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

OBJECTIVE: 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. SUMMARY BACKGROUND DATA: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Treatment of solid sarcomas in immunocompetent mice with novel, oncolytic herpes simplex viruses.

OBJECTIVE: Attenuated, replication-competent herpes simplex viruses (HSVs) have shown promise as antitumor agents for cancer therapy. In this study, we sought to develop a novel type of oncolytic HSV with more potent antitumor activity for use in localized malignant tumors. STUDY DESIGN: A new, attenuated multimutated HSV (termed HL) was developed, and then a highly metastatic murine fibrosarcoma cell line, NfSa Y83, was injected into the necks or flanks of immunocompetent C3H mice. The mice were treated with attenuated HSV mutants by intratumoral injection, and antitumor efficacy was assessed by measuring tumor dimensions and overall survival rates. RESULTS: Treatment with intratumoral injection of HL resulted in marked regression of tumors. In fact, roughly 75% of flank tumors and 50% of neck tumors were completely eradicated. CONCLUSION: A novel type of attenuated HSV recombinant HL demonstrated a remarkable antitumor efficacy in a localized tumor model in mice.     

Effective treatment of pancreatic tumors with two multimutated herpes simplex oncolytic viruses

Pancreatic cancer is an aggressive, rapidly fatal disease against which current nonsurgical therapy has minimal impact. This study evaluates the efficacy of two novel, replication-competent, multimutated herpes viruses (G207 and NV1020) in an experimental model of pancreatic cancer. Four human pancreatic carcinoma cell lines were exposed to G207 or NV1020, and cell survival and viral progeny production were determined. Flank tumors in athymic mice were subjected to single or multiple injections of 1 X 10⁷ G207 or NV1020, and tumor volume was evaluated over time. For all of the cell lines, G207 and NV1020 produced infection, viral replication, and cell lysis (P <0.05). NV1020 resulted in a higher production of viral progeny compared to G207. The efficacy of viral tumor cell kill was greatest in those cells with the shortest in vitro doubling time. For flank tumors derived from hs766t, single or multiple injections of both viruses were equally effective and significantly reduced flank tumor burden (P <0.05). Complete hs766t flank tumor eradication was achieved in 25% (5 of 20) of animals treated with G207 and 40% (8 of 20) of animals treated with NV1020. In vivo efficacy correlated with in vivo tumor doubling time. There were no adverse effects related to viral administration observed in any animal. NV1020 and G2O7 effectively infect and kill human pancreatic cancer cells in vitro and in vivo. Given the lack of effective nonoperative treatments for pancreatic cancer, oncolytic herpes viruses should be considered for clinical evaluation. Presented in part at the Forty-First Annual Meeting of The Society for Surgery of the Alimentary Tract, San Diego, Calif., May 21–24, 2000.     

Efficacy of multiagent herpes simplex virus amplicon-mediated immunotherapy as adjuvant treatment for experimental hepatic cancer

OBJECTIVE: To evaluate the use of herpes simplex viral (HSV) amplicon vectors for production of tumor vaccines and to determine if such vaccines expressing combinations of immunostimulatory agents may be effective in the treatment of experimental liver cancer. METHODS: A hepatic metastatic tumor model using CT-26 colorectal cancer in syngeneic Balb/C mice was utilized. Tumor vaccines were produced by brief (20 minutes) exposure of irradiated tumor cells to herpes amplicon vectors carrying the transgene for RANTES, B7.1, or GM-CSF. The antitumor efficacy of vaccination using tumor cells secreting GM-CSF (single agent) or a combination of RANTES/B7.1/GM-CSF (multiagent) was tested. The effect of 60% hepatectomy or T-cell depletion was also tested in this model. RESULTS: In vitro assays confirmed high-level cytokine or costimulatory molecule production by cells transduced with amplicons. Antitumor efficacy was observed with single-agent or multiagent treatment. Without hepatectomy, immunization with single-agent or multiagent vaccine therapy appears equivalent. When administered in the setting of hepatectomy, multiagent regimens produced a higher cure rate than single-agent therapy (50% vs. 12.5%, =.03). Animals treated with GM-CSF alone had an average nodule count of 40 +/- 19 ( <.006 vs. Hep control 232 +/- 30), while animals treated with multiagent therapy had an average nodule count of 11 +/- 7 ( <.0004 vs. control). CD4 and CD8 lymphocyte blockade abrogated observed efficacy, confirming a lymphocyte-mediated response. CONCLUSIONS: Tumor vaccines produced using HSV amplicon-mediated gene transfer may be useful in the treatment of liver malignancies. In the setting of hepatectomy, multiagent vaccine therapy offers an advantage over single-agent therapy. These data encourage consideration of such HSV-based neoadjuvant immunotherapy for treatment of liver malignancies.     

