Immunotherapy of Cancer

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who were treated with HD-AdghApoA-I (1X10E13 vp/kg), we observed prolonged expression of ApoA-I, increase of HDL-C, and a modest decrease of aortic atherosclerosis. In contrast, the administration of a first generation vector expressing human Apo A-I (AdDE1BOShApoA-I) in ApoE-deficient mice produced very high toxicity and short duration of expression ApoA-I. These data suggest that over-expression of Apo A-I with a HD Ad vector has potential application for treatment of atherosclerosis especially in patients with reduced HDL-C levels.

707. Characterization of the Complex Physiological Response to Constitutive VEGF Gene Expression

Matthew L. Springer*, Randall Lee†, Gonzalo Hortelano‡, Donna Bouley§, William E. Blanco-Bose*, Helen M. Blau* *Stanford University, Dept. of Molecular Pharmacology †University of California, San Francisco, Department of Medicine ‡McMaster University, Dept. of Pathology and Molecular Medicine §Stanford University, Dept. of Comparative Medicine We have examined the effects of location and mode of consitutive vascular endothelial growth factor (VEGF) gene delivery on tissues at four different sites. VEGF constantly expressed in non-ischemic adult tissue results in a complex cellular response involving the appearance of both true blood vessels and a mixture of endothelial cells, smooth muscle cells, and inflammatory cells. Depending on the mode and site of gene introduction, this process can lead to the formation of hemangiomas. Implantation of VEGF-overexpressing myoblasts in skeletal muscle of the leg leads ultimately to large hemangiomas within 1.5 months, whereas implantation of the same cells into the heart leads to small myocardial hemangiomas that are fatal within 2 weeks. By contrast, when these cells are encapsulated in alginate and implanted subcutaneously, there is inflammation and substantial recruitment of cells including smooth muscle cells to the implantation sites, accompanied by angiogenesis. Intraperitoneal implantation of these encapsulated cells leads to similar inflammation and cell recruitment, along with angiogenesis and vascular leakage into the peritoneum. Closer examination of intramuscular implantation sites using confocal microscopy or injectable non-leaky tracer bead suspensions reveals these implantation sites to consist both of capillaries that are continuous with the vasculature and of individual endothelial cells that may correspond to circulating endothelial precursor cells. In some cases there is a visible capillary-rich zone directly adjacent to the implantation sites, but there is no significant increase in capillary density either outside that zone or in neighboring muscles. Work is ongoing to determine whether or not the increase in capillaries is more pronounced in ischemic muscle and if regulatable expression of the VEGF transgene can lead to the orderly growth of non-leaky vessels without the associated deleterious effects of overexpression.

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708. Effective Particle-mediated Vaccination Against Mouse Melanoma by Co-administration of Plasmid DNA Encoding gp100 and Granulocyte-macrophage Colony Stimulation Factor

Alexander L. Rakhmilevich*, Michael Imboden*, Zhengling Hao*, Michael D. Macklin†, Timothy Roberts*, Kelly M. Wright*, Mark R. Albertini*, Paul M. Sondel*, Ning-Sun Yang*‡ *Comprehensive Cancer Center, University of Wisconsin-Madison, Madison, WI †PowderJect Vaccines Inc., Madison, WI ‡Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, R. O. C. DNA vaccination by particle-mediated gene delivery was used to immunize mice against melanoma. Mice were immunized

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with an expression vector for cDNA coding for the human melanoma-associated antigen, gp100. Murine B16 melanoma, stably transfected with human gp100 cDNA, was used as a tumor model. Particle-mediated delivery and expression of gp100 cDNA in the skin of naive mice resulted in significant tumor protection from subsequent tumor challenge. In comparison, co-delivery of granulocyte macrophage colony-stimulating factor (GM-CSF) cDNA together with gp100 cDNA consistently resulted in a significantly greater level of protection from tumor challenge. The inclusion of GM-CSF cDNA with the gp100 cDNA vaccine also allowed a drastic reduction in the gp100 cDNA dose required for antitumor efficacy, to as little as 62.5 ng per vaccination. Tumor protection induced by the gp1001GM-CSF gene combination was T cell-mediated, as it was abrogated in the vaccinated mice treated with anti-CD4 plus anti-CD8 monoclonal antibodies. In a more clinically relevant experimental system, DNA cancer vaccine was given to mice bearing established (7 day-old) tumors. Administration of the gp1001GM-CSF DNA vaccine to these tumor-bearing mice resulted in a significant suppression of tumor growth and extended survival in the test animals. These results indicate that inclusion of GM-CSF cDNA can augment the efficacy of particle-mediated vaccination with gp100 cDNA, and warrants testing as cancer vaccine therapy of human melanoma. This work was partially supported by the U. S. Army Medical Research Acquisition Activity Grant under DAMD17-96-26017.

709. IL-12-enhanced Anti-tumor DNA Vaccination Studied in Gene Knockout Mice

K. Song, Y. Chang, G. J. Prud’homme Department of Pathology, McGill University, 3775 University Street, Room B13, Montreal, Qc, Canada H3A 2B4 Object: A major limitation of anti-tumor vaccination is the poor imunogenicity of most tumor-associated antigens (TAAs). In this study, we used intramuscular (i.m.) injection of a plasmid encoding human carcinoembryonic antigen (CEA) to generate protective immunity against tumors. To analyze the immune mechanisms of tumor rejection we immunized various immune-gene knockout (KO) mice. Materials and Methods. We immunized mice of C57BL/6-derived immune-gene KO strains, deficient in either CD3, CD4, CD8, interferon g (IFNg), perforin or Fas ligand (FasL). Mice were immunized by i.m. injection of an expression plasmid encoding CEA, with or without coinjection of an IL-12 p35/p40 bicistronic plasmid. Following 5 weekly i.m. injections of plasmids, immunized mice were injected s.c. in the flank with syngeneic (C57BL/6) CEA-positive Lewis lung carcinoma (CEA/LLC) cells. Results. We show that i.m. injection of wild type (Wt) mice with a plasmid encoding CEA elicits both humoral and cellular antiCEA immunity in mice, and partial resistance to transplanted CEA/LLC cells. Nonetheless, tumors still appear in all the mice. Coinjection of the IL-12 plasmid markedly enhances anti-CEA humoral, T-helper-1 (Th1) and cytotoxic T lymphocyte (CTL) responses, as well as resistance to a CEA/LLC tumor challenge such that 80% of mice remain tumor free. In gene KO mice, we find that protective immunity is entirely CD3 (T-cell) dependent. Only mice expressing both CD4 and CD8, which appear equally important, and injected with a mixture of the CEA and IL-12 vectors, can fully resist a tumor challenge. Coinjection of the IL-12 vector improves resistance to a tumor challenge in all strains tested, except CD3-KO mice. IL-12 increases IgG antiCEA levels in a CD3/CD4-restricted manner. It promotes antiCEA/LLC delayed-type hypersensitivity (DTH) which is CD3/ CD4-dependent, and CTL activity which is CD3/CD8/perforindependent. FasL-KO mice have completely normal responses, indicating only the perforin cytolytic pathway is involved. MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

IMMUNOTHERAPY CEA-stimulated IFNg production occurs in either CD4- or CD8-KO (but not CD3-KO) mice and is enhanced by IL-12. In IFNg-KO mice, IL-12 only partially restores impaired DTH and tumor resistance. Summary and Conclusion. DNA vaccination can induce protective immune responses against tumors, and these responses can be markedly enhanced by codelivery of cytokine expression plasmids. Mice were immunized by i.m. injection of a plasmid encoding human CEA, with or without coinjection of a plasmid encoding murine IL-12. CD3 (restricted to T cells) was essential for protective immunity, and CD41 and CD81 cells appeared equally important. CTL activity was mediated via perforin, but not FasL. IL-12 augmented anti-CEA antibody production, provided CD3 and CD4 were expressed. IFNg-deficient mice had impaired anti-CEA DTH and reduced tumor resistance, which was only partially restored by IL-12. Only mice inoculated with both vectors and expressing all of CD3, CD4, CD8, perforin and IFNg could completely eliminate tumors. The results suggest that protective DNA vaccination depends on multiple effector components, which are all enhanced by IL-12.

710. Induction of Antitumor Immunity by Adenovirus Vectors Encoding Melanoma-associated Antigens

J. Kaplan*, M. Perricone*, Q. Yu*, K. Claussen*, L. Woodworth*, W. Siders*, K. Pavelka*, K. Hubley*, P. Gauthier*, K. Smith*, M. Goldberg†, B. Roberts† *Genzyme Corporation †Genzyme Molecular Oncology The immunizing and anti-tumor potential of recombinant adenovirus (Ad) vectors encoding various melanomaassociated antigens (gp100, TRP-1, TRP-2, MART-1) was tested in the murine B16 melanoma model. Comparison of various immunization protocols indicated that optimal induction of anti-tumor responses was obtained following the injection of vector at multiple intradermal sites. The potency of anti-tumor protection varied for the different antigens tested and ranked as follows: murine TRP-2 . human gp100 . murine TRP-1 . MART-1 . murine gp100 5 empty vector. The intradermal mode of Ad delivery resulted in transduction of Langerhans cells in the skin, as determined by immunohistochemistry, and a single administration of vector was sufficient to induce a dose-dependent cytotoxic T lymphocyte (CTL) response against the encoded tumor antigen. The induced CD81 effector cells exhibited cytolytic activity against B16 tumor cells and antigen-transduced target cells in a chromium release assay, and produced interferon-gamma upon antigenic stimulation in an ELISPOT assay. Antigen-reactive CTLs were detectable in the spleen, lymph nodes and peripheral blood of immunized mice. An overall correlation was observed between the dose of vector, the magnitude of the CTL and ELISPOT responses induced and the level of protection achieved against a lethal challenge with B16 tumor cells. However, the antitumor effect elicited by Ad vector immunization was abolished, not only in mice with deficient CD81 T cell activity (B2-microglobulin knock-out), but also in CD4 knock-out mice and in mice with defective NK cell activity (beige mice) indicating that each of these cell populations was required for the development of optimal anti-tumor immunity. In a therapeutic setting, immunization with Ad vector was successful in preventing or delaying established tumor cell growth but multiple administrations of vector were required. In conclusion, our results indicate that recombinant Ad vectors encoding tumor-associated antigens MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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711. Clinical Studies of Immunogene Therapy Using Autologous GM-CSF Transduced Tumor Vaccines (GVAX) for Stage IV Renal Cell Cancer

Kenzaburo Tani*, Yukoh Nakazaki*, Hidenori Hase*, Keisuke Takahashi*, Maki Monna*, Fumihiko Komine*, Reiko Kitamura*, Utako Machida*, Junko Ohata*, Yasushi Soda*, Kiyoshi Watari*, Atsuko Masunaga*, Noriharu Satoh*, Arinobu Tojo*, Naohide Yamashita*, Taira Maekawa*, Masazumi Eriguchi*, Shinji Tomikawa*, Kisaburo Hanazawa†, Yoshiaki Wakumoto†, Koji Kawai‡, Hirofumi Hamada§, Miyuki Azuma††, Koh Okumura†, Tadao Kakizoe¶, Hideyuki Akaza‡, Makoto Fujime†, Richard C. Mulligan\, Shirley Clift**, Dale Andoh**, Stephan Sherwin**, Shigetaka Asano* *The Institute of Medical Science, The University of Tokyo †Juntendo University, School of Medicine, ‡University of Tsukuba, School of Medicine, §Sapporo Medical University, ¶ National Cancer Center, \Harvard Medical School, **Cell Genesys ††Tokyo Medical and Dental University, School of Medicine There is no effective treatment for patients with Stage IV renal cell cancer (RCC). The introduction of new therapy is needed. Vaccination with autologous GM-CSF transduced renal tumor cells (GVAX) is one of promising choices to overcome this situation according to preclinical animal studies. Our preliminary data showed that we could safely produce lethally irradiated retroviral transduced autologous RCC secreting GMCSF at the University of Tokyo’s Gene Therapy Unit. We now have treated two Japanese men suffering from Stage IV RCC with metastases with autologous GVAX vaccines. The 1st patient was a 60 year old male with multiple lung and liver metastases. He was injected intradermally with a total of 2.2x108 cells over 10 vaccinations. During the course of vaccination, the growth of the largest lung hilar mass deccelerated; this was volumetrically calculated on CT scan. At the time of 10th injection, he was treated with low dose interleukin-2 (IL-2) intravenously. A week after starting IL-2, his right hilar mass lesion became smaller and 30% of the total volume disappeared in 1 month. Unfortunately, he died of multiple RCC metastases at 9 months after nephrectomy and 7 months after initiation of GVAX. Autopsy findings demonstrated that he had multiple metastasis to lungs, pleura, liver, kidney and paraaortic lymph nodes. Of interest is that lung tissue arround the necrosis was infiltrated with CD8 positive T lymphocytes. T cell receptor Vb CDR length analysis using PCR-SSCP method demonstrated the oligoclonal expansion of T cell clones at the site of necrotic RCC lung metastasis. The combination effects of GM-CSF transduced autologous RCC vaccines and low dose IL-2 might have contributed to the reduction of the large hilar mass lesion. Our 2nd patient was 71 year old man with large sacral metastasis. One month after the local irradiation of 30Gy, he was nephrectomized and the GVAX was produced. He was injected with a total of 3.7x108 cells over 17 vaccinations. During the course of vaccination, the sacral tumor remained stable on CT scan. A thallium scan showed decreased uptake of Tl at the tumor site. His peripheral blood lymphocytes showed persistent cytotoxic activity to his own tumor cells. GVAX treatment in StageIV RCC was safely performed and some immunological antitumor reaction was obtained. Further clinical studies are required to elucidate the clinical significance of GVAX treatment.

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712. CD40-targeted Adenoviral Gene Transfer to Human Cutaneous Dendritic Cells In Situ

Tanja de Gruijl*‡, Sylvia Luykx-de Bakker*‡, Bryan Tillman†, Fons van den Eertwegh*‡, Hidde Haisma*, David T Curiel*†, Rik Scheper‡, Herbert Pinedo*, Winald Gerritsen* *Division of Gene Therapy,Department of Medical Oncology,University Hospital Vrije Universiteit, Amsterdam †Gene Therapy Center,University of Alabama, Birmingham, AL ‡Department of Pathology,University Hospital Vrije Universiteit, Amsterdam As transduction of non-professional antigen presenting cells accompanying genetic vaccination may lead to T cell tolerance, the aim of this study was the targeted and selective transduction of Dendritic Cells (DC) in situ for optimal cytotoxic T lymphocyte (CTL) activation. Adenoviral vectors (Ad) complexed to a bispecific antibody conjugate, which neutralized the receptor-binding site on the Ad and agonistically bound to CD40 on the DC surface, were used to achieve targeted transduction and simultaneous activation of cutaneous DC in ex vivo cultured skin explants. Transduction was visualized by b-Galactosidase (b-Gal) activity after intradermal injection of CD40-targeted Ad containing the LacZ gene together with 100ng Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). Intradermal injection of 108 pfu untargeted Ad-LacZ and GMCSF led to the expression of b-Gal in large numbers of cells in the dermis with less than 0.1% of these consisting of CD1a1 DC. By contrast, injection of an equal amount of CD40-targeted Ad-LacZ resulted in a 100-fold reduction of the absolute number of transduced cells in the dermis with over 50% of the remaining cells consisting of CD1a1 DC. These cells retained their capacity to migrate out of the dermis, as observed in a 48-hour migration assay. In addition, in vitro tests indicated a superior ability of CD40-Ad transduced monocyte-derived DC (MoDC) to prime specific CTL, recognizing an HLA-A2 binding epitope derived from the Her-2/neu sequence. The more selective in situ transduction of cutaneous CD1a1 DC and the in vitro observation of an enhanced activation state and CTL stimulatory capacity of transduced MoDC, make CD40targeted Ad vectors a promising modality for tumor vaccination.

713. Genetic Approaches to Sustain the Function of Tumor–specific T Lymphocytes: Comparison of Tumor Antigen–specific, Chimeric CD28 and 4-1BB Co–receptors in Genetically Modified Human Primary CD41 and CD81 T Lymphocytes Anja Krause, Michael Gong, Cuiwen Tan, Michel Sadelain Departments of Human Genetics and Immunology Program, Memorial Sloan-Kettering Cancer Center

The difficulty to generate tumor-specific lymphocytes and sustain the activity of cultured lymphocytes upon infusion in the recipient are severe obstacles to the success of adoptive cell therapies directed against cancer. Effective T cell function and survival critically depend on T cell receptor (TCR)–mediated activation and provision of a second costimulatory signal, such as that elicited by engagement of CD28 with its natural ligands, B7.1 and B7.2, or of alternative costimulatory molecules like 4-1BB. Adoptively transferred lymphocytes are prone to cell death and/or anergy secondary to cytokine withdrawal or lack of costimulatory ligands at the tumor site. We have previously shown that antigen– dependent CD28 signaling by a chimeric CD28 coreceptor specific for GD2 ganglioside selectively enhances survival and proliferation in genetically modified human primary CD81 T cells (Krause et al, 1998). We also investigated the 4-1BB coreceptor because it appeared to inhibit activation–induced cell death preferentially in CD81 T cells, acting in both CD28 dependent and independent manners. To provide antigen-dependent costimulation, we expressed in human peripheral blood lymphocytes 4-1BB and CD28 fusion molecules (PBB-1 and

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P28-1) that are engaged by PSMA, an alternative ligand highly expressed by prostate cancer cells. Recognition of PSMA was provided by a single-chain antibody derived from the PSMA– specific monoclonal antibody (mAb) J591. Primary human T lymphocytes were transduced with gibbon ape leukemia viruspseudotyped recombinant retroviruses encoding the P28-1, PBB-1 fusion protein or P28TR, a control molecule truncated of the cytoplasmic domain. All constructs coexpressed the enhanced green fluorescence protein eGFP allowing to monitor efficiency of gene transfer in human PBL (20-40%) by FACS analysis and sort transduced T cell populations to high purity. Antigen-dependent reactivation studies in primary T lymphocytes cocultured on PSMA expressing fibroblasts led to preferential activation of IFN g secretion rather than IL2 secretion in 4 –1BB–transduced CD81 T cells. The opposite pattern was obtained with the CD28 –transduced CD81 T cells. In both instances, cytokine secretion was PSMA–specific and PSMA– dependent. Most importantly, a sustained proliferative response and expansion was obtained in the PBB-1 transduced CD81 T cells, beyond that obtained in the P28-1 transduced CD81 T cells. The effect of PBB-1 in CD41 T cells was more moderate in comparison to that of P28-1 in transduced CD41 T cells. Thus, the PBB-1 fusion receptor provides a long term survival signal to activated human primary T cells, distinct from and beyond that afforded by CD28 signaling. T lymphocytes given such an antigen-dependent survival advantage may be useful in the context of adoptive cell therapies. In vivo studies are under–way

714. In Vitro Immunogenicity of a P210BCR-ABL Fusion Domain Candidate DNA Vaccine Targeted to Human Dendritic Cells by an rAAV Vector JY Sun, S Chatterjee, SJ Forman, D Senitzer, I Sniecinski, KK Wong Jr.* (*corresponding author) Departments of Hematology and Bone Marrow Transplantation, Virology and Transfusion Medicine, City of Hope National Medical Center, Duarte, CA

Chronic myelogenous leukemia (CML) is characterized by a t(9; 22) translocation which results in the expression of chimeric, fusion transcripts consisting of cellular bcr and abl genes. BCRABL fusion transcripts and corresponding oncoproteins are both characteristic and unique to the leukemic clones. Furthermore, of hematologic malignancies, the protective role of host immune responses has been most convincingly demonstrated for CML. Thus, methods to exploit and specifically enhance these responses could reduce the risk of relapse following hematopoietic stem cell transplantation (HSCT) for CML. As a strategy to augment anti-leukemic immune responses, peptides derived from the fusion domain of the P210BCR-ABL oncoproteins have been intensively studied as immunogenic, tumor-specific antigens. Although promising and demonstrating the feasibility of this approach, results to date have been somewhat variable. To circumvent potential problems with the use of peptide immunogens, we developed a candidate DNA vaccine based on recombinant adeno-associated virus (rAAV) vectors which targets the P210BCRABL fusion region. We previously demonstrated that primary human dendritic cells (DCs), the most potent antigen presenting cells currently known, could be efficiently transduced with rAAV vectors. An 835 bp cDNA fragment containing the P210BCR-ABL b3a2 variant fusion domain and flanking sequences was inserted into an rAAV vector under control of the RSV promoter, yielding pCWRF8. 293 cells transfected with pCWRF8 expressed an appropriately sized transcript, as well as the predicted 25 KDa truncated protein. Using an institutionally approved protocol, PBMCs from healthy donors were primed and restimulated in vitro with autologous DCs transduced with vCWRF8 (DC/F8), vCWRAP, an equivalent control vector encoding human placental alkaline phosphatase (hu-PLAP)(DC/AP), or pulsed with a 25-amino acid peptide (Pb3) corresponding to the p210 BCR-ABL fusion domain (DC/b3). No specific responses were generated using DC/AP, the negative transduction control. In contrast, MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

IMMUNOTHERAPY DC/F8 was capable of priming autologous T cells to an equivalent extent as DC/b3. Proliferative responses were demonstrable in three donors with MHC alleles previously described in the literature as capable of responding to the BCR-ABL fusion domain, but not in two donors with divergent alleles. Furthermore, DC/F8 were also capable of presenting immunogenic epitopes present in sequences flanking the BCR-ABL fusion domain. All antigen specific T cell lines generated to date were phenotypically CD41 and Th1. Importantly, several T cell lines demonstrating antigen specific (BCR-ABL b3a2 subtype) cytotoxicity were identified. A cytotoxic T cell clone obtained from a representative cell line primed with DC/F8 recognized a cytotoxic epitope presented by HLA-DR 15 (DRB1*1501 allele). Thus, the construct developed herein may serve as an important candidate for gene-based immunotherapy of CML. Key words: Chronic Myelogenous Leukemia (CML), Dendritic Cells (DC), and recombinant Adeno-Associated Virus (rAAV).

