer therapeutic

RNAi has been successfully applied in genomic research, and it also holds considerable promise as a therapeutic approach to suppress disease-causing gene expression. Here, we show that attenuated S. typhimurium were capable of delivering shRNA-expressing vectors to mammalian cells and inducing RNAi in vitro and in vivo. Upon oral administration, S. typhimurium carrying shRNA-expressing vectors targeting bcl2 induced significant gene silencing in murine melanoma cells that led to a remarkably delayed tumor growth and prolonged survival in the mouse model. These results suggest that bacteria mediated RNAi may be a new potent approach to the treatment of cancers.     

Development of a Listeria monocytogenes-based vaccine against hepatocellular carcinoma

Live attenuated Listeria monocytogenes (LM) is a promising bacterial vector able to induce a T-cell response to tumor-associated antigens and demonstrates great potential for use in vaccine development. A novel recombinant LM-based vaccine (Lmdd (LM ΔdalΔdat)-MPFG (multiple peptide fusing genes)) was developed with the ability to express and secrete hepatocellular carcinoma (HCC)-related tumor-associated antigens fragments due to the insertion of hepatitis B virus (HBV)-X protein (HBx)-derived epitopes HBx(52-60) and HBx(140-148), the universal T-helper epitope, alpha-fetoprotein (AFP) epitope AFP(158-166), and melanoma antigen gene (MAGE)-3(271-279) into the HBV core protein. Following immunization with the Lmdd-MPFG vaccine, macrophages exhibited uptake of the bacteria; the vaccine was then nearly cleared 3 days after the first administration. It disappeared even more quickly following subsequent vaccinations. However, recombinant Lmdd-MPFG allowed for the full development of an antitumor response towards the human leukocyte antigen (HLA)-A0201 epitopes of MPFG. Each epitope stimulated an augmented T-cell proliferation and enhanced the supernatant level of interferon (IFN)-γ in vitro. In addition, IFN-γ-producing CD8(+) T cells as well as in vivo cytolytic activity were significantly increased in HLA-A2 transgenic mice. Additionally, the Lmdd-MPFG developed a strong antitumor response, as indicated by the significant resistance of immunized mice to MPFG-positive Hepa1-6 cell challenge in both a prophylactic and therapeutic setting. Tumor regression was accompanied by an enhanced cytotoxic T lymphocyte response and a decrease of regulatory T cells in the tumor. Collectively, these results suggest that utilizing attenuated LM as a vaccine vector, able to carry the MPFG gene, presents a potentially feasible strategy for prevention of HCC.     

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。     

In vivo long-term imaging and radioiodine therapy by sodium-iodide symporter gene expression using a lentiviral system containing ubiquitin C promoter

To establish stable and long-term gene expression in vitro and in vivo, we developed a lentiviral vector system carrying sodium iodide symporter (hNIS) gene under UbC promoter, and transfected this into a colon cancer cell line. The in vitro and in vivo kinetics of radioiodine and [99mTc]-pertechnetate were then investigated, and the therapeutic effect of 1-131 was evaluated in this system. The hNIS gene was transferred into CT26 cells using lentivirus containing UbC promoter. In vitro iodide uptake and efflux were measured in CT26-hNIS cells at various time points. In addition, scintigraphic images were acquired at 30 min after injecting [99mTc]-pertechnetate i.p. into Balb/C mice for 27 days after CT26-hNIS induction. Biodistribution studies were performed at 10 and 30 min and at 1.5, 6 and 24 h after [99mTc]-pertechnetate injections, and the therapeutic effects of radioiodine were investigated by measuring tumor size using a caliper or by quantifying tumor radioactivity levels in scintigraphic images. The iodide uptakes of CT26-hNIS tumors were 10-fold greater than those of CT26 tumors. In addition, iodide uptake was completely blocked by 100 microM potassium perchlorate. The accumulation of [99mTc]-pertechnetate in hNIS expressing tumor cells was found to be positively related to tumor growth. In biodistribution studies, the %ID/g values of CT26-hNIS were 84.0 +/- 4.5 at 1.5 h and 40.8 +/- 3.9 at 24 h and these were approximately 60 times greater than those of CT26 at these time points. Tumor growth in mice treated with 131I was retarded until 46 days post-tumor challenge. The devised lentiviral vector system carrying hNIS controlled by UbC promoter was found to be suitable for the long-term monitoring and radionuclide therapy of cancer in living organism.     

Comparison of two cancer vaccines targeting tyrosinase: plasmid DNA and recombinant alphavirus replicon particles.

PURPOSE: Immunization of mice with xenogeneic DNA encoding human tyrosinase-related proteins 1 and 2 breaks tolerance to these self-antigens and leads to tumor rejection. Viral vectors used alone or in heterologous DNA prime/viral boost combinations have shown improved responses to certain infectious diseases. The purpose of this study was to compare viral and plasmid DNA in combination vaccination strategies in the context of a tumor antigen. EXPERIMENTAL DESIGN: Using tyrosinase as a prototypical differentiation antigen, we determined the optimal regimen for immunization with plasmid DNA. Then, using propagation-incompetent alphavirus vectors (virus-like replicon particles, VRP) encoding tyrosinase, we tested different combinations of priming with DNA or VRP followed by boosting with VRP. We subsequently followed antibody production, T-cell response, and tumor rejection. RESULTS: T-cell responses to newly identified mouse tyrosinase epitopes were generated in mice immunized with plasmid DNA encoding human (xenogeneic) tyrosinase. In contrast, when VRP encoding either mouse or human tyrosinase were used as single agents, antibody and T-cell responses and a significant delay in tumor growth in vivo were observed. Similarly, a heterologous vaccine regimen using DNA prime and VRP boost showed a markedly stronger response than DNA vaccination alone. CONCLUSIONS: Alphavirus replicon particle vectors encoding the melanoma antigen tyrosinase (self or xenogeneic) induce immune responses and tumor protection when administered either alone or in the heterologous DNA prime/VRP boost approaches that are superior to the use of plasmid DNA alone.     

人T细胞识别的鳞状细胞癌抗原3基因疫苗肿瘤免疫研究

目的观察人T细胞识别的鳞状细胞癌抗原3（SART3）基因的DNA疫苗能否诱导小鼠产生针对表达该基因肿瘤细胞的免疫反应。方法构建表达人SART3基因的C3H小鼠肿瘤细胞LM8-SART3。以空载体为对照,注射疫苗,分离小鼠脾细胞,体外检测CTL反应。于疫苗免疫后2周接种肿瘤细胞,比较免疫组与空载体组肿瘤长出及出瘤后肿瘤生长情况。结果体外可检测到疫苗诱导的小鼠脾细胞CTL活性;疫苗注射后小鼠成瘤率降低,肿瘤生长慢于对照组。结论表达人SART3基因的DNA疫苗可在一定程度上预防表达该基因的肿瘤细胞LM8-SART3在小鼠体内的成瘤及生长。     

Effective particle-mediated vaccination against mouse melanoma by coadministration of plasmid DNA encoding Gp100 and granulocyte-macrophage colony-stimulating factor.

Particle-mediated gene delivery was used to immunize mice against melanoma. Mice were immunized with a plasmid cDNA coding for the human melanoma-associated antigen, gp100. Murine B16 melanoma, stably transfected with human gp100 expression plasmid, was used as a tumor model. Particle-mediated delivery of gp100 plasmid into the skin of na?ve mice resulted in significant protection from a subsequent tumor challenge. Co-delivery of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) expression plasmid together with the gp100 plasmid consistently resulted in a greater level of protection from tumor challenge. The inclusion of the GM-CSF plasmid with the gp100 DNA vaccine allowed a reduction in the gp100 plasmid dose required for antitumor efficacy. Protection from tumor challenge was achieved with as little as 62.5 ng of gp100 DNA per vaccination. Tumor protection induced by the gp100 + GM-CSF gene combination was T cell mediated, because it was abrogated in vaccinated mice treated with anti-CD4 and anti-CD8 monoclonal antibodies. In addition, administration of the gp100 + GM-CSF DNA vaccine to mice bearing established 7-day tumors resulted in significant suppression of tumor growth. These results indicate that inclusion of GM-CSF DNA augments the efficacy of particle-mediated vaccination with gp100 DNA, and this form of combined gp100 + GM-CSF DNA vaccine warrants clinical evaluation in melanoma patients.     

Tumor vaccine therapy against recrudescent tumor using dendritic cells simultaneously transfected with tumor RNA and granulocyte macrophage colony-stimulating factor RNA

Recently, dendritic cells (DC) transfected with tumor RNA have been used as a cancer vaccine. The efficacy of a cancer vaccine using DC transfected tumor RNA was examined. Of particular interest was whether a vaccine using DC transfected with recrudescent tumor RNA is effective for the treatment of a regrowing tumor after prior immunotherapy. In addition, the usefulness of co-transfection of granulocyte macrophage colony-stimulating factor (GM-CSF) mRNA to augment the DC vaccine was examined. CT26 tumor-bearing mice were immunized by s.c. injection with DC transfected with CT26 mRNA (DC-CT26). The cytotoxic activity against CT26 in mice immunized with DC-CT26 was significantly higher than that in the control group ( P  < 0.001) and was augmented by GM-CSF mRNA co-transfection ( P  < 0.05), resulting in remarkable therapeutic efficacy in CT26 s.c. tumor models. Cytotoxic T lymphocytes induced by the vaccination using DC transfected with mRNA from the recrudescent tumor showed a potent cytotoxicity against the recrudescent CT26 tumor cells, which was significantly higher than the cytotoxicity induced by the vaccination using DC-CT26 ( P  < 0.05). In addition, in a recrudescent tumor model, this vaccination suppressed the regrowing s.c. tumors, and was augmented by GM-CSF mRNA co-transfection ( P  < 0.05). These results suggested that vaccination therapy using DC simultaneously transfected with whole tumor RNA and GM-CSF mRNA could generate therapeutic immune responses even against recrudescent tumor after prior vaccination. ( Cancer Sci 2008; 99: 407–413)     

DNA vaccination with AFP-encoding plasmid DNA prevents growth of subcutaneous AFP-expressing tumors and does not interfere with liver regeneration in mice

The oncofetal alpha-fetoprotein (AFP) is reexpressed in the majority of hepatocellular carcinomas and may be used as a target molecule for an immunotherapy or prophylaxis against this tumor. We investigated the potential of DNA vaccination with AFP-expressing plasmid DNA to induce an immune response against AFP-expressing tumor cells in DBA/2 mice. 62.5% of mice vaccinated with AFP-expressing plasmid DNA, rejected subcutaneous syngeneic AFP-expressing P815 tumors, whereas only 16.7% of mice vaccinated with control plasmid rejected these tumor cells (P=.03). Mean survival of mice after challenge with subcutaneous AFP-expressing tumor cells was prolonged for 8 days in mice vaccinated with AFP-expressing DNA (35 days) compared to mice vaccinated with control plasmid (27 days). To rule out possible autoimmune reactions against regenerating liver, which also reexpresses AFP, we evaluated the influence of AFP-specific DNA vaccination on liver regeneration in DBA/2 mice. Histologic quantification of proliferating hepatocytes and of the amount of necrotic liver tissue in carbon tetrachloride-damaged liver did not reveal statistically significant differences in mice vaccinated with AFP-expressing plasmid compared to control mice. These data suggest that AFP-specific DNA vaccination represents a useful tool to inhibit growth of AFP-expressing tumors in mice that does not affect liver regeneration.     

Th2-dominated antitumor immunity induced by DNA immunization with the genes coding for a basal core peptide PDTRP and GM-CSF

Our previous study showed that DNA vaccination with a plasmid vector encoding a core peptide of mucin1 (PDTRP) provided modest protection against challenge with tumor cells that expressed mucin1 protein. We report here that a DNA vaccine comprising a modified PDTRP plasmid and GM-CSF coding sequence at the C-terminus induced better protection against tumor challenge. The increased protection was directly correlated with a stronger PDTRP-specific immune response induced by the GM-CSF fusion plasmid. The plasmid encoding GM-CSF and the target PDTRP antigen induced a greater PDTRP-specific Th proliferation, antibodies, and cytotoxicity. Interestingly, the modified plasmid vaccine predominantly enhanced the type 2 immune responses manifested by an increased IgG1 to IgG2a antibody ratio and a greater induction of GATA-3 and IL-4 mRNA than that of T-bet and IFN-gamma mRNA in spleen cells from vaccinated mice. In addition, protection against tumor challenge in vaccinated mice showed that there was no significant change in mice survival after in vivo CD8+CTL depletion, indicating that antitumor immunity augmented by plasmid encoding GM-CSF and target PDTRP gene vaccine was dominated by Th2 immune response.     

The targeted expression of interleukin-2 in human hepatocellular carcinoma cells

To improve the safety and efficiency of human hepatocellular carcinoma (HCC) gene therapy, we explored the use of a liver-specific promoter and a tumor-specific enhancer to achieve regular IL-2 gene expression for treatment of HCC. The human alpha-fetoprotein (AFP) enhancer [E(AFP)] and the albumin promoter [P(ALB)] were amplified from human genomic DNA. We used eukaryotic expression vector pcDNA-3 for the delivery of the IL-2 gene because this plasmid is a non-transient, fast-selection expression vector. A recombinant plasmid was constructed including the selectable marker neoR gene and the human IL-2 gene derived by the E(AFP) - P(ALB). The liver-predominant expression pattern of the IL-2 gene was observed in the medium of the transfected cells. When human HCC cell lines displaying different levels of AFP and non-hepatocyte tumor cell lines were transfected with the recombinant plasmid, IL-2 was expressed highly in AFP and albumin-positive HCC cells, but low in nonhepatocyte tumor cells. Moreover, the expression level of IL-2 gene was positively proportional to the level of AFP expression in the transfected cells.     

腺病毒介导IL-2基因转染的瘤苗体内抗肿瘤作用

目的 :探讨重组腺病毒介导的 IL- 2基因转染的瘤苗的体内抗肿瘤作用及其免疫学机制。方法 :应用腺病毒介导的鼠 IL- 2基因转染 CT2 6小鼠结肠癌细胞 ,灭活后用作瘤苗治疗荷瘤小鼠 ,观察皮下肿瘤生长及其存活期。采用乳酸脱氢酶释放法检测荷瘤小鼠脾细胞 CTL、L AK、NK细胞的杀伤活性。结果 :鼠 IL- 2基因转染瘤苗治疗能显著抑制荷瘤小鼠皮下肿瘤生长并明显延长其存活期 (P<0 .0 1)。体内免疫功能检测表明 ,鼠 IL- 2基因转染疫苗治疗组小鼠脾细胞 CTL 活性、L AK活性和 NK活性显著高于对照组 (P<0 .0 1)。结论 :腺病毒介导鼠 IL- 2基因转染的瘤苗体内具有较强的抗肿瘤效应 ,其机制可能是提高了荷瘤小鼠特异性和非特异性抗肿瘤免疫反应     

Inhibition of tumor growth using salmonella expressing Fas ligand

Intravenous administration of bacteria leads to their accumulation in tumors and to sporadic tumor regression. We therefore explored the hypothesis that Salmonella typhimurium engineered to express the proapoptotic cytokine Fas ligand (FasL) would exhibit enhanced antitumor activity. Immunocompetent mice carrying tumors derived from syngeneic murine D2F2 breast carcinoma or CT-26 colon carcinoma cells were treated intravenously with FasL-expressing S. typhimurium or with phosphate-buffered saline (PBS; control). Treatment with FasL-expressing S. typhimurium inhibited growth of primary tumors by an average of 59% for D2F2 tumors and 82% for CT-26 tumors (eg, at 25 days after initial treatment, mean volume of PBS-treated CT-26 colon carcinomas = 1385 mm(3) and of S. typhimurium FasL-treated CT-26 tumors = 243 mm(3), difference = 1142 mm(3), 95% confidence interval = 800 mm(3) to 1484 mm(3), P < .001). Pulmonary D2F2 metastases (as measured by lung weight) were reduced by 34% in S. typhimurium FasL-treated mice compared with PBS-treated mice. FasL-expressing S. typhimurium had similar effects on growth of murine B16 melanoma tumors in wild-type mice but not in lpr/lpr mice, which lack Fas, or in mice with disrupted host inflammatory responses. Antitumor activity was achieved without overt toxicity. These preclinical results raise the possibility that using attenuated S. typhimurium to deliver FasL to tumors may be an effective and well-tolerated therapeutic strategy for some cancers.     

Apoptotic,but not necrotic,tumor cell vaccines induce a potent immune response in vivo

Prophylactic tumor vaccination against subsequent tumor challenge depends on effective cross-priming in vivo. Professional APCs process tumor antigens from whole tumor cells and present them to CD4(+) and CD8(+) T cells. Data suggest that dendritic cells process antigens more efficiently from necrotic cells than from apoptotic cells in vitro. We compared the effect of apoptosis vs. necrosis in vivo using different tumor models (CT26, RENCA, B16 and CT26-HA). Apoptosis was induced by gamma-irradiation prior to injection and verified in vivo. Apoptotic CT26-HA, CT26-wt or RENCA prevented tumor outgrowth in 100%, 75% and 100%, respectively, of mice for more than 30 days after challenge. In contrast, injection of necrotic tumor cells led to protection of no more than 0-30%. Prolonged tumor-free survival was also observed in mice after vaccination with irradiated B16 cells. In vivo protection experiments correlated very well with in vitro cytotoxicity assays. Immunohistochemical analysis of the vaccine site showed a strong CD4(+) and CD8(+) T-cell response after injection of apoptotic cells, which was accompanied by the presence of dendritic cells. In contrast, necrotic cell vaccines attracted a strong local macrophage response. Our data clearly demonstrate that only apoptotic tumor cell vaccines induce a potent antitumor immune response.     

Ad介导人AFP和IFN-у协同诱发抗小鼠肝癌免疫效应

背景与目的:原发性肝癌(hepatocellular carcinoma,HCC)是常见的恶性肿瘤之一,目前对HCC的治疗尚无行之有效的手段。本研究探讨腺病毒(Adenovirus,Ad)载体介导异种甲胎蛋白(Alpha-fetoprotein,AFP)和酌干扰素(Interferon-gamma,IFN-γ酌)的协同抗肝癌效应。方法:用RT-PCR(reverse transcri-ptase-polymerase chain reaction)方法克隆小鼠IFN-γ酌基因并构建复制缺损型腺病毒编码人AFP和小鼠IFN-γ酌联合表达载体(Ad-hAFP/IFN-γ酌)。皮内免疫C57BL/6小鼠7d后,取脾细胞行51Cr释放实验检测特异性细胞毒T淋巴细胞(Cytotoxic Tlymphocytes,CTLs)杀伤活性;或给免疫小鼠皮下接种Hepa1-6肝癌细胞,观察荷瘤小鼠成活情况。结果:51Cr释放实验显示,Ad-hAFP/IFN-γ酌免疫小鼠1周后其诱导产生的特异性CTL杀伤活性明显强于Ad-hAFP或Ad-IFN-γ酌单独免疫,在效∶靶比(E∶T)为10∶1时,Ad-hAFP/IFN-γ酌、Ad-hAFP和Ad-IFN-γ酌诱发的CTL杀伤率分别(43.8±5.5)%、(28.2±3.2)%和(12.8±1.9)%;30∶1时,为(79.6±6.4)%、(51.9±4.3)%和(15.6±2.3)%以及90∶1时(88.2±6.3)%、(62.5±4.8)%和(26.5±2.4)%。荷瘤试验表明,Ad-hAFP或Ad-IFN-γ酌单独免疫小鼠后1周接种5×106Hepa1-6肝瘤细胞,观察2个月,Ad-hAFP免疫组80%的小鼠荷瘤,Ad-IFN-γ酌免疫组小鼠则100%荷瘤;而Ad-hAFP/IFN-γ酌免疫小鼠在接种同样数量的Hepa1-6细胞,2个月无小鼠荷瘤,小鼠100%存活。结论:腺病毒载体介导异种AFP能有效地诱发针对小鼠AFP的特异性细胞免疫反应,IFN-γ酌能明显增强这种效应。