Developing Immunomodulators:IL-12 and ONO-4007

IL-12:Interleikin-12(IL-12)was discovered as a cytokine with aunique heterodimeric structure.It was originally found in cultere su-pematant from B lymphobastoid cell lines as natural killer cell stimu-latory factor by M.Kobayashi,et al.in 1989.Recent studies haveshown that the majority of IL-12 is produced by macrophages/mono-cytes following appropriate stimulation.IL-12 Palys a pivotal role inthe regulation of cell-mediated immunity,exerting pleiotropic effectson T cells and natural killer cells.This function is promoted by theIL-12 induced production of interferon-γ from both resting and acti-vated NK and T cells,by the proliferative activity of IL-12 on acti-vated NK and T cells,by enhancing the cytotoxic activity of NKcells,and by supperting cytotoxic T lymphocyte generation.IL-12also promotes the development of naive murine CD4~ T cells into Thelper type 1(Th1)cells in response to antigen.Based on thesepotent immunomodulatory activities,IL-12 has been shown to havetherapeutic activity in a variety     

Immunotherapy of spontaneous metastatic lung cancer with tumor antigen-pulsed,interleukin-12 gene-modified dendritic cells

Interleukin-12 (IL-12), with the ability of inducing production of interferon-gamma and enhancing of NK activity and Th1 response, has potent antitumor role and has been used in treatment of tumors[1-7]. Dendritic cells (DC) are the uniquely potent APCs involved in the initiation of immune responses. As adjuvants for Ag delivery, DC pick up Ags in the periphery and carry them to T cells area in lymphoid organs to prime the immune responses. With the development of the methods for propaga…     

急性髓系白血病细胞对树突状细胞分化、成熟和调节性T细胞生成的影响

Objective To investigate the effects of soluble factors secreted by acute myeloid leukemia (AML) cells on the phenotypical and functional properties of DCs derived from normal mononuclear cells. Methods Mononuclear cells were cultured with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF), in the presence or absence of 24 h culture supernatants from fresh primary AML cells, to generate immature DCs. The maturation of DCs was induced by cytokines IL-1beta, IL-6, tumor necrosis factor-alpha (TNF-alpha), and prostaglandin-2 (PGE-2). The phenotypic alterations of DCs and DCs-primed CD4 T cells were evaluated using flow cytometry. Precursor frequency (PF) was calculated to monitor the allostimulatory effects of DCs on CD4 and CD8 T cells. ResultsAML cell supernatant-treated DCs showed significantly lower expression of co-stimulatory molecules CD80 and CD86, and reduced response to cytokines IL-1beta, IL-6, TNF-alpha,and PGE-2. The allostimulatory effects of AML cell supernatant-treated DCs on CD4 and CD8 T cells were significantly lower than those of normal mature DCs [PF (1.8±0.5)% vs. (5.2±1.6)% for CD4 T cells, (2.1±0.6)% vs. (6.5±2.0)%for CD8 T cells, P＜0.01]. These AML supematant-induced DCs could also induce allogeneic CD4 T cells to differentiate into CD4 CD25high T cells, which had immunophenotyping characteristics of regulatory T cells, i.e. they expressed Foxp3 but not active maker CD69. Conclusion This study demonstrates that soluble factors secreted by AML cells can inhibit development and functions of DCs. In addition, AML supematant-induced DCs can induce the generation of CD4 CD25high T cells from CD4 T cells, which may be a mechanism of increased prevalence of CD4 CD25high regulatory T cells and immune dysfunction in AML patients.     

INDUCEMENT OF ANTITUMOR－IMMUNITY BY DC ACTIVATED BY HSP70－H22 TUMOR ANTIGEN PEPTIDE

Antigen peptides from tumor cells, bound with heat shock protein 70 (Hsp70), can induce specific antitumor immunity in vivo[1]. On the surface of dentritic cells (DC), the most powerful antigen-presenting cells in vivo, there is CD91[2] which is high-affinity receptor of Hsp70. Hsp70 from tumor cells can induce the maturation of DC[3]. After capturing tumor antigen, DC can be used as specific vaccine for immunotherapy of tumor[4]. Through DC-presenting pathway, limited amount of antig…     

