- Email: [email protected]
242. Electrochemogenetherapy with Bleomycin and Interleukin 12 for the Treatment of Hystocytic Sarcoma and Acanthomatous Epulis in Dogs
CANCER - IMMUNOTHERAPY I 240. A Recombinant Adenovirus Type 35 Fiber Knob Protein Sensitizes Lymphoma Cells to Rituximab Therapy
Hongjie Wang,1 Ying Liu,1 Zongyi Li,1 Xiaolong Fan,2 Akseli Hemminki,3 Andre Lieber.1 1 Medicine, University of Washington, Seattle, WA; 2University of Lund, Lund, Sweden; 3Cancer Gene Therapy Group, University of Helsinki, Helsinki, Finland. Monoclonal antibodies (mAbs) exert anti-tumor effects in cancer patients through complement-dependent cytotoxicity (CDC). However, the therapeutic potential of anti-cancer mAbs is often significantly limited due to the ability of cancer cells to block CDC by the expression of membrane complement regulatory proteins such as CD46. Our central hypothesis is that transient removal of CD46 from the surface of cancer cells would render them more susceptible to mAb therapy. In a previous study, we used an E.coli expression library of adenovirus type 35 fiber knobs with random mutations to identify a knob mutant with high affinity to CD46 (Ad35K++). Here we show that incubation of tumor cells with Ad35K++ leads to transient CD46 internalization, which in turn sensitizes cancer cells to rituximab-mediated CDC. Notably, Ad35K++ treatment allowed for CDC in lymphoma cells that were resistant to rituximab. Removal of surface CD46 and stimulation of CDC was not efficient with mAbs specific to CD46. In an in vivo xenograft model with systemically injected human lymphoma cells, intravenous injection of Ad35K++ in combination with rituximab remarkably increased the medium survival of animals. (The medium survival time for untreated mice, rituximab-treated mice, and Ad35K++/rituximab treated mice, was 15.5, 16.5, and 24 days, respectively). Intravenous injection of Ad35K++ was not associated with detectable toxic side effects. Furthermore, we provided data that support the possibility of repeated Ad35K++ application. Considering the central role of CDC in mAb cancer therapy, Ad35K++ might also be beneficial in combination with other mAbs specific to other types of cancer. Our Ad35K++ -based approach has clear advantages over other approaches for down-regulation of complement regulatory proteins (MCPs). We demonstrated that it was more efficient than monoclonal antibodies against CD46. Because Ad35K++ can easily be produced in E.coli and purified by affinity columns, our approach is more feasible than approaches based on synthetic MCP-siRNA or siRNA gene transfer.
241. Phase II Study of Adenoviral Vector (Ad3f35) Transduced Autologous Dendritic Cells in Combination with Celecoxib in Patients with Metastatic Nasopharyngeal Carcinoma
Marissa Teo,1 Peter Wang,1 John Chia,1 Li Sun,3 Stephen Gottschalk,2 Han Chong Toh.1 1 Medical Oncology, National Cancer Centre, Singapore, Singapore; 2Pediatric and Immunology, Baylor College of Medicine, Houston; 3Clinical Research, Singapore General Hospital, Singapore, Singapore.
Background: Nasopharyngeal Carcinoma (NPC) is an EBV associated malignancy expressing LMP1 and LMP2 viral antigens. This is a first-in-man phase II trial involving sixteen advanced NPC patients vaccinated with autologous monocyte-derived dendritic cells (DCs) transduced with Ad5f35 vector expressing a truncated form of LMP-1 and full-length LMP-2 genes (Ad5f35-LMPd1-2). Materials and methods: Peripheral blood mononuclear cells (PBMC) were obtained from venesected patients. DCs were cultured and transduced with Ad5f35-LMPd1-2, and cytokine cocktail of IL-1, IL-6, TNFa and PGE-2. Each patient received up to 5 intradermal vaccinations (3-5 x 106 cells/dose) at biweekly intervals. The vaccine was administered in combination with Celecoxib, known to have immune Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
modulating properties, following stable disease or progression on lines of chemotherapy. Patients were followed for clinical benefit, response, disease-free (PFS) and overall survival (OS). Plasma EBV DNA levels and peripheral regulatory T cells (Treg), defined as CD4+CD25HiCD127Lo/-Foxp3+ cells, were also analyzed. Results: Median age was 49yrs (range 36 to 58 yrs) and median sites of disease were 3 (range 1 to 7). All except one patient had prior RT. Median ECOG performance status was 1 (range 0-3) and median lines of chemotherapy was 3.5 (1 to 8) and median chemotherapy agents 4.5 (range 1 to 8). Thirteen patients completed at least 4 of 5 vaccinations, of which 9 patients completed all 5 vaccinations. Median total dose of vaccine administered per patient was 22.7x 106 cells. The vaccine was well tolerated with minimal toxicity mainly related to local reaction at the vaccination site. The majority of patients (81%) progressed during the vaccination phase. Two patients (12.5%) had disease stabilization for more than 18 weeks and one patient experienced a partial response leading to an overall clinical benefit rate of 19%. The median PFS was 2 months (95% CI, 1.8-7.4 mths). Nine patients have died and median OS was 4.6 mths (95% CI, 1.5-12.5 mths). This study was terminated early due to lack of efficacy. Analysis for peripheral Treg cells was completed in nine patients. Downregulation of Treg cells was seen in four patients; two were responders,one partial response (PR) and one stable disease (SD) and two were non-responders (progressed disease, PD). The patient who experienced PR, had sustained suppressed Treg levels for 3 months after the first vaccination. In another patient who experienced SD, an initial 6-fold down-regulation of Treg levels was observed at 1.5 months post-vaccine, with recovery to pre-vaccination level at 3 months. Three patients (3/14) had a reduction of EBV DNA level post-vaccination, of which one had SD. Conclusion: Our study showed that Ad535 transduced DCs was safe in NPC patients, with modest clinical efficacy in advanced heavily-pretreated NPC patients. ELISPOT and pentamer assays are underway to assess EBV-specific immune response and will be presented at the poster session.
242. Electrochemogenetherapy with Bleomycin and Interleukin 12 for the Treatment of Hystocytic Sarcoma and Acanthomatous Epulis in Dogs
Jeffry Cutrera,1 Giselle Hosgood,2 Julia Buchholz,2 Tracy Gieger,2 Keijiro Shiomitsu,2 Shulin Li.1 1 Comparative Biomedical Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA; 2Veterinary Clinical Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA. Several combinations of cytokine gene therapies and chemotherapies have been shown to increase the efficacy of treatments compared to either treatment alone. Individually, bleomycin (BLM) and interleukin 12 (IL-12) plasmid DNA have been tested clinically to treat tumors. Delivery of BLM via intratumoral electroporation eradicates large tumors but fails to prevent tumor redevelopment in animal models (Clin Can Res, 2005). Injection of IL-12 plasmid DNA via intratumoral electroporation stimulates an anti-tumor immune response and inhibits tumor metastasis and redevelopment, but the IL-12 treatment cannot eradicate large volume tumors. In preclinical mouse models, the combinatorial treatment of intratumoral injection of Bleomycin and IL-12 plasmid DNA followed by electroporation can completely eradicate syngeneic tumors. Also, we have previously reported the treatment and complete cure of three dogs bearing spontaneous head and neck squamous cell carcinoma. Here, we present two new unique cases bearing different types of cancer: histocytic sarcoma located on the knee joint and acanthomatous epulis in the oral cavity. This treatment successfully regressed the histocytic sarcoma which had recurred after surgery. The survival time was extended without the need of amputation plus post-amputational chemotherapy. The success of this treatment prevented the suffering due to amputation and improved the quality of life of this dog. Likewise, this treatment S95
CANCER - IMMUNOTHERAPY I successfully regressed the primary acanthomatous epulis tumors without recurrence up to date. Also, there is no side effect on the tooth or other tissues adjacent to where tumors were located. Blood and urine profile analyses indicate that no toxicities were found after either two or three administrations, indicating that co-administration of BLM and IL12 encoding DNA via electroporation is safe. This report shows the potential for the use of electrochemogenetherapy with Bleomycin and IL-12 plasmid DNA in multiple types of spontaneous tumors localized on areas other than the head and neck.
243. Vaccination Encorporating a Selectively Replicating HSV-1 That Expresses IL-12 after IntraTumoral Virus Administration Prolongs Survival in a Murine Model of Neuroblastoma David F. Bauer,1 Jacqueline N. Parker,1 James M. Markert.1 Neurosurgery, University of Alabama, Birmingham, AL.
1
High grade intracranial neoplasms carry poor prognosis with current therapy. Novel strategies, including oncolytic viral therapy using conditionally replicating, tumor-selective herpes simplex virus type 1 vectors (HSV-1), have been investigated and have had encouraging results in Phase 1 clinical trials in both the U.S. and the U.K. M002 is a gamma-1-34.5 deleted HSV-1 vector that expresses IL-12, and selectively replicates in tumor cells. We have previously reported that M002 significantly improves survive of tumor-bearing mice in multiple experimental murine brain tumor models. The current study aimed to ascertain whether tumor cells previously infected with M002 could be used as a vaccine against developing tumors in the brain. For vaccination, Neuro-2a cells were infected using a high multiplicity of infection (m.o.i.= 5) of M002, treated with 10 Gray gamma radiation, then injected into the gastrocnemius muscle of A/J mice. A boost vaccination was administered 7 days later. Cells from the murine neuroblastoma cell line, Neuro-2a, were stereotactically injected into the right basal ganglia of syngeneic A/J mice. Intratumoral injection of 1x10E7 PFU M002 was done through the original burr hole at 7 days following initial tumor implantation. When given preemptively, the Neuro-2a/M002 vaccine significantly prolonged survival in our orthotopic, murine model of intracranial neuroblastoma (p<0.01). No improved survival was observed in mice that were vaccinated one week following tumor implantation (p=0.9851). However, when intracranial tumors were treated with M002 seven days after implantation, followed by intramuscular vaccination one day later, survival was significantly improved as compared to mice whose tumors were treated with M002 alone (p<0.0001). A selectively replicating HSV-1 that expresses IL-12 shows efficacy in a tumor vaccine when combined with direct treatment of a malignant intracranial neoplasm. Pilot data has demonstrated a vaccine induced Th1 mediated immune response against the tumor, a response which may be enhanced by the expression of IL-12.
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244. High-Capacity Adenovirus VectorMediated Transfer of Conditional Cytotoxic Herpes Simplex Type 1-Thymidine Kinase [HSV1-TK] and Immunostimulatory Fms-Like Tyrosine Kinase 3 Ligand [Flt3L] Transgenes Elicits ImmuneMediated Long-Term Survival in a Syngeneic Intracranial GBM Model Mariana Puntel,1,2 A. K. M. Muhammad,1,2 Kurt M. Kroeger,1,2 Marianela Candolfi,1,2 Kader Yagiz,1,2 Chunyan Liu,1,2 Alireza Salem,1,2 Maricel Gozo,1,2 Gregory J. Baker,1,2 Yilan Shi,1,2 David Foulad,1,2 Weidong Xiong,1,2 Donna Palmer,3 Phil Ng,3 Pedro R. Lowenstein,1,2 Maria G. Castro.1,2 1 Board of Governors Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA; 2Departments of Medicine, and Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, CA; 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.
Glioblastoma multiforme (GBM) is a malignant primary brain tumor associated with 5% survival at 5 years post-diagnosis. Adenoviral (Ad)-mediated transfer of conditional cytotoxic, HSV1TK and immunostimulatory Flt3L transgenes elicited immunemediated long-term survival in a syngeneic intracranial GBM model in rodents. Helper dependent, high-capacity Ad vectors (HC-Ads) have a significantly favorable immunological profile due to the lack of Ad encoding sequences. Even in the presence of a preexisting systemic anti-Ad immune response, transgene expression from HCAds remains stable for up to 1 year. In order to test efficacy, transgene expression, distribution of vector genomes, and potential toxicity of the high capacity vectors for the treatment of GBM we performed intratumoral delivery of the combined therapy using HC-Ad-TK and HC-Ad-TetON-Flt3L co-administered with gancyclovir and doxicycline. Three doses of the HC-Ads were administered intratumorally and analyzed at 5 days, 30 days and 6 months after injection. Neuropathology studies were conducted by immunocytochemistry of brain sections; distribution of HC-Ads genomes were analyzed by qPCR as genome copy numbers within 13 different tissues including brain injection side and contra-lateral side, spleen, and liver; FLt3L expression in brain tissue and plasma. Approximately 70% of the rats treated survived for long term when treated with the combined therapy. At 5 days after treatment Flt3L was detected in brain tissue and serum; strong infiltration of immune cells into the tumor mass was observed; an average of 7.4x105, 1x106, and 7.2 x106 total vector genomes were detected for the doses 5x108, 1x109 and 5x109 vp, respectively; the vector genomes were exclusively restricted to the brain injection side for all the doses tested. At 30 days after treatment, Flt3L was not detected in neither brain tissue nor serum; infiltration of immune cells into the tumor mass was observed limited to the scar region within the striatum; vector genomes were not detected at the doses tested. Also, no toxic effects were found as consequence of the treatment. Our data indicate that the combined administration of HC-Ad vectors encoding TK and Flt3L was efficacious and safe for the treatment of glioma in a syngeneic, intracranial rodent model. These results will form the basis for further developments towards the implementation of HC-Ad mediated combined gene therapy for the treatment of GBM in human patients.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Hongjie Wang,1 Ying Liu,1 Zongyi Li,1 Xiaolong Fan,2 Akseli Hemminki,3 Andre Lieber.1 1 Medicine, University of Washington, Seattle, WA; 2University of Lund, Lund, Sweden; 3Cancer Gene Therapy Group, University of Helsinki, Helsinki, Finland. Monoclonal antibodies (mAbs) exert anti-tumor effects in cancer patients through complement-dependent cytotoxicity (CDC). However, the therapeutic potential of anti-cancer mAbs is often significantly limited due to the ability of cancer cells to block CDC by the expression of membrane complement regulatory proteins such as CD46. Our central hypothesis is that transient removal of CD46 from the surface of cancer cells would render them more susceptible to mAb therapy. In a previous study, we used an E.coli expression library of adenovirus type 35 fiber knobs with random mutations to identify a knob mutant with high affinity to CD46 (Ad35K++). Here we show that incubation of tumor cells with Ad35K++ leads to transient CD46 internalization, which in turn sensitizes cancer cells to rituximab-mediated CDC. Notably, Ad35K++ treatment allowed for CDC in lymphoma cells that were resistant to rituximab. Removal of surface CD46 and stimulation of CDC was not efficient with mAbs specific to CD46. In an in vivo xenograft model with systemically injected human lymphoma cells, intravenous injection of Ad35K++ in combination with rituximab remarkably increased the medium survival of animals. (The medium survival time for untreated mice, rituximab-treated mice, and Ad35K++/rituximab treated mice, was 15.5, 16.5, and 24 days, respectively). Intravenous injection of Ad35K++ was not associated with detectable toxic side effects. Furthermore, we provided data that support the possibility of repeated Ad35K++ application. Considering the central role of CDC in mAb cancer therapy, Ad35K++ might also be beneficial in combination with other mAbs specific to other types of cancer. Our Ad35K++ -based approach has clear advantages over other approaches for down-regulation of complement regulatory proteins (MCPs). We demonstrated that it was more efficient than monoclonal antibodies against CD46. Because Ad35K++ can easily be produced in E.coli and purified by affinity columns, our approach is more feasible than approaches based on synthetic MCP-siRNA or siRNA gene transfer.
241. Phase II Study of Adenoviral Vector (Ad3f35) Transduced Autologous Dendritic Cells in Combination with Celecoxib in Patients with Metastatic Nasopharyngeal Carcinoma
Marissa Teo,1 Peter Wang,1 John Chia,1 Li Sun,3 Stephen Gottschalk,2 Han Chong Toh.1 1 Medical Oncology, National Cancer Centre, Singapore, Singapore; 2Pediatric and Immunology, Baylor College of Medicine, Houston; 3Clinical Research, Singapore General Hospital, Singapore, Singapore.
Background: Nasopharyngeal Carcinoma (NPC) is an EBV associated malignancy expressing LMP1 and LMP2 viral antigens. This is a first-in-man phase II trial involving sixteen advanced NPC patients vaccinated with autologous monocyte-derived dendritic cells (DCs) transduced with Ad5f35 vector expressing a truncated form of LMP-1 and full-length LMP-2 genes (Ad5f35-LMPd1-2). Materials and methods: Peripheral blood mononuclear cells (PBMC) were obtained from venesected patients. DCs were cultured and transduced with Ad5f35-LMPd1-2, and cytokine cocktail of IL-1, IL-6, TNFa and PGE-2. Each patient received up to 5 intradermal vaccinations (3-5 x 106 cells/dose) at biweekly intervals. The vaccine was administered in combination with Celecoxib, known to have immune Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
modulating properties, following stable disease or progression on lines of chemotherapy. Patients were followed for clinical benefit, response, disease-free (PFS) and overall survival (OS). Plasma EBV DNA levels and peripheral regulatory T cells (Treg), defined as CD4+CD25HiCD127Lo/-Foxp3+ cells, were also analyzed. Results: Median age was 49yrs (range 36 to 58 yrs) and median sites of disease were 3 (range 1 to 7). All except one patient had prior RT. Median ECOG performance status was 1 (range 0-3) and median lines of chemotherapy was 3.5 (1 to 8) and median chemotherapy agents 4.5 (range 1 to 8). Thirteen patients completed at least 4 of 5 vaccinations, of which 9 patients completed all 5 vaccinations. Median total dose of vaccine administered per patient was 22.7x 106 cells. The vaccine was well tolerated with minimal toxicity mainly related to local reaction at the vaccination site. The majority of patients (81%) progressed during the vaccination phase. Two patients (12.5%) had disease stabilization for more than 18 weeks and one patient experienced a partial response leading to an overall clinical benefit rate of 19%. The median PFS was 2 months (95% CI, 1.8-7.4 mths). Nine patients have died and median OS was 4.6 mths (95% CI, 1.5-12.5 mths). This study was terminated early due to lack of efficacy. Analysis for peripheral Treg cells was completed in nine patients. Downregulation of Treg cells was seen in four patients; two were responders,one partial response (PR) and one stable disease (SD) and two were non-responders (progressed disease, PD). The patient who experienced PR, had sustained suppressed Treg levels for 3 months after the first vaccination. In another patient who experienced SD, an initial 6-fold down-regulation of Treg levels was observed at 1.5 months post-vaccine, with recovery to pre-vaccination level at 3 months. Three patients (3/14) had a reduction of EBV DNA level post-vaccination, of which one had SD. Conclusion: Our study showed that Ad535 transduced DCs was safe in NPC patients, with modest clinical efficacy in advanced heavily-pretreated NPC patients. ELISPOT and pentamer assays are underway to assess EBV-specific immune response and will be presented at the poster session.
242. Electrochemogenetherapy with Bleomycin and Interleukin 12 for the Treatment of Hystocytic Sarcoma and Acanthomatous Epulis in Dogs
Jeffry Cutrera,1 Giselle Hosgood,2 Julia Buchholz,2 Tracy Gieger,2 Keijiro Shiomitsu,2 Shulin Li.1 1 Comparative Biomedical Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA; 2Veterinary Clinical Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA. Several combinations of cytokine gene therapies and chemotherapies have been shown to increase the efficacy of treatments compared to either treatment alone. Individually, bleomycin (BLM) and interleukin 12 (IL-12) plasmid DNA have been tested clinically to treat tumors. Delivery of BLM via intratumoral electroporation eradicates large tumors but fails to prevent tumor redevelopment in animal models (Clin Can Res, 2005). Injection of IL-12 plasmid DNA via intratumoral electroporation stimulates an anti-tumor immune response and inhibits tumor metastasis and redevelopment, but the IL-12 treatment cannot eradicate large volume tumors. In preclinical mouse models, the combinatorial treatment of intratumoral injection of Bleomycin and IL-12 plasmid DNA followed by electroporation can completely eradicate syngeneic tumors. Also, we have previously reported the treatment and complete cure of three dogs bearing spontaneous head and neck squamous cell carcinoma. Here, we present two new unique cases bearing different types of cancer: histocytic sarcoma located on the knee joint and acanthomatous epulis in the oral cavity. This treatment successfully regressed the histocytic sarcoma which had recurred after surgery. The survival time was extended without the need of amputation plus post-amputational chemotherapy. The success of this treatment prevented the suffering due to amputation and improved the quality of life of this dog. Likewise, this treatment S95
CANCER - IMMUNOTHERAPY I successfully regressed the primary acanthomatous epulis tumors without recurrence up to date. Also, there is no side effect on the tooth or other tissues adjacent to where tumors were located. Blood and urine profile analyses indicate that no toxicities were found after either two or three administrations, indicating that co-administration of BLM and IL12 encoding DNA via electroporation is safe. This report shows the potential for the use of electrochemogenetherapy with Bleomycin and IL-12 plasmid DNA in multiple types of spontaneous tumors localized on areas other than the head and neck.
243. Vaccination Encorporating a Selectively Replicating HSV-1 That Expresses IL-12 after IntraTumoral Virus Administration Prolongs Survival in a Murine Model of Neuroblastoma David F. Bauer,1 Jacqueline N. Parker,1 James M. Markert.1 Neurosurgery, University of Alabama, Birmingham, AL.
1
High grade intracranial neoplasms carry poor prognosis with current therapy. Novel strategies, including oncolytic viral therapy using conditionally replicating, tumor-selective herpes simplex virus type 1 vectors (HSV-1), have been investigated and have had encouraging results in Phase 1 clinical trials in both the U.S. and the U.K. M002 is a gamma-1-34.5 deleted HSV-1 vector that expresses IL-12, and selectively replicates in tumor cells. We have previously reported that M002 significantly improves survive of tumor-bearing mice in multiple experimental murine brain tumor models. The current study aimed to ascertain whether tumor cells previously infected with M002 could be used as a vaccine against developing tumors in the brain. For vaccination, Neuro-2a cells were infected using a high multiplicity of infection (m.o.i.= 5) of M002, treated with 10 Gray gamma radiation, then injected into the gastrocnemius muscle of A/J mice. A boost vaccination was administered 7 days later. Cells from the murine neuroblastoma cell line, Neuro-2a, were stereotactically injected into the right basal ganglia of syngeneic A/J mice. Intratumoral injection of 1x10E7 PFU M002 was done through the original burr hole at 7 days following initial tumor implantation. When given preemptively, the Neuro-2a/M002 vaccine significantly prolonged survival in our orthotopic, murine model of intracranial neuroblastoma (p<0.01). No improved survival was observed in mice that were vaccinated one week following tumor implantation (p=0.9851). However, when intracranial tumors were treated with M002 seven days after implantation, followed by intramuscular vaccination one day later, survival was significantly improved as compared to mice whose tumors were treated with M002 alone (p<0.0001). A selectively replicating HSV-1 that expresses IL-12 shows efficacy in a tumor vaccine when combined with direct treatment of a malignant intracranial neoplasm. Pilot data has demonstrated a vaccine induced Th1 mediated immune response against the tumor, a response which may be enhanced by the expression of IL-12.
S96
244. High-Capacity Adenovirus VectorMediated Transfer of Conditional Cytotoxic Herpes Simplex Type 1-Thymidine Kinase [HSV1-TK] and Immunostimulatory Fms-Like Tyrosine Kinase 3 Ligand [Flt3L] Transgenes Elicits ImmuneMediated Long-Term Survival in a Syngeneic Intracranial GBM Model Mariana Puntel,1,2 A. K. M. Muhammad,1,2 Kurt M. Kroeger,1,2 Marianela Candolfi,1,2 Kader Yagiz,1,2 Chunyan Liu,1,2 Alireza Salem,1,2 Maricel Gozo,1,2 Gregory J. Baker,1,2 Yilan Shi,1,2 David Foulad,1,2 Weidong Xiong,1,2 Donna Palmer,3 Phil Ng,3 Pedro R. Lowenstein,1,2 Maria G. Castro.1,2 1 Board of Governors Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA; 2Departments of Medicine, and Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, CA; 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.
Glioblastoma multiforme (GBM) is a malignant primary brain tumor associated with 5% survival at 5 years post-diagnosis. Adenoviral (Ad)-mediated transfer of conditional cytotoxic, HSV1TK and immunostimulatory Flt3L transgenes elicited immunemediated long-term survival in a syngeneic intracranial GBM model in rodents. Helper dependent, high-capacity Ad vectors (HC-Ads) have a significantly favorable immunological profile due to the lack of Ad encoding sequences. Even in the presence of a preexisting systemic anti-Ad immune response, transgene expression from HCAds remains stable for up to 1 year. In order to test efficacy, transgene expression, distribution of vector genomes, and potential toxicity of the high capacity vectors for the treatment of GBM we performed intratumoral delivery of the combined therapy using HC-Ad-TK and HC-Ad-TetON-Flt3L co-administered with gancyclovir and doxicycline. Three doses of the HC-Ads were administered intratumorally and analyzed at 5 days, 30 days and 6 months after injection. Neuropathology studies were conducted by immunocytochemistry of brain sections; distribution of HC-Ads genomes were analyzed by qPCR as genome copy numbers within 13 different tissues including brain injection side and contra-lateral side, spleen, and liver; FLt3L expression in brain tissue and plasma. Approximately 70% of the rats treated survived for long term when treated with the combined therapy. At 5 days after treatment Flt3L was detected in brain tissue and serum; strong infiltration of immune cells into the tumor mass was observed; an average of 7.4x105, 1x106, and 7.2 x106 total vector genomes were detected for the doses 5x108, 1x109 and 5x109 vp, respectively; the vector genomes were exclusively restricted to the brain injection side for all the doses tested. At 30 days after treatment, Flt3L was not detected in neither brain tissue nor serum; infiltration of immune cells into the tumor mass was observed limited to the scar region within the striatum; vector genomes were not detected at the doses tested. Also, no toxic effects were found as consequence of the treatment. Our data indicate that the combined administration of HC-Ad vectors encoding TK and Flt3L was efficacious and safe for the treatment of glioma in a syngeneic, intracranial rodent model. These results will form the basis for further developments towards the implementation of HC-Ad mediated combined gene therapy for the treatment of GBM in human patients.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy