mIL-12 Gene Research Articles

Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma

BackgroundInterleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer.MethodsSolid SA-1 fibrosarcoma tumors, on the back of A/J mice, were treated intratumorally by mIL-12 gene electrotransfer and 24 h later irradiated with a single dose. Treatment effectiveness was measured by tumor growth delay and local tumor control assay (TCD50 assay). With respect to therapeutic index, skin reaction in the radiation field was scored. The tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured. Besides single, also multiple intratumoral mIL-12 gene electrotransfer before and after tumor irradiation was evaluated.ResultsSingle intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral but not serum mIL-12 and mIFNγ concentrations, and had good antitumor (7.1% tumor cures) and radiosensitizing effect (21.4% tumor cures). Combined treatment resulted in the radiation dose-modifying factor of 2.16. Multiple mIL-12 gene electrotransfer had an even more pronounced antitumor (50% tumor cures) and radiosensitizing (86.7% tumor cures) effect.ConclusionsSingle or multiple intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral mIL-12 and mIFNγ cytokine level, and may provide an efficient treatment modality for soft tissue sarcoma as single or adjuvant therapy to tumor irradiation.

Potentiation of electrochemotherapy by intramuscular IL-12 gene electrotransfer in murine sarcoma and carcinoma with different immunogenicity

Background.Electrochemotherapy provides good local tumor control but requires adjuvant treatment for increased local response and action on distant metastasis. In relation to this, intramuscular interleukin-12 (IL-12) gene electro-transfer, which provides systemic shedding of IL-12, was combined with local electrochemotherapy with cisplatin. Furthermore, the dependence on tumor immunogenicity and immunocompetence of the host on combined treatment response was evaluated.Materials and methods.Sensitivity of SA-1 sarcoma and TS/A carcinoma cells to electrochemotherapy with cisplatin was tested in vitro. In vivo, intratumoral electrochemotherapy with cisplatin (day 1) was combined with a single (day 0) or multiple (days 0, 2, 4) intramuscular murine IL-12 (mIL-12) gene electrotransfer. The antitumor effectiveness of combined treatment was evaluated on immunogenic murine SA-1 sarcoma in A/J mice and moderately immunogenic murine TS/A carcinoma, in immunocompetent BALB/c and immunodeficient SCID mice.Results.Electrochemotherapy in vitro resulted in a similar IC50 values for both sarcoma and carcinoma cell lines. However, in vivo electrochemotherapy was more effective in the treatment of sarcoma, the more immunogenic of the tumors, resulting in a higher log cell kill, longer specific tumor growth delay, and also 17% tumor cures compared to carcinoma where no tumor cures were observed. Adjuvant intramuscular mIL-12 gene electrotransfer increased the log cell kill in both tumor models, potentiating the specific tumor growth delay by a factor of 1.8-2 and increasing tumor cure rate by approximately 20%. In sarcoma tumors, the potentiation of the response by intramuscular mIL-12 gene electrotransfer was dose-dependent and also resulted in a faster onset of tumor cures. Comparison of the carcinoma response to the combined treatment modality in immunocompetent and immunodeficient mice demonstrated that the immune system is needed both for increased cell kill and for attaining tumor cures.Conclusions.Based on the comparison of the antitumor effectiveness of electrochemotherapy to intratumoral cisplatin administration, we can conclude that the fraction of cells killed and the tumor cure rate are higher in immunogenic sarcoma tumor compared to moderately immunogenic carcinoma tumor. The tumor cell kill and cure rate depend on the immune response elicited by the destroyed tumor cells, which might depend on the tumor immunogenicity. The effect of adjuvant intramuscular mIL-12 gene electrotransfer is dependent on the amount of IL-12 in the system and the immune competence of the host, as demonstrated by the dose-dependent increase in the cure rate of SA-1 tumors after multiple intramuscular mIL-12 gene electrotransfer and in the differential cure rate of TS/A tumors growing in immunocompetent and immunodeficient mice.