TCRb REPERTOIRE IN TIL AND PBL OF PATIENTS WITH COLORECTAL CANCER

It has been demonstrated that T lymphocytes play an important role in anti-tumor immunology[1]. Solid tumors are often characterized by tumor-infiltrating lymphocyte (TIL) in primary lesions, which in several cases, has been associated with a good prognosis[2]. The activated T cells in PBL can also affect the prognosis of the patients with cancers[3]. It is possible that the TIL can transfer from tumor tissue into the blood circulation. Tumor cells carry the antigenic peptides, but it is …     

贲门癌患者外周血CD4+CD25hiCD127low调节性T细胞水平及其临床意义

Objective To analyse the dysfunction of immunity and clinical significance in patients with cardiac cancer.Methods The level of CD4+ CD25hi CD127low Treg cells were detected by flow cytometry (FCM),and serum IL-10 and TGF-β1 levels were determined by enzyme linked immunosorbent assay (ELISA) kit in 56 patients with cardiac cancer.15 healthy volunteers were tested as normal controls.The clinical data of each patient were collected and analyzed. Results There was a significantly higher percentage of CD4+ CD25hi CD127low Treg cells in patients with cardiac cancer (5.73±1.56)% than that (4.45±1.06)% of healthy volunteers (P<0.01).The IL-10 and TGF-β1 levels in the serum of patients with cardiac cancer were also significantly higher than that of healthy volunteers (P<0.05).There was a positive correlation between levels of IL-10.TGF-β1 and the levels of CD4+ CD25hi CD127low Treg cells.The number of CD4+ CD25hi CD127low regulatory T cells in the peripheral blood of cardiac cancer patients were significantly correlated with clinical stages and metastasis lymph node.Conclusion The CD4+ CD25hi CD127low Treg cells in the peripheral blood of cardiac cancer patients is significantly increased in comparison with that in healthy volunteers,and was also correlated with different stages.The abnormal levels of CD4+ CD25hi CD127low Treg cells may be related to tumor progression in patients with cardiac cancer.     

贲门癌患者外周血CD4+CD25hiCD127low调节性T细胞水平及其临床意义

Objective To analyse the dysfunction of immunity and clinical significance in patients with cardiac cancer.Methods The level of CD4+ CD25hi CD127low Treg cells were detected by flow cytometry (FCM),and serum IL-10 and TGF-β1 levels were determined by enzyme linked immunosorbent assay (ELISA) kit in 56 patients with cardiac cancer.15 healthy volunteers were tested as normal controls.The clinical data of each patient were collected and analyzed. Results There was a significantly higher percentage of CD4+ CD25hi CD127low Treg cells in patients with cardiac cancer (5.73±1.56)% than that (4.45±1.06)% of healthy volunteers (P<0.01).The IL-10 and TGF-β1 levels in the serum of patients with cardiac cancer were also significantly higher than that of healthy volunteers (P<0.05).There was a positive correlation between levels of IL-10.TGF-β1 and the levels of CD4+ CD25hi CD127low Treg cells.The number of CD4+ CD25hi CD127low regulatory T cells in the peripheral blood of cardiac cancer patients were significantly correlated with clinical stages and metastasis lymph node.Conclusion The CD4+ CD25hi CD127low Treg cells in the peripheral blood of cardiac cancer patients is significantly increased in comparison with that in healthy volunteers,and was also correlated with different stages.The abnormal levels of CD4+ CD25hi CD127low Treg cells may be related to tumor progression in patients with cardiac cancer.     

转染CD40配体的卵巢癌细胞株OVHM抗卵巢癌肝转移机制的研究

Objective To examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murinc ovarian cancer OVUM cells (CD4OL-OVHM) injected into the spleen on liver metastasis in mice. Methods OVUM cells were inoculated into the spleen of 6 ～ 8 week-old female B6C3F1 (C57BL/6N × C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVUM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-γ, TNF-α, IL-12, IL4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded. Results The mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rote in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-γ, TNF-α and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (P < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05). Conclusion The data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Thl cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.     