Herpes simplex virus based gene therapy enhances the efficacy of mitomycin C for the treatment of human bladder transitional cell carcinoma

PURPOSE: Oncolytic replication competent herpes simplex virus type-1 (HSV) mutants have the ability to replicate in and kill malignant cells. We have previously reported the ability of replication competent HSV to control bladder cancer growth in an orthotopic murine model. We hypothesized that the combination of a chemotherapeutic agent used for intravesical treatment, namely mitomycin C (MMC) (Bristol-Myers Squibb Oncology, Princeton, New Jersey), and oncolytic HSV would exert a synergistic effect for the treatment of human transitional cell carcinoma. MATERIALS AND METHODS: We used mutant HSV NV1066 (Medigene, San Diego, California), which is deleted for viral genes ICP0 and ICP4, and selectively infects cancer cells, to treat the transitional cell carcinoma lines KU19-19 and SKUB. Cell survival was determined by lactate dehydrogenase assay for each agent as well as for drug-viral combinations from days 1 to 5. The isobologram method and the combination index method of Chou-Talalay were used to assess the synergistic effect. RESULTS: NV1066 enhanced MMC mediated cytotoxicity at all combinations tested for KU19-19 and SKUB. The combination of the 2 agents demonstrated a synergistic effect and allowed dose reduction by 12 and 10.4 times (NV1066), and by 3 and 156 times (MMC) for the treatment of KU19-19 and SKUB, respectively, while achieving an estimated 90% cell kill. CONCLUSIONS: These data provide the cellular basis for the clinical investigation of combined MMC and oncolytic HSV therapy for the treatment of bladder cancer.     

Ionizing radiation potentiates the antitumor efficacy of oncolytic herpes simplex virus G207 by upregulating ribonucleotide reductase

BACKGROUND: Replication-competent herpes simplex virus-1 (HSV-1) mutants have an oncolytic effect on human and animal cancers. The aim of this study was to determine whether G207, an HSV-1 mutant, can be combined with ionizing radiation (IR) to increase antitumor activity while decreasing treatment-associated toxicity. METHODS: This study was performed by using G207, a replication-competent HSV-1 mutant deficient in viral ribonucleotide reductase (RR) and the gamma(1)34.5 neurovirulence protein. The antitumor activity of G207 or IR was tested against HCT-8 human colorectal cancer cells in vitro and in an in vivo mouse subcutaneous tumor model. RESULTS: We demonstrated that G207 has significant oncolytic effect on HCT-8 cells in vitro in a cytotoxicity assay and in vivo in a mouse flank tumor model and that these effects are improved with low-dose IR. We further illustrated that the increased tumoricidal effect is dependent on the up-regulation of cellular RR by IR measured by a functional bioassay for RR activity. Chemical inhibition of RR by hydroxyurea abrogates the enhanced effect. In contrast to G207, R3616, the parent virus of G207 that expresses functional RR, does not exhibit enhanced oncolysis when combined with IR. CONCLUSIONS: These data encourage clinical investigation of combination radiation therapy and HSV oncolytic therapy.     

Potent antitumor activity after systemic delivery of a doubly fusogenic oncolytic herpes simplex virus against metastatic prostate cancer

BACKGROUND: Although conventional radiation therapy and surgery are potentially curative treatments for organ-confined prostate cancer, there are few effective treatments for metastatic disease. Oncolytic viruses have shown considerable promise for the treatment of solid tumors including prostate cancer. We recently demonstrated that incorporation of a cell membrane fusion capability into an oncolytic herpes simplex virus (HSV) can significantly increase the antitumor potency of the virus. METHODS: We used a mouse model of primary and metastatic human prostate cancer established from PC-3M-Pro4 to evaluate three different types of oncolytic HSVs: non-fusogenic Baco-1, singly fusogenic Synco-2, and doubly fusogenic Synco-2D. RESULTS: Our results show that Synco-2D has greater oncolytic activity than either Baco-1 or Synco-2 virus. Against lung metastases of human prostate cancer xenografts, intravenous administration of Synco-2D had produced a significant reduction of tumor nodules by day 40 post-inoculation as compared with Synco-2 (P < 0.05), Baco-1 (P < 0.01), and PBS control (P < 0.01). CONCLUSIONS: We conclude that the doubly fusogenic Synco-2D is an effective therapeutic agent for human metastatic prostate cancer.     

Utilizing tumor hypoxia to enhance oncolytic viral therapy in colorectal metastases

OBJECTIVE: To determine the effects of hypoxia-induced ribonucleotide reductase (RR) production on herpes oncolytic viral therapy. SUMMARY BACKGROUND DATA: Hypoxia is a common tumor condition correlated with therapeutic resistance and metastases. Attenuated viruses offer a unique cancer treatment by specifically infecting and lysing tumor cells. G207 is an oncolytic herpes virus deficient in RR, a rate-limiting enzyme for viral replication. METHODS: A multimerized hypoxia-responsive enhancer was constructed (10xHRE) and functionally tested by luciferase assay. 10xHRE was cloned upstream of UL39, the gene encoding the large subunit of RR (10xHRE-UL39). CT26 murine colorectal cancer cells were transfected with 10xHRE-UL39, incubated in hypoxia (1% O2) or normoxia (21% O2), and infected with G207 for cytotoxicity assays. CT26 liver metastases, with or without 10xHRE-UL39, were created in syngeneic Balb/C mice (n = 40). Livers were treated with G207 or saline. Tumors were assessed and stained immunohistochemically for G207. RESULTS: 10xHRE increased luciferase expression 33-fold in hypoxia versus controls (P < 0.001). In normoxia, 10xHRE-UL39 transfection did not improve G207 cytotoxicity. In hypoxia, G207 cytotoxicity increased 87% with 10xHRE-UL39 transfection versus nontransfected cells (P < 0.001). CT26 were resistant to G207 alone. Combining 10xHRE-UL39 with G207 resulted in a 66% decrease in tumor weights (P < 0.0001) and a 65% reduction in tumor nodules (P < 0.0001) versus G207 monotherapy. 10xHRE-UL39-transfected tumors demonstrated greater viral staining. CONCLUSIONS: Hypoxia-driven RR production significantly enhances viral cytotoxicity in vitro and reduces tumor burden in vivo. G207 combined with RR under hypoxic control is a promising treatment for colorectal cancer, which would otherwise be resistant to oncolytic herpes virus alone.     

5-Fluorouracil and gemcitabine potentiate the efficacy of oncolytic herpes viral gene therapy in the treatment of pancreatic cancer

Oncolytic herpes viruses are attenuated, replication-competent viruses that selectively infect, replicate within, and lyse cancer cells and are highly efficacious in the treatment of a wide variety of experimental cancers. The current study seeks to define the pharmacologic interactions between chemotherapeutic drugs and the oncolytic herpes viral strain NV1066 in the treatment of pancreatic cancer cell lines. The human pancreatic cancer cell lines Hs 700T, PANC-1, and MIA P_aC_a-2 were treated in vitro with NV1066 at multiplicities of infection (MOI; ratio of the number of viral particles per tumor cell) ranging from 0.01 to 1.0 with or without 5-fluorouracil (5-FU) or gemcitabine. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Viral replication was measured using a standard plaque assay. Six days after combination therapy, 76% of Hs 700T cells were killed compared with 43% with NV1066 infection alone (MOI = 0.1) or 0% with 5-FU alone (2 βmol/L) (P < .01). Isobologram and combination-index analyses confirmed a strongly synergistic pharmacologic interaction between the agents at all viral and drug combinations tested (LD5 to LD95) in the three cell lines. Dose reductions up to 6- and 78-fold may be achieved with combination therapy for NV1066 and 5-FU, respectively, without compromising cell kill. 5-FU increased viral replication up to 19-fold compared with cells treated with virus alone. Similar results were observed by combining gemcitabine and NV1066. We have demonstrated that 5-FU and gemcitabine potentiate oncolytic herpes viral replication and cytotoxicity across a range of clinically achievable doses in the treatment of human pancreatic cancer cell lines. The potential clinical implications of this synergistic interaction include improvements in efficacy, treatment-associated toxicity, tolerability of therapeutic regimens, and quality of life. These data provide the cellular basis for the clinical investigation of combined oncolytic herpes virus therapy and chemotherapy in the treatment of pancreatic cancer. Presented at the Forty-Sixth Annual Meeting of The Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 141-18, 2005 (oral presentation). Supported in part by training grant T 32 CA09501 (D.P.E. and K.J.H.), AACR-AstraZeneca Cancer Research and Prevention fellowship (P.S.A), grants RO1 CA 76416 and RO1 CA/DK80982 (Y.F.) from the National Institutes of Health, grant BC024118 from the U.S. Army (Y.F.), grant IMG0402501 from the Susan G. Komen Foundation (Y.F.), and grant 032047 from Flight Attendant Medical Research Institute (Y.F.).     

Cisplatin augments cytotoxic T-lymphocyte-mediated antitumor immunity in poorly immunogenic murine lung cancer

OBJECTIVE: Many tumors are poorly immunogenic and resistant to cytotoxic T-lymphocyte-mediated cell lysis. Because cisplatin has been demonstrated to increase tumor cell Fas receptor expression, we hypothesized that cisplatin will enhance cytotoxic T-lymphocyte tumor cell killing and augment the antitumor effect of an active immunotherapy strategy in a poorly immunogenic murine lung cancer model. METHODS: Lewis lung carcinoma cells were exposed to cisplatin in vitro, and Fas receptor expression and apoptosis in response to an agonistic anti-Fas antibody were quantified using flow cytometry. Wild-type and Fas ligand-deficient mice bearing Lewis lung carcinoma flank tumors were then treated with intraperitoneal cisplatin as well as an intratumoral injection of an adenovirus gene transfer vector encoding CD40 ligand. End points included tumor size, animal survival, and Fas expression (determined using immunofluorescence). Cytotoxicity assays were performed using splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated animals as effectors and cisplatin-treated Lewis lung carcinoma cells as targets. RESULTS: Cisplatin induced heightened expression of Fas receptor on Lewis lung carcinoma cells in vitro and in vivo and enhanced apoptosis in cells exposed to an agonistic anti-Fas antibody. In vivo, the combination of 1 dose of intraperitoneal cisplatin and intratumoral adenovirus gene transfer vector encoding CD40 ligand inhibited tumor growth and prolonged survival compared with adenovirus gene transfer vector encoding CD40 ligand alone, resulting in a higher cure rate. This effect was lost in Fas ligand-deficient mice. Splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated wild-type mice lysed cisplatin-treated Lewis lung carcinoma cells more efficiently than untreated Lewis lung carcinoma cells, an effect lost in splenocytes from Fas ligand-deficient mice. CONCLUSION: Cisplatin augments the antitumor effect of a cytotoxic T-lymphocyte-mediated immunotherapy strategy, resulting in a higher cure rate than seen with immunotherapy alone. This effect is associated with the enhanced ability of cytotoxic T lymphocytes to lyse tumor cells that have been exposed to cisplatin through Fas/Fas ligand interactions.     

Intravesikale Therapie nicht muskelinvasiver Blasentumoren mit onkolytischen Vesikular-Stomatitisviren

Patients with high-risk bladder cancer who do not respond to bacillus Calmette-Guerin (BCG) immunotherapy represent a significant therapeutic challenge. The addition of interferon to BCG has recently evolved as a second-line treatment option; however, many high-grade tumors are nonresponsive to interferon. Thus, replication-competent oncolytic vesicular stomatitis viruses (VSV) that selectively target interferon-refractory tumors are promising intravesical agents. In vitro, wild-type VSV as well as a mutant variant (AV3) that has an impaired ability to shut down innate immunity preferentially killed undifferentiated, interferon-nonresponsive bladder cancer cells. Testing of these viruses in an orthotopic murine model of high-grade bladder cancer, which we have recently validated, revealed that both AV3 and wild-type VSV significantly inhibited orthotopic tumor growth. Despite the use of immunocompromised nude mice, there was no evidence of toxicity. In conclusion, VSV instillation therapy demonstrated strong antitumor activity and safety in an orthotopic model of high-risk disease. These findings provide preclinical proof-of-principle for the intravesical use of VSV, especially in interferon-refractory patients.     

The replication-competent oncolytic herpes simplex mutant virus NV1066 is effective in the treatment of esophageal cancer

BACKGROUND: The oncolytic herpes simplex-1 virus, NV1066, is a replication-competent virus that has been engineered to infect and lyse tumor cells selectively and to carry a transgene for enhanced green fluorescent protein (EGFP). The purpose of this study was to determine viral cytotoxicity in an esophageal cancer cell line and to determine whether EGFP expression could be used as a marker of viral infection. METHODS: BE3 esophageal adenocarcinoma cells were infected with NV1066 in vitro to determine cell kill and viral replication. EGFP expression was assessed by flow cytometry. The in vivo anti-tumor activity of NV1066 was tested in subcutaneous and intraperitoneal xenograft models. EGFP expression was localized in vivo by fluorescent microscopy and fluorescent laparoscopy. RESULTS: NV1066 effectively replicated within and killed BE3 cells in vitro and in vivo. EGFP expression identified infected tumor cells. After NV1066 treatment in vivo, EGFP expression localized to the tumor. In an intraperitoneal tumor model, EGFP could be visualized endoscopically using a laparoscope with a fluorescent filter. CONCLUSIONS: NV1066 has oncolytic activity against the BE3 cell line and may be a useful therapy against esophageal cancer. EGFP expression localizes the virus and may help to identify tumor deposits in vivo. Oncolytic activity with NV1066 against gastrointestinal cancers may potentially be tracked by endoscopy.     

Adoptive transfer of TRAIL-expressing natural killer cells prevents recurrence of hepatocellular carcinoma after partial hepatectomy

BACKGROUND: Antitumor activity of the liver natural killer (NK) cells reportedly decreases after partial hepatectomy, suggesting that patients with such depressed immune status are susceptible to the recurrence of hepatocellular carcinoma (HCC). We hypothesize that adoptive immunotherapy using activated NK cells can be a novel strategy to improve the depressed immune status in patients with HCC after hepatectomy or partial liver transplantation. In the present study, we have tested this hypothesis by using a mouse model. METHODS: Intraportal injection of 1-5 x 10(6) Hepa1-6 cells (hepatoma cell line) did not result in liver metastases in untreated B6 mice, but led to the growth of liver metastases after extensive partial hepatectomy. Utilizing this murine HCC metastasis model, we investigated the antitumor activity of both remnant liver and exogenously transferred NK cells. RESULTS: The anti-HCC activity of liver NK cells significantly decreased after partial hepatectomy. The expression of CD69 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on liver NK cells was temporarily downregulated. The adoptive transfer of NK cells, including a TRAIL-expressing fraction, extracted from the liver perfusates of poly I:C-stimulated B6 mice inhibited the growth of liver metastasis in B6 or (B6xBALB/c) F1 (B6CF1) mice that underwent hepatectomy and received intraportal Hepa1-6 injection. CONCLUSIONS: These findings indicate that adoptive immunotherapy using activated NK cells extracted from normal liver perfusates may be a novel technique for reconstituting the depressed immune status in cases of living donor liver transplantation involving HCC patients, recipients of a partial liver graft.     

Oncolytic viral gene therapy for prostate cancer using two attenuated,replication-competent,genetically engineered herpes simplex viruses

BACKGROUND: Attenuated, replication-competent herpes simplex virus mutants offer an exciting new modality in cancer therapy through their ability to selectively replicate within and kill malignant cells with minimal harm to normal tissues. METHODS: This study investigates the efficacy of two such viruses, G207 and NV1020, in human prostatic carcinoma. In vitro studies were performed on four human prostatic carcinoma cell lines, and in vivo single/multiple dose studies were undertaken on mice by using two human cell types. Tumor volume, histopathology at necropsy, and serum prostate specific antigen (PSA) were used as measures of antiproliferative effect in the in vivo experiments. RESULTS: Both viruses were effective in producing cytolytic effects in vitro at various multiplicities of infection in all cell lines tested. Both viruses demonstrated antitumor effects in vivo with a statistically significant decrease in serum PSA and inhibition of growth of both PC-3 and C4-2 subcutaneous xenografts. Tumor-free animals at necropsy were observed in the treated groups but not in control animals. CONCLUSION: These results display impressive activity against human prostate cancer and offer promise for the use of this modality in the future.     