715. Intratumoral Injection of Dendritic Cells Genetically Modified to Express Interleukin-12 Induces Antitumor Immunity and Inhibits Established Tumor Growth and Spontaneous Metastases

Yasuhiko Nishioka*, Naoki Nishimura*, Paul B Robbins†, Michael T Lotze†, Saburo Sone*, Hideaki Tahara‡ *Third Department of Internal Medicine, University of Tokushima School of Medicine, Tokushima 770-8503, Japan †Department of Surgery and Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA ‡Department of Surgery, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan Effective antitumor immunity might be induced with the delivery of dendritic cells (DCs) to the site of tumor antigen. However, DC functions could be disturbed within the tumor microenvironment. In this study, we investigated the antitumor effects of intratumoral injection of murine bone marrow-derived DCs (BMDCs) transduced with IL-12 gene. Murine BM-DCs generated with GM-CSF and IL-4 were transduced with DFG-mIL-12 retroviral supernatant produced by BOSC23 producer cells using centrifugal method on day 2, 3 and 4 culture days. IL-12 genemodification of DCs was comfirmed by RT-PCR for IL-12 p40 transgene. IL-12 gene-transduced DCs produced bioactive IL-12 protein (4-80 ng/10*6 cells/48h). FACS analysis showed that IL-12 gene-transduced DCs have an increased level of MHC class I and classII expressions. Antitumor effect of IL-12 gene-modified DCs examined against Lewis lung carcinoma (LLC), MCA205 fibrosarcoma and B16 melanoma cells. Intratumoral injection of nontransduced DCs or Zeo-transduced DCs did not show any effect for tumor growth. However, intratumoral administration of IL-12 gene-modified DCs resulted in regression of day 7 subcutaneously established tumors in all cases. Next, to examine antimetastatic effect of this approach, IL-12 gene-modified DCs were injected into LLC tumor established (day 10) in foot pad and their tumor-bearing leg were amputated. Three weeks later, mice were sacrificed and metastatic nodules and lung weight were evaluated. Treatment with IL-12 gene-trasduced DCs significantly reduced the number of lung metastastatic colonies. These antimetastatic effects were more profound than that of treatment with the same dose of recombinant IL-12. Furthermore, intratumoral injection with IL-12 gene-modified DCs induced Th1 responses (IFN-gamma production) to tumor in regional lymph nodes and spleen. These responses were, at least in part, specific for tumors used. CTL activity induced by treatment with IL-12 gene-transduced DCs was significantly higher than that observed in other groups. Immunohistological analysis showed the marked infiltration of CD41 and CD81 T cells into tumors treated with IL-12 gene-modified DCs. These results indicated that this strategy is effective in inducing systemic and therapeuMOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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tic antitumor immunity and support clinical application for patients with various tumors.

716. Development of a Herpes Virus Vector-mediated Approach to Cancer Immunotherapy Using Transduced Human Dendritic Cells Laila Samady*, Katherina Speidel†, Benjamin Chain†, Jonathan Ledermann‡, David Latchman§, Robert Coffin*¶ *Deparment of Molecular Pathology, Windeyer Institute, University College London †Department of Immunology, Windeyer Institute, University College London ‡Department of Oncology, Windeyer Institute, University College London §Institute of Child Health ¶ Neurovex Ltd, University College London

Dendritic cells (DC) are specialised antigen presenting cells that play an important role in the initiation of antigen-specific T cell immune response. Thus DC might be used for immunisation following delivery of genes encoding for tumour-associated antigens towards which a specific immune response might be raised. DC can be generated from human peripheral blood mononuclear cells by culture in selective medium containing GM-CSF and IL-4. However, for the approach described a significant problem has been to efficiently transduce DC. We have previously shown that an essentially wild type herpes simplex virus (HSV-1) can be used to transfer genes to DC. However, in order to use HSV-1 as a vector for gene delivery, it must be disabled for safety and to minimise toxic effects. A panel of disabled HSV-1 vectors containing reporter genes GFP and LacZ have been tested in DC in order to identify an optimal vector for gene delivery. Two vectors have been identified, either partially or fully disabled, which give the best results in DC at low multiplicity of infection (MOI51; respectively 82% and at least 30% gene delivery). No cell death was observed. The phenotype of infected DC assessed by FACS analysis remained unchanged as compared to uninfected cells. T-cell proliferation assays using exogenous CD3 antigens showed that infected DC retained an ability to stimulate CD81 cells. Transduced DC also secrete normal levels of IL-12 following stimulation. Based on these results, we have now constructed two novel HSV-1 vectors expressing hepatitis B surface antigen (HbsAg) in place of the reporter genes. These vectors are being used as a model to infect DC generated from human peripheral blood of vaccinated donors and to test their ability to raise a HbsAg-specific T-cell response. Vectors containing tumour-associated antigens are also under construction and further experiments are underway to confirm the functionality of transduced DC.

717. Long-term Remission of Pre-established Hepatic Metastases from Colorectal and Breast Cancer by In Vivo Adenoviral-mediated Transfer of the IL-12 and 4-1BB Ligand Genes. Olivier Martinet*†, Bernhard V. Sauter*, Vanadyia Ermekova*, Celia M. Divino*†, Savio L.C. Woo*, Shu-Hsia Chen* *Institute for Gene Therapy and Molecular Medicine †Department of Surgery, Mount Sinai School of Medicine, New York, NY

IL-12 is a cytokine that has been shown to generate a potent NK and Th1 cell response to a variety of cellular antigens. In animal models of liver metastases from colon and breast carcinoma, we have previously shown the efficacy of in vivo adenovirus-mediated IL-12 gene transfer. NK cells were the early and major effectors of the IL-12-induced anti-tumor immune response. However, only a small percentage of the treated mice developed a CTL response required for long-term systemic anti-tumor immunity, and most animals succumbed to relapsing tumors. 4-1BB is a costimulatory molecule expressed on the surface of activated T cells. Interaction with its natural ligand (4-1BBL) has been shown

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to amplify T cell (especially CD81) mediated immunity. This study was designed to investigate the potential of an in vivo adenoviral-mediated combined IL-12 and 4-1BBL gene therapy in syngeneic metastatic colon and breast cancer models. Liver metastases were induced in BALB/c mice by direct intrahepatic injection of MCA26 colon or JC breast cancer cells. 7 days later, various combinations of replication-defective adenoviruses expressing IL-12 and 4-1BBL genes were injected in the established tumors. In a long-term survival study, 62% (colon cancer model) to 78% (breast cancer model) of mice treated with the combination IL-12/4-1BBL were alive more than 120 days after tumor inoculation, while 70% of IL-12 treated animals (P,0.02) and all the animals in 4-1BBL alone and control groups died within 30 to 50 days (P,0.001). In vivo NK or CD81 T cell depletion completely abolished the long-term survival advantage of the IL-12/ 4-1BBL treated animals (P,0.002), whereas CD41 T cell depletion did not modify it. Long-term survivors (.120 days) rejected s.c. challenge with parental tumor cell, while in vivo NK or T cell depletion allowed tumor growth in 88% and 100% of the animals respectively (P,0.004). Moreover, the induced anti-tumor immunity was tumor specific since tumor growth was observed in 75 to 88% of long-term survivors receiving a s.c. challenge of unrelated tumor cells. The combination of IL-12 and 4-1BBL is synergistic in the generation of a systemic and specific antitumor immunity. NK and CD81 T cells are the major immune effector cells. This work was partially supported by NCI grants RO1-CA75175 and R29-CA70337 to S.H. Chen.

718. Generating Cytotoxic T Lymphocytes Against Cervical Carcinoma with Lentivirus-transduced Antigen-presenting Cells H.T. Spencer*†, A. Galloway†, L. Pirisi-Creek*, J. Evans‡, V.J. Garcia‡, F. van Rhee† *University of South Carolina †South Carolina Cancer Center ‡University of Texas Southwestern Medical Center

A causative relation between Human Papilloma Virus (HPV) and cervical carcinoma is well established; HPV serotype 16 is most commonly associated with cervical carcinoma. HPV infects keratinocytes which then present foreign viral antigens, making cervical carcinoma an attractive target for immunotherapy. The 98 aa HPV oncoprotein E7 is constitutively overexpressed and required for immortalization of transformed keratinocytes. Cytotoxic T-cell responses have been generated to peptides derived from E7 in the context of several HLA-A molecules making E7 an excellent antigenic target for immunotherapy. However, HPV infected keratinocytes do not express co-stimulatory molecules and are, therefore, poor inducers of immunologic T-cell responses, and professional antigen presenting cells such as dendritic cells are required to stimulate patient derived T-cells. The E7 protein has 2 Cys-X-X-Cys zinc finger motifs at position 58-61 and 91-94 that are critical for transformation ability. A double mutant of E7, C58G/C91G, has been reported to have abrogated transforming ability and be more unstable, resulting in increased processing and presentation by HLA-molecules. We have made single, double, and triple mutants of C58G, C91G and C94V in order to abrogate the transforming ability of wt-E7. The peptide E786-94 has been reported to be immunogenic in the context of HLA-*A201 and we substituted the Cys at position 94 with Val, based on HLA-*A201 anchor position affinities, which predicts to increase the binding affinity of this mutant fourteen-fold. The relative expression and stability of these mutant proteins compared to wt-E7 is currently being tested in stably transfected homozygous HLA-*0201 Epstein-Barr virus transformed B-cells (LCLs). We have also generated a T-cell clone specific to HLA*0201 that recognizes E711-20 which is used to confirm normal processing and presentation of mutant E7 peptides and to determine which mutant E7 protein is best presented at the cell surface. In parallel studies we have used various gene transfer

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methods and measured transduction efficiencies of GFP into antigen presenting cells. We observed .40% transduction efficiency into dendritic cells differentiated from transduced CD341 cells and 60-85% transduction efficiency into LCLs using VSV-G pseudotyped recombinant human immunodeficiency virus. We propose, therefore, to use mutant E7 transduced dendritic cells for priming of T-cell lines and use patient derived mutant E7 transduced LCLs for re-stimulation. LCLs will provide a readily available large resource of gene modified antigen presenting cells, are highly immunogenic, and express critical co-stimulatory and adhesion molecules. We believe that mutant E7 specific CTLs can be generated ex vivo using lentivirus-modified patient derived CD341 cells and LCLs, and that these CTLs can be used in the treatment of cervical carcinoma.

719. Identification of the In Vivo Antitumor Effector Molecules in the GM-CSF-immuno Gene Therapy

Hidenori Hase*, Kenzaburo Tani*, Yukoh Nakazaki*, Arinobu Tojo*, Yusuke Nakamura†, Shigetaka Asano* *The Department of Hematology / Oncology, The Institute of Medical Science, The University of Tokyo, Japan. †Human genome center, The Institute of Medical Science, The University of Tokyo, Japan. GM-CSF (granulocyte macrophage colony stimulating factor) is considered to be one of the most potent cytokines that induces anti-tumor immunity in vivo. In this mechanism, GM-CSF activate professional antigen-presenting cells (APC), such as dendritic cells, by up-regulating their migration, maturation, antigen capturing, processing and presenting pathways. Thus antigens addressed to APC in the presence of GM-CSF are considered to stimulate effectively the proliferation of antigen-specific helper T lymphocytes and the subsequent expansion of anti-tumor effector cells. Few reports, however, describe the substantial molecular mechanism working at the anti-tumor immunity in vivo. By using the GM-CSF transduced tumor cells, we have recently established several mice models to study in vivo anti-tumor immunity. For instance, subcutaneously injected wild-type WEHI 3B cells in immunocompetent syngeneic BALB/c mice (WTB) proliferated to make large tumor causing death. But GM-CSF gene transduced WEHI 3B (GMCSF/WEHI) cells lost their original tumorigenicity in immunocompetent BALB/c mice (GMB). On the other hand, inoculated GMCSF/WEHI cells into immunodeficient BALB/c (nu/nu) mice (GMN) retained their tumorigenicity. Based on this background, we analyzed and compared the gene expression profile by serial analysis of gene expression (SAGE) method in tumor tissue in the above three mice tumor model system. As a result, a total of 90,000 tags was sequenced and most of the tags did not show any remarkably different expression. However, 9 tags were expressed at least 20-fold greater in the suppressive state (GMB) than other progressive states (GMN or WTB), and 2 tags were expressed at least 50-fold higher in the progressive states than suppressive state. The genes highly expressed in suppressive state were considered to play important roles in GM-CSF included tumor regression in vivo.

