Interleukin-12 Gene Therapy Research Articles

Evaluation of Toxicity following Electrically Mediated Interleukin-12 Gene Delivery in a B16 Mouse Melanoma Model

Interleukin-12 (IL-12) has potential as an immunotherapeutic agent for the treatment of cancer but is unfortunately associated with toxicity. Delivery of a plasmid encoding IL-12 with electroporation induces an antitumor effect in the B16 mouse melanoma model without serious side effects. To translate this observation to the clinic, an evaluation of toxicity was done in the mouse model. Weight change, tumor response, blood chemistry and hematology values, and serum IL-12 levels were evaluated. Multiple tissues were analyzed histopathologically. A pronounced reduction in tumor volume, including a large percentage of complete regressions, was observed after electrically mediated gene therapy. No significant increases in serum IL-12 levels were detected. Tumor-bearing mice showed an increased number of atypical hematology values when compared with normal naive controls. Statistically significant differences in chemistry and hematology values were observed sporadically in most of the standard chemistry and hematology categories in all groups. The only histopathologic abnormality specific to the animals receiving both plasmid and electroporation was inflammation associated with the kidney at the last time point. In general, mice that received both plasmid and electroporation showed the least abnormal histopathologic findings and were found to be in the best health, reflecting the reduced burden of disease. No significant toxic effects due to the IL-12 gene therapy were observed.

Stat1 Deficiency in the Host Enhances Interleukin-12–Mediated Tumor Regression

Signal transducer and activator of transcription 1 (Stat1) is considered a key transcription factor that inhibits tumorigenesis, and Stat1 activation in the host is required for interleukin-12 (IL-12)-mediated generation of CTL activity. Using syngeneic Stat1-/- C3H mice bearing SCCVII tumors in this study, we discovered opposite results. Stat1 deficiency in the host significantly enhances IL-12-mediated tumor regression, resulting in tumor eradication from 60% of SCCVII tumor-bearing mice and significant inhibition of tumor growth when compared with control treatment (P < 0.01). This effect is independent of both Stat1-activating cytokine IFN-gamma and Stat1-downstream effector molecule FasL because neither neutralization of IFN-gamma nor knocking out of FasL enhances or inhibits IL-12-mediated tumor regression. IL-12 induces a high intensity of tumor-specific CTL activity in Stat1-deficient mice (P < 0.01), increases the CD8 T-cell density in tumor bearing Stat1-/- mice, and induces a T-cell-dependent tumor regression. The increased CTL activity and the high-intensity infiltration of T cells into the tumors in IL-12-treated Stat1-/- mice are likely due to the longer survival than the same cells from wild-type mice. Together, the data show that inhibition of Stat1 expression in the host enhances tumor-local IL-12 gene therapy for regressing tumors. This conclusion provides a new concept for designing an effective treatment strategy.

622: Intravesical Interleukin-12 Gene Therapy in an Orthotopic Bladder Cancer Model

You have accessJournal of UrologyDiscussed Poster, Monday, May 22, 2006, 9:00 am - 12:00 pm1 Apr 2006622: Intravesical Interleukin-12 Gene Therapy in an Orthotopic Bladder Cancer Model Minoru Horinaga, Kelley Harsch, Ryuichi Fukuyama, Warren Heston, and William Larchian Minoru HorinagaMinoru Horinaga More articles by this author , Kelley HarschKelley Harsch More articles by this author , Ryuichi FukuyamaRyuichi Fukuyama More articles by this author , Warren HestonWarren Heston More articles by this author , and William LarchianWilliam Larchian More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)32868-4AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "622: Intravesical Interleukin-12 Gene Therapy in an Orthotopic Bladder Cancer Model." The Journal of Urology, 175(4S), pp. 200–201 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 175Issue 4SApril 2006Page: 200-201 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Minoru Horinaga More articles by this author Kelley Harsch More articles by this author Ryuichi Fukuyama More articles by this author Warren Heston More articles by this author William Larchian More articles by this author Expand All Advertisement PDF DownloadLoading ...

Chemotherapy, IL-12 gene therapy and combined adjuvant therapy of HPV 16-associated MHC class I-proficient and -deficient tumours

Moderately immunogenic HPV 16-associated murine tumour cell line mimicking human HPV 16-associated neoplasms TC-1 (MHC class I(+)) and its variants, TC-1/P3C10 and TC-1/A9, with a marked down-regulation of MHC I molecules, were used to examine the effect of local interleukin 12 (IL-12) gene therapy for the treatment of early tumour transplants and minimal residual tumour disease obtained after cytoreductive chemotherapy (CMRTD). Experiments were designed to examine whether down-regulation of MHC class I molecules plays a role during chemotherapy and gene therapy of early tumour transplants. It was found that peritumoral administration of IL-12-producing tumour cell vaccines (single dose, day 8 after tumour cell administration) inhibited the growth of both TC-1 (MHC class I positive) tumours and their MHC class I-deficient variants. To investigate the antitumour effects in a clinically relevant setting, IL-12 gene therapy was utilised for the treatment of minimal residual tumour disease after cytoreductive chemotherapy. Intra-peritoneal treatment of tumour-bearing mice with ifosfamide derivative, CBM-4A, produced a significant tumour-inhibitory effect. This treatment was followed by peritumoral s.c. administration of genetically modified TC-1 (MHC class I positive) or MK16/I/IIIABC (MHC class I negative) vaccines producing IL-12 (single dose, day 7 after chemotherapy) or with recombinant interleukin 12 (rIL-12) in two cycles of 5 daily doses (days 8-19) after chemotherapy. This combined therapy significantly inhibited the growth of TC-1 and TC-1/A9 (MHC class I-) tumours. When the combined therapy of TC-1 (MHC class I positive) tumours was followed by peritumoral administration of bone marrow dendritic cell (BMDC) vaccines, the IL-12-mediated inhibitory effect was significantly boosted. In the next set of experiments, the impacts of chemotherapy and IL-12 adjuvant therapy on MHC class I surface expression were assessed. Chemotherapy and gene therapy of tumours led to the up-regulation of MHC I expression on MHC class I-deficient tumours (TC-1/A9 and TC-1/P3C10) and to down-regulation on MHC I-proficient tumours (TC-1). These findings indicate that the MHC I phenotype is not stable during tumour progression and treatment. Collectively, these results illustrate the efficacy of IL-12 gene therapy in combination with chemotherapy on HPV-associated tumours regardless of the level of MHC class I expression on the tumour cells.

In vivo electrogene transfer of interleukin‐12 inhibits tumor growth and lymph node and lung metastases in mouse mammary carcinomas

Human breast cancer metastasizes mainly to lymph nodes, lungs, liver, and bone; in the majority of cases, it is the development of metastases which leads to death. In order to suppress mammary cancer metastasis, we applied in vivo electrogene transfer (non-viral method) as a means of interleukin-12 (IL-12) gene therapy on highly metastatic murine mammary cancer model. Metastatic mammary tumors induced by inoculation in BALB/c female mice were treated by intratumoral injections of either a plasmid vector containing IL-12 or empty vector and then subjected to in vivo electrogene transfer once a week for 8 weeks. Treatment with IL-12 resulted in elevation of both IL-12 and IFNgamma levels in mammary tumors and in serum and intratumoral levels of CD4 and CD8 proteins were also increased. Tumor volumes and lymphatic and pulmonary metastases were significantly reduced. The histopathological changes induced by IL-12 characteristically included marked inflammation, increased apoptosis, decreased DNA synthesis, peripheral influx of significantly greater numbers of active macrophages, and reduced blood microvessel density, and apoptotic vascular endothelial cells were frequently seen. Western blotting showed decreases in VEGFR-3 of tumors exposed to IL-12 gene therapy. In adjuvant immunofluorescence studies, the CD31-positive endothelial cells of microvessels showed decreased VEGFR-3 expression in IL-12-treated tumors. However, apparent alterations in VEGFR-3 expression of podoplanin-positive lymphatic endothelial cells were not observed in IL-12-treated tumors. Although recombinant IL-12 did not inhibit tubular formation of human umbilical vein endothelial cells in a Matrigel assay, recombinant IFNgamma did completely suppress the tubular formation. In vivo electrogene transfer of IL-12 exerts strong anti-tumorigenic and anti-metastatic effects likely due to T-cell-mediated immune responses as well as anti-angiogenic action.

Intravesical interleukin-12 gene therapy in an orthotopic bladder cancer model

Objectives To evaluate the antitumor effect of intravesical cationic liposome-mediated interleukin-12 (IL-12) gene delivery in an orthotopic murine bladder cancer model, and to investigate the immunologic memory against tumors between IL-12 gene therapy and bacille Calmette-Guérin (BCG) therapy. Methods Orthotopic murine bladder tumors were established by implanting 5 × 10 5 MBT-2 cells into the bladder of syngeneic female C3H mice. Intravesical IL-12 gene therapy was evaluated at varying doses: 0 μg (control) and 3, 5, and 10 μg (n = 8 for each group). Intravesical treatments were performed every 3 days and repeated six times beginning 5 days after tumor implantation. To compare the long-term, tumor-specific immunity between IL-12-treated mice (n = 18) and BCG-treated mice (n = 20), the animals surviving at day 60 and 10 new control mice were rechallenged with MBT-2 cells and received no additional treatment. On day 120, all surviving mice were killed and underwent necropsy. Results In the IL-12 groups at doses of 0, 3, 5, and 10 μg, 0, 2, 3, and 3 mice survived, respectively. Mice in the 5-μg and 10-μg IL-12 groups survived significantly longer than did the control group. All mice cured by IL-12 treatment successfully rejected the rechallenge with MBT-2 cells; however, mice cured by BCG and the new control mice died of the rechallenged bladder tumors. Conclusions Intravesical IL-12 gene therapy, which induced long-lasting tumor-specific immunologic memory compared with BCG therapy, improved survival in an orthotopic bladder cancer model.

Dendritic cells transduced with an adenovirus vector encoding interleukin-12 are a potent vaccine for invasive pulmonary aspergillosis

Invasive pulmonary aspergillosis (IPA) is a common and devastating pneumonia. We developed a novel antiinfective vaccine that couples the potent Ag-presenting capacity of dendritic cells (DCs) with paracrine delivery of interleukin-12 (IL-12) to local immune response sites. Our results showed that DCs engulfed Aspergillus conidia through coiling phagocytosis. Transfection of DCs with adenovirus encoding the cDNA of IL-12 did not affect their morphology and capacity to engulf conidia. The transduced DCs secreted IL-12, which was biologically active, to induce the production of gamma interferon (IFN-gamma) from spleen cells. Adoptive transfer of DCs pulsed with heat-inactivated Aspergillus fumigatus (HAF) to naive mice induced the Ag-specific production of IFN-gamma; the transduced HAF-pulsed DCs augmented this immune response further. Animals receiving HAF-pulsed DCs had lower fungal burdens, a more than three-fold higher survival rate at day 3. This protection was associated with a pronounced enhancement in the Aspergillus-specific IFN-gamma response. IL-12-engineered DCs augmented this protection strikingly as judged by a higher survival, and almost no Aspergillus could be detected in the lung of mice that had received IL-12-transduced HAF-pulsed DCs. These results suggest that antigen-pulsed DCs and IL-12 gene therapy could be used as adjunct therapy for aspergillosis.

P.1 Combination nonviral interleukin-2,interleukin-12 gene therapy and radiotherapy for head and neck squamous cell carcinoma

P.1 Combination nonviral interleukin-2,interleukin-12 gene therapy and radiotherapy for head and neck squamous cell carcinoma

Local, non-viral IL-12 gene therapy using a water soluble lipopolymer as carrier system combined with systemic paclitaxel for cancer treatment

Development of improved gene transfer methods is needed for gene therapy to achieve its clinical potential. The use of biocompatible polymeric gene carriers has shown effectiveness in overcoming the current problems associated with viral vectors in safety, immunogenicity and mutagenesis. Previous work has demonstrated that repeated, local, non-viral interleukin-12 (IL-12) gene delivery successfully slows down tumor progression, while improving immunogenicity. Combining IL-12 gene delivery with systemic paclitaxel (PCT) chemotherapy as a treatment for various subcutaneous mouse mammary carcinomas, we used PCT with either a biodegradable polymeric solubilizer, HySolv™ or Cremophor EL® for systemic treatment and injected water soluble lipopolymer (WSLP)/plasmid-encoding IL-12 gene (p2CMVmIL-12) complexes local once every week. The amount of lung metastases being essential for survival as well as subcutaneous tumor volume were compared against untreated controls. We showed inhibition of tumor growth and decreased lung metastases in the combined WSLP/p2CMVmIL-12/HySolv™ group compared to the controls and the PCT only treated groups. Compared to Cremophor, HySolv™ performed better alone or in combination with IL-12. Using polymeric vectors as gene carrier systems in combination with improved systemic therapies provide evidence for the efficacy and feasibility of polymer-based drug delivery systems. Especially local cytokine gene delivery showed augmentation of systemic chemotherapy while reducing the hosts risk for further systemic toxicity.

Treatment of experimental breast cancer using interleukin-12 gene therapy combined with anti–vascular endothelial growth factor receptor-2 antibody

We have shown previously that interleukin-12 (IL-12) gene therapy induced strong antitumor effects in several syngeneic murine tumor models including 4T1 mammary adenocarcinoma. Antiangiogenic treatment with a monoclonal antibody (mAb) directed against the vascular endothelial growth factor receptor-2 (VEGFR-2) is another promising treatment approach that can cause transient suppression of tumor growth. We hypothesized that the combination of IL-12 gene therapy and anti-VEGFR-2 mAb will achieve better antitumor and antimetastatic effects against 4T1 adenocarcinoma than each treatment alone via implementation of different mechanisms. Administration of anti-VEGFR-2 mAb into BALB/c mice bearing s.c. 4T1 tumors induced significant suppression of tumor growth, as did intratumoral administration of naked IL-12 DNA. The combined treatment with anti-VEGFR-2 mAb and IL-12 DNA resulted in significantly enhanced inhibition of tumor growth as compared with each treatment alone. This combination was also effective against spontaneous lung metastases. In T-cell-deficient nude mice, both IL-12 DNA and anti-VEGFR-2 mAb were effective in suppressing tumor growth. In T-cell- and natural killer cell-deficient scid/beige mice, only anti-VEGFR-2 mAb was effective, suggesting that natural killer cells are involved in the antitumor effects induced by IL-12 DNA. In both types of immunodeficient mice, the combination of anti-VEGFR-2 mAb and IL-12 DNA was as effective in suppressing 4T1 tumor growth as anti-VEGFR-2 mAb alone. Antitumor effects of anti-VEGFR-2 mAb were associated with the inhibition of angiogenesis within the tumors, whereas the antiangiogenic effect of IL-12 gene therapy was not detected. Our results show a therapeutic benefit of combining IL-12 gene therapy and anti-VEGFR-2 mAb for cancer treatment.

Antitumor and antiangiogenic effects of interleukin 12 gene therapy in murine head and neck carcinoma model

Interleukin-12 (IL-12) plays a critical role in producing an immune response, as indicated in many ways, e.g., induction of interferon-γ (IFN-γ), and augmentation of the cytotoxic activity of resting activated T cells and natural killer (NK) cells. In this study, we examined whether intratumoral injection of a recombinant retrovirus vector expressing IL-12s induce antitumor and antiangiogenic effects in a murine model using a murine head and neck squamous cell carcinoma (NR-S1). In vitro the levels of vascular endothelial growth factor (VEGF) mRNA and protein expression were decreased in IL-12 gene transfected NR-S1 cell. in vivo direct IL-12 gene therapy resulted in significantly remarkable inhibition of tumor growth compared to the control group. The tumor regression by direct IL-12 gene therapy was also associated with decreased vessel density, and apoptosis and increased infiltration of CD8 + T cells and CD56 + NK cells in the tumor increased. Also, the number of IFN-γ expressed cells of spleen cells was increased in the treatment group compared with the control group. These results suggested that direct IL-12 gene therapy appears to be effective in reducing tumor growth by triggering both antiangiogenic effects and an immunological enhancing mechanism through induction of IFN-γ.

Combination of local, nonviral IL12 gene therapy and systemic paclitaxel treatment in a metastatic breast cancer model

Repeated, local, nonviral IL12 (interleukin-12) gene delivery decreased tumor progression and increased immunogenicity. We combined our IL12 gene delivery with systemic paclitaxel chemotherapy as a treatment for paclitaxel (PCT)-resistant 4T1 subcutaneous mouse mammary carcinomas and PCT-sensitive, immunogenic/nonimmunogenic tumors. We mixed PCT with either a biodegradable polymeric solubilizer, HySolv, or Cremophor EL for bimonthly systemic treatments and injected water-soluble lipopolymer (WSLP)/p2CMVmIL-12 (plasmid encoding IL12 gene) complexes locally every week. We compared treated subcutaneous tumor volume and lung metastasis with controls. HySolv alone performed better compared to Cremophor EL in combination with WSLP/p2CMVmIL-12. We showed inhibition of 4T1 tumor growth and lung metastases in the combined WSLP/p2CMVmIL-12/HySolv group compared to the controls and the paclitaxel-only treated groups. In parallel experiments we also demonstrated additive responses for tumor growth and number of lung metastases within other PCT-sensitive mammary tumor models using this combination strategy. Our combination therapy provides evidence for the efficacy and feasibility of improved drug delivery systems. Local cytokine gene delivery can augment local and systemic chemotherapy without placing the host at risk for further systemic toxicity.

713: Urinary Interleukin-2 or -10 Levels Following Intravesical Liposome-Mediated Interleukin-2 Gene Therapy Plus BCG in Orthotopic Murine Bladder Cancer

713: Urinary Interleukin-2 or -10 Levels Following Intravesical Liposome-Mediated Interleukin-2 Gene Therapy Plus BCG in Orthotopic Murine Bladder Cancer

YW3-04 Intravesical Liposome-mediated Interleukin-2 Gene Therapy plus BCG in an Orthotopic Murine Bladder Cancer Model (Young Urologist Research Workshop)

YW3-04 Intravesical Liposome-mediated Interleukin-2 Gene Therapy plus BCG in an Orthotopic Murine Bladder Cancer Model (Young Urologist Research Workshop)

IL-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma.

Interleukin-12 (IL-12) has been evaluated in both preclinical and clinical immunotherapy protocols as a potential therapy for melanoma. However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. This study investigated the therapeutic effect of delivering a plasmid encoding IL-12 followed by electroporation on primary and secondary tumors. Three treatments of intratumoral (i.t.) plasmid injection and electroporation resulted in 80% of mice with B16.F10 melanoma tumors being tumor free for >100 days (cure). The "cured animals" were resistant to challenge with B16 cells. In a separate experiment, B16 cells were injected on the opposite flank of the treated tumor on the day of treatment. Eighty-seven percent of control mice developed a distant tumor while only 43.8% of mice receiving two or three i.t. electroporation treatments developed a distant tumor. For examination of tumor development in the lungs, mice were injected intravenously with B16.F10 cells then treated with i.m. injections of plasmid with or without electroporation. Only 37.5% of mice receiving i.m. injections and electroporation developed nodules in the lungs compared to 87.5% of mice in the no-treatment group. The results show that administration of a plasmid encoding IL-12 with electroporation has a therapeutic effect on primary tumors as well as distant tumors and metastases.

Safety of interleukin-12 gene therapy against cancer: a murine biodistribution and toxicity study.

As a prerequisite for a human clinical trial using interleukin (IL)-12 gene therapy, the biodistribution and safety of IL-12, administered as an intradermal naked DNA injection, was evaluated in mice. The pNGVL3-mIL12 plasmid used in this study is a nonviral vector designed to induce a high level of IL-12 protein expression during a transient transfection of the host cell. The biodistribution was evaluated by a polymerase chain reaction (PCR) assay that is capable of detecting less than 100 copies of the plasmid in the context of host DNA. Twenty-four hours after three intradermal injections of 0.5 microg or 5 microg of pNGVL3-mIL12 plasmid, the plasmid was detectable in various internal organs, the blood, and the injection site. The plasmid was detectable in the gonads of only one animal at the high-dose treatment 24 hr after the injections. In the majority of the organs the plasmid was undetectable throughout the study. Possible side effects were monitored by histology and clinical chemistry, and the level of IL-12 protein expression was assessed by enzyme-linked immunosorbent assay (ELISA). No treatment-related histologic abnormalities were detected and the blood chemistry parameters showed no toxicity. The IL-12 protein was undetectable at all times at the injection site and interferon (IFN)-gamma levels at the injection site and in the serum were at background levels. The results of this murine safety study indicate that based on the distribution pattern of the plasmid in the body and the undetectable toxicities in the tissues, the use of the pNGVL3-hIL12 plasmid in cancer gene therapy clinical trials can be considered as safe.

Total vascular exclusion of the liver enhances the efficacy of retroviral-mediated associated thymidine kinase and interleukin-2 genes transfer against multiple hepatic tumors in rats

Backround. Recent advances in gene transfer technology render gene therapy an attractive treatment of disseminated liver metastases for which other treatments remain disappointing. In this setting, total vascular exclusion of the liver could improve gene transfer to cancer cells and prevent extrahepatic vector spreading during portal infusion of therapeutic genes. We evaluate the efficiency of combined herpes simplex virus type-1 thymidine kinase (HSV1-TK) and interleukin-2 retrovirus-mediated gene transfer through the portal vein, under total vascular exclusion of the liver, in a model of macroscopic multiple liver metastases in rats. Methods. Multifocal liver metastases were established in BDIX rats with intraportal injection of DHDK12 colon cancer cells. On randomization, rats received either vector-producing cells or saline solution under total vascular exclusion of the liver. Vector-producing cells released retroviral vectors encoding Lac-Z in marking studies or HSV1-TK or interleukin-2 in therapeutic studies. Rats were either killed for pathologic studies, or followed for survival. Results. Total vascular exclusion of the liver markedly improved gene transfer efficacy in marking studies. In therapeutic studies we observed a significant reduction in tumor volume of treated rats compared with untreated controls (2170 ± 310 mm3). Although singular HSV1-TK or interleukin-2 gene transfer showed significant efficacy, the greatest tumor volume regression was observed in rats treated with combined HSV1-TK + interleukin-2 gene therapy (145 ± 60 mm3; P =.0001 vs control). This translated into an increased median survival rate compared with either control rats (P =.006) or rats treated with single gene therapy. Conclusion. In a rat model, a significant antitumoral effect against macroscopic multifocal liver metastases can be observed after retrovirus-mediated HSV1-TK and interleukin-2 gene transfer through the portal vein under total vascular exclusion of the liver, followed by ganciclovir administration. We believe that this well-tolerated and efficient therapeutic approach deserves clinical evaluation in patients with disseminated colorectal liver metastases. (Surgery 2003;133:669-77)

430. Urinary Interleukin-2 Levels Following Intravesical Liposome-Mediated Interleukin-2 Gene Therapy Plus BCG in Orthotopic Murine Bladder Cancer Model

430. Urinary Interleukin-2 Levels Following Intravesical Liposome-Mediated Interleukin-2 Gene Therapy Plus BCG in Orthotopic Murine Bladder Cancer Model

Interferon-alpha and interleukin-12 gene therapy of cancer: interferon-alpha induces tumor-specific immune responses while interleukin-12 stimulates non-specific killing.

Cytokine gene therapy is applied in clinical studies of tumors, and IFN-alpha and IL-12 are widely used for cancer immunotherapy. Using a poorly immunogenic murine colorectal cancer cell line, MC38, we compared antitumor effects of IFN-alpha and IL-12. Transduced MC38 cell lines expressing IFN-alpha or IL-12 (MC38-IFNalpha or MC38-IL12, respectively) were established using retroviral vectors. Transduction of IFN-alpha or IL-12 gene to MC38 cells significantly reduced tumorigenicity in immunocompetent mice. When tumor-free mice initially injected with MC38-IFNalpha or MC38-IL12 cells were reinjected contralaterally with wild-type MC38 cells (MC38-WT) after 35 days, 7 of 12 or 2 of 12 mice rejected MC38-WT cells, respectively. In therapy-model mice with established tumor derived from MC38-WT cells, inoculation of gene-transduced cells significantly suppressed growth of the tumor in MC38-IFNalpha-inoculated groups, but not in the IL-12-inoculated group. Immunohistologic and flow cytometric analyses showed marked infiltration of CD8(+) cells in wild-type tumors of mice inoculated with IFN-alpha-expressing cells. Leukocyte-depletion experiments implicated CD8(+) T cells in tumor rejection induced by IFN-alpha-transduction; both CD8(+) T cells and natural killer cells were implicated in the more modest antitumor effect from IL-12 expression. To investigate induction of tumor-specific immune responses, we stimulated splenocytes from tumor-free mice twice in vitro with genetically modified MC38 cells. In vitro stimulations with MC38-IFNalpha cells induced definite MC38-specific lysis, but not stimulations with MC38-IL-12 cells. Injecting combination of MC38-IFNalpha and MC38-IL-12 cells caused an additive antitumor effect in the therapy model. These data suggested that IFN-alpha induces cytotoxic T lymphocytes and elicits long-lasting tumor-specific immunity, whereas IL-12 seems to stimulate non-specific killing. With additional refinements, combined IFN-alpha and IL-12 gene therapy might warrant clinical trials.

Development of Anti-tumor Immunity against a Non-immunogenic Mammary Carcinoma through Somatic GM-CSF, IL-2, and HSVtk Combination Gene Therapy

Gene therapy for cancer using suicide genes such as the herpes simplex virus thymidine kinase gene (HSVtk) has been explored extensively in preclinical and clinical studies. We have improved the use of HSVtk by combining it with two cytokine genes encoding granulocyte/macrophagecolony-stimulating factor (GM-CSF) and interleukin-2 (IL-2), and determined their additive/synergistic effects on tumor regression and inhibition of metastases in the non-immunogenic, spontaneously metastatic mammary tumor model, 4T1. Two adenoviral vectors (AV) were constructed, one carrying HSVtk (AV-TK) and the second (AV-GM/IL2) carrying Gm-CSf and Il2. Only the combination of AV-TK/GCV and AV-GM/IL2 showed a significant decrease in tumor growth and reduction of distant metastases with 25% of the tumors undergoing complete regression. When surgical excision of primary tumors was included in the regimen, local treatment with AV-TK/GCV plus AV-GM/IL2 further enhanced long-term survival. A fraction of the treated mice developed anti-tumor immunity and survived a second challenge with 4T1. Functional analyses demonstrated infiltration of lymphocytes within the tumor and a strong tumor-specific cytotoxic T lymphocyte response in TK- plus cytokine-treated animals. These data indicate that the coexpression of GM-CSF and IL-2 can augment the effect of HSVtk suicide gene therapy.