Inhibition of Host Signal Transducer and Activator of Transcription Factor 6 Results in Cure With Cyclophosphamide and Interleukin 12 Immunotherapy

BACKGROUND: Interleukin (IL)-12 immunotherapy is highly effective against established immunogenic tumors. However, nonimmunogenic tumors fail to respond to IL-12 therapy. Analysis of tumor rejection of the immunogenic tumors shows that a preexisting antitumor immune response is required for an effective IL-12 response. It is not known whether this lack of a preexisting host antitumor immune response is a limiting factor for the lack of response to IL-12 therapy by nonimmunogenic tumors. METHODS: Experiments were done using the spontaneously arising nonimmunogenic metastatic murine breast 4T1 carcinoma in normal and STAT6 knockout BALB/c mice. RESULTS: 4T1 is nonimmunogenic in normal mice, and established subcutaneous tumors are resistant to immunotherapy with cyclophosphamide (Cy) plus IL-12. However, in STAT6 knockout mice, 4T1 becomes immunogenic, and established 4T1 tumors are eradicated by Cy plus IL-12. Adoptive transfer of spleen cells from normal mice into STAT6 knockout mice before tumor inoculation reduces both the immunogenicity and response to Cy plus IL-12 immunotherapy of 4T1 in the recipient mice. CONCLUSIONS: Cy plus IL-12 immunotherapy can eradicate nonimmunogenic tumors as long as a preexisting immunity is established in the tumor-bearing host. Furthermore, the STAT6 pathway is likely involved in the suppression of the development of host antitumor immunity.     

表达白细胞介素-18的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

目的 观察表达IL-18的溶瘤腺病毒(ZD55-IL18)对裸鼠肾癌移植瘤生长及血管形成的抑制作用.方法 荷肾癌裸鼠随机分4组,每组8只.瘤体内注射ZD55-IL18、溶瘤腺病毒ZD55-EGFP、表达IL-18的增殖缺陷腺病毒Ad-IL18及PBS,每次注射病毒7×108PFU/只,连续注射3 d.注射后第7天,每组处死3只取肿瘤组织,免疫组织化学检测肿瘤E1A、IL-18、CD34、VEGF表达及凋亡.第50天时处死动物测量肿瘤体积.结果 ZD55-IL18、ZD55-EGFP、Ad-IL18及生理盐水处理组肿瘤体积(mm3)分别为:299.7±52.9、536.8±90.3、570.3±99.0、766.1±145.8,ZD55-IL18与各组之间差异有统计学意义(P<0.01).Ad-IL18处理组肿瘤无E1A蛋白表达,ZD55-IL18处理组有大量E1A蛋白表达,表明病毒复制.ZD55-IL18处理组肿瘤IL-18表达及凋亡细胞阳性率均显著高于Ad-IL18处理组.ZD55-IL18处理组肿瘤CD34、VEGF表达均显著低于Ad-IL18处理组.结论 表达IL-18的溶瘤腺病毒ZD55-IL18比增殖缺陷腺病毒Ad-IL18具有更强的抑制肾癌生长及血管形成作用.     

Administration of interleukin-12 and -18 enhancing the antitumor immunity of genetically modified dendritic cells that had been pulsed with Semliki forest virus-mediated tumor complementary DNA

OBJECT: Immunogene therapy for malignant gliomas was further investigated in this study to improve its therapeutic efficacy. METHODS: Dendritic cells (DCs) were isolated from bone marrow and pulsed with phosphate-buffered saline or Semliki Forest virus (SFV)-mediated 203 glioma complementary (c)DNA with or without systemic administration of interleukin (IL)-12 and IL-18 to treat mice bearing the 203 glioma. To study the immune mechanisms involved in tumor regression, the authors investigated tumor growth of an implanted 203 glioma model in T cell subset-depleted mice and in interferon (IFN) gamma-neutralized mice. To examine the protective immunity produced by tumor inoculation, a repeated challenge of 203 glioma cells was given by injecting the cells into the left thighs of surviving mice and the growth of these cells was monitored. The authors demonstrated that the combined administration of SFV-cDNA, IL-12, and IL-18 produced significant antitumor effects against the growth of murine glioma cells in vivo and also can induce specific antitumor immunity. The synergic effects of the combination of SFV-cDNA, IL-12, and IL-18 in vivo were also observed to coincide with markedly augmented IFN-gamma production. The antitumor effects of this combined therapy are mediated by CD4+ and CD8+ T cells and by NK cells. These results indicate that the use of IL-18 and IL-12 in DC-based immunotherapy for malignant glioma is beneficial. CONCLUSIONS: Immunogene therapy combined with DC therapy, IL-12, and IL-18 may be an excellent candidate in the development of a new treatment protocol. The self-replicating SFV system may therefore provide a novel approach for the treatment of malignant gliomas.     

肿瘤浸润性淋巴细胞在白细胞介素-2、-4协同下局部过继免疫治疗膀胱癌的实验研究

目的　观察膀胱癌肿瘤浸润性淋巴细胞 (TIL)联合不同细胞因子瘤灶内过继免疫抗癌的效应及对机体全身抗肿瘤免疫机制的影响。方法　建立BTT73 9动物模型 ,分离、培养TIL。采用正交设计实验方法 ,将TIL、白细胞介素 (IL) 2、 4及三因素交互组合悬液分别直接注射至瘤体内 ,定期测量肿瘤体积 ,免疫治疗 2周后检测NK细胞活性、T淋巴细胞转刺激指数 ,观察组织学及超微结构变化。结果　比较对照组 ,治疗 2周时各TIL相关组均不同程度抑制了膀胱肿瘤体积的增长 ,且NK细胞活性及T淋巴细胞转化增殖能力得以提高 (P <0 .0 5 )。TIL/IL 2疗法明显抑制了瘤体的增长 ,免疫治疗 1周后即表现出协同增强效应 (P <0 .0 5 ) ,而NK细胞活性及T淋巴细胞转刺激指数也显著提高 (P <0 .0 5 )。TIL/IL 2 /IL 4组获得了较强的抗癌功效 ,但与TIL/IL 2组差异无显著性 (P >0 .0 5 )。超微结构变化显示出TIL强烈的溶癌现象。结论　TIL在细胞因子特别是IL 2协同下瘤灶内注射的局部免疫疗法 ,具有较强的抗膀胱癌效应 ,并显著提高了机体全身抗瘤免疫功能。     

Oncolytic herpes viral therapy is effective in the treatment of hepatocellular carcinoma cell lines

The rising incidence of hepatocellular carcinoma (HCC) in western countries, along with the poor prognosis offered by present-day treatment modalities, makes novel therapies for this disease necessary. Oncolytic herpes simplex viruses (HSV) are replication-competent viruses that are highly effective in the treatment of a wide variety of experimental models of human malignancies. This study seeks to investigate the effectiveness of oncolytic herpes viruses in the treatment of primary HCC cell lines. Sixteen commercially available human HCC cell lines were studied. G207 is an attenuated, replication-competent, oncolytic HSV engineered to selectively replicate within cancer cells. Cell lines were tested for viral sensitivity to G207 and their ability to support viral replication using standard cytotoxicity and viral replication assays. Eleven of 16 cell lines were moderately to highly sensitive to G207 viral oncolysis. HCC cell lines additionally demonstrated the ability to support viral replication in vitro with as high as 800-fold amplification of the administered viral dose observed. G207 is cytotoxic to, and efficiently replicates within, HCC cell lines in vitro. From these data, we suggest that oncolytic HSV therapy may have a role in the treatment of HCC, and in vivo studies are warranted. Presented in part at the 2005 American Hepato-Pancreato-Biliary Association Congress, Hollywood, Florida, April 14–17, 2005. Supported by grants R01CA75461 and R01CA72632 from the National Institutes of Health, and by grant MBC-99366 from the American Cancer Society (Yuman Fong).     

Current status of clinical trials assessing oncolytic virus therapy for urological cancers

Oncolytic virus therapy has recently been recognized as a promising new option for cancer treatment. Oncolytic viruses replicate selectively in cancer cells, thus killing them without harming normal cells. Notably, T‐VEC (talimogene laherparepvec, formerly called OncoVEX^GM‐CSF), an oncolytic herpes simplex virus type 1, was approved by the US Food and Drug Administration for the treatment of inoperable melanoma in October 2015, and was subsequently approved in Europe and Australia in 2016. The efficacies of many types of oncolytic viruses against urological cancers have been investigated in preclinical studies during the past decade, and some have already been tested in clinical trials. For example, a phase I trial of the third‐generation oncolytic Herpes simplex virus type 1, G47Δ, in patients with prostate cancer was completed in 2016. We summarize the current status of clinical trials of oncolytic virus therapy in patients with the three major urological cancers: prostate, bladder and renal cell cancers. In addition to Herpes simplex virus type 1, adenoviruses, reoviruses, vaccinia virus, Sendai virus and Newcastle disease virus have also been used as parental viruses in these trials. We believe that oncolytic virus therapy is likely to become an important and major treatment option for urological cancers in the near future.