Clinical trial links oncolytic immunoactivation to survival in glioblastoma.

Immunotherapy failures can result from the highly suppressive tumour microenvironment that characterizes aggressive forms of cancer such as recurrent glioblastoma (rGBM)1,2. Here we report the results of a first-in-human phase I trial in 41 patients with rGBM who were injected with CAN-3110-an oncolytic herpes virus (oHSV)3. In contrast to other clinical oHSVs, CAN-3110 retains the viral neurovirulence ICP34.5 gene transcribed by a nestin promoter; nestin is overexpressed in GBM and other invasive tumours, but not in the adult brain or healthy differentiated tissue4. These modifications confer CAN-3110 with preferential tumour replication. No dose-limiting toxicities were encountered. Positive HSV1 serology was significantly associated with both improved survival and clearance of CAN-3110 from injected tumours. Survival after treatment, particularly in individuals seropositive for HSV1, was significantly associated with (1) changes in tumour/PBMC T cell counts and clonal diversity, (2) peripheral expansion/contraction of specific T cell clonotypes; and (3) tumour transcriptomic signatures of immune activation. These results provide human validation that intralesional oHSV treatment enhances anticancer immune responses even in immunosuppressive tumour microenvironments, particularly in individuals with cognate serology to the injected virus. This provides a biological rationale for use of this oncolytic modality in cancers that are otherwise unresponsive to immunotherapy (ClinicalTrials.gov: NCT03152318 ).

Perturbing DDR signaling enhances cytotoxic effects of local oncolytic virotherapy and modulates the immune environment in glioma.

CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409 in vitro using cytotoxicity, cytokine, and fluorescence-activated cell sorting (FACS) assays in glioma cell lines and in vivo with an orthotopic syngeneic murine glioma model. Tumor immune infiltrates were analyzed by cytometry by time of flight (CyTOF). In vitro, we observed a significant increase in the DNA-damage marker γH2AX and decreased expression of PD-L1, pro-tumorigenic cytokines (interleukin-1ß [IL-1ß], IL-4), and ligand NKG2D after combination treatment compared with monotherapy or control. In vivo, long-term survival was increased after combination treatment (66.7%) compared with CAN-2409 (50%) and control. In a tumor re-challenge, long-term immunity after combination treatment was not improved. Our results suggest that ATR inhibition could amplify CAN-2409's efficacy in glioblastoma through increased DNA damage while having complex immunological ramifications, warranting further studies to determine the ideal conditions for maximized therapeutic benefit.

Leveraging external data in the design and analysis of clinical trials in neuro-oncology.

Integration of external control data, with patient-level information, in clinical trials has the potential to accelerate the development of new treatments in neuro-oncology by contextualising single-arm studies and improving decision making (eg, early stopping decisions). Based on a series of presentations at the 2020 Clinical Trials Think Tank hosted by the Society of Neuro-Oncology, we provide an overview on the use of external control data representative of the standard of care in the design and analysis of clinical trials. High-quality patient-level records, rigorous methods, and validation analyses are necessary to effectively leverage external data. We review study designs, statistical methods, risks, and potential distortions in using external data from completed trials and real-world data, as well as data sources, data sharing models, ongoing work, and applications in glioblastoma.

Neoadjuvant Gene-Mediated Cytotoxic Immunotherapy for Non-Small-Cell Lung Cancer: Safety and Immunologic Activity.

Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 1012 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8+ T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8+ T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8+ T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies.

Phase I study of gene-mediated cytotoxic immunotherapy with AdV-tk as adjuvant to surgery and radiation for pediatric malignant glioma and recurrent ependymoma.

BACKGROUND: Gene-mediated cytotoxic immunotherapy (GMCI) is a tumor-specific immune stimulatory strategy implemented through local delivery of aglatimagene besadenovec (AdV-tk) followed by anti-herpetic prodrug. GMCI induces T-cell dependent tumor immunity and synergizes with radiotherapy. Clinical trials in adult malignant gliomas demonstrated safety and potential efficacy. This is the first trial of GMCI in pediatric brain tumors. METHODS: This phase I dose escalation study was conducted to evaluate GMCI in patients 3 years of age or older with malignant glioma or recurrent ependymoma. AdV-tk at doses of 1 × 1011 and 3 × 1011 vector particles (vp) was injected into the tumor bed at the time of surgery followed by 14 days of valacyclovir. Radiation started within 8 days of surgery, and if indicated, chemotherapy began after completion of valacyclovir. RESULTS: Eight patients (6 glioblastoma, 1 anaplastic astrocytoma, 1 recurrent ependymoma) were enrolled and completed therapy: 3 on dose level 1 and 5 on dose level 2. Median age was 12.5 years (range 7-17) and Lansky/Karnofsky performance scores were 60-100. Five patients had multifocal/extensive tumors that could not be resected completely and 3 had gross total resection. There were no dose-limiting toxicities. The most common possibly GMCI-related adverse events included Common Terminology Criteria for Adverse Events grade 1-2 fever, fatigue, and nausea/vomiting. Three patients, in dose level 2, lived more than 24 months, with 2 alive without progression 37.3 and 47.7 months after AdV-tk injection. CONCLUSIONS: GMCI can be safely combined with radiation therapy with or without temozolomide in pediatric patients with brain tumors and the present results strongly support further investigation. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov NCT00634231.

Phase I Study of Intrapleural Gene-Mediated Cytotoxic Immunotherapy in Patients with Malignant Pleural Effusion.

Gene-mediated cytotoxic immunotherapy (GMCI) is an immune strategy implemented through local delivery of an adenovirus-based vector expressing the thymidine kinase gene (aglatimagene besadenovec, AdV-tk) followed by anti-herpetic prodrug valacyclovir. A phase I dose escalation trial of GMCI followed by chemotherapy was conducted in patients with malignant pleural effusion (MPE). AdV-tk was administered intrapleurally (IP) in three cohorts at a dose of 1 × 1012 to 1013 vector particles. Primary endpoint was safety; secondary endpoints included response rate, progression-free survival, and overall survival. Nineteen patients were enrolled: median age 67 years; 14 with malignant mesothelioma, 4 non-small-cell lung cancer (NSCLC), and 1 breast cancer. There were no dose limiting toxicities. All 3 patients in cohort 2 experienced transient cytokine release syndrome (CRS). Addition of celecoxib in cohort 3 reduced the incidence and severity of CRS (none > grade 2). Three patients are alive (23-33 months after GMCI), and 3 of 4 NSCLC patients had prolonged disease stabilization; one is alive 29 months after GMCI, 3.6 years after initial diagnosis. GMCI was safe and well tolerated in combination with chemotherapy in patients with MPE and showed encouraging response. Further studies are warranted to determine efficacy.

Preclinical investigation of combined gene-mediated cytotoxic immunotherapy and immune checkpoint blockade in glioblastoma.

Background: Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods: The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results: GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions: Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.

Immunotherapy and gene therapy as novel treatments for cancer.

The immune system interacts closely with tumors during the disease development and progression to metastasis. The complex communication between the immune system and the tumor cells can prevent or promote tumor growth. New therapeutic approaches harnessing protective immunological mechanisms have recently shown very promising results. This is performed by blocking inhibitory signals or by activating immunological effector cells directly. Immune checkpoint blockade with monoclonal antibodies directed against the inhibitory immune receptors CTLA-4 and PD-1 has emerged as a successful treatment approach for patients with advanced melanoma. Ipilimumab is an anti-CTLA-4 antibody which demonstrated good results when administered to patients with melanoma. Gene therapy has also shown promising results in clinical trials. Particularly, Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (TK) gene to tumor cells in combination with ganciclovir (GCV) may provide an effective suicide gene therapy for destruction of glioblastomas, prostate tumors and other neoplasias by recruiting tumor-infiltrating lymphocytes into the tumor. The development of new treatment strategies or combination of available innovative therapies to improve cell cytotoxic T lymphocytes trafficking into the tumor mass and the production of inhibitory molecules blocking tumor tissue immune-tolerance are crucial to improve the efficacy of cancer therapy.

El sistema inmune interactúa íntimamente con los tumores durante el proceso del desarrollo de la enfermedad y su progresión a metástasis. Esta compleja comunicación entre el sistema inmune y las células tumorales puede prevenir o promover el crecimiento del tumor. Los nuevos enfoques terapéuticos que aprovechan los mecanismos inmunológicos, ya sea por el bloqueo de señales inhibitorias o por la activación directa de células efectoras, han mostrado resultados prometedores. El bloqueo de puntos de control inmunológicos immune-checkpoints con anticuerpos monoclonales dirigidos contra receptores que normalmente inhiben el sistema inmune, como CTLA-4 o PD-1, ha resultado ser un tratamiento exitoso para pacientes con melanoma avanzado. El fármaco ipilimumab es un anticuerpo anti-CTLA-4 que ha demostrado buenos resultados terapéuticos en pacientes con melanoma. Por otro lado, la terapia génica también ha mostrado resultados prometedores en ensayos clínicos. En especial, la administración de la enzima timidina quinasa del virus Herpes simplex (HSV-TK) en combinación con el fármaco ganciclovir (GCV) ha mostrado ser una terapia suicida muy efectiva para la destrucción de diferentes neoplasias incluyendo glioblastomas y tumores prostáticos, por un mecanismo que involucra el reclutamiento de linfocitos infiltrantes de tumor. Es importante la búsqueda de nuevas estrategias o la combinación de terapias innovadoras, con el fin de involucrar tanto la atracción de linfocitos citotóxicos así como el empleo de moléculas que inhiban la inmunotolerancia del tejido tumoral para mejorar la eficiencia de los tratamientos contra el cáncer.

Immunotherapy and gene therapy as novel treatments for cancer / Inmunoterapia y terapia génica como nuevos tratamientos contra el cáncer

Abstract The immune system interacts closely with tumors during the disease development and progression to metastasis. The complex communication between the immune system and the tumor cells can prevent or promote tumor growth. New therapeutic approaches harnessing protective immunological mechanisms have recently shown very promising results. This is performed by blocking inhibitory signals or by activating immunological effector cells directly. Immune checkpoint blockade with monoclonal antibodies directed against the inhibitory immune receptors CTLA-4 and PD-1 has emerged as a successful treatment approach for patients with advanced melanoma. Ipilimumab is an anti-CTLA-4 antibody which demonstrated good results when administered to patients with melanoma. Gene therapy has also shown promising results in clinical trials. Particularly, Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (TK) gene to tumor cells in combination with ganciclovir (GCV) may provide an effective suicide gene therapy for destruction of glioblastomas, prostate tumors and other neoplasias by recruiting tumor-infiltrating lymphocytes into the tumor. The development of new treatment strategies or combination of available innovative therapies to improve cell cytotoxic T lymphocytes trafficking into the tumor mass and the production of inhibitory molecules blocking tumor tissue immune-tolerance are crucial to improve the efficacy of cancer therapy.

Resumen El sistema inmune interactúa íntimamente con los tumores durante el proceso del desarrollo de la enfermedad y su progresión a metástasis. Esta compleja comunicación entre el sistema inmune y las células tumorales puede prevenir o promover el crecimiento del tumor. Los nuevos enfoques terapéuticos que aprovechan los mecanismos inmunológicos, ya sea por el bloqueo de señales inhibitorias o por la activación directa de células efectoras, han mostrado resultados prometedores. El bloqueo de puntos de control inmunológicos (immune-checkpoints) con anticuerpos monoclonales dirigidos contra receptores que normalmente inhiben el sistema inmune, como CTLA-4 o PD-1, ha resultado ser un tratamiento exitoso para pacientes con melanoma avanzado. El fármaco ipilimumab es un anticuerpo anti-CTLA-4 que ha demostrado buenos resultados terapéuticos en pacientes con melanoma. Por otro lado, la terapia génica también ha mostrado resultados prometedores en ensayos clínicos. En especial, la administración de la enzima timidina quinasa del virus Herpes simplex (HSV-TK) en combinación con el fármaco ganciclovir (GCV) ha mostrado ser una terapia suicida muy efectiva para la destrucción de diferentes neoplasias incluyendo glioblastomas y tumores prostáticos, por un mecanismo que involucra el reclutamiento de linfocitos infiltrantes de tumor. Es importante la búsqueda de nuevas estrategias o la combinación de terapias innovadoras, con el fin de involucrar tanto la atracción de linfocitos citotóxicos así como el empleo de moléculas que inhiban la inmunotolerancia del tejido tumoral para mejorar la eficiencia de los tratamientos contra el cáncer.

Phase II multicenter study of gene-mediated cytotoxic immunotherapy as adjuvant to surgical resection for newly diagnosed malignant glioma.

BACKGROUND: Despite aggressive standard of care (SOC) treatment, survival of malignant gliomas remains very poor. This Phase II, prospective, matched controlled, multicenter trial was conducted to assess the safety and efficacy of aglatimagene besadenovec (AdV-tk) plus valacyclovir (gene-mediated cytotoxic immunotherapy [GMCI]) in combination with SOC for newly diagnosed malignant glioma patients. METHODS: Treatment cohort patients received SOC + GMCI and were enrolled at 4 institutions from 2006 to 2010. The preplanned, matched-control cohort included all concurrent patients meeting protocol criteria and SOC at a fifth institution. AdV-tk was administered at surgery followed by SOC radiation and temozolomide. Subset analyses were preplanned, based on prognostic factors: pathological diagnosis (glioblastoma vs others) and extent of resection. RESULTS: Forty-eight patients completed SOC + GMCI, and 134 met control cohort criteria. Median overall survival (OS) was 17.1 months for GMCI + SOC versus 13.5 months for SOC alone (P = .0417). Survival at 1, 2, and 3 years was 67%, 35%, and 19% versus 57%, 22%, and 8%, respectively. The greatest benefit was observed in gross total resection patients: median OS of 25 versus 16.9 months (P = .0492); 1, 2, and 3-year survival of 90%, 53%, and 32% versus 64%, 28% and 6%, respectively. There were no dose-limiting toxicities; fever, fatigue, and headache were the most common GMCI-related symptoms. CONCLUSIONS: GMCI can be safely combined with SOC in newly diagnosed malignant gliomas. Survival outcomes were most notably improved in patients with minimal residual disease after gross total resection. These data should help guide future immunotherapy studies and strongly support further evaluation of GMCI for malignant gliomas. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov NCT00589875.

Gene-mediated cytotoxic immunotherapy as adjuvant to surgery or chemoradiation for pancreatic adenocarcinoma.

BACKGROUND: While surgical resection of pancreatic adenocarcinoma provides the only chance of cure, long-term survival remains poor. Immunotherapy may improve outcomes, especially as adjuvant to local therapies. Gene-mediated cytotoxic immunotherapy (GMCI) generates a systemic anti-tumor response through local delivery of an adenoviral vector expressing the HSV-tk gene (aglatimagene besadenovec, AdV-tk) followed by anti-herpetic prodrug. GMCI has demonstrated synergy with standard of care (SOC) in other tumor types. This is the first application in pancreatic cancer. METHODS: Four dose levels (3 × 10(10) to 1 × 10(12) vector particles) were evaluated as adjuvant to surgery for resectable disease (Arm A) or to 5-FU chemoradiation for locally advanced disease (Arm B). Each patient received two cycles of AdV-tk + prodrug. RESULTS: Twenty-four patients completed therapy, 12 per arm, with no dose-limiting toxicities. All Arm A patients were explored, eight were resected, one was locally advanced and three had distant metastases. CD8(+) T cell infiltration increased an average of 22-fold (range sixfold to 75-fold) compared with baseline (p = 0.0021). PD-L1 expression increased in 5/7 samples analyzed. One node-positive resected patient is alive >66 months without recurrence. Arm B RECIST response rate was 25 % with a median OS of 12 months and 1-year survival of 50 %. Patient-reported quality of life showed no evidence of deterioration. CONCLUSIONS: AdV-tk can be safely combined with pancreatic cancer SOC without added toxicity. Response and survival compare favorably to expected outcomes and immune activity increased. These results support further evaluation of GMCI with more modern chemoradiation and surgery as well as PD-1/PD-L1 inhibitors in pancreatic cancer.

Therapeutic endoscopic ultrasonography: intratumoral injection for pancreatic adenocarcinoma.

Pancreatic adenocarcinoma is an aggressive disease that has poor outcomes despite maximal traditional therapies. Thus, treatment of this cancer demands innovative strategies to be used in addition to standing therapies in order to provide new avenues of care. Here, we describe the technique of using endoscopic ultrasound in order to directly inject both novel and conventional therapies into pancreatic tumors. We detail the rationale behind this strategy and the many benefits it provides. We then describe our technique in detail, including our experience injecting the AdV-tk adenoviral vector to create an in situ vaccine effect.

The spectrum of vaccine therapies for patients with glioblastoma multiforme.

OPINION STATEMENT: Glioblastoma multiforme (GBM) is the most common primary malignant tumor of the central nervous system (CNS) and one of the most lethal cancers in adults and children. Despite aggressive treatment with surgery, radiation, and chemotherapy, median survival is less than 15 months and overall survival is less than 10 % at 5 years. Development of therapeutics for malignant gliomas has been hampered by their natural complexity as well as protective mechanisms unique to the CNS. Better understanding of the pathogenesis of GBM is opening the path to novel, specific-targeted therapies. Recently, multiple immunotherapy approaches have been acquiring substantial indication of therapeutic efficacy with a very safe profile. Examples of the leading clinical approaches for GBM will be discussed in detail in this review.

Phase IB study of gene-mediated cytotoxic immunotherapy adjuvant to up-front surgery and intensive timing radiation for malignant glioma.

PURPOSE: Despite aggressive therapies, median survival for malignant gliomas is less than 15 months. Patients with unmethylated O(6)-methylguanine-DNA methyltransferase (MGMT) fare worse, presumably because of temozolomide resistance. AdV-tk, an adenoviral vector containing the herpes simplex virus thymidine kinase gene, plus prodrug synergizes with surgery and chemoradiotherapy, kills tumor cells, has not shown MGMT dependency, and elicits an antitumor vaccine effect. PATIENTS AND METHODS: Patients with newly diagnosed malignant glioma received AdV-tk at 3 × 10(10), 1 × 10(11), or 3 × 10(11) vector particles (vp) via tumor bed injection at time of surgery followed by 14 days of valacyclovir. Radiation was initiated within 9 days after AdV-tk injection to overlap with AdV-tk activity. Temozolomide was administered after completing valacyclovir treatment. RESULTS: Accrual began December 2005 and was completed in 13 months. Thirteen patients were enrolled and 12 completed therapy, three at dose levels 1 and 2 and six at dose level 3. There were no dose-limiting or significant added toxicities. One patient withdrew before completing prodrug because of an unrelated surgical complication. Survival at 2 years was 33% and at 3 years was 25%. Patient-reported quality of life assessed with the Functional Assessment of Cancer Therapy-Brain (FACT-Br) was stable or improved after treatment. A significant CD3(+) T-cell infiltrate was found in four of four tumors analyzed after treatment. Three patients with MGMT unmethylated glioblastoma multiforme survived 6.5, 8.7, and 46.4 months. CONCLUSION: AdV-tk plus valacyclovir can be safely delivered with surgery and accelerated radiation in newly diagnosed malignant gliomas. Temozolomide did not prevent immune responses. Although not powered for efficacy, the survival and MGMT independence trends are encouraging. A phase II trial is ongoing.

Cytotoxic immunotherapy strategies for cancer: mechanisms and clinical development.

Traditional therapies for cancer include surgery, chemotherapy, and radiation. Chemotherapy has widespread systemic cytotoxic effects against tumor cells but also affects normal cells. Radiation has more targeted local cytotoxicity but is limited to killing cells in the radiation field. Immunotherapy has the potential for systemic, specific killing of tumor cells. However, if the immune response is specific to a single antigen, tumor evasion can occur by down-regulation of that antigen. An immunotherapy approach that induces polyvalent immunity to autologous tumor antigens can provide a personalized vaccine with less potential for immunologic escape. A cytotoxic immunotherapy strategy creates such a tumor vaccine in situ. Immunogenic tumor cell death provides tumor antigen targets for the adaptive immune response and stimulates innate immunity. Attraction and activation of antigen presenting cells such as dendritic cells is important to process and present tumor antigens to T cells. These include cytotoxic T cells that kill tumor cells and T cells which positively and negatively regulate immunity. Tipping the balance in favor of anti-tumor immunity is an important aspect of an effective strategy. Clinically, immunotherapies may be most effective when combined with standard therapies in a complimentary way. An example is gene-mediated cytotoxic immunotherapy (GMCI) which uses an adenoviral vector, AdV-tk, to deliver a cytotoxic and immunostimulatory gene to tumor cells in vivo in combination with standard therapies creating an immunostimulatory milieu. This approach, studied extensively in animal models and early stage clinical trials, is now entering a definitive Phase 3 trial for prostate cancer.

Biological response determinants in HSV-tk + ganciclovir gene therapy for prostate cancer.

The limitations of current forms of prostate cancer therapy have driven researchers to search for new alternatives. Previously we showed cytopathic effect related to HSV-tk in prostate cancer. In this study we present initial results of a neoadjuvant HSV-tk gene therapy trial and address some of the potential mechanistic aspects of its effect in human tissues. We enrolled 23 men with clinically localized prostate cancer but high risk for recurrence in this Phase I-II trial. Intraprostatic viral injections (one to four) were followed by 2 weeks of ganciclovir and prostatectomy 2-4 weeks later. Toxicity was modest. Surgical specimens were embedded fully and whole-mount slides were imaged and analyzed for areas of cytopathic effect. The larger the tumor the greater the cytopathic effect. The effect also seems to be related to areas of high CAR expression. However, the number of injection sites did not influence effect. Local (CD8+ cells and macrophages) and systemic immune response (CD8+ and activated CD8+, IL-12) was increased in patients treated with HSV-tk. Increased apoptosis and decreased microvessel density were also noted in these patients. The results suggest a tumor-specific effect mediated by systemic and local immune response, antiangiogenic effect, and modulation of apoptosis.

Sustained long-term immune responses after in situ gene therapy combined with radiotherapy and hormonal therapy in prostate cancer patients.

PURPOSE: To explore long-term immune responses after combined radio-gene-hormonal therapy. METHODS AND MATERIALS: Thirty-three patients with prostate specific antigen 10 or higher or Gleason score of 7 or higher or clinical stage T2b to T3 were treated with gene therapy that consisted of 3 separate intraprostatic injections of AdHSV-tk on Days 0, 56, and 70. Each injection was followed by 2 weeks of valacyclovir. Intensity-modulated radiation therapy was delivered 2 days after the second AdHSV-tk injection for 7 weeks. Hormonal therapy was initiated on Day 0 and continued for 4 months or 2.3 years. Blood samples were taken before, during, and after treatment. Lymphocytes were analyzed by fluorescent antibody cell sorting (FACS). RESULTS: Median follow-up was 26 months (range, 4-48 months). The mean percentages of DR+CD8+ T cells were increased at all timepoints up to 8 months. The mean percentages of DR+CD4+ T cells were increased later and sustained longer until 12 months. Long-term (2.3 years) use of hormonal therapy did not affect the percentage of any lymphocyte population. CONCLUSIONS: Sustained long-term (up to 8 to 12 months) systemic T-cell responses were noted after combined radio-gene-hormonal therapy for prostate cancer. Prolonged use of hormonal therapy does not suppress this response. These results suggest the potential for sustained activation of cell-mediated immune responses against cancer.

Expanding the therapeutic index of radiation therapy by combining in situ gene therapy in the treatment of prostate cancer.

The advances in radiotherapy (3D-CRT, IMRT) have enabled high doses of radiation to be delivered with the least possible associated toxicity. However, the persistence of cancer (local recurrence after radiotherapy) despite these increased doses as well as distant failure suggesting the existence of micro-metastases, especially in the case of higher risk disease, have underscored the need for continued improvement in treatment strategies to manage local and micro-metastatic disease as definitively as possible. This has prompted the idea that an increase in the therapeutic index of radiotherapy might be achieved by combining it with in situ gene therapy. The goal of these combinatorial therapies is to maximize the selective pressure against cancer cell growth while minimizing treatment-associated toxicity. Major efforts utilizing different gene therapy strategies have been employed in conjunction with radiotherapy. We reviewed our and other published clinical trials utilizing this combined radio-genetherapy approach including their associated pre-clinical in vitro and in vivo models. The use of in situ gene therapy as an adjuvant to radiation therapy dramatically reduced cell viability in vitro and tumor growth in vivo. No significant worsening of the toxicities normally observed in single-modality approaches were identified in Phase I/II clinical studies. Enhancement of both local and systemic T-cell activation was noted with this combined approach suggesting anti-tumor immunity. Early clinical outcome including biochemical and biopsy data was very promising. These results demonstrate the increased therapeutic efficacy achieved by combining in situ gene therapy with radiotherapy in the management of local prostate cancer. The combined approach maximizes tumor control, both local-regional and systemic through radio-genetherapy induced cytotoxicity and anti-tumor immunity.

Treatment with human metalloproteinase-8 gene delivery ameliorates experimental rat liver cirrhosis.

BACKGROUND & AIMS: An extrahepatic human neutrophil collagenase complementary DNA (matrix metalloprotease-8) cloned in an adenovirus vector was used as a therapeutic agent in cirrhosis. METHODS: A high titer of clinical-grade AdMMP8 was obtained. RESULTS: HeLa cells transduced with AdMMP8 expressed recombinant matrix metalloprotease-8 messenger RNA and matrix metalloprotease-8 protein. Matrix metalloprotease-8 in culture sups showed enzymatic activity against native collagen type I, which was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline, and tissue inhibitor of metalloprotease-1. In vivo transduction showed matrix metalloprotease-8 activity, and studies to establish the efficacy of this characterized vector were performed in CCl(4) and bile duct-ligated cirrhotic rats. Transduction with 3 x 10(11) viral particles per kilogram resulted in hepatic detection of both messenger RNA and protein matrix metalloprotease-8. A consistent response in fibrosis reversal was observed in CCl(4) rats. Liver fibrosis in bile duct-ligated cirrhotic animals was decreased in 45%, along with diminished hydroxyproline content, after AdMMP8 treatment. The expression of matrix metalloprotease-2 and matrix metalloprotease-3 was up-regulated in AdMMP8 rats. Free tissue inhibitor of metalloprotease-1, as an indirect measurement of active uncomplexed matrix metalloproteases, was also increased in the AdMMP8 groups. Transforming growth factor-beta messenger RNA was diminished, and matrix metalloprotease-9 and hepatocyte growth factor increased. Treatment in both models correlated with improvements in ascites, functional hepatic tests, and gastric varices, indicating diminished intrahepatic blood pressure in animals injected with AdMMP8. CONCLUSIONS: Therefore, therapy with the matrix metalloprotease-8 gene is promising for use in a clinical setting.

The combination of adenoviral HSV TK gene therapy and radiation is effective in athymic mouse glioblastoma xenografts without increasing toxic side effects.

OBJECT: In mouse models of prostate and breast cancer therapeutic effects are enhanced when adenoviral HSV TK gene therapy is combined with ionizing radiation. In the present study, we adopted this approach for the treatment of human glioblastoma xenografts in an athymic mouse model and assessed treatment results as well as toxic side effects. METHODS: About 72 nude mice received intracerebral inoculations of 2 x 10(5) U87deltaEGFR cells. On day 7 after tumor implantation the study population was randomized into six treatment arms: (1) intratumoral buffer inoculation on day 7, (2) intratumoral adenoviral vector injection (2 x 10(9) vp) on day 7, (3) single dose radiation (2.1 Gy) on day 9, (4) adenoviral injection + radiation, (5) adenoviral injection + ganciclovir (GCV) (20 microg/g twice daily from day 8 to 17), (6) adenoviral injection + GCV + radiation. On day 21 half of the animals were sacrificed for histological evaluation of the brain tumors, the other half was assessed for survival. RESULTS: This study showed significantly prolonged median survival time of 5 days for the GCV treated groups. The addition of radiation decreased the frequency of neurological symptoms and delayed the onset of deficits without altering the expression of thymidine kinase in the tumor cells. CONCLUSIONS: We conclude that adenoviral HSV TK gene therapy in combination with adjuvant radiotherapy does not generate increased toxic side effects in glioblastoma treatment. The prolonged survival time of animals receiving gene therapy and the reduced occurrence of neurological symptoms in irradiated mice constitute promising features of the combined treatment.