313 Results of Clinical Study Phase 1 with Adenovirus Interferon γ (TG1042) in Primary Cutaneous T and B Cell Lymphomas Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© ����[.]
CANCER IMMUNOTHERAPY 310 Pancreatic Cancer Escape Variants That Evade Immuno-Gene Therapy through Loss of Sensitivity to IFN-γγ Induced Apoptosis 312 Phase I Trial of Repeated Intralesional Injection of TG1024 (Adenovirus-Interleukin-2) in Melanoma and Accessible Solid Tumours Guillermo Mazzolini, Iñigo Narvaiza, Alfonso Martinez-Cruz, Ainhoa Arina, Miguel Barajas, Juan Carlos Galofre, Cheng Qian, Jose Maria Mato, Jesus Prieto, Ignacio Melero Gene Therapy Unit FIMA, University of Navarra, Pamplona, Navarra, Spain Rochlitz Christoph,1 Morcinek Jessica,3 Reuter Juergen,1 Slos Philippe,2 Squiban Patrick,2 Dummer Reinhard.3 Medical Oncology, Kantonsspital, Basel, Switzerland; Transgene, Strasbourg, France; 3Dermatology, University Hospital, Zurich, Switzerland Combined injections into experimental tumor nodules of adenovirus encoding IL-12 and certain chemokines are capable to induce immune-mediated complete regressions In this study we found that the combination of two adenoviruses, one encoding IL12 and other MIP3 α (AdCMVIL-12+AdCMVMIP3 α) was very successful in treating CT-26 derived colon carcinomas However, in experimental tumors generated from the pancreatic carcinoma cell line Panc02 such combined treatment induces 50% of macroscopic complete regressions, although local relapses within one week are almost constant We derived cell lines from such relapsing tumors and found that experimental malignancies derived from their inoculum were not amenable to treatment in any case with AdCMVIL12+AdCMVMIP-3 α Importantly, relapsing cell lines were insensitive to in vitro induction of apoptosis by IFNγ, in clear contrast with the original Panc02 cells Comparative analyses by cDNA arrays of relapsing cell lines versus wild type Panc02 were performed revealing an important number of genes (383) whose expression levels were modified more than two-fold These changes grouped in certain gene ontology categories and should harbor the mechanistic explanations of the acquired selective resistance to IFNγ Interestingly, the expression at the RNA level of the apoptosisrelated protein clusterin is completely lost in the studied IFNγresistant cell variants Many solid tumors cannot be completely removed by surgery and not respond to radio or chemotherapy However, they are accessible to direct injection of agents such as gene therapy vectors A percentage of some advanced solid tumors, melanoma and renal cell cancer respond to systemic Interleukin-2 (IL-2) and/or interferon However, these are associated with significant toxicities Local production of IL-2, by direct injection of a gene transfer agent, should result in local production of IL2, and associated local biological impact, but with lower systemic toxicities The TG1024 product is made of a suspension of non-replicating (E1 and E3 regions deleted) recombinant adenoviral particles containing a human IL-2 cDNA insert We undertook a phase I, open-label, doseescalating trial of repeated intratumoral administration of TG1024 in patients with advanced melanoma and other accessible solid tumors Twenty patients (12 melanoma and other solid tumors were enrolled in successive cohorts at the following TG1024 doses: 3x10E8 total particles (tp), 3x10E9 tp, 3x10E10 tp, 8x10E10 and 3x10E11 Patients received intratumoral injections of TG1024 into the designated lesions on days 1, 21, and 42 (1 treatment cycle) and thereafter up to cycles, if there was no evidence of progressive disease (PD) Blood samples were taken at baseline, before injection, hours after injection and week after injection and cytokine levels evaluated A dose dependent production of circulating IL2 was noted IL2 production was particularly striking in melanoma patients Repeated administration was associated with repeated cycles of detectable IL2 in the circulation of several patients Treatment was well tolerated Most common adverse event were injections site reactions Only grade III (fever, transient lymphopenia) adverse events were noted and 3x10E11 has been considered as the maximum tolerated dose Evaluations of the patients clinical responses are still are ongoing and will be presented Our results show that local administration of an IL2 gene transfer agent can result in the production of significant levels of IL2, but does not result in the severe toxicities associated with systemic administration of IL2 This information is of importance in the design of new gene delivery-based therapeutics in cancer Principal Investigator and Consultant 311 An Evaluation of the Use of IL-1H4 Adenovirus To Induce Protein Production and Reduce Tumor Burden Mary Donahee,1 Karen Kozarsky,1 Deborah Welham.1 Protein Agents and Human Gene Therapy, GlaxoSmithKline, King of Prussia, PA IL-1H4/IL-1F7 is a 22 kD cytokine in the Interleukin(IL)-1 family that shares significant sequence homology with IL-18 Like IL-18, IL-1H4 lacks a characteristic leader peptide but contains a propeptide domain that is cleavable by members of the caspase family IL-18 has significant antitumor effects in mouse models; therefore, IL1H4 was evaluated as an antitumor agent Adenovirus vectors encoding IL-1H4 and IL-18 were used to obtain sustained protein expression in mice injected with a mouse sarcoma line Prior to in vivo testing, verification of protein expression was performed in vitro Lysates from A549, Huh-7 and HeLa cells that were transduced with Ad.IL-1H4 showed both the 22 kD mature form of the IL-1H4 protein as well as the 26 kD pro form while only low levels of secreted IL-1H4 were detected in the media In vivo, MCA205 tumor cells were introduced into C57Bl/6 male mice days prior to treatment with x 10 11 viral particles of adenovirus The treatment groups receiving the Ad.IL-1H4 or Ad.IL-18 showed a day delay in tumor growth over the control groups Subsequent experimentation was performed using a dual infection with both Ad.IL-1H4 and Ad.IL-18 Again a trend toward tumor growth reduction was seen The group receiving Ad.IL-1H4 showed a day delay in tumor growth vs the control group while the Ad.IL-18 showed a day delay The group receiving both Ad.IL-1H4 and Ad.IL-18 also showed a day delay in tumor growth, indicating no obvious synergistic effect when combining the viruses Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy 313 Results of Clinical Study Phase with Adenovirus-Interferon-γγ (TG1042) in Primary Cutaneous T and B Cell Lymphomas Urosevic Mirjana,1 Mayer Tania,1 Morcinek Jessica,1 Acres Bruce,2 Slos Philippe,2 Squiban Patrick,2 Burg Gunter,1 Dummer Reinhard.1 Dermatology, University Hospital, Zurich, Switzerland; Transgene, Strasbourg, France During the progression of primary cutaneous lymphomas (CL), production of the Th1 cytokines decreases with a shift towards the immunosuppressive Th2 phenotype This group of diseases has been therefore successfully treated with interferons (IFNs), counterbalancing the Th2-skewing state We undertook a phase I, open-label, dose-escalating trial of repeated, intratumoral administration of TG1042 in patients with advanced primary cutaneous T cell lymphomas (CTCL) and multilesional cutaneous B cell lymphomas (CBCL) TG1042 product is a suspension of non-replicating (E1 and E3 regions deleted) recombinant adenoviral S123 CANCER IMMUNOTHERAPY particles containing human IFN-γ cDNA insert Nine patients (7 CTCL, CBCL) were enrolled in successive cohorts at the following TG1042 doses: 3x10E9 total particles (tp), 3x10E10 and 3x10E11 Patients received intratumoral injections of TG1042 into the designated lesions on days 1, 8, and 15 with no injection in the fourth week (1 treatment cycle) and thereafter up to cycles, if there was no evidence of progressive disease (PD) The injected lesion was biopsied at baseline and after injections and assessed for changes in lesion’s morphology, immunohistochemical changes of T and B-cell (CD3, 4, 8, 20, 21, 79a), NK (CD56), cytotoxic (TIA-1), and dendritic cell markers (CD1a) as well as for the expression of coxsackie-adenovirus receptor (CAR) and HLA class I molecules In addition, the lesions were evaluated for the expression of transgene-derived IFN-γ as well as for total cytokine production (IFN-γ, IL-2, IL-13, IL-10) with respect to CD4 and CD8 T cells population by quantitative PCR Clinical responses and stabilizations were observed in the majority of the patients Injection site reaction was the most commonly observed adverse event Following TG1042 injections, histology demonstrated pronounced changes in infiltrate pattern differing from initial lymphoma finding, with signs of vasculitis and increase in eosinophil and neutrophil numbers Immunohistochemistry revealed increase in CD8 and TIA-1 immunoreactivity in various cell types All lesions demonstrated clear up-regulation of CAR following injection of TG1042 Quantitative PCR showed decrease in CD4/CD8 ratio in 6/8 patients Transgene-derived IFN-γ mRNA could be detected in injected lesions, implying successful IFN-γ gene transfer Our results reveal for the first time the in situ immunological changes following the administration of adenoviral vector expressing IFN-γ in primary CTCL and CBCL, offering new insight and information of importance to the design of new gene delivery-based therapeutics in cancer Principal investigator and consultant 314 Evaluation of Cytokine Gene Transfer Mediated by a Novel Cationic Liposome Preparation in an Orthotopic Bladder Cancer Model Qinghui Wu,1 Ratha Mahendran,1 Kesavan Esuvaranathan.1 Surgery, National University of Singapore, Singapore Purpose: To assess cytokine gene expression by tumor cells in vitro and in vivo in an orthotopic mouse bladder cancer model after liposome-mediated gene transfer To evaluate the treatment effect of cytokines on the growth of orthotopic tumor Materials and Methods: The murine bladder cancer cell lines MB49 was maintained in RPMI 1640 X 105 cells was instilled into the bladder of C57BL/6 mice after electric cautery to establish the tumor model The plasmid vectors were constructed by inserting the coding sequences of IFN-α1 and GM-CSF into plasmid vector pBudCE4.1 Transient transfection was performed using a cationic lipid DOTAP and methyl-b-cyclodextrin solubilized cholesterol (MBC) The expression level of IFN-α1 and GM-CSF in culture medium was checked by ELISA The effects of cytokine gene transfer on tumor cell proliferation and some surface marker expression such as MHC-I and II were evaluated The presence of the tumors was confirmed by histological examination and MRI scan of the mouse bladders The expression of the transgene in situ was confirmed by immunohistochemistry and x-gal staining Four groups of animals with orthotopic bladder tumor were treated with control vector, GM-CSF, interferon-alpha or both cytokines respectively twice a week by intravesical instillation of the gene tranfer mixture Results: Superficial bladder tumors were consistently established by intravesical instillation of MB49 cells The tumors were detectable by MRI scan The expression levels of both cytokines in transfected cell lines were increased significantly The inhibition of in vitro tumor cells proliferation and up-regulation of some surface markers S124 were observed In situ gene transfer to bladder tumors was accomplished via intravesical instillation of plasmid DNA/DOTAP/ MBC beta-galactosidase (b-gal) after a single 2h in situ transfection Using immunohistochemistry, it could be clearly seen that cytokine was produced by the urothelial cells The survival of animals in treatment group was increased as compare to control group, some tumors were even cured Conclusion: The orthotopic bladder cancer model is very useful tool to study the feasibility of potential cytokine gene therapy We demonstrated here that the orthotopic mouse bladder cancer can be transfected with a single instillation of liposome-DNA complexes The results also suggest that our liposome-mediated cytokine transfection system appears to be a potential promising non-viral gene treatment modality to bladder cancer in vivo 315 Therapeutic Interleukin-10 Gene Expression in Mammary Tumors Modulates Multiple Malignant Phenotypes and Reduces Metastatic Burden Van Tsai,1 Anja Muller,2 Albert Zlotnick,2 Brian Helmich,1 Iqbal Ahmed,1 Robert Ralston,1 Daniel Maneval,1 Drake LaFace.1 Canji, Inc., a Division of Schering-Plough, San Diego, CA; DNAX Research Institute, Palo Alto, CA We report here the use of recombinant adenoviral (rAd) vectors to induce expression of human IL-10 in established tumors to examine the capacity for suppressing tumor growth and spontaneous metastasis in a murine mammary carcinoma model Prior reports of the effect of IL-10 expression on the metastatic phenotype have relied on transfection or pre-transduction prior to tumor implantation Thus IL-10 expression had preceded the tissue responses required for establishing a solid tumor and promoting metastasis Treatment of established tumors with rAd-empty control vector resulted in modest tumor growth inhibition, whereas, tumor regression was observed in the rAd/IL-10 treated mice In Addition, spontaneous metastasis to axillary lymph nodes and lungs was markedly inhibited in rAd/IL-10 treated mice Immunohistochemical analysis revealed markedly reduced CD31 expression in rAd/IL-10 treated tumors but not in rAd/empty vector control treated tumors Moreover, rIL10 inhibited endothelial cell migration induced by activated macrophages These results support the hypothesis that IL-10 can inhibit tumor metastasis by suppressing macrophage-mediated induction of angiogenesis Additional mechanisms for inhibition of tumor growth and metastasis associated with expression of IL-10 were elucidated using a TaqMan RT-PCR based molecular profiling method We performed an analysis of differential expression profiles of 121 genes for interdependent expression profiles associated with IL-10 expression that correlated with the capacity to suppress metastasis Treatment with rAd/empty control vector induced very few changes in gene expression, whereas, rAd/IL-10 modulated expression of numerous genes that regulate tissue remodeling, angiogenesis and immune responses Importantly, IL-10 suppressed gene expression of several key metalloproteinases and pro-metastatic cytokines that have been demonstrated to promote tumor progression and metastasis in breast carcinomas The capacity of rAd/IL-10 to modulate expression of multiple mediators of this complex multistep progression may circumvent the conundrum of redundant molecular mechanisms promoting malignant phenotypes Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy ... and 3x10E 11 Patients received intratumoral injections of TG1042 into the designated lesions on days 1, 8, and 15 with no injection in the fourth week (1 treatment cycle) and thereafter up to cycles,... lines MB49 was maintained in RPMI 16 40 X 10 5 cells was instilled into the bladder of C57BL/6 mice after electric cautery to establish the tumor model The plasmid vectors were constructed by inserting... intravesical instillation of the gene tranfer mixture Results: Superficial bladder tumors were consistently established by intravesical instillation of MB49 cells The tumors were detectable by