转染CD40配体的卵巢癌细胞株OVHM抗卵巢癌肝转移机制的研究

Objective To examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murinc ovarian cancer OVUM cells (CD4OL-OVHM) injected into the spleen on liver metastasis in mice. Methods OVUM cells were inoculated into the spleen of 6 ～ 8 week-old female B6C3F1 (C57BL/6N × C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVUM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-γ, TNF-α, IL-12, IL4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded. Results The mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rote in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-γ, TNF-α and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (P < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05). Conclusion The data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Thl cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.     

转染CD40配体的卵巢癌细胞株OVHM抗卵巢癌肝转移机制的研究

Objective To examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murinc ovarian cancer OVUM cells (CD4OL-OVHM) injected into the spleen on liver metastasis in mice. Methods OVUM cells were inoculated into the spleen of 6 ～ 8 week-old female B6C3F1 (C57BL/6N × C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVUM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-γ, TNF-α, IL-12, IL4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded. Results The mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rote in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-γ, TNF-α and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (P < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05). Conclusion The data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Thl cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.     

树突状细胞与髓性白血病的免疫治疗 *

目的：我们应用逆转录病毒构建了ＩＬ－１２,Ｂ－７和ＧＭ－ＣＳＦ表达载体,以研究基因修的肿瘤细胞的癌疫苗作用。方法：将３种表达载体分别转染ＥＬ－４胞腺瘤细胞并研究了该基因导入细胞的抗肿瘤免疫效果。结果：当接种子ＥＬ４／ＩＬ－１２细胞后,在Ｃ５７ＰＬ／６同系鼠中其基因导入细胞的肿瘤原性比较ＥＬ４／Ｗｔ和ＥＬ－４／Ｎｅｏ组明显减少（Ｐ〈０．０１）。在ＥＬ４／ＩＬ－１２被排斥后,体内试验中诱发了实验动物抗     

GM-CSF gene or B7-1 gene modified murine EL-4 cells vaccine

Since 1990, gene transduced tumor vaccine has been studied. Many articles reported that tumor cells transduced with some cytokine or costimulatory molecule could induce system antitumor immunity[1,2] In this study, EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene and B7-1 gene, respectively. The effect of gene transduction on antitumor immunity was investigated.MATERIALS AND METHODSMice and Cell Lines Female C57BL/6 mice were bought from …     

ANTITUMOR EFFECTS INDUCED BY B7-1 GENE MODIFIED EL-4 LYMPHOMA COOPERATED WITH IL-2 IN VIVO AND IN VITRO

ＡＮＴＩＴＵＭＯＲＥＦＦＥＣＴＳＩＮＤＵＣＥＤＢＹＢ７１ＧＥＮＥＭＯＤＩＦＩＥＤＥＬ４ＬＹＭＰＨＯＭＡＣＯＯＰＥＲＡＴＥＤＷＩＴＨＩＬ２ＩＮＶＩＶＯＡＮＤＩＮＶＩＴＲＯＷｕＡｉｍｉｎ武爱民ＺｈａｎｇＹｕｅｊｉａｎ张跃建ＱｉｎＨｕｉｌｉａｎ秦慧...     

抗胃癌生物活性肽对荷胃癌裸鼠酯酶同工酶代谢的影响

本文观察了协同刺激分子B7-1某因导入小鼠EL-4淋巴瘤细胞后在小鼠体内诱导的抗瘤效应.结果表明,逆转录病毒(PLXSN)载体重组的小鼠B7-1基因表达质粒导入小鼠EL-4淋巴瘤细胞,经有限稀释法克隆后获得高表达的B7-1~ EL-4细胞.B7-1~ 瘤细胞的形态,体外增殖能力及MHC Ⅰ类分子表达水平与野生型肿瘤细胞无显著差别,但致瘤性显著降低,用野生型肿瘤致死剂量接种C57BL/6小鼠完全排斥.同时免疫原性明显增强,以X-线灭活的B7-1~ 肿瘤细胞免疫后小鼠获得了对随后致死剂量野生型细胞攻击的免疫保护作用.以X-线灭活的肿瘤细胞作为瘤苗进行实验性免疫治疗,对早期(接种7天)形成的肿瘤有一定的治疗效果,但时晚期(接种14天)肿瘤.B7-1和B7-1~ 瘤细胞都未显示出明显的治疗效果.上述结果提示肿瘤细胞表达B7-1分子可有效激发机体的抗肿瘤免疫应答.     