720. Treatment of Mouse Transplantable Hepatoma by Intravenous Injection of a Polyplex Comprising a Novel Receptor - mediated Composite Polypeptide and p21/GM-CSF DNA

Xiang Liu*, Pei-Kun Tian*, Xue-Tao Cao†, Jian-Ren Gu* *National Laboratory of Oncogenes and Related Genes, Shanghai, P.R.China †Institute of Immunology, The Second Military Medical University, Shanghai, P.R.China Object: The in vivo targetability and therapeutic effect of a novel gene delivery system with p21 and murine GM-CSF cDNA were examined in mouse cancer model. In our laboratory, a novel non-viral vector system has been constructed. The system is a polypeptide composed of 3 elements: a ligand MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

IMMUNOTHERAPY oligopeptide (LOP) for recognition and binding of the relevant receptor(s), a polycationic polypeptide (PCP) for binding of DNA from exogenous gene in an expression vector and an endosome-releasing oligopeptide (EROP) for endosomolysis to escape from lysosome fusion and subsequent enzyme degradation. We designed LOP E5 for receptor IGF IR and IGF II R, GV1 and GV2 for VEGF R and GE7 for EGF R recognition. HA20, a 20 amino acid oligopeptide of Influenza virus hemagglutinin domain is used as EROP. Protamine or polylysine can be used as PCP. The polypeptide system could be in the form of a mixture of LOP-PCP and EROP-PCP or in the form of LOP-PCP-EROP. Materials and Methods: Using the GE7 system, a polyplex comprising plasmid DNA of pCMV-p21 and pCMV-mGM-CSF was intravenoulsy injected via tail vein of mice harboring subcutaneously transplanted hepatoma. Plasmid DNA or polypeptide vector alone was administrated in the same route as control. Results: The polyplex containing either p21 or GM-CSF single gene gave some inhibitory effect on tumor growth, but the survival rate at 3 months was lower than 20%. Only the polyplex comprising p21 combined with mGM-CSF gave 75% survival rate at 3 months. Conclusion: These results indicated that the GE7 system could target therapeutic genes into cancer in vivo by intraveous injection. The combined effect of cell cycle inhibitor and immune regulatory genes gave dramatic results in treatment of cancer in animal.

721. Lipid-complexed Intratumor Lymphokine/Superantigen Immunotherapy for Spontaneous Canine Soft-tissue Sarcoma

D Thamm*, I Kurzman*, R Feinmehl†, E MacEwen*, S Longhofer‡, T Towell‡, G Frank‡, D Stinchcomb‡ *University of Wisconsin-Madison, School of Veterinary Medicine †Veterinary Medical Referral Service Ltd, Riverwoods, IL ‡Heska Corporation, Ft. Collins, CO Recent studies have demonstrated the efficacy and safety of local gene therapy with interleukin-2 and superantigen genes in rodent tumors and dogs with spontaneous oral melanoma. Advantages of non-viral systems for in vivo gene therapy include the relative simplicity of plasmid construction, lack of immunogenicity of the DNA plasmid and safety of synthetic lipid-based gene delivery systems. This study sought to determine the safety and efficacy of lipid-complexed immunotherapy with a construct encoding canine interleukin-2 (cIL-2) and Staphylococcus aureus superantigen A (SEA). Client-owned dogs with histologically confirmed soft-tissue sarcomas of various histotypes accessible by injection were treated weekly. Initial dosage of formulated plasmid was based on tumor size, and ranged from 240 mcg to 480 mcg per injection. Tumor measurement in 3 orthogonal planes was obtained weekly, and response assessed according to standard oncologic criteria. Dose-escalation was performed after 4 and 8 weeks of therapy if the patient experienced stable disease (SD), minimal response (MR) or partial response (PR). If SD, MR, or PR persisted for 12 weeks, tumors were surgically excised and assessed histologically. Fourteen patients have been treated to date, and 13 evaluated for response to therapy at the time of writing. Complete responses (CR) were seen in 2 patients (low-grade fibrosarcomas), and PR was documented in 2 patients (fibrosarcoma, hemangiopericytoma). SD was documented at 12 weeks in 1 patient (hemangiopericytoma), and progressive disease (PD) occurred in 8 (myxosarcoma, hemangiopericytoma, anaplastic sarcoma (2), spindle cell sarcoma, malignant nerve sheath tumor (3)). Histologically, evidence of lymphoplasmacytic/ granulomatous inflammation was observed in 3 of 4 tumors of patients that experienced CR or PR, whereas these changes were not evident in tumors from patients with PD or SD. MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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Overall, the treatment has been well tolerated, with several patients experiencing transient and self-limiting (24-48 hours) lethargy, inappetance, and perceived “stiffness” when ambulating. One patient with a hemangiopericytoma on a limb experienced transient lameness on the tumor-bearing leg after several injections. Side effects were not dose-limiting in any patient, and did not increase in severity with dose escalation. Owners did not note subjective changes in tumor appearance in the period immediately following injection. In conclusion, this interim report suggests that intratumor immunotherapy with lipid formulated SEA and cIL-2 genes is well tolerated, and can stimulate tumor regression in dogs with spontaneous soft-tissue sarcomas. (The authors would like to acknowledge Valentis, Inc., The Woodlands, TX for the lipid-based gene delivery system.)

722. Fate of Human T Cells Expressing a Chimeric Receptor against EGP-2 Antigen Following EGP-21 Tumor Exposure Lifen RenHeidenreich*, Katrina T. Trevor†, Ann V. LeFever* *St Lukes Medical Center †Arizona Cancer Center

Adoptive immunotherapy with T cells expressing chimeric T cell receptors (cTCR) directed against tumor could overcome limitations associated with insufficient numbers or ineffective responses of endogenous, tumor specific T cells. We have studied a cTCR (GAHg) that targets EGP-2, a tumorassociated antigen overexpressed on a wide variety of human carcinomas (1). The molecule encodes a single chain antibody (scFv) based on the murine anti-EGP-2 antibody GA733.2 along with a hinge extracellular spacer region and the FcRIg transmembrane/cytoplasmic domains. The FcRIg subunit mediates T cell signaling and activation upon scFv tumor recognition. T cells transduced with these cTCR recognize and lyse EGP-21 tumor cells (LS174T colon cancer cells) and exhibit increased cytokine production (1). Clinical efficacy of cTCRexpressing T cells could depend upon the ability of these cells to recycle their lytic capacity against multiple tumor targets. In these experiments, GAHg-T cells were applied to LS174T tumor cells 14 days after retrovirus-cTCR transduction (transduction efficiency was ;70%) and removed from the tumor cells the next day. T cell populations were maintained in X-VIVO 15 medium with 5% human serum and 100 IU of IL-2. Lytic activity, IFN-g cytokine production and growth/viability were evaluated for this transduced population relative to transduced cells that had not been exposed to tumor and served as control. Six days after the first tumor exposure, 51Cr release assays indicated that the tumor lytic activity was increased ;2-fold: 32% versus 15% for the control population (E: T52.5:1, 4 hr assay) and IFN-g production was 416 pg/ml/106 T cells compare to 260 pg/ml/106 T cells respectively (ELISA). By day 45 of culture, the viability of GAHg-T cells exposed to tumor remained ;80% while that of cells that had never seen tumor was only ;20%. In addition, the number of cells had increased 7-fold after exposure to tumor. Our data demonstrate that the GAHg-T cells are capable of recycling antitumor activity against EGP-21 tumor cells, which could benefit clinical outcome. More importantly, initial tumor cell exposure appears to enhance the anti-tumor potential of the cTCR-T cells against subsequent tumor targets, as indicated by increased lytic activity, IFN-g production and growth. Most likely, initial tumor cell exposure augments the general activation status of the T cell, as mediated by the cTCR, resulting in improved T cell function and activity against additional tumor cells.

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(1)Ren, L., Hayman, GT., and Trevor, KT., Specific Targeting of EGP-21 Tumor Cells by Primary Lymphocytes Modified with Chimeric T Cell Receptors, Human Gene Therapy, 11(1):9-19, 2000.

723. Tumor Suppression with Microencapsulated NonAutologous Cells Secreting a Recombinant Fusion Protein Directed against the HER-2/neu Gene Product

Pasquale Cirone*, Richard Austin†, Patricia Chang*‡ *McMaster University, Department of Biology, Hamilton, Ontario, Canada. †Civic Hospitals Research Centre and Department of Pathology, McMaster University, Hamilton, Ontario, Canada. ‡McMaster University, Department of Pediatrics, Hamilton, Ontario, Canada. Gene-immunotherapy of cancer is currently one of the most promising cancer treatments in research. Despite this, several concerns are raised involving both the cytokines being delivered as well as the vehicles for the delivery of such cytokines. Such problems include the short half-life and systemic morbid effects of cytokines as well as the transient delivery by the vectors delivering the gene therapy. Interleukin-2 (IL-2) has been shown to stimulate an anti-tumor immune response. Due to its short half-life, several groups have sought to conjugate this cytokine to larger molecules to stabilize it. In addition, due to its systemic toxicity at higher dosages, similar efforts have been made to target IL-2 directly to tumors. In following both of these criteria, a gene fusion protein consisting of a single-chain variable region of a humanized antibody against the HER-2/neu gene product p185 (Chiron Corporation, Emeryville, CA) fused to human IL-2 has been constructed (denoted as sFvIL-2). We wanted to study the efficacy of delivering this immunoconjugate protein via a potentially cost effective and benign form of gene therapy using non-autologous microencapsulated cells. This paradigm for gene therapy utilizes a “universal” cell line (genetically modified to express the transgene) that would subsequently be immunoprotected via encapsulation in alginate-based beads. A murine myoblast cell line was created expressing the immunoconjugate (sFvIL-2 expressed from C2C12 cells) and encapsulated in alginatepoly-L-lysine-alginate microcapsules. Both in vitro and after implantation into C57/BL6 mice, it was shown that the secretion of sFvIL-2 from the microcapsules remained constant for up to 21 days post implantation. Biological activity of the IL-2 portion of the immunoconjugate in serum was confirmed by CTLL-2 assay while the antibody portion was confirmed for affinity to p185 by assaying its ability to titer out sFvIL-2 from standard media samples. Antibody production in response to sFvIL-2, a xenogeneic human protein in mice, was quantified by neutralization of IL-2 in an ELISA and qualitatively detected via Western Blot. Host animals were induced to tumor formation by injection with B16/neu tumor cells, and subsequently implanted with our microcapsules at Day 14 post tumor injection. These treated mice showed increased survival (mice were sacrificed when the tumor attained a volume of .1 cm3) and retarded tumor progression by almost a week before resuming the normal kinetics observed in the untreated animals. Neither the mock-treated controls (mice implanted with microcapsules containing the background C2C12-untransfected cell line), nor the untreated mice showed improvement in survival or statistically significant difference in tumor progression. From these trials we conclude that the encapsulated cells can sustain production of a biologically active sFv / cytokine conjugate protein and most importantly, that the microcapsules secreting the sFvIL-2 immunoconjugate can retard tumor progression and promote survival.

