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695. Enhanced Tumor Immunogenicity through Antigen Epitope-Modified Interferon-γ Gene Transfer
CANCER VACCINES 694. The mda-7 Gene Encodes Both Tumor Suppressor and TH1 Cytokine (IL-24) Activities – Prospects for Systemic Therapy Abner Mhashilkar,1 Suhendan Ekmekcioglu,2 Alexis Stewart,1 John Mumm,2 Kerry Sieger,1 Bryan Sutton,3 Jack Roth,2 Ramesh Rajagopal,2 Elizabeth Grimm,2 Sunil Chada.1 1 Introgen Therapeutics, Houston, TX, United States; 2The University of Texas, MD Anderson Cancer Center, Houston, TX, United States; 3Dept. of Biochemistry and Biophysics, University of Texas Medical Branch, Galveston, TX, United States. Introduction of the melanoma differentiation-associated gene 7 (mda-7) into cells using a replication-deficient adenovirus (Ad-mda7) results in growth inhibition and apoptosis in a broad spectrum of cancer cells, including those of breast, lung, colorectal, and melanoma origins. The cell killing activity of Ad-mda7 is tumor-selective as normal human cells are resistant to Ad-mda7-induced cell death. The anti-tumor activity of Ad-mda7 has been confirmed using xenograft models in nude mice. Accumulated data suggests that MDA-7 activates genes important for the onset of apoptosis. Lysates from Ad-mda7 transduced cells showed the predicted intracellular MDA-7 protein, however, a soluble larger form of MDA7 was also secreted into the media. Sequence and structural analyses revealed MDA-7 protein is a novel member of the interleukin-10 (IL-10) family. The mda-7 gene locus neighbors the IL-10 locus at 1q31/32. At a structural level, the MDA-7 protein appears to share the 6-helical configuration of IL-10. Human MDA-7 protein does not share the immuno-suppressive properties of IL-10, and appears to function in a Th1 manner. MDA-7 expression is induced in LPSand PHA-stimulated lymphocytes. Cell fractionation studies indicate that MDA-7 is expressed in NK and B-cell subsets. r.hMDA-7 treatment of human PBMC results in secretion of IL-6, gammaIFN, IL-12, TNF-a and GM-CSF. Secretion of these Th1 cytokines is inhibited by IL-10. Ad-mda7 transduction or r.hMDA-7 treatment of human melanoma cells activates STAT3 and induces secretion of IL-6 and gamma IFN. Therefore, mda-7, recently classified as IL24, appears to be a novel Th1 IL-10 homolog with pro-apoptotic properties. This unique combination of apoptosis induction and immune stimulation may provide a powerful approach for systemic treatment of cancer. Dr. Jack A. Roth is Chairman of the SAB of Introgen
695. Enhanced Tumor Immunogenicity through Antigen Epitope-Modified Interferon-γγ Gene Transfer Xianghui He,1 Tom C. Tsang,1 Phoebe Luo,1 Tong Zhang,1 David T. Harris.1 1 Gene Therapy Group, Dept. of Microbiology & Immunology, The University of Arizona, Tucson, AZ, United States. The density of tumor antigen in conjunction with major histocompatibility complex (MHC) class I molecules on the cell surface affects cytotoxic T cell (CTL) function in an active antitumor immune response. Thus, methods to enhance antigen expression/presentation could augment the effect of cancer immune therapy. In previous study, we have shown that it is feasible to modify a cytokine signal peptide with a tumor antigenic epitope. These modified signal peptides functioned as indicated by cytokine secretion, and the antigenic epitope within the modified signal peptide could be processed properly and presented on tumor cell surface. Interferon (IFN)-γ has been shown to be capable of enhancing class I MHC expression in many tumor cells. Therefore, we hypothesized that IFN-γ could synergize with the antigen epitope contained within the signal peptide to enhance tumor immunogenicity. In the current study, the signal peptide of mouse IFN-γ was replaced by tyrosinase-related protein (TRP)-2 antigen Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
epitope containing signal peptide through genetic modification. Mouse melanoma B16 tumor cells were transfected with different constructs, including epitope alone, IFN-γ alone, and antigen epitope-containing IFN-γ expression plasmids. Results showed that tumor cells transfected with antigen epitope-containing IFN-γ gene were more immunogenic as indicated by increased susceptibility to CTL lysis in vitro and decreased tumor growth in vivo, as compared with the epitope alone or IFN-γ alone gene modified tumor cells. The ability of the antigen epitope-containing IFN-γ expression plasmid to serve as a DNA vaccine is currently under investigation.