GM—CSF基因修饰的肿瘤细胞疫苗对荷瘤小鼠的抗肿瘤作用

目的研究GM-CSF基因修饰的肿瘤细胞作为瘤苗的可行性.方法通过逆转录病毒载体,将小鼠GM-CSF基因导入EL-4淋巴瘤细胞中,研究EL-4/GM-CSF在同系C57BL/6小鼠中的成瘤性及其诱导抗肿瘤免疫的效果.结果EL-4/GM-CSF细胞在小鼠中的成瘤性下降,50%小鼠无瘤生存.射线灭活的EL-4/GM-CSF瘤苗能有效地保护野生型肿瘤细胞的攻击,在肿瘤生长的早期对实验性荷瘤小鼠进行治疗,能延长荷瘤宿主的存活时间(33.1±1.8)与对照组EL-4/Wt(23.2±1.5天)比差异有显著性(P＜0.01).基因修饰的瘤苗作用明显增强.结论GM-CSF基因修饰的肿瘤疫苗可诱导较强的抗肿瘤免疫反应,导致肿瘤的部分根除,本实验为基因修饰的肿瘤疫苗的临床应用提出了一定的实验依据.     

Inhibitory effects of B cells on antitumor immunity

B-cell functions in antitumor immunity are not well understood. In this study, we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag), D5 mouse melanoma, or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments, spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors), IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells.     

Antitumor effects of the combination therapy with TNF-alpha gene-modified tumor cells and interleukin 12 in a melanoma model in mice

In the present study, TNF-alpha gene-transduced B78 melanoma cells (B78/TNF) were used as a vaccine and combined with interleukin (IL)-12 in the treatment of B78 melanoma-bearing mice. The combined administration of genetically modified melanoma cells and IL-12 induced specific protective antitumor immunity resulting in a decreased rate of the tumor take following a rechallenge with parental B78 cells. When used therapeutically, intratumoral injections of irradiated B78/TNF melanoma cells and IL-12 exerted strong antitumor effects and led to complete regression of established tumors in 50% of mice. Injections of irradiated B78/TNF cells alone did not influence tumor development and IL-12 itself significantly delayed tumor growth but without curative effect. FACS analysis of parental B78 melanoma cells and its B78/TNF genetically modified variant showed that a proportion of cells of both cell lines expressed 87-1 (CD80) costimulatory molecule and that the expression of this molecule was increased during incubation with IFN-gamma. Moreover, IFN-gamma markedly augmented expression of major histocompatibility class (MHC) class I and II molecules on B78/TNF cells that were primarily MHC class I and II negative with no substantial effect on MHC-negative parental B78 melanoma. IFN-gamma also synergized in cytostatic/cytotoxic effects with TNF-alpha against B78 melanoma in vitro. Lymphocyte depletion studies in vivo showed reduction of the antitumor response in mice treated with anti - NK monoclonal antibodies (mAbs) as well as in mice treated with anti-CD4+ anti-CD8 mAbs. The results suggest that, when used therapeutically, IL-12 and a vaccine containing TNF-alpha gene-transduced tumor cells may reciprocally augment their overall antitumor effectiveness by facilitating development of systemic antitumor immunity and by stimulating local effector mechanisms of the tumor destruction.     

Interleukin-12-secreting human papillomavirus type 16-transformed cells provide a potent cancer vaccine that generates E7-directed immunity

The development of a vaccine that would be capable of preventing or curing the (pre)cancerous lesions induced by genital oncogenic human papillomaviruses (HPVs) is the focus of much research. Many studies are presently evaluating vaccines based on the viral E6 and E7 oncoproteins, both of which are continually expressed by tumor cells. The success of a cancer vaccine relies, in large part, on the induction of a tumor-specific Th1-type immunity. In this study, we have evaluated the ability of B7-related and/or interleukin-12 (IL-12)-expressing, non-immunogenic murine HPV16-transformed BMK-16/myc cells, to achieve this goal. BMK-16/myc cells engineered to express surface B7-1 or B7-2 molecules remain tumorigenic in syngeneic BALB/c mice, suggesting that expression of these molecules alone is not sufficient to induce tumor regression. In contrast, mice injected with tumor cells engineered to secrete IL-12 remained tumor-free, demonstrating that IL-12 expression is sufficient to induce tumor rejection. IL-12-secreting BMK-16/myc cells were further shown to induce potent and specific long-term tumor resistance, even after irradiation. B7-1 was found to slightly but systematically improve anti-tumor immunity elicited by IL-12-secreting BMK-16/myc cells. Injection of irradiated B7-1/IL-12+ BMK-16/myc cells generates long-lasting, Th1-type, BMK-16/myc-directed immunity in tumor-resistant mice. These mice display a memory-type, E7-specific, cell-mediated immune response, which is potentially significant for clinical applications.     

Enhanced antitumor effect of oncolytic adenovirus expressing interleukin-12 and B7-1 in an immunocompetent murine model.

PURPOSE: We investigated whether an armed viral platform, where lytic property of a viral infection is coupled to viral-mediated delivery of therapeutic genes, could increase the therapeutic potential of a viral-based therapy. EXPERIMENTAL DESIGN: We generated interleukin (IL)-12-expressing oncolytic adenovirus (YKL-IL-12) and IL-12- and B7-1-expressing (YKL-IL12/B7) oncolytic adenovirus. Therapeutic efficacy of these newly engineered adenoviruses was then evaluated in vivo using an immunocompetent mouse bearing murine melanoma B16-F10 tumors. Overall survival was assessed with the Kaplan-Meier method. The induction of immune cell cytotoxicity was assessed by CTL assay, IFN-gamma enzyme-linked immunospot assay, and immunohistochemical studies. RESULTS: YKL-IL12/B7 oncolytic adenovirus, expressing both IL-12 and B7-1, showed a higher incidence of complete tumor regression compared with the analogous oncolytic adenovirus, YKL-1, or IL-12-expressing, YKL-IL12. Significant survival advantage was also seen in response to YKL-IL12/B7. Moreover, IL-12 and IFN-gamma levels produced in tumors treated with YKL-IL12/B7 was significantly greater than those treated with YKL-IL12. The enhanced survival advantage was mediated by the induction of immune cell cytotoxicity. In agreement with these results, massive infiltration of CD4(+) and CD8(+) T cells into tissues surrounding the necrotic area of the tumor was observed following in situ delivery of YKL-IL12/B7. CONCLUSION: Combination of oncolysis and the enhancement of antitumor immune response by oncolytic adenovirus expressing both IL-12 and B7-1 elicits potent antitumor effect and survival advantage.     

Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: long- term remission of liver metastases in a mouse model

BACKGROUND: The success of immunomodulatory cancer therapy is frequently hampered by the transient nature of the antitumor immune response. We have shown previously in a mouse model that interleukin 12 (IL-12) generates a strong natural killer (NK) cell-mediated antitumor response and reduces liver metastases induced by a colon carcinoma cell line. However, only a small percentage of the treated animals developed the cytotoxic T-lymphocytic response required for a long-term systemic antitumor immunity. 4-1BB is a co-stimulatory molecule expressed on the surface of activated T cells. Interaction of 4-1BB with its natural ligand (4-1BBL) has been shown to amplify T-cell (especially CD8+)-mediated immunity. In this study, we investigated the effects of adenovirus-mediated gene therapy delivering both IL-12 and 4-1BBL genes on mice with hepatic metastases induced by colon cancer cells. METHODS: Syngeneic BALB/c mice received intrahepatic injection of poorly immunogenic MCA26 colon cancer cells. Various combinations of replication-defective adenoviruses expressing IL-12 and 4-1BBL genes were injected into the established liver tumors. Changes in tumor size and animal survival were then monitored. All statistical tests were two-sided. RESULTS: The long-term survival rate of mice treated with the combination of IL-12 and 4-1BBL was significantly improved over that of animals in the control group (P =.0001). In vivo depletion of NK cells or CD8+ T cells completely abolished the long-term survival advantage of the IL-12 plus 4-1BBL-treated animals (P<.002). Moreover, the systemic immunity induced by this combination treatment protected these animals against a subcutaneous challenge with parental MCA26 cells. CONCLUSION: Adenovirus-mediated transfer of IL-12 and 4-1BBL genes directly into liver tumors resulted in tumor regression that required both NK and CD8+ T cells and generated a potent, long-lasting antitumor immunity.