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724. Intracutaneous Genetic Vaccination with Autologous Melanoma-associated Antigen Pme117/gp100 Induces a Specific and Protective Antimelanoma Immune Response Stephan N. Wagner*, Christine Wagner*, Petra Luehrs†, Tatjana K. Weimann*, Raphaela Kutil†, Manfred Goos*, Georg Stingl†, Achim Schneeberger† *Dept. of Dermatology, University School of Medicine Essen, Germany †Dept. of Dermatology, University School of Medicine Vienna, Austria

We and others have demonstrated recently that genetic immunization against the melanoma associated antigen (MAA) Pmel17/gp100 can lead to a protective in vivo anti-tumor immune response (IR) in the experimental B16/C57BL/6 (H2-b) melanoma model. However, in this model protective IRs were generated exclusively by xenoimmunization with human Pmel17/gp100 due to induction of a crossreacting T cell response by the human peptide epitope Pmel17/gp10025-33 that exhibits significant sequence variation to the respective MHC class I (H2b)-restricted mouse epitope. Thus, xenoimmunization in this tumor model can be regarded as a result of the fortuitous existence of peptide homologues with H2-b-restriction that is not predictive for the human system and needs reevaluation in experimental tumor models with different genetic background. Therefore, we used DBA/2 (H2-d) mice that were genetically immunized by direct i.c. injection of autologous H2-d-derived Pmel17/gp100 pDNA and scored for complete protection against subsequent challenge with syngeneic Cloudman M3 melanoma cells. Immunized mice showed significant protection against subsequent melanoma challenge with Pmel17/gp100-high expressing M3 melanoma cells as compared to control mice immunized with vector pDNA alone but not against challenge with Pmel17/ gp100-low expressing M3 variants. Even successfully immunized mice that had previously rejected M3 melanoma cells were not protected against a subsequent challenge with Pmel17/gp100negative histogenetically unrelated syngeneic tumor cells. Relevant immunological mechanisms underlying this protection were analyzed as follows: (i) in vivo CD41/CD81 T cell depletion experiments resulted in loss of this protection; (ii) CTL assays with splenocytes from immunized mice demonstrated induction of a Pmel17/gp100-specific T cell response; and (iii) immunohistochemical analysis of tumor escape variants in immunized mice demonstrated loss of Pmel17/gp100 expression. Taken together, these results demonstrate for the first time that i.c. genetic immunization with autologous MAA Pmel17/gp100 pDNA can lead to a protective anti-melanoma immune response in vivo as the result of the induction of a MAA-specific T cell response.

725. Adenovirus Vector Administration Inhibits the Progression of Lymphatic Metastasis of Murine Metastatic Tumors Manesh Ailawadi*, Jay M. Lee*, Sang Lee*, Ronald G. Crystal‡, Robert J. Korst*‡ *Memorial Sloan-Kettering Cancer Center, New York, NY †Weill Medical College of Cornell University, New York, NY

Gene therapy utilizing adenovirus (Ad) vectors encoding for a variety of therapeutic transgenes has been shown to cause tumor regression in many experimental animal models. In addition, Ad vector administration, independent of transgene expression, is associated with an immunological response which involves, but is not limited to, the lymph nodes draining the injection site. Given this background, we hypothesized that Ad vector administration might suppress the progression of regional lymph node metastasis in an established murine metastatic model. To assess this concept, a murine mammary carcinoma cell line (MM48) known to develop metastases was used to initiate footpad tumors in C3h/He mice. Two weeks later, 108 pfu of an Ad vector encoding for no transgene (AdCMV.Null) was injected in a peritumoral MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

IMMUNOTHERAPY location (n515), and the diameter of the popliteal lymph node was measured periodically. As a control, animals with tumors were injected with saline. Eighteen days after treatment, significantly more popliteal lymph nodes from sham injected animals reached 1 cm in greatest dimension, compared to nodes obtained from AdCMV.Null injected animals (77% vs 27%, p,0.005). Since heterologous transgene products resulting from Ad vector administration often elicit an immune response in animal models, intradermal administration of an Ad vector encoding for the E.coli cytosine deaminase cDNA (AdCMV.CD, n513) was compared to AdCMV.Null infection (n515) using the same experimental design. In the absence of 5-fluorocytosine (the prodrug for CD), the presence of the exogenous transgene did not inhibit progression of lymphatic metastases more significantly than AdCMV.Null (15% vs 27%, p . 0.05). To test whether this inhibition of lymphatic metastasis was a regional or systemic effect, bilateral footpad tumors were initiated (n58), and AdCMV.Null was injected peritumorally on one side. Eighteen days after treatment, the popliteal lymph nodes on the untreated side (54 6 64 mm2) were significantly larger than those on the Ad- treated side (11 6 8 mm2, p ,0.05). In this murine metastatic tumor model, we conclude that: (1) Ad vector administration inhibits the progression of regional lymph node metastases in the absence of a therapeutic transgene; (2) although many heterologous transgene products elicit an immune response, the addition of a nontherapeutic, exogenous transgene does not potentiate this effect; and (3) the inhibition of lymphatic metastases by Ad vectors is a regional, not systemic, effect. These data suggest that Ad vectors may possess an added benefit unrelated to the therapeutic transgene which may be useful in cancer gene therapy strategies.

726. Suppression of Growth of Established Macroscopic Tumors by Intratumoral Administration of Dendritic Cells and an Adenovirus Vector Expressing TNFa

Reza Kianmanesh, Toshiaki Kikuchi, Antje Koller, Neil R. Hackett, Ronald G. Crystal Weill Medical College of Cornell University, New York, NY Although tumor necrosis factor-a (TNFa), a potent cytokine, has a myriad of innate immune anti-tumor properties, its systemic administration is associated with significant toxicity, limiting the use of the TNFa protein as an anti-tumor therapeutic. Based on the knowledge that dendritic cells (DC) play a central role in initiating anti-tumor adaptive immune responses, we hypothesized that intra-tumoral administration of low doses of an adenovirus vector expressing TNFa (AdTNFa) together with syngeneic DC would act synergistically to promote suppression of preexisting tumors. As a model tumor, we chose murine CT26 colon carcinoma cells, a tumor line syngeneic to Balb/c mice known to be resistant to TNFa in vitro. In vitro, CT26 cells infected with AdTNFa showed no cell killing, but production of large quantities of functional TNFa as shown by ELISA and cytotoxicity of L292 cells. Administration of AdTNFa alone to palpable subcutaneous tumors (7 days after implantation of 2x105 CT26 cells to Balb/c mice) at 107, 108 or 109 pfu showed no significant suppression of tumor growth at 107 or 108 pfu, compared to 109 pfu control AdNull administration (n54/group; day 21, p . 0.05). Although the 109 pfu dose of AdTNFa alone mediated an antitumoral effect, it was associated with significant toxicity, including cachexia and a high mortality rate. In contrast, intratumoral administration of the lowest dose (107 pfu) of AdTNFa at day 7 into palpable subcutaneous tumors, followed by intratumoral injection of DC (2x105) at day 9 showed remarkable suppression of tumor growth compared to controls (AdTNFa alone, AdNull1DC, or PBS; n55/group; day 21, p , 0.05). No adverse effects were noted in the AdTNFa1DC treated mice. Strikingly, AdTNFa1DC treated mice challenged with CT26 (5x105 cells, subcutaneous, day 21) did not generate tumors. These observations demonstrate that local intratumoral gene transfer of TNFa, MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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together with dendritic cells, can evoke marked suppression of tumor growth without concomitant toxicity, providing a new paradigm for the use of TNFa as an antitumor therapeutic.

727. Development of a Broad-scope DNA Vaccine against Human Papilloma Virus-induced Cervical Dysplasia

Nazneen Aziz, Firoz Ahmed, Renee Hallack, Una McKeever, T. Hao, Yu-Ching Wu, Robert Urban, M L Hedley Zycos Inc. Human papilloma viral infections of the cervix and/or anal mucosa are known to be a predisposing risk factor in epithelial neoplasia and cancer. There are 23 strains of HPV, but type 16 and 18 strains are most commonly found in cervical high grade precancer and invasive cancers. Previously, immunization of mice with a HPV16 E7 synthetic peptide has been shown to induce CTL-mediated immunity against subsequent challenge with a lethal dose of HPV 16 transformed tumor. We are developing a multi-epitope DNA vaccine which encodes over 50 peptides fused together in a plasmid expression vector. These peptides which constitute regions of the E6 and E7 protein of HPV 16 and HPV 18 were selected because they bind to one of 5 allotypes of HLA-A, are naturally processed or activate human T cells. The DNA construct has been determined to express the expected 29 kDa multi-epitope polypeptide by an in vitro transcription and translation assay. The tumorigenic residues of the transforming E6 and E7 genes have been excluded in this construct. Furthermore, the tumorigenic potential of the entire multiepitope polypeptide have been tested using soft agar transformation assay in NIH 3T3 cells and have been determined to be completely non-transforming. Humoral response to the multi-epitope vaccine construct has been tested in three different strains of mice. Antibody production in these mice strains, after injection of the naked DNA vaccine was tested by ELISA assay. High titer antibodies (.1:2,500) were present in DNA vaccine injected mice vs. the non-responding population which received the empty expression vector. Cellular immune response to this DNA vaccine is being evaluated in animal studies and will be presented.

728. Secondary Lymphoid Tissue, SLC, a CC Chemokine with a Significant Antitumor Activity in Murine Lymphoma

Khaled Tolba*, Howard Federoff†, Shannon Hilchey*, Vicki HouseKnecht*, Qiang Lou*, John Facciponte*, Pia Challita-Eid*, Corliss Newman*, Joseph Rosenblatt* *University of Rochester Cancer Center, Hematology-Oncology Unit †Division of Molecular Medicine and Gene Therapy, University of Rochester Chemokines represent a family of small molecular weight proteins that play an important role in leukocyte trafficking and homing. SLC is a CC chemokine with selective chemotactic activity for T-cells, dendritic cells as well as an anti-angiogenic effect. We have tested SLC in a murine pre-B cell lymphoma model, A20. SLC cDNA was cloned into a HSV amplicon vector and packaged (HSVSLC) for direct intratumoral delivery. A20 tumor cells were transduced with either HSVSLC or a control vector, HSVlac encoding ß-galactosidase, and in vitro production of SLC was confirmed by ELISA (10.5 ng/ml/106 cells). In vitro activity of the recombinant protein was tested in a two chamber chemotactic assay. Supernatant from HSVSLC transduced A20 cells showed selective recruitment of purified mouse T-cells in vitro as compared to supernatant from HSVlac or uninfected A20 cells. HSVSLC was used to transduce pre-established A20 tumors in Balb/C mice and compared to mock and HSVlac infected mice. Immunohistochemistry of HSVSLC and HSVlac treated A20 tumors stained for CD4 and CD8 T-cell markers showed selective recruitment of CD81 T-cells into HSVSLC treated tumors. Four out of five HSVSLC treated mice were eradicated pre-established A20 tumors, as opposed to none of the four tumor bearing mice

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on each of the two control arms, mock and HSVlac-infected tumors. Rechallenge experiments with parental A20 cells in immune mice are ongoing. We are currently studying this antitumor activity to determine whether it is immune mediated and/or directly related to the anti-angiogenic or anti-proliferative effects of the SLC. Levels of cytolytic T-cell activity and analysis of cellular infiltration of regressing tumors will be presented.

729. IFN-a Gene Therapy for Squamous Cell Carcinoma Using Muscle Electroporation Delivery

S. Li, X. Zhang, R. Breau, J. Suen, E. Hanna Department of Otolaryngology-Head and Neck Surgery University of Arkansas for medical Sciences, Little Rock, AR 72205 Objective of Study: Gene therapy is currently being investigated worldwide as a promising new treatment for head and neck cancer. Significant progress in gene therapy has been made during the past decade, but several challenges remain. The most prominent challenge to reaching a therapeutic outcome is to achieve efficient delivery of genes into the cell, and to maintain high levels of gene expression for an adequate period of time. Although direct intratumoral injection of genes is easy, gene transfection efficiency and levels of gene expression are low. These disadvantages limit the therapeutic effectiveness of intratumoral gene therapy. Skeletal muscle provides an accessible target for direct gene delivery and allows stable expression, but the resulting expression level is low. The purpose of this study is to develop an efficient gene transfection method for cancer treatment. Methods: A novel method that we demonstrated here is called electroporation (EP) delivery. We used a caliper electrode to pulse the muscle following the injection of gene into the muscle using a 28-gauage needle syringe. The optimized EP paramaters for delivery of gene to muscle were 300 v/cm, 3 pulses and 20 msec duration with 200 usec interval between pulses. The SCC tumorbearing model was used. The volume of tumor growth was monitored, the NK, CD4,CD8 T cell and NK cell population were analyzed using Flowcytometry, the muscle damage was analyzed by H&E staining, the transfection efficiency was determined by GFP expression, and the IFN-alpha biological activity was determined by cell inhibition assay. Result and Conclusion: The level and duration of gene expression in muscle was increased by a magnitude of 2-4 logs, and more than 80% of the muscle fibers in the treated muscle were transfected. Delivery of IFN-alpha gene therapy as less as 10 ug to the hind limb tibialis muscle by electroporation once a week for 3 times significantly reduced the growth of squamous cell carcinoma tumor in an immunocompetent mouse model. This is the first time in our knowledge to demonstrate that IFN-alpha gene therapy using muscle EP delivery reduced the tumor growth. The reduction of tumor growth is associated with increased bio-functional IFN-alpha production in muscle, increased NK cell population and decreased CD4 T cell population in the spleen. Because the muscle accounts for 65-85% of body mass, and the muscle delivery is easy to perform, thus, we conclude that this is a convenient method to deliver gene for distant tumor treatment.

730. Safety Issues of Interleukin-12 Gene Therapy Using Adenoviral Vectors B Sangro, A Gil, L Diaz, J Ruiz, M Cordoba, Miguel Barajas, P Alzuguren, M Bustos, I Melero, C Qian, J Prieto Gene Therapy Unit.University of Navarra. Pamplona. Spain.

We have observed an antitumor effect of adenoviral-based IL-12 gene therapy against gastrointestinal neoplasms when given either intratumorally or systemically. Hence, we have evaluated the safety of systemic and intratumoral (IT) administration of first generation adenoviral vectors encoding for murine and human interleukin-12 (Ad.mIL12 and Ad.hIL12). For that purpose, an experimental model of orthotopic liver cancer was established

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by subcapsular injection of 2 z 10e6 MCA-RH7777 cells into syngenic Buffalo rats. Male Wistar rats and male Macaca fascicularis were used as healthy rodents and non-human primates, respectively. First, healthy rats received an IV injection of either saline or Ad.mIL12 in doses ranging from 10e7 to 10e10 pfu. At days 7, 30 and 90 after injection, blood samples were obtained and a complete necropsy was performed. Secondly, both tumorbearing and healthy animals were studied 7 days after an IT or IV injection of 10e10 pfu of Ad.lacZ, Ad.mIL12 or saline. Finally, five healthy primates were given an IV injection of Ad.hIL12 in doses ranging from 10e9 to 10e12 pfu. A comprehensive clinical evaluation was performed including hematological and biochemical studies, blood pressure, ECG, chest films and abdominal ultrasonography at days 0, 3, 7, 15, 30, 60 and 90 after injection. Systemic administration of Ad.mIL12 to healthy rats produced a transient, dose-dependant elevation of liver function tests, anemia, leucocytosis and thrombocytopenia. Pathological examination showed mild hepatitis, marked splenomegaly, peribronchial lymphoid infiltrate and lymphoid follicular hyperplasia in the bowel and mesenteric lymph nodes. Most of these changes had subsided by day 90. One out of 20 animals receiving 10e10 pfu/kg died unexpectedly at day 6 from an undisclosed cause. Compared with the IV route, intratumoral injection of Ad.mIL12 resulted in much modest toxicity with a similar profile. Non-human primates receiving Ad.mIL12 in doses up to 5 z 10e11 pfu had no apparent toxicity other than a transient, mild splenomegaly. However, after having received 10e12 pfu one animal developed weight loss, diarrhea and poor general condition. The analytical profile was that of an intense rhabdomiolysis (CPK: 6370 UI/l; LDH: 7060 UI/l) with acute renal failure, and slightly altered liver function tests. This occurred together with a marked increase in serum levels of interferon-g. The animal developed severe metabolic acidosis and shock, and was finally euthanized on day 8 after injection. In summary, intratumoral and systemic administration of Ad.mIL12 to rodents produces a similar profile of side effects, although the intensity was lower after intratumoral injection. In non-human primates, the toxic profile seems to be different and muscular damage may be a limiting toxicity. Acute toxicity is reversible in both species and there is no hint of chronic toxicity. Maximal tolerated doses in rodents and primates were 4 z 10e10 pfu/kg and 1.25 z 10e11 pfu/kg, respectively. These findings should be taken into consideration in the design of clinical trials using Ad.hIL12.

731. Gene Therapy of Murine Neuroblastoma Using Retroviral Vectors Containing IL-16 Gene

Hyun-Sang Cho*, Chong Young Park*†, Yeon Soo Kim*† *Department of Pediatrics, Hallym University College of Medicine, Seoul, Korea †Research Institute for Bioscience and Biotechnology, Taejon, Korea Interleukin(IL)-16 is a potent chemoattractant factor for CD41 T cells, monocytes, and eosinophils and up-regulates IL-2R on CD41 T lymphocytes and regulates the function of antigen presenting cell. We used retroviral-mediated gene transfer of the human IL-16 gene into murine neuroblastoma cells to investigate whether locally secreted IL-16 might generate an anti-tumor immune responses. We evaluated that the local secretion of IL-16 from the genetically-modified tumor cells would affect their tumorigenicity in vivo, and then, IL-16 transfected neuroblastoma cells would protect mice from tumor development after wild-type tumor cell challenge, and finally, the local secretion of IL-16 would affect the growth of pre-established large tumor. We performed a histological examination of the tumors and tunnel assay for the evaluation of apoptosis. The IL-16 gene-transduced neuro-2a clones secreted 120.25-177.3 ng of IL-16 per ml per 106 cells during 24 hr. None of the mice (N56) which injected with 2 x 106 of irradiated, IL-16 gene-transfected neuro-2a cells developed tumors within 6 weeks while all of the mice(N56) which injected with wild-type neuro-2a cells developed tumors. Immunization of mice(N56) with 2 x 106 IL-16 gene-transfected, irradiated neuro-2a cells protected these animals against a subseMOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

IMMUNOTHERAPY quent challenge with 2 x 106 wild-type tumor cells for a long time. The growth of large neuroblastoma was delayed after 2 x 106 IL-16-secreting neuro-2a cell injection into mice(N56) compared with the control group which injected with 2 x 106 IL-16nontransduced neuro-2a cells into mice(N56). Microscopically, the tumor that had been injected with IL-16-secreting neuro-2a cells revealed numerous tunel positive cells, which showed dark blue colored nuclear stain. In conclusion, local secretion of IL-16 gene-transduced tumor cells abrogated their tumorigenicity and induced protective immunity and may inhibit the growth of neuroblastoma. However, the higher the tumor burden, the less efficient tumor regression by IL-16 transduction into the neuro 2a cell line in A/J mice was observed.

732. Intratumoral Coinjection of Two Adenoviruses, One Encoding the Chemokine IP-10 and Another Encoding Interleukin-12 Results in Marked Antitumoral Synergy

In ˜ igo Narvaiza*, Guillermo Mazzolini*, Miguel Barajas*, Marina Duarte*, Mikel Zaratiegui‡, Cheng Qian*, Ignacio Melero*, Jesu´s Prieto* *Gene Therapy Unit.Department of Medicine.School of Medicine. University of Navarra. Pamplona. Spain †Department of Genetics.School of Medicine. University of Navarra. Pamplona. Spain INTRODUCTION: IP-10 is a CXC chemokine which mainly attracts activated CD41 lymphocytes and that has been shown to mediate angiostatic activity in vivo. It is induced in many cell types upon stimulation with IFN-gamma. IL-12 is a cytokine with a remarkable antitumor activity due to its ability to enhance the cellular antitumor immune response and to inhibit angiogenesis. MATERIALS AND METHODS: Construction of recombinant adenoviral vectors. A first generation recombinant adenovirus carrying murine IP-10 cDNA under the control of CMV promoter was constructed using a Cre/lox-based system. Recombinant adenovirus carrying IL-12 (AdCMVIL-12) in an expression cassette under the control of CMV promoter was constructed encompassing IL-12 p35 cDNA, an internal ribosomal entry site (IRES), IL-12 p40 cDNA and a polyadenylation signal. An adenovirus carrying LacZ reporter gene under the control of CMV promoter (AdCMVLacZ) was used as control adenovirus. Chemotaxis assay. Chemotactic activity was measured by migration assays of Con A T-cell blasts across polycarbonate membranes. In vivo gene therapy of established tumors. Tumors were established by subcutaneous or intrahepatic implantation of CT26 cells by injection of cell suspension. Tumors were treated by intratumor injection of the different adenovirus combinations. Citotoxicity assay. Cytotoxicity was analyzed in conventional 5 h 51Cr-release assays. RESULTS AND CONCLUSIONS:We have constructed a recombinant defective adenovirus which expresses functional murine IP-10 chemokine (AdCMVIP-10). Injection of AdCMVIP-10 into subcutaneous tumor nodules derived from the CT26 murine colorectal adenocarcinoma cell line displayed some antitumor activity but it was not curative in most cases. Previous studies have shown that injection of similar subcutaneous CT26 tumor nodules with adenovirus encoding IL-12 (AdCMVIL12) induces tumor regression in nearly 70% of the cases in association with generation of antitumor CTL activity. AdCMVIP-10 synergizes with the antitumor effect of suboptimal doses of AdCMVIL-12 reaching 100% of tumor eradication not only against injected but also against distant non-injected tumor nodules. Colocalization of both adenovirus at the same tumor nodule was required for the local and distant therapeutic effects. Importantly, intratumoral gene transfer with IL-12 and IP-10 generated a powerful tumor specific CTL response in a synergistic fashion, while both CD4 and CD8 T-cells appeared in the infiltrate of regressing tumors. Moreover, the antitumor activity of IP-10 1 IL-12 combined gene therapy was greatly diminished by simultaneous in vivo depletion of CD41 and CD81 T-cells but was largely unafMOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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fected by single depletion of each T-cell subset. An important role for NK cells was also suggested by asialo GM1 depletion experiments. From a clinical point of view, the effects of IP-10 permit to lower the required gene transfer level of IL-12, thus preventing dose-dependent IL-12 mediated toxicity while improving the therapeutic efficacy of the elicited antitumor response.

733. Reduced Immune Response by E4-modified Adenoviruses: Evaluation of Pharmacopathology and Tumor Efficacy D. Kang*, D. La Face*, V. Tsai*, M. Lusky†, M. Mehtali†, K. Wills*, J. Avanzini*, D. Maneval*, M. Zepeda* *Canji Inc. San Diego, CA USA †Transgene S.A. Strasbourg France

One of the challenges in adenoviral gene therapy is the expression of late viral genes, which may elicit host immune responses. Cellular immune responses can lead to cell destruction and removal of transduced cells, translating to the loss of transgene expression. The adenovirus E4 region encodes viral proteins that are involved in viral DNA replication, late viral mRNA accumulation, protein synthesis, host cell “shut-off”, virus assembly, and E2 expression. Although essential for viral replication and assembly, E4 functions can be provided in trans by producer cell lines. Our hypothesis is that deletions in the E4 region will help reduce late viral gene expression; thus, reducing immunological responses for longer gene expression. A series of adenoviral vectors were constructed, which retained various open reading frames (ORF) in the E4 region. These constructs were compared to complete E4 vectors containing the human tumor suppressor gene, p53. p53 expression levels and apoptosis from each construct were similar to each other, when compared on cell lines with varying p53 status. In vivo studies focused on an E4 deleted adenovirus that contained only ORF 3 and ORF 4 of this region. Liver morphology, serum transaminase levels, immunohistochemistry (IHC), cytotoxic T-lymphocyte (CTL), and T-helper lymphoproliferation assays suggested that rAd-p53-E4 ORF 3,41 constructs resulted in reduced pharmacopathology and adaptive host immune responses. Liver morphology showed a reduction in hypertrophy (loss of cording), necrosis of hepatocytes, and immune cell infiltrates from the rAd-p53-E4 ORF 3,41 treated animals. The rAd-E4 modified vectors generated lower serum transaminase levels than the rAd-E41 vectors. The E4 modified vectors also showed a reduced cellular immune response when analyzed by CTL and lymphoproliferation assays. Furthermore, these constructs resulted in a reduction in CD81 cellular infiltrates in the liver. Lastly, tumor efficacy studies were performed in both immunocompromised and immunocompetent animal models.

734. The Effect of Neutralizing Antibodies on Adenovirus-mediated Gene Transfer in Tumors MC Esandi, SFF Verlinden, DW van Bekkum IntroGene BV Leiden, The Netherlands

Most of the population have neutralizing antibodies (nab) against adenovirus serotype 5, recombinants of which are commonly employed as vectors in clinical gene therapy studies. The presence of nab limits effective gene transfer in many tissues including the brain (Y.Ohmoto et al Gene Therapy 1999, 6:471). Interestingly, JL Bramson et al. (Gene Therapy 1997, 4:1069) reported that the response of subcutaneous tumors in mice to intratumor injection of ad5.IL-12 was unaffected in immunized mice, although expression of the marker gene luciferase (luc) was reduced 2-10 fold. We show that the injection of ad5.luc into subcutaneously growing tumors ( bronchial carcinoma L42) results in 2 log lower expression of luciferase in the tumors of immunized rats as compared to naı¨ ve animals. A reduction of at least 100 fold was also seen in immunized animals of the expression of the rat IL-3 gene, as measured by leukocytosis. However, as in the case of Bramson et al.with the ad5. IL-12 vector, the therapeutic effect of the ad5.IL-3 upon intratumoral administration was similar in immunized and naı¨ve rats.

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Thus, in the presence of high titre nab the intratumoral administration of ad5.IL-3 is effective in causing tumor regression, but the leukocytosis which is an unwanted side-effect in naı¨ve rats, does not occur. The mechanism by which the vector is allowed to escape from the neutralizing effects of the antibody in tumor tissue has not been unraveled. We found that following i.v injection of ad5.luc, the luciferase expression in tumors is not reduced in immunized animals in contrast to more than 3 log decrease in the liver. When we neutralized the ad5.luc vector with high titre antiserum prior to intratumor administration in naı¨ve animals, we could detect significant luciferase activity in the tumor tissue after 48 hours. In constrast, no activity was detected following intravenous administration of the complex. We hypothesize that the tumor contains proteolytic enzymes which break the binding site between the vector and the antibody, thus causing local reactivation. Another interesting feature is that the leakage to the liver following intratumor administration - which is in our experiments 0.1 percent of the tumor dose in naı¨ ve animals - was reduced to undetectable levels in immunized animals. The percentage of people with high and low titres of nab varies in different areas of the world. Our results confirm those of Bramson et al. and underline the necessity for establishing the nab titre in patients- and if low to boost them- prior to gene transfer with ad5 vectors. Patients with high titres are expected to tolerate higher doses of vector by intratumor administration because of reduced side effects. It still has to be determined if the retainment of the therapeutic effect applies to other agents than cytokine genes. Finally, standardization of titration of nab should have the highest priority.

735. Gene Therapy of Colon Carcinomas (CC) and Hepatocellular Carcinomas (HCC) by Adenovirusmediated In Vivo Gene Transfer of CD40 Ligand

Volker Schmitz, Miguel Barajas, Yonglian Sun, Dacheng Peng, Cheng Qian, Jesu´s Prieto Gene Therapy Unit, Department of Medicine, School of Medicine, University of Navarra, Pamplona, Spain Introduction: Both, the cellular and the humoral immune response can be initiated by the interaction of CD40 ligand (CD40L, CD154) and its corresponding receptor CD40 on antigen presenting cells. Activation of the CD40 receptor can be achieved either by anti-CD40 antibodies, by recombinant soluble CD40L or by gene transfer of CD40L. This CD40 receptor activation can induce antitumoral immune responses. In this current study, we investigated the efficacy of treatment by intratumoral injection of adenovirus expressing CD40L in two experimental models: established subcutaneous CC in mice and established intrahepatic HCC in rats. Material and Methods: Recombinant adenoviruses (rAd) carrying the gene encoding mouse CD40L or LacZ reporter under the control of the CMV promotor were constructed (AdCMVmCD40L and AdCMVLaZ). Murine CC cells (CT-26) were injected subcutaneously to establish the murine CC model. HCC were established by intrahepatic injection of rat HCC cells (McA-RH 7777) in rats. Treatment was carried out by intratumoral injection of rAd when the tumors reached 0.4 cm to 0.6 cm or 0.4 cm to 1 cm in diameter for CC and HCC, respectively. Results: Tumor cells expressed CD40L only when they were infected with AdCMVmCD40L in vitro, but not when infected with AdCMVLacZ. The in vivo experiments showed that all tumor bearing mice which had received either AdCMVLacZ or PBS had a progressive tumor disease and all of them died within 25 days. By contrast 13 out of 16 mice treated with AdCMVmCD40L (109 pfu/ animal) showed complete tumor regression and long-term survival for more than 100 days. In the low dosage group of 108 pfu/animal 4 out of 12 animals presented tumor regression with long-term survival. Preliminary results in the HCC model demonstrate that rats bearing tumors, which had been treated in the control group (AdCMVLacZ 5 x 109 pfu/animal), showed a progressive tumor stage two weeks after treatment. By contrast, all tumor bearing rats

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treated with the high dose of AdCMVmCD40L (1010 pfu/animal) showed tumor regression three weeks after rAd injection and complete tumor eradication five weeks later. All of them had long-term survival. In the low dosage group of AdCMVmCD40L (109 pfu/ animal) one animal had complete tumor regression and long-term survival. Conclusion: This study demonstrates that CC and HCC could be treated effectively by adenovirus mediated in vivo gene transfer of CD40L into tumors. in vivo

736. Pre-existing Humoral Immunity against Adenovirus in Immunologically Naive Mice Limits Effective Gene Transfer and Intratumoral Gene Expression

Andres Hernandez, Hugo Barrera, Estuardo Aguilar-Cordova Center for Cell and Gene Therapy, Texas Children’s Hospital, Baylor College of Medicine, Houston Texas The humoral immune response to adenoviral antigens may be a limitation to the use of adenoviral vectors for gene therapy in some applications especially when repeated injections are necessary. However, since solid tumors are often scantily vascularized, and somewhat isolated from the systemic circulation, they may be less sensitive to adenoviral immunity and thus more susceptible to gene therapy. To investigate the effect of the immune response on the intratumoral expression of reporter genes carried by adenoviral vectors, 45 BALB/c mice were subcutaneously inoculated with 106 mammary tumor cells. After 20 days, mice were divided into three groups of 15 each and immunized as follows: 1) subcutaneous saline (control), 2) subcutaneous AdGFP, and 3) intratumoral Ad-GFP. Two weeks after immunization (day 14) all the animals were treated by intratumoral injection with Ad-luciferase. On day 16, 21 and 28, five animals of each group were sacrificed and liver, spleen, ovary, skin, and tumor samples were analyzed. In this report we show that pre-existing Ad-immunity decrease the effectiveness of gene transfer in mice after one single injection of a replication-defective adenovirus. Two routes of immunization, subcutaneous and intratumoral, were compared for the response induced against the adenovirus particles. A single immunization with the Ad-vector induces a long and persistent overproduction of neutralizing antibodies, no difference between the antibodies titers was observed in both routes of immunization. A time course of gene expression showed that in control animals the greatest level of expression was seen in the liver followed by tumor, spleen and ovary. However, the Luciferase and GFP activity in preimmunized animals decreased during the sixteenth day following Ad-injection, then declined even more in day 21 and disappeared by day 28. There was no difference between subcutaneous or intratumoral immunization. Conclusion: although there was a significant decrease in the level and duration of gene expression in pre-immunized animals, it was detected primarily in the tumor. Thus, pre-immunization may increase the systemic safety of adenoviral gene therapy.

737. Synergistic Antitumoral Effect of the Immunomodulator AS101 1 Paclitaxel: Association with Ras-dependent Signal Transduction Pathways

Benjamin Sredni*, Dan Longo†, Raphael Catane‡, Adi Shani§, Yona Kalechman¶ *CAIR Institute, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel †Gerontology Research Center, NIA, NIH, Baltimore, MD, USA ‡Department of Oncology. Shaare Zedek Medical Center, Jerusalem, Israel §Department of Oncology, Kaplan Hospital, Rehovot, Israel ¶ CAIR Institute, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

Optimal doses of Taxol combined with the immunomodulator AS101, previously shown to have antitumoral effects, adminMOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

IMMUNOTHERAPY istered to B16 melanoma-bearing mice, decreased tumor volume and resulted in over 60% cure. Taxol1AS101 directly inhibited clonogenicity of B16 melanoma cells in a synergistic dose-dependent manner. We suggest that this results from both the reduced Taxol-induced BM toxicity and the induction of differential signal transduction pathways which lead to apoptosis of tumor cells. Taxol1AS101 synergistically activated c-raf-1 and the MAPK ERK1 and ERK2. This activation was essential for the synergistic induction of p21waf protein. Cell cycle analysis of B16 cells treated with both compounds revealed an increased accumulation of the cells in G2M, although AS101 alone produced a significant G1 arrest. These activities were ras-dependent. AS1011Taxol induced a significant synergistic phosphorylation (inactivation) of the antiapoptotic protein Bcl-2. Whereas phosphorylation of Bcl-2 by Taxol was only raf-dependent, the synergistic effect of both compounds together was both ras-, raf- and MAPK-dependent. No effect of the combined treatment on the Bax protein expression was observed. We suggest that AS101 renders more cells susceptible to Bcl-2 phosphorylation by Taxol, possibly by increasing the accumulation of Taxol-induced cells in G2M. Exposure of B16 cells to clinically achievable concentrations of Taxol1AS101 increased the rate of apoptosis of treated cells. Apoptosis induced by AS101 alone was both raf- and MAPKdependent, while that induced by Taxol was only raf-dependent.

