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272. Induction of Anti-Tumoral Immune Response and IFN-Regulated Factors by Delivery of Control DNA
CANCER-IMMUNOTHERAPY: GENETICALLY MODIFIED ANTIGEN PRESENTING CELLS AND ADJUNCTS 271. Interactions between Replicating Virotherapy and Tumor Growth in the Presence of an Intact Immune System
272. Induction of Anti-Tumoral Immune Response and IFN-Regulated Factors by Delivery of Control DNA
Michael Forshaw,1 Caroline Cole,1,2 Timothy Kottke,1 Jill Thompson,1 Jian Qiao,1 Peter Selby,3 Alan Melcher,3 Richard Vile.1,2 1 Molecular Medicine Program, Mayo Clinic, Rochester, MN; 2 Department of Immunology, Mayo Clinic, Rochester, MN; 3 Cancer Research UK, St James’ Hospital, Leeds, United Kingdom.
Shulin Li, Miles Wilkinson, Xueqing Xia, Michael David, Lihong Xu. 1 Comparative Biomedical Sciences, Louisiana State University, Baton Rouge, LA; 2Immunology, MD Anderson Cancer Center, Houston, TX.
An intact immune system provides potential opportunities for, and barriers against, effective virotherapy for cancer using replication competent viruses. On one hand, anti-viral and anti-tumor immune responses may allow greater levels of immune-mediated tumor killing than viral oncolysis alone; on the other hand, the same immune responses may inhibit viral replication and spread, thereby diminishing the efficacy of the virotherapy itself. We have investigated the complex interactions between the immune system, viral replication and tumor growth by treating established subcutaneous (s.c.) B16ova murine melanomas with the Western Reserve strain of Vaccinia Virus (VV). In SCID mice, intratumoral (i.t.) injection of 1x108 pfu of VV into 8-day-old established B16.ova tumor enhanced survival (median 19 days) compared to inactivated virus (median 13.5 days) (p=0.003). The lack of a functional immune system permitted systemic spread of VV and the mice succumbed to treatment-associated disease 11 days following injection. In C57Bl/6 immunocompetent mice, a single i.t. injection of 1x108 pfu of VV led to a much smaller survival advantage of only 4 days compared to controls (p=0.01). The intact immune system did, however, protect the mice from systemic toxicity. We have analysed these two models to characterize the immune effectors involved in controlling the balance between anti-tumor efficacy and systemic toxicity. Eleven days following injection of VV, viral titres recovered from whole tumors from immunodeficient mice were significantly higher (6.9x108 pfu) than from immunocompetent mice (4.9x107 pfu) (p=0.01). In C57Bl/6 mice treated with VV, a VV-specific CD8 response was generated but C57Bl/6 mice depleted of CD8 cells did not develop systemic toxicity and there was no difference in titre of i.t. virus recovered compared to non-depleted C57Bl/6 mice. Following treatment with VV, there were elevated titres of neutralising antibody to VV in serum from C57Bl/6, but not from immunodeficient mice. Although additional daily sequential i.t. injections had no further therapeutic effects, interestingly, two i.t. injections of VV separated by 5 days, led to complete tumor regression in 30% of C57Bl/6 mice without any associated systemic toxicity. The cured mice were protected long term (>90 days) against rechallenge with 1x106 B16.ova cells. In summary, complex interactions are established in immunocompetent animals, between tumor, VV and the immune system. Neutralising Abs against VV may be a double-edged sword, which protect the host from toxicity but impede oncolysis. However, a prime-boost strategy may modulate immunity directed against viral and/or tumor antigens that can shift the equilibrium towards increased tumor cell clearance to achieve tumor regression and longterm protection. We are currently defining the immune mediators involved in this prime-boost strategy.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
Delivery of DNA encoding therapeutic genes in vivo has great potential for treating malignancy as well as genetic diseases. Delivery of placebo DNA without a transgene is used as a control in gene therapy studies. It is tacitly assumed by most investigators that the protein expressed from the transfected DNA has phenotypic consequences, but that the consequences are not from the DNA itself. Here, we demonstrate that transfection of control plasmid DNA (that does not express a gene product) into tumor cell lines induces a dramatic (>10-fold) increase in the expression of the interferon (IFN)-regulated genes IRF7, STAT1, CXCL9, MHCI and CD11a (ITGAL) in tumor cell lines. Induction of these genes inhibits tumor development and tumor growth in immunocompetent mice that are immunized with apoptotic tumor cells. The antibody depletion study indicates that the underlying mechanism by which transfection of control DNA induces IFN-regulated genes is the induction of a secreting factor such as IFN-beta. Three lines of evidence indicate that DNA transfection-mediated induction of IFNregulatory genes is independent of TLR9. The three lines of evidence are : 1) TLR9 is not expressed in either SCCVII or 4T1 cell line, 2) activation of TLR9 downstream signaling molecules is not associated with the induction of gene expression, and 3) the secretion factor (s) obtained from the conditioned medium of DNA-transfected SCCVII tumor cells induces the same type of gene expression in the 4T1 tumor cell line, which is resistant to the DNA transfection-mediated induction of IFN-regulated genes, can be used to determine the real therapeutic gene function.
273. Effects of Pre-Existing Immunity on Gene Therapy Mediated by First Generation or High Capacity Adenoviral Vectors Expressing Flt3L in a Syngeneic Glioma Model Gwendalyn D. King,1 James F. Curtin,1 Weidong Xiong,1 Chunyan Liu,1 Donna Palmer,2 Philip Ng,2 Pedro R. Lowenstein,1 Maria G. Castro.1 1 Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, CA; 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. In preclinical models, replication deficient adenovirus (RAd) expressing Herpes Simplex Virus type 1-thymidine kinase (TK) plus ganciclovir are highly efficient in causing glioma regression. While most preclinical models utilize microscopic tumors, clinical tumors are usually very large at the time of diagnosis. We have developed a macroscopic glioma model in immunocompetent, syngeneic rats. In this model, combined treatment with RAds expressing TK and human soluble FMS-like tyrosine kinase 3 ligand (hsFlt3L) cause tumor regression in 80% of rats. While adenoviral gene therapy may be a viable treatment for glioma the majority of adults have been exposed to adenovirus and are immune making long-term expression of gene therapy vectors and transgenes problematic. Here we sought to test whether pre-existing immunity to adenovirus would prevent gene therapeutic mediated tumor regression with RAd vectors but not high capacity, gutless vectors (HC). Upon injection in pre-immunized or naive macroscopic glioma bearing rats survival studies indicate that pre-existing immunity to adenovirus blocked therapeutic effects of RAd TK/hsFlt3L. Neutralizing antibody assays verified the presence or absence of S107
272. Induction of Anti-Tumoral Immune Response and IFN-Regulated Factors by Delivery of Control DNA
Michael Forshaw,1 Caroline Cole,1,2 Timothy Kottke,1 Jill Thompson,1 Jian Qiao,1 Peter Selby,3 Alan Melcher,3 Richard Vile.1,2 1 Molecular Medicine Program, Mayo Clinic, Rochester, MN; 2 Department of Immunology, Mayo Clinic, Rochester, MN; 3 Cancer Research UK, St James’ Hospital, Leeds, United Kingdom.
Shulin Li, Miles Wilkinson, Xueqing Xia, Michael David, Lihong Xu. 1 Comparative Biomedical Sciences, Louisiana State University, Baton Rouge, LA; 2Immunology, MD Anderson Cancer Center, Houston, TX.
An intact immune system provides potential opportunities for, and barriers against, effective virotherapy for cancer using replication competent viruses. On one hand, anti-viral and anti-tumor immune responses may allow greater levels of immune-mediated tumor killing than viral oncolysis alone; on the other hand, the same immune responses may inhibit viral replication and spread, thereby diminishing the efficacy of the virotherapy itself. We have investigated the complex interactions between the immune system, viral replication and tumor growth by treating established subcutaneous (s.c.) B16ova murine melanomas with the Western Reserve strain of Vaccinia Virus (VV). In SCID mice, intratumoral (i.t.) injection of 1x108 pfu of VV into 8-day-old established B16.ova tumor enhanced survival (median 19 days) compared to inactivated virus (median 13.5 days) (p=0.003). The lack of a functional immune system permitted systemic spread of VV and the mice succumbed to treatment-associated disease 11 days following injection. In C57Bl/6 immunocompetent mice, a single i.t. injection of 1x108 pfu of VV led to a much smaller survival advantage of only 4 days compared to controls (p=0.01). The intact immune system did, however, protect the mice from systemic toxicity. We have analysed these two models to characterize the immune effectors involved in controlling the balance between anti-tumor efficacy and systemic toxicity. Eleven days following injection of VV, viral titres recovered from whole tumors from immunodeficient mice were significantly higher (6.9x108 pfu) than from immunocompetent mice (4.9x107 pfu) (p=0.01). In C57Bl/6 mice treated with VV, a VV-specific CD8 response was generated but C57Bl/6 mice depleted of CD8 cells did not develop systemic toxicity and there was no difference in titre of i.t. virus recovered compared to non-depleted C57Bl/6 mice. Following treatment with VV, there were elevated titres of neutralising antibody to VV in serum from C57Bl/6, but not from immunodeficient mice. Although additional daily sequential i.t. injections had no further therapeutic effects, interestingly, two i.t. injections of VV separated by 5 days, led to complete tumor regression in 30% of C57Bl/6 mice without any associated systemic toxicity. The cured mice were protected long term (>90 days) against rechallenge with 1x106 B16.ova cells. In summary, complex interactions are established in immunocompetent animals, between tumor, VV and the immune system. Neutralising Abs against VV may be a double-edged sword, which protect the host from toxicity but impede oncolysis. However, a prime-boost strategy may modulate immunity directed against viral and/or tumor antigens that can shift the equilibrium towards increased tumor cell clearance to achieve tumor regression and longterm protection. We are currently defining the immune mediators involved in this prime-boost strategy.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
Delivery of DNA encoding therapeutic genes in vivo has great potential for treating malignancy as well as genetic diseases. Delivery of placebo DNA without a transgene is used as a control in gene therapy studies. It is tacitly assumed by most investigators that the protein expressed from the transfected DNA has phenotypic consequences, but that the consequences are not from the DNA itself. Here, we demonstrate that transfection of control plasmid DNA (that does not express a gene product) into tumor cell lines induces a dramatic (>10-fold) increase in the expression of the interferon (IFN)-regulated genes IRF7, STAT1, CXCL9, MHCI and CD11a (ITGAL) in tumor cell lines. Induction of these genes inhibits tumor development and tumor growth in immunocompetent mice that are immunized with apoptotic tumor cells. The antibody depletion study indicates that the underlying mechanism by which transfection of control DNA induces IFN-regulated genes is the induction of a secreting factor such as IFN-beta. Three lines of evidence indicate that DNA transfection-mediated induction of IFNregulatory genes is independent of TLR9. The three lines of evidence are : 1) TLR9 is not expressed in either SCCVII or 4T1 cell line, 2) activation of TLR9 downstream signaling molecules is not associated with the induction of gene expression, and 3) the secretion factor (s) obtained from the conditioned medium of DNA-transfected SCCVII tumor cells induces the same type of gene expression in the 4T1 tumor cell line, which is resistant to the DNA transfection-mediated induction of IFN-regulated genes, can be used to determine the real therapeutic gene function.
273. Effects of Pre-Existing Immunity on Gene Therapy Mediated by First Generation or High Capacity Adenoviral Vectors Expressing Flt3L in a Syngeneic Glioma Model Gwendalyn D. King,1 James F. Curtin,1 Weidong Xiong,1 Chunyan Liu,1 Donna Palmer,2 Philip Ng,2 Pedro R. Lowenstein,1 Maria G. Castro.1 1 Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, CA; 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. In preclinical models, replication deficient adenovirus (RAd) expressing Herpes Simplex Virus type 1-thymidine kinase (TK) plus ganciclovir are highly efficient in causing glioma regression. While most preclinical models utilize microscopic tumors, clinical tumors are usually very large at the time of diagnosis. We have developed a macroscopic glioma model in immunocompetent, syngeneic rats. In this model, combined treatment with RAds expressing TK and human soluble FMS-like tyrosine kinase 3 ligand (hsFlt3L) cause tumor regression in 80% of rats. While adenoviral gene therapy may be a viable treatment for glioma the majority of adults have been exposed to adenovirus and are immune making long-term expression of gene therapy vectors and transgenes problematic. Here we sought to test whether pre-existing immunity to adenovirus would prevent gene therapeutic mediated tumor regression with RAd vectors but not high capacity, gutless vectors (HC). Upon injection in pre-immunized or naive macroscopic glioma bearing rats survival studies indicate that pre-existing immunity to adenovirus blocked therapeutic effects of RAd TK/hsFlt3L. Neutralizing antibody assays verified the presence or absence of S107