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486. AdCD40L Gene Therapy Enhances the Efficacy of Chemotherapy in Experimental Bladder Cancer
CANCER - IMMUNOTHERAPY II 485. Liver Specic, Inducible, AAV-Mediated IL12 Delivered Resulted in a Strong Anti Tumoral Inmune Response in a Murine Model of Liver Metastasis
Lucia Vanrell,1 Laura Blanco,1 Pedro Berraondo,1 Marianna Di Scala,1 Astrid Paneda,1 Victor Baldim,2 Jesus Prieto,1 Gloria González-Aseguinolaza.1 1 Gene Therapy and Hepatology, FIMA, Pamplona, Spain; 2 Institute of Chemistry, UNICAMP, Campinas, Brazil.
Interleukin-12 (IL12) is a multifunctional cytokine that stimulates both innate and adaptive immunity, acting as a key regulator of cell-mediated immune responses. The immunomodulating and antiangiogenic functions of IL12 have provided the rationale for exploiting this cytokine as an anticancer agent. The promising data obtained by the administration of IL12 recombinant protein in preclinical animal models of cancer raised hopes that recombinant IL12 could be a powerful therapeutic agent. However, clinical trials revealed a modest clinical response that was limited by the development of severe toxicity when high doses of this cytokine were used. Gene therapy can signicantly increase cytokine expression in the target organ without excessively elevating systemic cytokine levels, which leads to an increased efcacy/toxicity ratio. Early clinical trials with short-term IL12 expression vectors have set the proof-of-concept that local production of IL12 inside a tumor can stimulate tumor inltration by effector immune cells, sometimes followed by tumor regression. New vectors that might achieve regulated, long-term production of this cytokine might have better results and merit clinical testing. Recombinant adeno-associated viral (AAV) vectors have unique properties, which make them suitable vectors for long-term gene transfer. In this work we have developed a liver-specic and inducible adeno-associated vectors delivering IL12 and we have analyzed their performance on immunotherapy. To that end we used the single chain mouse IL12 transgene, and set up a prevention protocol of colorectal cancer liver metastases, using the C57BL/6-MC38 murine tumor model. The vector induced viral dosedependent serum levels of mIL12 and mIFNγ, showed no signicant toxicity, and was highly efcient in preventing establishment of colorrectal metastasis in the liver. Moreover, it induced an efcient T cell memory response to MC38 cell line.
486. AdCD40L Gene Therapy Enhances the Efcacy of Chemotherapy in Experimental Bladder Cancer
Lina S. E. Liljenfeldt,1 Lisa H. Christiansson,1 Katerina Gkirtzimanaki,2 Angelica S. I. Loskog,1 Aristides G. Eliopoulos.2 1 Division of Clinical Immunology, Uppsala University, Uppsala, Sweden; 2Foundation for Research and Technology – Hellas (FORTH), Crete, Greece.
Chemotherapy is standard cancer treatment but it is rarely curative and adjuvant therapies are warranted. We have shown that adenoviral CD40L gene therapy is successful in bladder cancer. CD40L is a strong inducer of anti-tumor immune responses. It is also inducing growth inhibition and apoptosis in CD40 positive tumors. We have shown that the combination of 5-Fluorouracil (5-FU) and AdCD40L enhances the apoptotic effect in human bladder cancer cells. In this study we combined AdCD40L and 5-FU to treat murine orthotopic bladder cancer. AdCD40L and 5-FU synergized to inhibit the proliferation of MB49 cells in vitro and up-regulated cell surface molecules and immunoproteasome components associated with tumor antigen processing and presentation, such as MHCI, Fas, B7.1 and TAP1/2. MB49 cells were implanted into C57BL/6 mice. Mice received 3 local treatments of AdCD40L (1x10e8 ffu), 5-FU (50mg/ kg), or the combination. Survival of mice, systemic immunity and the presence of activated tumor inltrating T cells were monitored. S188
The combination of AdCD40L and 5-FU was superior to either agent alone. Thus, the combination therapy cured 68% of large orthotopic tumors while AdCD40L alone cured 32%. Cured animals were rechallenged with a s.c. injection of MB49. All cured mice treated with the combination showed systemic MB49-specic immunity. To investigate the role of the immune system, the treatment regiments were tested on MB49 cells growing in NUDE mice. Although the combination treatment could enhance survival, no mice were cured. In conclusion, AdCD40L gene therapy is promising as adjuvant treatment to conventional chemotherapy such as 5-FU.
487. Xenograft Expanded FANG Autologous Tumor Cell Vaccine Development and Manufacturing for Clinical Use
Phillip B. Maples,1 Padmasini Kumar,1 Yang Yu,1 Beena O. Pappen,1 David Monsma,2 Stephanie Scott,2 Dawna Dylewski,2 Craig Webb,2 Neil Senzer,1,3,4,5 John Nemunaitis.1,3,4,5 1 Gradalis, Inc., Dallas, TX; 2Van Andel Research Institute, Grand Rapids, MI; 3Mary Crowley Cancer Research Centers, Dallas, TX; 4 Baylor Sammons Cancer Center, Dallas, TX; 5Texas Oncology, P.A., Dallas, TX. Many tumors are of insufcient size or otherwise inaccessible for surgical ressection to allow successful autologous vaccine manufacturing. However, small accessible lesions can be obtained or small samples obtained more safely by needle biopsy. We have previously demonstrated with our TAG vector that small quantities of tumor can be expanded in immunodecient mice and harvested for clinical tumor vaccine production (P. Kumar, et al, BioProcessing J, 8(1): 30-36, 2009; BB-IND 13401). We have developed the FANG expression vector which we believe, when transfected into tumor cells, will evoke an enhanced immune recognition /stimulation versus our previous TAG vaccine vector. The FANG nonviral vector system expresses both GM-CSF and a proprietary bifunctional shRNA to furin. Preclinical data demonstrated that blocking furin protein expression in turn blocked the activation of both TGFβ1 and TGFβ2. In contrast, our TAG vector expressed both GM-CSF and a TGFβ2 antisense. Data from our TAG Phase I autologous vaccine clinical trial and others indicate that TFGβ1 overexpression is present in a wide range of cancers. In fact our data suggest that TGFβ1 expression may be up to tenfold higher than TGFβ2 in the more than thirty tumors we examined in that study. So while the TAG vector blocked TGFβ2 expression, there was no effect on TGFβ1 expression. The FANG expression vector is identical to the TAG vector except that the TGFβ2 antisense coding sequence has been replaced with the furin shRNA sequence. FANG plasmid DNA was GMP-S manufactured. The rst clinical grade xenograft FANG vaccine was manufactured from a xenograft expanded breast cancer tumor. The tumor was expanded at the Van Andel Research Institute using their colony of athymic nude mice under conditions optimized for clinical xenograft expansion. The breast cancer tumor is ER, PR negative and Her-2/ neu +++. From the time of surgery to removal of 6 F1 tumors for vaccine manufacturing was 323 days. The combined weight of the tumors was 6.2g. The cGMP manufacturing is a 2 day process that consists of tissue disaggregation into a single cell suspension, purging of murine cells, electroporation of FANG vector, overnight incubation, gamma irradiation, cryopreservation and quality control testing. The prefreeze cell viability was 90%. The transfected tumor cell GM-CSF expression was 259pg/1x106 cells/ml. Nontransfected TFGβ1 expression was 860pg/1x106 cells/ml, the transfected TFGβ1 expression was 142pg/1x106 cells/ml and the TGFβ1 knockdown was 83%. Nontransfected TGFβ2 expression was 479pg/1x106 cells/ ml, the transfected TGFβ2 expression was 127pg/1x106 cells/ml and the TGFβ2 knockdown was 73%. These data are consistent with
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Lucia Vanrell,1 Laura Blanco,1 Pedro Berraondo,1 Marianna Di Scala,1 Astrid Paneda,1 Victor Baldim,2 Jesus Prieto,1 Gloria González-Aseguinolaza.1 1 Gene Therapy and Hepatology, FIMA, Pamplona, Spain; 2 Institute of Chemistry, UNICAMP, Campinas, Brazil.
Interleukin-12 (IL12) is a multifunctional cytokine that stimulates both innate and adaptive immunity, acting as a key regulator of cell-mediated immune responses. The immunomodulating and antiangiogenic functions of IL12 have provided the rationale for exploiting this cytokine as an anticancer agent. The promising data obtained by the administration of IL12 recombinant protein in preclinical animal models of cancer raised hopes that recombinant IL12 could be a powerful therapeutic agent. However, clinical trials revealed a modest clinical response that was limited by the development of severe toxicity when high doses of this cytokine were used. Gene therapy can signicantly increase cytokine expression in the target organ without excessively elevating systemic cytokine levels, which leads to an increased efcacy/toxicity ratio. Early clinical trials with short-term IL12 expression vectors have set the proof-of-concept that local production of IL12 inside a tumor can stimulate tumor inltration by effector immune cells, sometimes followed by tumor regression. New vectors that might achieve regulated, long-term production of this cytokine might have better results and merit clinical testing. Recombinant adeno-associated viral (AAV) vectors have unique properties, which make them suitable vectors for long-term gene transfer. In this work we have developed a liver-specic and inducible adeno-associated vectors delivering IL12 and we have analyzed their performance on immunotherapy. To that end we used the single chain mouse IL12 transgene, and set up a prevention protocol of colorectal cancer liver metastases, using the C57BL/6-MC38 murine tumor model. The vector induced viral dosedependent serum levels of mIL12 and mIFNγ, showed no signicant toxicity, and was highly efcient in preventing establishment of colorrectal metastasis in the liver. Moreover, it induced an efcient T cell memory response to MC38 cell line.
486. AdCD40L Gene Therapy Enhances the Efcacy of Chemotherapy in Experimental Bladder Cancer
Lina S. E. Liljenfeldt,1 Lisa H. Christiansson,1 Katerina Gkirtzimanaki,2 Angelica S. I. Loskog,1 Aristides G. Eliopoulos.2 1 Division of Clinical Immunology, Uppsala University, Uppsala, Sweden; 2Foundation for Research and Technology – Hellas (FORTH), Crete, Greece.