Abstract 4412: Combining cytokine gene therapy with conventional cancer chemotherapyfor treatment of metastatic renal cell carcinoma

Abstract Renal cell carcinoma (RCC) accounts for approximately 3% of adult malignancies and 85-90% of kidney cancer. Treatment was generally aggressive involving nephrectomy and excision of the metastatic lesion. The current study was designed to evaluate a new strategy for treatment of RCC, using combination of cytokine gene therapy with conventional cancer chemotherapy. The study established tumor metastasis in Balb/c mice using Renca cells as a model1. Treatment was performed on tumor bearing mice with hydrodynamic gene delivery of plasmids containing either mIL-2 or mIL-12 gene, separately, or in combination with or without two weekly injection of 5-flououracil (5-FU) (20 mg/kg, ip). The results showed that the lung, liver and kidney were the primary sites of tumor growth. 5-FU is capable of inhibiting tumor growth in the liver and kidney and effective in slowing down tumor growth in the lung when treatment started early and with high frequency. mIL-2 gene therapy inhibited tumor growth in the liver and kidney. The effect became more obvious if treatment started early. However, IL-2 alone is unable to eliminate all liver tumors. mIL-2 gene therapy had almost no effect on tumor growth in lung. mIL-12 gene therapy significantly blocked cancer-induced weight loss. mIL-12 inhibited tumor growth in the liver and kidney, but IL-12 alone is unable to eliminate all liver and kidney tumors. In addition, IL-12 inhibited lung swelling induced by tumor, especially when treatment was started early. Cytokine gene therapy combined with chemotherapy is effective in blocking tumor-induced weight loss, and tumor growth in the liver, lung and kidney. These results provide direct evidence in support that combined therapy of cytokine gene therapy with traditional chemotherapy is effective in treating RCC. Key words: Renal cell carcinoma, gene therapy, IL-12, IL-2, 5- flououracil 1 Jianfeng Li, Qian Yao and Dexi Liu (2011) Hydrodynamic cell delivery for simultaneous establishment of tumor growth in mouse lung, liver and kidney. Cancer Biol Ther 12:737-741. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4412. doi:1538-7445.AM2012-4412

Bifidobacterium as an oral delivery carrier of interleukin-12 for the treatment of Coxsackie virus B3-induced myocarditis in the Balb/c mice

IL-12 plays an important role in the treatment of many infectious diseases by being administered intravenously or intramuscularly. However, intravenous or intramuscular administration is difficult and inconvenient and may cause side effects. The aim of this study is to develop a novel oral delivery system for IL-12 using genetically engineered Bifidobacterium longum as the carrier and further investigate the efficacy of IL-12-expressed B. longum on the coxsackie virus B3 (CVB3)-induced myocarditis in mice. A mIL-12 gene expression vector pBBADs-IL-12 for B. longum was constructed and transformed into Bifidobacterium. Subsequently, the expression of mIL-12 in the engineered B. longum was identified in vitro by western blot and enzyme-linked immunosorbent assay (ELISA) after l-arabinose induction. Moreover, our data indicated that oral administration of IL-12-expressed B. longum for two weeks after CVB3 infection in the Balb/c mice could downregulate the severity of virus-induced myocarditis, markedly reduce the virus titers in the heart and induce a Th1 pattern in the spleen and heart compared with the controls. In conclusion, a novel oral delivery system of Bifidobacterium for murine IL-12 has been successfully established. Oral administration of mIL-12-transformed B. longum may play a therapeutic role in the treatment of CVB3-induced myocarditis in the mice.

Intramuscular delivery of mIL-12 gene reduces the expression of CD44/CD49d on pulmonary leucocytes and inhibits ovalbumin-induced airway hyperreactivity

To investigate the effects of murine IL-12 (mIL-12) gene on the expression of CD44 and CD49d on pulmonary leukocytes. BALB/c mice (n = 24) were sensitized to ovalbumin (OVA) by intraperitoneal injection and challenged with OVA aerosol for 7 consecutive days. mIL-12 plasmid was then intramuscularly administered in eight BALB/c mice every second day for three times during OVA challenge. Administration of mIL-12 plasmid significantly reduced airway hyperreactivity, the expression of CD44 and CD49d on pulmonary leukocytes, pulmonary infiltration of inflammatory cells, Th2 cytokines production and goblet cell hyperplasia (P < 0.05). Importantly, the percentages of CD44+/CD49d+ leucocytes showed positive correlations with numbers of inflammatory cells, goblet cells hyperplasia and levels of IL-4 and IL-5 (P < 0.01). Our study suggests that CD44 and CD49d might contribute to the preventive effects of mIL-12 gene in asthma.

Therapy of cancer by cytokines mediated by gene therapy approach

Gene therapy offers a new approach for treatment of cancer. Transfer of genes encoding immunostimulatory cytokines has been used with remarkable success to eliminate cancer in animals. However, clinical trials in patients with this strategy had limited efficacy. Therefore, improvement of gene transfer vector system is necessary. A hybrid viral vector, consisting of SFV replicon with either murine IL-12 or reporter LacZ gene, was constructed. This hybrid vector showed specificity and high level of expression in HCC both in vitro and in vivo. In a rat orthotropic liver tumor model, treatment of established tumors by the hybrid vector with mIL-12 gene resulted in a strong anti-tumor activity without accompanying toxicity. Subsequently, a helper-dependent adenovirus vectors containing a mifepristone (RU486) inducible system was constructed for controlled and liver-specific expression of human interleukin 12 (hIL-12) (HD-Ad/RUhIL-12) and mouse IL-12 (mIL-12) (HD-Ad/RUmIL-12). Data showed that high and sustained serum levels of hIL-12 could be attained by continuing administration of RU486 every 12 or 24 h. Repetitive induction of hIL-12 could be obtained over, at least, a period of 48 weeks after a single injection of HD-Ad/RUhIL-12. Treatment of liver metastases with of HD-Ad/RUmIL-12 plus RU846 resulted in complete tumor regression in all animals. Then, different cytokine genes were inserted into conditional replicative adenoviruses vectors (also called oncolytic adenovirus). Replication of adenovirus in tumor cells would kill tumor cells and release viruses, which infect surrounding tumor cells. The combination of cytopathic effect by oncolytic adenovirus and biological effect of transgene would exert strong antitumor activity. These new types of vectors may provide a potent and safe tool for cancer gene therapy.

Increased Efficacy and Safety in the Treatment of Experimental Liver Cancer with a Novel Adenovirus-Alphavirus Hybrid Vector

An improved viral vector for cancer gene therapy should be capable of infecting tumors with high efficiency, inducing specific and high-level expression of transgene in the tumor and selectively destroying tumor cells. In the design of such a vector to treat hepatocellular carcinoma, we took advantage of (a) the high infectivity of adenoviruses for hepatic cells, (b) the high level of protein expression and proapoptotic properties that characterize Semliki Forest virus (SFV) replicon, and (c) tumor selectivity provided by alpha-fetoprotein (AFP) promoter. We constructed a hybrid viral vector composed of a helper-dependent adenovirus containing an SFV replicon under the transcriptional control of AFP promoter and a transgene driven by SFV subgenomic promoter. Hybrid vectors containing murine interleukin-12 (mIL-12) genes or reporter gene LacZ showed very specific and high-level expression of transgenes in AFP-expressing hepatocellular carcinoma cells, both in vitro and in an in vivo hepatocellular carcinoma animal model. Infected hepatocellular carcinoma cells were selectively eliminated due to the induction of apoptosis by SFV replication. In a rat orthotopic liver tumor model, treatment of established tumors with a hybrid vector carrying mIL-12 gene resulted in strong antitumoral activity without accompanying toxicity. This new type of hybrid vectors may provide a potent and safe tool for cancer gene therapy.

Combination Nonviral Murine Interleukin 2 and Interleukin 12 Gene Therapy and Radiotherapy for Head and Neck Squamous Cell Carcinoma

To demonstrate that the combination of nonviral murine interleukin (mIL) 2 and mIL-12 gene therapy and external beam radiation therapy (XRT) have an enhanced therapeutic effect for the treatment of head and neck squamous cell carcinoma (HNSCC) in an orthotopic murine model and to elucidate the mechanism of action. A randomized, controlled study in a murine HNSCC model. Tumors were established in the floor of the mouth in C3H/HeJ immunocompetent mice with the SCC VII cell line. These tumors were directly injected with single lipid-formulated mIL-2 or single polymer-formulated mIL-12 or a combination of them and with phosphate-buffered saline or vector without mIL-2 and mIL-12 gene as controls. Then the local tumor was radiated twice with a dose of 1 Gy the next day and injected again 4 days later. Antitumor responses, cytokine expression, and natural killer cell and cytolytic T-lymphocyte activity were assayed. Meanwhile, tumor sizes were measured before and after treatment and compared among the different treatment groups and the controls. The combination mIL-2 + mIL-12 + XRT demonstrated a significant increase in antitumor effects compared with single therapy or controls. Increased expression levels of primary and secondary cytokines were found in the group treated with mIL-2 + mIL-12, and this effect was preserved when mIL-2 and mIL-12 treatments were combined with XRT. Combination therapy significantly increased antitumor effects, T-lymphocyte infiltration of CD4(+)and CD8(+), and the numerous necroses compared with monotherapy. Combination mIL-2 and mIL-12 gene therapy and XRT generates potent antitumor immune responses against HNSCC and significantly increases necrosis (apoptosis) in an orthotopic murine model of HNSCC. The nonviral mIL-2 and mIL-12 gene delivery system was well tolerated. Further optimization of treatment strategy for patients with HNSCC is warranted as well as consideration for human clinical trials.

IL-12 Gene Therapy Is an Effective Therapeutic Strategy for Hepatocellular Carcinoma in Immunosuppressed Mice

Immunosuppressive therapy for organ transplantation is essential for controlling rejection. When liver transplantation is performed as a therapy for hepatocellular carcinoma (HCC), recurrent HCC is one of the most fatal complications. In this study, we show that intratumoral murine IL-12 (mIL-12) gene therapy has the potential to be an effective treatment for malignancies under immunosuppression. C3H mice (H-2(k)), injected with FK506 (3 mg/kg) i.p., were s.c. implanted with 2.5 x 10(6) MH134 cells (H-2(k)) and we treated the established HCC with electroporation-mediated gene therapy using mIL-12 plasmid DNA. Intratumoral gene transfer of mIL-12 elevated intratumoral mIL-12, IFN-gamma, and IFN-gamma-inducible protein-10, significantly reduced the number of microvessels and inhibited the growth of HCC, compared with HCC-transferred control pCAGGS plasmid. The inhibition of tumor growth in immunosuppressed mice was comparable with that of mIL-12 gene therapy in immunocompetent mice. Intratumoral mIL-12 gene therapy enhanced lymphocytic infiltration into the tumor and elicited the MH134-specific CTL response even under FK506. The dose of FK506 was sufficient to prevent the rejection of distant allogenic skin grafts (BALB/c mice, H-2(d)) and tumors, B7-p815 (H-2(d)) used as transplants, during mIL-12 gene therapy against MH134. Ab-mediated depletion studies suggested that the inhibition of tumor growth, neovascularization, and spontaneous lung metastasis by mIL-12 was dependent almost entirely on NK cells and partially on T cells. These results suggest that intratumoral mIL-12 gene therapy is a potent effective strategy not only to treat recurrences of HCC in liver transplantation, but also to treat solid malignant tumors in immunosuppressed patients with transplanted organ.

Combined therapy with flk1-based DNA vaccine and interleukin-12 results in enhanced antiangiogenic and antitumor effects

In this study, we investigated the anti-vasculature 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 interleukin-12 (mIL-12) gene. In combination, flk1 based DNA vaccine and mIL-12 slowed down tumor growth more effectively than either one alone. Splenocytes from the combined group were showed a strong CTL response against both the flk1 and tumor cells. Automated image analysis revealed that the mean microvessel density was significantly reduced after administering either flk1 based DNA vaccine or mIL-12. In addition, the combination of flk1 based DNA vaccine and mIL-12 appeared more effective at reducing the microvessel density of tumor ( P<0.01, both comparisons). In summary, the antivasculature effect and the anti-tumor effect were better when the combination of flk1 based DNA vaccine and IL-12 was administered in comparison to the individual treatment groups, suggesting the synergistic action of flk1 based DNA vaccine and mIL-12.

A Combination of flk1-Based DNA Vaccine and an Immunomodulatory Gene (IL-12) in the Treatment of Murine Cancer

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. 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. 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. The therapy of attenuated Salmonella typhimurium vaccine strain encoding flk1 combined with the interleukin-12 gene has significant synergistic effect against tumors.

Muscle-targetedinterleukin-12gene therapy of orthotopic hepatocellular carcinoma in mice usingin vivoelectrosonoporation

AbstractWe developed a new potent nonviral gene transfer method into mouse muscles in vivo named “electrosonoporation.” We tried in this report to treat murine orthotopic hepatocellular carcinoma (HCC) by muscle-targeted mouse interleukin-12 (mIL-12) gene transfer using in vivo electrosonoporation. I.m. administration of the mIL-12 gene with electrosonoporation elevated serum IL-12 and IFN-γ and significantly prolonged the survival periods with both growth inhibition of orthotopic HCC and inhibition of spontaneous lung metastasis. The IL-12 gene therapy reduced the number of microvessels and induced more Mac-1-positive cells into HCC. These results show that muscle-targeted mIL-12 gene therapy for orthotopic HCC using in vivo electrosonoporation is very efficient and is thus promising for further clinical trial.

INTERLEUKIN-12 GENE THERAPY IS AN EFFECTIVE THERAPEUTIC STRATEGY FOR HEPATOCELLULAR CARCINOMA IN IMMUNOSUPPRESSED MICE.

P1140 Aims: Immunosuppressive therapy for organ transplantation is essential for controlling rejection. In particular, when liver transplantation is performed as a therapy for hepatocellular carcinoma (HCC), recurrent HCC is one of the most common fatal complications. In this study, we evaluated the efficacy of the intratumoral murine Interleukin 12 (mIL-12) gene therapy on HCC under immunosuppression. Methods: C3H mice (H-2k), injected with FK506 (3 mg/kg) intraperitoneally, were subcutaneously implanted with 2.5x106 MH134 cells (H-2k) in the right flank. About 7 days after tumor cell inoculation, s.c. HCCs were established. The established HCC was treated with electroporation-mediated gene therapy using mIL-12 plasmid DNA. The mIL-12 expression plasmid vector, designated pCAGGS-mIL-12, for IL-12-gene therapy and empty plasmid vector, desinated pCAGGS, for control vector were used. Results: Intratumoral gene transfer of mIL-12 elevated intratumoral mIL-12, IFN-γ, and IP-10, significantly reduced the number of microvessels and inhibited the growth of HCC, compared with HCC-transferred control pCAGGS plasmid. The inhibition of tumor growth in immunosuppressed mice was comparable with that of mIL-12 gene therapy in immunocompetent mice. Intratumoral mIL-12 gene therapy enhanced lymphocytic infiltration into the tumor and elicited the MH134-specific CTL response even under FK506. The dose of FK506 was sufficient to prevent the rejection of distant allogenic tumor, B7-p815 (H-2d) used as transplants, during mIL-12 gene therapy against to MH134. Antibody-mediated depletion studies suggested that the inhibition of tumor growth, neovascularlization, and spontaneous lung metastasis by mIL-12 gene therapy was dependent almost entirely on NK cells and partially on T cells. Conclusions: These results suggest that intratumoral mIL-12 gene therapy is a potent effective strategy not only to treat recurrences of HCC in liver transplantation, but also to treat solid malignant tumors in immunosuppressed patients with transplanted organ.

Antitumor immunity and vaccine effect induced by IL-12 synergizes B7-1 gene transfected cells

Objective: To study the synergic effects of IL-12 and B7-1 transfectant on antitumor immunity in vivo. Methods: The retrovirus vector encoding mIL-12 and mB7-1 gene was tranfected into EL-4 thymic lymphoma cells respectively. The cells were used as tumor vaccine and the therapeutic effect was observed. Results: In contrast to the mice immunized with EL-4/Wt or EL-4/Neo groups, the tumorigenicity of EL-4/IL-12 transfectant was decreased (P<0.001). The EL-4/IL-12 and EL-4/B7-1 cells irradiated with 60Co showed significant systematic protective effects against the rechallenge of EL-4/Wt. 60Co irradiated EL-4/IL-12 cells delayed the occurrence of tumor and prolonged the survival period of tumor bearing mice. Combination of the vaccines of EL-4/IL-12 and EL-4/B7-1 resulted in the enhanced therapeutic effect compared with each single transfectant group (P<0.001). Conclusion: The results showed that IL-12 transduced cells could enhance the antitumor immunity of host as cancer vaccine. Combination of the EL-4/IL-12 and EL-4/B7-1 transfectant could improve immunity of host and is a prospect cancer vaccine.

Continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy.

To explore the anti-tumor immunity against CT26 colon tumor of the microencapsulated cells modified with murine interleukine-12 (mIL-12) gene. Mouse fibroblasts (NIH3T3) were stably transfected to express mIL-12 using expression plasmids carrying mIL-12 gene (p35 and p40), and NIH3T3-mIL-12 cells were encapsulated in alginate microcapsules for long-term delivery of mIL-12. mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells was confirmed using ELISA assay. Transplantation of the microencapsulated NIH3T3-mIL-12 cells was performed in the tumor-bearing mice with CT26 cells. The anti-tumor responses and the anti-tumor activities of the microencapsulated NIH3T3-mIL-12 cells were evaluated. Microencapsulated NIH3T3-mIL-12 cells could release mIL-12 continuously and stably for a long time. After the microencapsulated NIH3T3-mIL-12 cells were transplanted subcutaneously into the tumor-bearing mice for 21 d, the serum concentrations of mIL-12, mIL-2 and mIFN-gamma, the cytotoxicity of the CTL from the splenocytes and the NK activity in the treatment group were significantly higher than those in the controls. Moreover, mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells resulted in a significant inhibition of tumor proliferation and a prolonged survival of tumor-bearing mice. The microencapsulated NIH3T3-mIL-12 cells have a significant therapeutic effect on the experimental colon tumor by activating anti-tumor immune responses in vivo. Microencapsulated and genetically engineered cells may be an extremely versatile tool for tumor gene therapy.

Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T lymphocyte responses for DNA immunization.

Interleukin-12 (IL-12), consisting of p40 and p35 subunits, produces both p70 heterodimer and free p40. p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity, whereas p40 inhibits p70-mediated function. Here, we found that mutations introduced into N-glycosylation sites (N220 of murine p40 and N222 of human p40) reduced secretion of p40 but not p70. Co-immunization of N220 mutant mIL-12 gene with hepatitis C virus (HCV) E2 DNA significantly enhanced long-term E2-specific CD8+ T-cell response and protection against tumor challenge compared with that of wild type. Our results indicate that the ratio of p70 to p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an adjuvant for the generation of long-term memory T-cell responses.

Eradication of osteosarcoma lung metastases following intranasal interleukin-12 gene therapy using a nonviral polyethylenimine vector.

The use of adenoviral vectors for therapeutic delivery of genes via pulmonary application poses several problems in terms of immune responses. The purpose of this study was to determine whether polyethylenimine (PEI), a polycationic DNA carrier, can be used to deliver the IL-12 gene into the lungs of mice having microscopic osteosarcoma (OS) lung metastases. Incubation of SAOS-LM6 cells in vitro with PEI containing the murine IL-12 (mIL-12) gene (PEI:IL-12) resulted in expression of both the p35 and p40 subunits of IL-12 mRNA and production of mIL-12 protein. Using our newly developed OS nude mouse model, we demonstrated that treatment of mice using intranasal PEI:IL-12 resulted in significant IL-12 mRNA expression in the lung but not the liver. Furthermore, plasma IL-12 was undetectable after up to 4 weeks of intranasal PEI:IL-12 therapy given twice weekly. No IL-12 expression was seen following intranasal PEI therapy alone. The number of lung metastases in animals that received intranasal PEI:IL-12 twice weekly for 4 weeks starting 6 weeks after tumor inoculation was significantly decreased (median, 11; range, 0-47) compared with those that received PEI alone (median, 89; range, 2 to >200; P=.012). Also, the size of the nodules was significantly smaller in the PEI:IL-12-treated animals, with 90% measuring < or =0.5 mm in diameter compared with 56% in the PEI-alone group. Animals that received PEI alone also had numerous large nodules (3-6 mm) throughout the lungs. Intranasal therapy is a noninvasive way to administer agents and has the advantage of targeting the pulmonary region, resulting in higher concentrations in the tumor area. Additionally, delivery of IL-12 to the lung via the airway using PEI may avoid systemic toxicity. Because OS metastasizes almost exclusively to the lung, this may be a novel approach to the treatment of pulmonary OS metastases.

Enhancing antitumor by immunization with fusion of dendritic cells and engineered tumor cells.

A novel approach for a dentritic cells (DCs)-based tumor vaccine was developed for the formation of hybrid-engineered J558 after fusion with DCs. To make the hybrid-tumor vaccine generate more efficient specific CTL cytotoxicity against wild-type tumor cells, we genetically engineered tumor cells with mIL-12 gene prior to the cell fusion. mIL-12 was detected at 870 +/- 60 pg/(10(5) cells/ml) in the culture supernatants and the fusion ratio was about 30% by the co-focal microscopic analysis. Vaccination of mice with DCs fused with engineered J558 induced more efficient tumor-specific CTL cytotoxicity against wild-type tumor cells in vitro and with efficient antitumor immunity in vivo. These results suggest that this approach of using DCs fused with engineered tumor cells could be applied in clinical settings of DCs-based cancer vaccines.

Systemic administration of naked DNA encoding interleukin 12 for the treatment of human papillomavirus DNA-positive tumor.

Interleukin 12 (IL-12) is one of the most effective and promising cytokines for cancer therapy. Its therapeutic effects have been demonstrated in a variety of tumors in animal models when it is administrated locally or systemically. We describe here a systemic delivery of naked murine IL-12 (mIL-12) gene in vivo. Dose-dependent systemic production of mIL-12, with a serum level up to approximately 20 microg/ml, was observed 24 hr after systemic gene delivery. The apparent half-life in the circulation was about 5 hr. The result of a bioactivity assay (in vitro interferon gamma [IFN-gamma] release) indicated that the gene product in mice was as active as the purified recombinant murine IL-12 protein (rmIL-12). The circulating mIL-12 activated natural killer cells and stimulated IFN-gamma production in vivo. A single administration of mIL-12 gene resulted in prominent regression of established subcutaneous tumor in a human papillomavirus (HPV) DNA-positive tumor model (TC-1) in C57BL/6J mice. The antitumor effect of the single gene dose was comparable to repeated intraperitoneal administration of rmIL-12 (0.5 microg/day for consecutive 5 days). This systemic gene delivery is simple, economical, and highly efficient for the production of large amounts of cytokine in vivo. With this gene delivery method, we have demonstrated the therapeutic potential of IL-12 for the treatment of HPV DNA-positive tumor and the usefulness of the systemic gene delivery for assessing the therapeutic effect of a candidate gene.

Interleukin 12 secretion enhances antitumor efficacy of oncolytic herpes simplex viral therapy for colorectal cancer.

To assess the strategy of combining oncolytic herpes simplex virus (HSV) therapy with immunomodulatory therapy as treatment for experimental colon cancer. The oncolytic HSV recombinant NV1023 and the interleukin 12 (IL-12)-secreting oncolytic NV1042 virus were evaluated in vitro and in vivo with respect to antitumor efficacy. Genetically engineered, replication-conditional, attenuated HSVs have shown oncolytic activity against a wide variety of solid malignancies. Other strategies for treating cancer have involved immunomodulation and cytokine gene transfer using viral vectors. This study has combined both of these strategies by inserting the murine IL-12 gene into a replication-competent HSV. This approach allows oncolytic therapy to replicate selectively within and lyse tumor cells while providing the host immune system with the cytokine stimulus necessary to recruit and activate inflammatory cells needed to enhance the antitumor effect. NV1023 is a multimutant HSV based on the wild-type HSV-1 F strain. NV1042 was created by insertion of the mIL-12 gene into NV1023. Cytotoxicity and viral proliferation of both NV1023 and NV1042 within murine CT26 colorectal cancer cells were first shown. Cells infected with NV1042 were then shown to produce significant levels of IL-12. Using an experimental flank model of colon cancer, mice were treated with both high and low doses of NV1023 or NV1042 and were followed up for both cure and reduction in tumor burden. Both viruses could replicate within and kill CT26 cells in vitro, with 100% cytotoxicity achieved after infection by either virus. Only NV1042 could produce mIL-12. Therapy using high viral doses to treat animals in vivo showed equal efficacy between NV1023 and NV1042, with five of seven cures for each virus. When viral doses were lowered, only the cytokine-producing NV1042 virus could reduce tumor burden and cure animals of their disease. Both NV1023 and NV1042 have the oncolytic potential to kill colon cancer cells at higher doses. Cytokine production by NV1042 may allow lower doses of viral therapy to be used without losing antitumor efficacy. The combination of oncolytic viral therapy and immunomodulatory strategies should be further investigated as treatment for colon cancer.