696. Suicide Gene and Cytokines Combined Non Viral Gene Therapy for Canine Spontaneous Melanoma Liliana M. E. Finocchiaro,1 Edgardo M. Soberano,2 Pablo J. J. Castillo,2 Armando L. Karara,1 Gabriel L. Fiszman,1 Gerardo C. Glikin.1 1 Unidad de Transferencia Genetica, Instituto de Oncologia Angel H. Roffo - UBA, Buenos Aires, Capital Federal, Argentina; 2 CRABA, Buenos Aires, Capital Fedral, Argentina. Canine malignant melanoma, a spontaneous and highly malignant canine tumor, may probe to be a useful animal model of human malignant melanoma. We evaluated the safety, efficacy, and antitumoral effects of direct in vivo tumor injections of both, lipidcomplexed plasmid DNA encoding herpes simplex thymidine kinase (HSVtk) co-administrated with ganciclovir (GCV) and irradiated xenogenic cells genetically modified to secrete human granulocytemacrophage colony-stimulating factor (hGM-CSF) and human interleukin-2 (hIL-2) as adjuvant of X-ray radiotherapy in canine patients with malignant melanoma. This spontaneous and highly aggressive tumor that usually appears in the oral cavity, readily metastasizes to the lungs, being the primary cause of death for most of these patients. We treated 2 dogs with stage IV disease (primary tumor plus radiographically visible lung metastases), and 8 patients with stage III tumors (primary tumor 4 cm or greater in the longest dimension, plus lymph node spread). The study was designed as an initial 5-weeks treatment. Animals were X-ray exposed (4 Gy/field) and subsequently injected intratumorally twice a week with HSVtkcarrying lipoplexes, GCV and irradiated xenogenic cells secreting 20-30 μg/day of either hGM-CSF or hIL-2 respectively. Then, dogs were evaluated for treatment-associated toxicity, tumor responses, systemic responses and survival times. Toxicity was minimal or absent in all patients. Treatment did not induce systemic toxicity, organ dysfunction or fever in any treated patient, but dogs developed transient peritumoral edema after injection. There were no significant changes in complete blood counts or serum biochemical parameters during or at the completion of the study. We found that our complete treatment induced local antitumor immunity, as evidenced by tumor regression and the development of pronounced infiltration of immune cells. The overall response rate (complete or partial remissions) was 70% (7 out of 10), being highest in patients with smaller tumors. Furthermore, local tumor injection also induced systemic antitumor immunity, as evidenced by a stage IV case of complete remission of pulmonary metastases and other stage IV case of pulmonary stable, and by the significantly prolonged survival free of pulmonary metastases observed in the 8 stage III cases treated. The median survival time of these 10 cases was 153 days with a range from 72 to 375 days. Control animals treated with either surgical tumor excision, radiotherapy or suicide gene alone developed lung metastases earlier in the course of the disease, with median survival times of 65 days with a range of 34 to 87 days. In conclusion, the use of this treatment previously to the surgical removal of the remaining primary tumor, could prevent post-surgical recurrence and metastases.
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695. Enhanced Tumor Immunogenicity through Antigen Epitope-Modified Interferon-γγ Gene Transfer Xianghui He,1 Tom C. Tsang,1 Phoebe Luo,1 Tong Zhang,1 David T. Harris.1 1 Gene Therapy Group, Dept. of Microbiology & Immunology, The University of Arizona, Tucson, AZ, United States. The density of tumor antigen in conjunction with major histocompatibility complex (MHC) class I molecules on the cell surface affects cytotoxic T cell (CTL) function in an active antitumor immune response. Thus, methods to enhance antigen expression/presentation could augment the effect of cancer immune therapy. In previous study, we have shown that it is feasible to modify a cytokine signal peptide with a tumor antigenic epitope. These modified signal peptides functioned as indicated by cytokine secretion, and the antigenic epitope within the modified signal peptide could be processed properly and presented on tumor cell surface. Interferon (IFN)-γ has been shown to be capable of enhancing class I MHC expression in many tumor cells. Therefore, we hypothesized that IFN-γ could synergize with the antigen epitope contained within the signal peptide to enhance tumor immunogenicity. In the current study, the signal peptide of mouse IFN-γ was replaced by tyrosinase-related protein (TRP)-2 antigen Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
epitope containing signal peptide through genetic modification. Mouse melanoma B16 tumor cells were transfected with different constructs, including epitope alone, IFN-γ alone, and antigen epitope-containing IFN-γ expression plasmids. Results showed that tumor cells transfected with antigen epitope-containing IFN-γ gene were more immunogenic as indicated by increased susceptibility to CTL lysis in vitro and decreased tumor growth in vivo, as compared with the epitope alone or IFN-γ alone gene modified tumor cells. The ability of the antigen epitope-containing IFN-γ expression plasmid to serve as a DNA vaccine is currently under investigation.
696. Suicide Gene and Cytokines Combined Non Viral Gene Therapy for Canine Spontaneous Melanoma Liliana M. E. Finocchiaro,1 Edgardo M. Soberano,2 Pablo J. J. Castillo,2 Armando L. Karara,1 Gabriel L. Fiszman,1 Gerardo C. Glikin.1 1 Unidad de Transferencia Genetica, Instituto de Oncologia Angel H. Roffo - UBA, Buenos Aires, Capital Federal, Argentina; 2 CRABA, Buenos Aires, Capital Fedral, Argentina. Canine malignant melanoma, a spontaneous and highly malignant canine tumor, may probe to be a useful animal model of human malignant melanoma. We evaluated the safety, efficacy, and antitumoral effects of direct in vivo tumor injections of both, lipidcomplexed plasmid DNA encoding herpes simplex thymidine kinase (HSVtk) co-administrated with ganciclovir (GCV) and irradiated xenogenic cells genetically modified to secrete human granulocytemacrophage colony-stimulating factor (hGM-CSF) and human interleukin-2 (hIL-2) as adjuvant of X-ray radiotherapy in canine patients with malignant melanoma. This spontaneous and highly aggressive tumor that usually appears in the oral cavity, readily metastasizes to the lungs, being the primary cause of death for most of these patients. We treated 2 dogs with stage IV disease (primary tumor plus radiographically visible lung metastases), and 8 patients with stage III tumors (primary tumor 4 cm or greater in the longest dimension, plus lymph node spread). The study was designed as an initial 5-weeks treatment. Animals were X-ray exposed (4 Gy/field) and subsequently injected intratumorally twice a week with HSVtkcarrying lipoplexes, GCV and irradiated xenogenic cells secreting 20-30 μg/day of either hGM-CSF or hIL-2 respectively. Then, dogs were evaluated for treatment-associated toxicity, tumor responses, systemic responses and survival times. Toxicity was minimal or absent in all patients. Treatment did not induce systemic toxicity, organ dysfunction or fever in any treated patient, but dogs developed transient peritumoral edema after injection. There were no significant changes in complete blood counts or serum biochemical parameters during or at the completion of the study. We found that our complete treatment induced local antitumor immunity, as evidenced by tumor regression and the development of pronounced infiltration of immune cells. The overall response rate (complete or partial remissions) was 70% (7 out of 10), being highest in patients with smaller tumors. Furthermore, local tumor injection also induced systemic antitumor immunity, as evidenced by a stage IV case of complete remission of pulmonary metastases and other stage IV case of pulmonary stable, and by the significantly prolonged survival free of pulmonary metastases observed in the 8 stage III cases treated. The median survival time of these 10 cases was 153 days with a range from 72 to 375 days. Control animals treated with either surgical tumor excision, radiotherapy or suicide gene alone developed lung metastases earlier in the course of the disease, with median survival times of 65 days with a range of 34 to 87 days. In conclusion, the use of this treatment previously to the surgical removal of the remaining primary tumor, could prevent post-surgical recurrence and metastases.
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