Chemotherapy is standard cancer treatment but it is rarely curative and adjuvant therapies are warranted. We have shown that adenoviral CD40L gene therapy is successful in bladder cancer. CD40L is a strong inducer of anti-tumor immune responses. It is also inducing growth inhibition and apoptosis in CD40 positive tumors. We have shown that the combination of 5-Fluorouracil (5-FU) and AdCD40L enhances the apoptotic effect in human bladder cancer cells. In this study we combined AdCD40L and 5-FU to treat murine orthotopic bladder cancer. AdCD40L and 5-FU synergized to inhibit the proliferation of MB49 cells in vitro and up-regulated cell surface molecules and immunoproteasome components associated with tumor antigen processing and presentation, such as MHCI, Fas, B7.1 and TAP1/2. MB49 cells were implanted into C57BL/6 mice. Mice received 3 local treatments of AdCD40L (1x10e8 ffu), 5-FU (50mg/ kg), or the combination. Survival of mice, systemic immunity and the presence of activated tumor inltrating T cells were monitored. S188
The combination of AdCD40L and 5-FU was superior to either agent alone. Thus, the combination therapy cured 68% of large orthotopic tumors while AdCD40L alone cured 32%. Cured animals were rechallenged with a s.c. injection of MB49. All cured mice treated with the combination showed systemic MB49-specic immunity. To investigate the role of the immune system, the treatment regiments were tested on MB49 cells growing in NUDE mice. Although the combination treatment could enhance survival, no mice were cured. In conclusion, AdCD40L gene therapy is promising as adjuvant treatment to conventional chemotherapy such as 5-FU.
487. Xenograft Expanded FANG Autologous Tumor Cell Vaccine Development and Manufacturing for Clinical Use
Phillip B. Maples,1 Padmasini Kumar,1 Yang Yu,1 Beena O. Pappen,1 David Monsma,2 Stephanie Scott,2 Dawna Dylewski,2 Craig Webb,2 Neil Senzer,1,3,4,5 John Nemunaitis.1,3,4,5 1 Gradalis, Inc., Dallas, TX; 2Van Andel Research Institute, Grand Rapids, MI; 3Mary Crowley Cancer Research Centers, Dallas, TX; 4 Baylor Sammons Cancer Center, Dallas, TX; 5Texas Oncology, P.A., Dallas, TX. Many tumors are of insufcient size or otherwise inaccessible for surgical ressection to allow successful autologous vaccine manufacturing. However, small accessible lesions can be obtained or small samples obtained more safely by needle biopsy. We have previously demonstrated with our TAG vector that small quantities of tumor can be expanded in immunodecient mice and harvested for clinical tumor vaccine production (P. Kumar, et al, BioProcessing J, 8(1): 30-36, 2009; BB-IND 13401). We have developed the FANG expression vector which we believe, when transfected into tumor cells, will evoke an enhanced immune recognition /stimulation versus our previous TAG vaccine vector. The FANG nonviral vector system expresses both GM-CSF and a proprietary bifunctional shRNA to furin. Preclinical data demonstrated that blocking furin protein expression in turn blocked the activation of both TGFβ1 and TGFβ2. In contrast, our TAG vector expressed both GM-CSF and a TGFβ2 antisense. Data from our TAG Phase I autologous vaccine clinical trial and others indicate that TFGβ1 overexpression is present in a wide range of cancers. In fact our data suggest that TGFβ1 expression may be up to tenfold higher than TGFβ2 in the more than thirty tumors we examined in that study. So while the TAG vector blocked TGFβ2 expression, there was no effect on TGFβ1 expression. The FANG expression vector is identical to the TAG vector except that the TGFβ2 antisense coding sequence has been replaced with the furin shRNA sequence. FANG plasmid DNA was GMP-S manufactured. The rst clinical grade xenograft FANG vaccine was manufactured from a xenograft expanded breast cancer tumor. The tumor was expanded at the Van Andel Research Institute using their colony of athymic nude mice under conditions optimized for clinical xenograft expansion. The breast cancer tumor is ER, PR negative and Her-2/ neu +++. From the time of surgery to removal of 6 F1 tumors for vaccine manufacturing was 323 days. The combined weight of the tumors was 6.2g. The cGMP manufacturing is a 2 day process that consists of tissue disaggregation into a single cell suspension, purging of murine cells, electroporation of FANG vector, overnight incubation, gamma irradiation, cryopreservation and quality control testing. The prefreeze cell viability was 90%. The transfected tumor cell GM-CSF expression was 259pg/1x106 cells/ml. Nontransfected TFGβ1 expression was 860pg/1x106 cells/ml, the transfected TFGβ1 expression was 142pg/1x106 cells/ml and the TGFβ1 knockdown was 83%. Nontransfected TGFβ2 expression was 479pg/1x106 cells/ ml, the transfected TGFβ2 expression was 127pg/1x106 cells/ml and the TGFβ2 knockdown was 73%. These data are consistent with
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy