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.     

CCL21 (SLC) improves tumor protection by a DNA vaccine in a Her2/neu mouse tumor model

Secondary lymphoid-tissue chemokine (SLC/CCL21) is a CC chemokine that is constitutively expressed in various lymphoid tissues and binds to chemokine receptor CCR7 on mature dendritic cells (DCs) and distinct T-and B-cell sub-populations. In vivo, CCL21 regulates the encounters between DC and T cells and thus is a key regulator of adaptive immune responses. We asked whether CCL21 is able to augment immunogenicity of a DNA-based vaccine against Her2/neu in a Balb/c mouse model with syngeneic Her2/neu+ tumor cells (D2F2/E2). Mice were vaccinated intramuscularly with plasmid DNA (pDNA) on day 1 and boosted on day 15; tumor challenge was performed subcutaneously on day 25. Coexpression of CCL21 and Her-2/neu resulted in induction of a TH1-polarized immune response and substantial improvement of the protective effect of the DNA vaccine. Coexpression of tumor antigen pDNA(Her2/neu) with both pDNA(GM-CSF) and pDNA(CCL21) as adjuvants led to further improvement of protection by the vaccine (70% tumor-free mice on day 35 vs 40% with either adjuvant alone vs 5-10% with tumor antigen alone). Our results show that CCL21 is a potent adjuvant for DNA vaccination, particularly in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Clinical use of a pDNA(Her2/neu/CCL21/GM-CSF) vaccine might be particularly promising in minimal residual Her2/neu+ breast cancer.     

Genetic vaccination with "self" tyrosinase-related protein 2 causes melanoma eradication but not vitiligo

"Self" melanocyte differentiation antigens are potential targets for specific melanoma immunotherapy. Vaccination against murine tyrosinase-related protein (TRP)-1/gp75 was shown recently to cause melanoma rejection, which was accompanied by autoimmune skin depigmentation (vitiligo). To further explore the linkage between immunotherapy and autoimmunity, we studied the response to vaccination with a related antigen, TRP-2. i.m. inoculation of plasmid DNA encoding murine trp-2 elicited antigen-specific CTLs that recognized the B16 mouse melanoma and protected the mice from challenge with tumor cells. Furthermore, mice bearing established s.c. B16 melanomas rejected the tumor upon vaccination with a recombinant vaccinia virus encoding trp-2. Depletion experiments showed that CD8+ lymphocytes and natural killer cells were crucial for the antitumor activity of the trp-2-encoding vaccines. Mice that rejected the tumor did not develop generalized vitiligo, indicating that protective immunity can be achieved in the absence of widespread autoimmune aggression.     

Immunogenicity, including vitiligo, and feasibility of vaccination with autologous GM-CSF-transduced tumor cells in metastatic melanoma patients.

PURPOSE: To determine the feasibility, toxicity, and immunologic effects of vaccination with autologous tumor cells retrovirally transduced with the GM-CSF gene, we performed a phase I/II vaccination study in stage IV metastatic melanoma patients. PATIENTS AND METHODS: Sixty-four patients were randomly assigned to receive three vaccinations of high-dose or low-dose tumor cells at 3-week intervals. Tumor cell vaccine preparation succeeded for 56 patients (88%), but because of progressive disease, the well-tolerated vaccination was completed in only 28 patients. We analyzed the priming of T cells against melanoma antigens, MART-1, tyrosinase, gp100, MAGE-A1, and MAGE-A3 using human leukocyte antigen/peptide tetramers and functional assays. RESULTS: The high-dose vaccination induced the infiltration of T cells into the tumor tissue. Three of 14 patients receiving the high-dose vaccine showed an increase in MART-1- or gp100-specific T cells in the peripheral blood during vaccination. Six patients experienced disease-free survival for more than 5 years, and two of these patients developed vitiligo at multiple sites after vaccination. MART-1- and gp100-specific T cells were found infiltrating in vitiligo skin. Upon vaccination, the T cells acquired an effector phenotype and produced interferon-gamma on specific antigenic stimulation. CONCLUSION: We conclude that vaccination with GM-CSF-transduced autologous tumor cells has limited toxicity and can enhance T-cell activation against melanocyte differentiation antigens, which can lead to vitiligo. Whether the induction of autoimmune vitiligo may prolong disease-free survival of metastatic melanoma patients who are surgically rendered as having no evidence of disease before vaccination is worthy of further investigation.     

Improved therapeutic outcome following combination immunogene vaccination therapy in murine myeloma

Increasing evidence suggests a role for immunologic vaccination and therapy in the management of minimal residual myeloma. We have previously demonstrated a synergistic effect of combining the Th1 stimulating cytokine IL-12 with the co-stimulatory molecule CD80 in murine myeloma vaccination therapy. We reasoned that the efficacy of such treatment might be further improved by incorporating additional gene products which enhance the function of antigen presenting cells. Studies were therefore conducted with murine myeloma BM1 cells expressing Flt3L (membrane bound or soluble forms) or GM-CSF and the IL-12·CD80 combination. Single agent and combined therapeutic approaches were explored. All gene-modified BM1 cells, except BM1/IL-12·CD80, developed tumors when subcutaneously injected into BALB/c mice. As prophylactic tumor vaccines, the combined use of gene-modified BM1/sFlt3L+GM-CSF+IL-12·CD80 was most effective, providing 100% protection against subsequent parental BM1 tumor challenge. By comparison, only partial protection was observed with any single gene-engineered tumor vaccine. Notably, IL-12·CD80 coexpressing BM1 cell vaccines were the most effective therapeutic vaccine in a minimal disease model. Such protective vaccination was achieved by stimulation of lymphocyte proliferation and enhancement of cytotoxic lymphocyte activity.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by cDNA-transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF

Clinical cancer gene therapy trials have generally focused on the transfer of cytokine cDNA to tumor cells ex vivo and with the subsequent vaccination of the patient with these genetically altered tumor cells. This approach results in high local cytokine concentrations that may account for the efficacy of this technique in animal models. We hypothesized that the expression of certain cytokines by tumor cells would be a superior immune stimulant when compared with local delivery of exogenous cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a nonviral expression vector was inserted into MDA-MB-231 (human breast cancer), M21 (human melanoma), B16 (murine melanoma), and P815 (mastocytoma) cells by particle-mediated gene transfer. The ability of transfected tumor cells to generate a tumor-specific immune response was evaluated in an in vitro mixed lymphocyte-tumor cell assay and in an in vivo murine tumor protection model. Peripheral blood lymphocytes cocultured with human GM-CSF-transfected tumor cells were 3- to 5-fold more effective at lysis of the parental tumor cells than were peripheral blood lymphocytes incubated with irradiated tumor cells and exogenous human GM-CSF. Mice immunized with murine GM-CSF-transfected irradiated B16 murine melanoma cells or P815 mastocytoma cells were protected from subsequent tumor challenge, whereas mice immunized with the nontransfected tumors and cutaneous transfection of murine GM-CSF cDNA at the vaccination site developed tumors more frequently. The results indicate that GM-CSF protein expressed in human and murine tumor cells is a superior antitumor immune stimulant compared with exogenous GM-CSF in the tumor microenvironment.     

Therapeutic tumor immunity induced by polyimmunization with melanoma antigens gp100 and TRP-2

To improve the immunogenicity of melanoma self-antigens, we used a novel strategy of nonviral genetic vaccination coupled with muscle electroporation. Electroporation-enhanced immunization with plasmids encoding either human gp100 or mouse TRP-2 antigens induced only partial rejection of B16 melanoma challenge. However, immunization with a combination of these two antigens caused tumor rejection in 100% of the immunized mice. Splenocytes from combination-immunized animals killed syngeneic targets loaded with peptides derived from both gp100 and TRP-2. Immune cell depletion experiments identified CD8+ T lymphocytes as the primary effectors of antitumor immunity. Most importantly, polyimmunization led to the generation of a therapeutic immune response that significantly improved the mean survival time of mice bearing established lung metastases. These results validated the usefulness of electroporation-enhanced, nonviral genetic immunization for the active immunotherapy of cancer and indicated that using a combination of different tumor antigens may be a decisive strategy for a successful therapeutic vaccination.     

异种黑色素细胞疫苗诱导小鼠抗恶性黑色素瘤免疫反应

目的：研究异种黑色素细胞疫苗对小鼠恶性黑色素瘤生长的抑制新作用。方法制轩异种黑色素细胞疫苗,皮皮预防免疫小鼠后再接种恶性黑色素瘤,并以该疫苗皮下注射治疗恶性黑色素瘤鼠,随后测量肿瘤大小,采用^51Cr释放法,体外分析小鼠脾细胞的细胞毒性T细胞（CTL）活性,以间接ELISA法检测小鼠血清抗肿瘤抗 。结果预防免疫使90％小鼠恶性黑色素瘤生长受到抑制,疫苗治疗使50％小鼠肿瘤生长受到抑制,免疫组鼠较对照组CTL杀伤活性在20：1、10：1、5：1不同效靶比时,分别增高34．0％、24．1％及13．9％（P＜0．05）,体内出现抗恶性黑色素瘤抗体。结论：异种黑色素细胞疫羁可诱导小鼠抗恶性黑色素瘤特异性免疫反,抑制肿瘤生长。     

Adjuvanticity of plasmid DNA encoding cytokines fused to immunoglobulin Fc domains.

PURPOSE: Plasmid DNAs encoding cytokines enhance immune responses to vaccination in models of infectious diseases and cancer. We compared DNA adjuvants for their ability to enhance immunity against a poorly immunogenic self-antigen expressed by cancer. EXPERIMENTAL DESIGN: DNAs encoding cytokines that affect T cells [interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, and the chemokine CCL21] and antigen-presenting cells [granulocyte macrophage colony-stimulating factor (GM-CSF)] were compared in mouse models as adjuvants to enhance CD8+ T-cell responses and tumor immunity. A DNA vaccine against a self-antigen, gp100, expressed by melanoma was used in combination with DNA encoding cytokines and cytokines fused to the Fc domain of mouse IgG1 (Ig). RESULTS: We found that (a) cytokine DNAs generally increased CD8+ T-cell responses against gp100; (b) ligation to Fc domains further enhanced T-cell responses; (c) adjuvant effects were sensitive to timing of DNA injection; (d) the most efficacious individual adjuvants for improving tumor-free survival were IL-12/Ig, IL-15/Ig, IL-21/Ig, GM-CSF/Ig, and CCL21; and (e) combinations of IL-2/Ig+IL-12/Ig, IL-2/Ig+IL-15/Ig, IL-12/Ig+IL-15/Ig, and IL-12/Ig+IL-21/Ig were most active; and (f) increased adjuvanticity of cytokine/Ig fusion DNAs was not related to higher tissue levels or greater stability. CONCLUSIONS: These observations support the potential of cytokine DNA adjuvants for immunization against self-antigens expressed by cancer, the importance of timing, and the enhancement of immune responses by Fc domains through mechanisms unrelated to increased half-life.     

Murine dendritic cells transfected with human GP100 elicit both antigen-specific CD8(+) and CD4(+) T-cell responses and are more effective than DNA vaccines at generating anti-tumor immunity.

Dendritic cells (DCs) are potent inducers of cytotoxic T lymphocytes (CTLs) when pulsed with an antigenic peptide or tumor lysate. In this report, we have used liposome-mediated gene transfer to examine the ability of plasmid DNA encoding the human melanoma-associated antigen gp100 to elicit CD8(+) and CD4(+) T-cell responses. We also compared the efficacy between gp100 gene-modified DCs and naked DNA (pCDNA3/gp100)-based vaccines at inducing anti-tumor immunity. DCs were generated from murine bone marrow and transfected in vitro with plasmid DNA containing the gp100 gene. These gp100-modified DCs (DC/gps) were used to stimulate syngeneic naive spleen T cells in vitro or to immunize mice in vivo. Antigen-specific, MHC-restricted CTLs were generated when DC/gps were used to prime T cells both in vitro and in vivo. Thus, these CTLs were cytolytic for gp100-transfected syngeneic (H-2(b)) tumor MCA106 (MCA/gp) and vaccinia-pMel17/gp100-infected syngeneic B16 and MCA106, but not parental tumor MCA106 and B16, or gp100-transfected allogeneic tumor P815 (H-2(d)). Immunization with DC/gp protected mice from subsequent challenge with MCA/gp but not parental MCA106. Antibody-mediated T-cell subset depletion experiments demonstrate that induction of CTLs in vivo is dependent on both CD4(+) and CD8(+) T cells. Furthermore, DC/gp immunization elicits an antigen-specific CD4(+) T-cell response, suggesting that DC/gps present MHC class II epitopes to CD4(+) T cells. In addition, our data show that gene-modified, DC-based vaccines are more effective than the naked DNA-based vaccines at eliciting anti-tumor immunity in both prophylactic and therapeutic models. These results suggest that the use of DCs transfected with plasmid DNA containing a gene for TAA may be superior to peptide-pulsed DCs and naked DNA-based vaccines for immunotherapy and could provide an alternative strategy for tumor vaccine design.     

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。     

Clinical and immunologic results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells.

PURPOSE: To determine clinical and immunologic responses to a multipeptide melanoma vaccine regimen, a randomized phase II trial was performed. PATIENTS AND METHODS: Twenty-six patients with advanced melanoma were randomly assigned to vaccination with a mixture of four gp100 and tyrosinase peptides restricted by HLA-A1, HLA-A2, and HLA-A3, plus a tetanus helper peptide, either in an emulsion with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Montanide ISA-51 adjuvant (Seppic Inc, Fairfield, NJ), or pulsed on monocyte-derived dendritic cells (DCs). Systemic low-dose interleukin-2 (Chiron, Emeryville, CA) was given to both groups. T-lymphocyte responses were assessed, by interferon gamma ELIspot assay (Chiron, Emeryville, CA), in peripheral-blood lymphocytes (PBLs) and in a lymph node draining a vaccine site (sentinel immunized node [SIN]). RESULTS: In patients vaccinated with GM-CSF in adjuvant, T-cell responses to melanoma peptides were observed in 42% of PBLs and 80% of SINs, but in patients vaccinated with DCs, they were observed in only 11% and 13%, respectively. The overall immune response was greater in the GM-CSF arm (P <.02). Vitiligo developed in two of 13 patients in the GM-CSF arm but in no patients in the DC arm. Helper T-cell responses to the tetanus peptide were detected in PBLs after vaccination and correlated with T-cell reactivity to the melanoma peptides. Objective clinical responses were observed in two patients in the GM-CSF arm and one patient in the DC arm. Stable disease was observed in two patients in the GM-CSF arm and one patient in the DC arm. CONCLUSION: The high frequency of cytotoxic T-lymphocyte responses and the occurrence of clinical tumor regressions support continued investigation of multipeptide vaccines administered with GM-CSF in adjuvant.     

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.     

Interleukin-7-dependent expansion and persistence of melanoma-specific T cells in lymphodepleted mice lead to tumor regression and editing

Active-specific immunotherapy with dendritic cells loaded with peptide derived from the melanoma antigen, gp100, failed to mediate regression of established B16F10 melanoma in normal mice. Dendritic cell vaccination induced activation and subsequent deletion of adoptively transferred naive CD8+ T-cell receptor transgenic (pmel-1) T cells specific for gp100 in normal mice. In lymphodepleted mice, dendritic cell vaccination produced greater T-cell expansion, long-term persistence of memory T cells, and tumor regression. Most tumors that persisted in the presence of functional memory T cells had either lost or exhibited reduced expression of MHC class I or gp100 proteins. In contrast to other naive T cells, pmel-1 T cells adoptively transferred to lymphodepleted mice exhibited faster proliferation and a more differentiated phenotype after exposure to peptide-pulsed dendritic cells. Proliferation and persistence of pmel-1 T cells was highly dependent on interleukin-7 (IL-7) in irradiated mice, and IL-15 when IL-7 was neutralized, two critical homeostatic cytokines produced in response to the irradiation-induced lymphodepletion.     

The boosting effect of co-transduction with cytokine genes on cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen

The T-helper 1 (Th1) immune reaction is most important in dendritic cell (DC)-based immunotherapy. Interleukin 12 (IL-12) and granulocyte macrophage colony-stimulating factor (GM-CSF) play a pivotal role in inducing Th1 and cytotoxic T lymphocyte (CTL) responses. In this study, DCs expressing the natural tumor antigen gp70 of BALB/c-derived CT26 were adenovirally transduced with the IL-12 gene and/or GM-CSF gene, and it was examined whether vaccinations using these genetically engineered DCs can induce strong therapeutic antitumor immunity. Mice were immunized once by subcutaneous (s.c.) injection with genetically modified DCs. The cytotoxic activity of splenocytes against CT26 was assayed in a 51Cr-release assay 14 days after immunization. The therapeutic efficacy of the vaccination was examined in s.c. tumor models. The cytotoxic activity of CTLs against CT26 in mice immunized with DCs expressing gp70 (DC-AxCAgp70) was significantly augmented by co-transduction with the GM-CSF/IL-12 gene (p<0.0001) and remarkably reduced by the depletion of CD4+ or CD8+ cells (p<0.01). The cytotoxic activity against CT26 of the plain spleen cells in mice immunized with DC-AxCAgp70/GM-CSF/IL-12 was significantly higher than that in mice immunized with DC-AxCAgp70 (p<0.0001), and this activity decreased to almost 50% upon the depletion of NK cells. Vaccinations using DC-AxCAgp70/GM-CSF/IL-12 or DC-AxCAgp70/IL-12 could elicit potent therapeutic immunity in s.c. tumor models; tumor-free mice were observed in these vaccination groups. However, there was no significant difference between these two groups. A vaccination therapy using DCs co-transduced with the TAA gene and Th 1-type cytokine genes, especially the IL-12 gene, is ideal for immunotherapy in terms of the activation of DCs, NK cells, CD4+ T cells and CD8+ T cells, and may be useful in the clinical application of a cancer vaccine therapy.     

T-cell responses against tyrosinase 368-376(370D) peptide in HLA*A0201+ melanoma patients: randomized trial comparing incomplete Freund's adjuvant, granulocyte macrophage colony-stimulating factor, and QS-21 as immunological adjuvants.

We conducted a randomized trial in HLA*A0201+ patientswith American Joint Committee on Cancer stage III or IV melanoma immunized with tyrosinase 368-376(370D) peptide and gp100 209-217(210M) peptide to compare the potency of three different adjuvants. Patients received 3 monthly immunizations with 500 microg of each peptide either with incomplete Freund's adjuvant (IFA), QS-21, or granulocyte macrophage colony-stimulating factor (GM-CSF). The primary end point was induction of IFN-gamma release by CD8+ T cells against tyrosinase and gp100 peptides measured by enzyme-linked immunospot assays without in vitro prestimulation measured pretreatment, 2 and 8 weeks after the third vaccination. Four of 9 and 4 of 8 patients immunized using QS-21 and GM-CSF, respectively, developed increased frequencies of CD8+ T cells against tyrosinase 370D peptide compared with 0 of 9 patients immunized using IFA (P = 0.045). T-cell responses against a gp100-related peptide showed similar results, but their relevance to T-cell reactivity against native gp100 209-217 is uncertain. These results show that: (a) QS-21 and GM-CSF are superior to IFA as immunological adjuvants for vaccination against tyrosinase 370D peptide; and (b) with appropriate adjuvants, increased frequencies of peptide-specific T cells after vaccination can be detected by enzyme-linked immunospot without prolonged prestimulation in vitro.     

锚定表达GM-CSF疫苗的抑瘤效果研究

子宫颈癌是妇科比较常见的恶性肿瘤之一,其病因及发病机制不清.近年来,随着细胞分子生物学研究的深入,发现肿瘤的发生发展不仅取决于细胞的增殖速率,而且与细胞凋亡有一定关系,本实验通过对宫颈癌组织芯片进行凋亡相关基因bag-1检测,探讨宫颈癌的发病机制.Bag-1是一种已经被确认的多功能结合蛋白,具有抗细胞凋亡的能力,该蛋白质为一种由219个氨基酸残基组成的酸性蛋白质.     

Targeted interleukin 2 therapy enhances protective immunity induced by an autologous oral DNA vaccine against murine melanoma

We demonstrate that a mouse-human chimeric anti-ganglioside GD2-interleukin (IL)-2 fusion protein (ch14.18-IL2) substantially amplifies tumor-protective immunity against murine melanoma induced by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to murine melanoma peptide epitopes gp100(25-35) and TRP-2(181-188). This combination therapy led to the complete rejection of a lethal challenge with B78D14 murine melanoma cells in six of eight mice and a marked suppression of s.c. tumor growth in the two remaining animals. The tumor-protective immunity was mediated by MHC class I antigen- restricted CD8(+) T cells together with CD4(+) T cell help, which was required only for tumor cell killing in the effector phase of the immune response. A single oral vaccination with the DNA vaccine, which was carried by attenuated Salmonella typhimurium, was equally as effective as three such vaccinations applied at 2-week intervals. The immunological mechanisms involved in this antitumor effect were suggested by a decisively increased secretion of tumor necrosis factor alpha TNFTnTNa and IFN-gamma from CD4(+) and CD8(+) T cells and a markedly up-regulated expression on CD8(+) T cells of high-affinity IL-2 receptor alpha chain (CD25), costimulatory molecule CD28, and adhesion molecule lymphocyte function-associated antigen-2 (LFA-2/CD2). Additionally, the combination therapy induced increased expression of costimulatory molecules B7.1 and CD48 on murine antigen-presenting cells. Taken together, our results suggest that IL-2 targeted to the tumor microenvironment by a specific antibody-IL-2 fusion protein is a potent enhancer of tumor-protective immunity induced by an oral DNA vaccine that may ultimately enhance the chances of success in its clinical application.