738. Pre-Existent Adenovirus Antibody Inhibits Systemic Toxicity and Antitumor Activity of CN706 in the Nude Mouse LNCaP Xenograft Model: Implications and Proposals for Human Therapy Daniel Henderson, De-Chao Yu, Yu Chen, David Charlton Calydon, Inc., 1324 Chesapeake Terrace, Sunnyvale, CA 94089

Pre-existent humoral antibody to adenovirus potentially confounds human clinical trials involving intravascular administration of adenovirus. Using the LNCaP prostate cancer xenograft model in Balb/C nu/nu mice and the prostate-specific attenuated replication competent adenovirus (ARCA) CN706, we developed an animal model that systematically controls both the dose of intravascularly administered adenovirus and the titer of the pre-existent anti-Ad5 antibody, and then measures the virus induced toxicity as well as anti-tumor activity. We prepared hyper-immune sera to adenovirus in rabbits, passively injected the purified rabbit anti-Ad5 antibody into tumor bearing mice, and established measurable humoral antiAd5 antibody titers. CN706 was intravenously injected into the tail vein of animals 24 hrs after passive anti-Ad5 antibody administration. In the absence of pre-existent antibody, the lethal dose (LD100) for Balb/C nu/nu mice was 2.5 X 1011 CN706 particles, whereas 1 X 1011 CN706 particles was not lethal. However, in the presence of a 1:80 pre-existent titer of Ad5 neutralizing antibody (NAb), intravenous (i.v.) injection of 5 X 1011 CN706 particles was no longer lethal. In addition, pre-existent antibody also prevented anti-tumor activity in a dose dependent manner: 1 X 1011 CN706 particles prevented LNCaP xenograft tumor progression, but anti-tumor activity was eliminated by a pre-existent 1:80 NAb titer. These results led us to propose transient removal of pre-existent adenovirus antibody by immunoapheresis. An affinity column of cloned virus capsid proteins was constructed which was able to specifically remove adenovirus antibody from human clinical serum samples. A 5 minute disposable immunoassay was also developed to monitor the level of pre-existent antibody in sera before and after immunoapheresis. Clinically, this approach may enable controlled clinical studies of intravenously administered adenovirus in patients with pre-existent anti-adenovirus antibody. MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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739. Particle-mediated IL-12 Gene Transfer into the Skin Distant from Tumor Site Elicits Anti-metastatic Effect Equivalent to Local Gene Transfer K Oshikawa*, F Shi*, A L Rakhmilevich†, P M Dondel†, D M Mahvi*, N-S Yang†‡ *Department of Surgery, University of Wisconsin, Madison, Wisconsin †Comprehensive Cancer Center, University of Wisconsin, Madison, Wisconsin ‡Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan

Recently, we have reported that gene gun-mediated interleukin-12 (IL-12) gene transfer into the skin overlying the local tumor inhibits systemic metastases. To further characterize this effect, we compared the anti-tumor and anti-metastatic effects of gene gun treatment with IL-12 cDNA delivered at the local tumor site versus at a site distant from the primary tumor, in a spontaneous metastasis model of LLC-F5. This aggressive tumor displayed rapid growth of the primary tumor mass and high incidence of lung metastases. Local IL-12 gene delivery into the skin overlying the intradermal tumor (local IL-12 treatment) on day 7, 9 and 11 after tumor implantation resulted in the most suppression of the growth of LLC-F5 tumor, while IL-12 gene transfer into the skin distant from the tumor (distant IL-12 treatment) was far less effective for suppressing the tumor growth. In contrast, both local IL-12 and distant IL-12 treatment, followed by tumor excision, inhibited lung metastases to a similar extent, resulting in extended survival of test mice. Both local and distant IL-12 treatments also induced similar levels of interferon-gama (IFN-gama) production in the treated skin tissue in vivo and by immune cells in vitro. Furthermore, splenocytes and lymph node cells obtained from mice receiving local and distant IL-12 gene gun treatments showed similar activation of natural killer cells and cytotoxic T cells. These results suggest that gene gun mediated-IL-12 gene transfer can confer anti-metastatic activities, irrespective of the transfection site, via cytotoxic T cells and IFNgamaf3 dependent pathways in a spontaneous LLC-F5 metastasis model.

740. Antitumor Effect of Bone Marrow-derived Dendritic Cells Engineered to Produce Interleukin-2 by Adenoviral Vector

Yoshio Gunji*, Masatoshi Tagawa†, Hideaki Shimada*, Hisahiro Matsubara*, Takanori Shimizu*, Takao Suzuki*, Seiji Hori*, Hideki Hayashi*, Takenori Ochiai*, Shigeru Sakiyama† *Department of Surgery (II), School of Medicine, Chiba University †Division of Pathology, Chiba Cancer Center Res. Inst.Chiba,Japan Retroviral transduction of murine colon carcinoma (Colon 26) cells with IL-2 gene could induce systemic immune responses and subsequently caused significant growth suppression of the transduced cells in vivo, when they were inoculated into syngeneic mice. Secretion of both GM-CSF and IL-4 from Colon 26 cells also produced an antitumor effect possibly because it potentiated the antigen presenting activity of dendritic cells (DC). A number of reports showed that stimulation of DC was a critical step to initiate antitumor immunity. To examine the antitumor effects of DC in a systemic immune response, bone marrowderived DC were obtained using mouse IL-4 (1000 u/ml) and GM-CSF (1000 u/ml) and were genetically engineered to produce IL-2 by ex vivo infection with replication-defective adenovirus (AdCMVIL-2) at m.o.i. 30. IL-2 expressing DC (3x106 cells) were then injected subcutaneously into the vicinity of established Colon 26 tumors. Inoculation of the DC suppressed subsequent tumor growth profoundly and prolonged the survival of the DC-treated mice. The antitumor effect observed was correlated with the cell number of the transduced DC injected. In contrast, injection of unmodified DC into local tumor sites did not achieve any antitumor effects on growing tumors. The data suggest a possible gene therapy of cancer with cytokine-producing DC.

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Since an antitumor effect by the inoculation of IL-2-producing DC was not strong enough to eradicate all of the established tumors, additional procedures to enhance the activity of DC can be required. We are now testing the effect of soluble CD40 ligand which strongly activates DC in order to magnify the antitumor effect caused by IL-2-producing DC.

up to 10 days. We are currently evaluating this vaccine in a mouse model for neuroblastoma.

741. Antitumor Effect of In Vivo Electroporation of Plasmids Encoding Cytokines into B16 Mouse Melanomas

T. Seregina*, C. J. Link, Jr.*, R. K. Burt†, A. E. Traynor†, C. Taylor‡, W. H. Burns‡, S. T. Rosen†, M. Edleman*, E. Christensen*, W. R. Drobyski* *Human Gene Therapy Research Institute, John Stoddard Cancer Center, Iowa Methodist Medical Center, Des Moines, IA †Northwestern University School of Medicine, Chicago, IL ‡Medical College of Wisconsin, Milwaukee, WI

Loree Heller*, Carlos Pottinger†, Mark Jaroszeski*†, Richard Gilbert*‡, Richard Heller*† *Center for Molecular Delivery, University of South Florida †Department of Surgery, University of South Florida College of Medicine ‡College of Engineering, University of South Florida

When cancer cells are transfected to express a cytokine, irradiated, and injected into a subject, long term immunity to challenge with that cancer can be generated. Direct intratumor transfer of the cytokine plasmid in vivo, which induces the tumor cells to express the cytokine, is an alternative to this ex vivo approach. In vivo plasmid DNA delivery offers an alternative to viral delivery, which is associated with several problems such as the possibility of insertional mutagenesis and the induction of the host immune response. Although plasmid cDNA expression is often lower than viral expression, this expression can be increased by chemical means such as liposomes, or by physical means such as in vivo electroporation. In vivo electroporation of plasmids encoding the cytokines GMCSF or IFN alpha directly into B16 melanomas results in a decrease in tumor growth or tumor regression, while electroporation of a plasmid encoding IL2 has no effect. Cytokine effectiveness is augmented by combination with electrochemotherapy, the enhancement of the delivery of chemotherapeutic agents with electric pulses. This technique results in complete regression of the primary tumor. In the B16 mouse melanoma model, this regression is short lived, lasting only 10 days to 2 weeks. When electrochemotherapy is combined with GMCSF or IL2, a small number of complete, long term cures with resistance to challenge with parental tumor cells are generated. In order to increase the number of long term cures, the effect of in vivo electroporation of combinations of plasmids encoding cytokines is being explored.

742. Neuroblastoma Immunotherapy Using a Novel Vector System

743. Use of HSVtk/GCV System as a “Safety Feature” to Control Graft versus Host Disease in Patients Receiving Adoptive Immunotherapy for Treatment of Hematologic Disorders

We have been conducting a study evaluating safety and efficacy of donor lymphocyte infusions (DLI) in patients who relapsed with leukemia or lymphoma after allogeneic bone marrow transplantation. In some of the relapsed patients DLI has resulted in the cure, but has been complicated by the development of graft versus host disease (GvHD). We have been conducting a clinical trial in which donor lymphocytes are transduced with HSVtk gene prior to infusion into patients relapsed with malignancies. This “safety feature” allows for destruction of genetically modified lymphocytes by ganciclovir (GCV) injection if GvHD develops. An attempt to control GvHD by conventional anti-GvHD treatment prior to GCV administration is required by the study design. Patients with such hematologic malignancies as CML, AML, Hodgkin’s disease, non-Hodgkin’s lymphoma and CTCL have entered this study. No adverse events related to DLI were observed. Also, no replication competent retrovirus was detected in any patients blood samples during patient monitoring. All pre-infusion donor lymphocyte populations had at least 85% HSVtk positive cells. The percentage of detectable HSVtk positive lymphocytes in the peripheral blood after transfusion ranged from 0 to 3.8%. Two patients died of their underlying malignancy before adequate number of donor cells could be prepared. Cells from two donors could not be propagated ex vivo. One patient with AML responded to DLI with longer remission duration compared to prior remissions induced by chemotherapy. Two other patients (CTCL and AML) achieved remission following HSVtk DLI infusion. One patient thus far developed GvHD that did not respond to conventional treatment but resolved after 5 days of GCV administration. Three patients with CML did not respond to the administration of HSVtk-modified donor cells.

Konstantina Elena Siapati, Paul Brickell, Christine Kinnon, Anthony Michalski, Adrian Thrasher, S Hart Molecular Immunology Unit, Institute of Child Health, London WC1N 1EH

744. Reduced Graft-versus-Host Inducing Potential of Thymidine Kinase Transduced, Selected And Expanded T Cells: Studies in a Rat Model

Neuroblastoma is one of the commonest paediatric solid tumours and its treatment using conventional therapy has so far been of limited success. Immunotherapy in the form of tumour vaccination is an alternative therapeutic approach and involves the use of allogeneic or autologous patient tumour cells that have been manipulated ex vivo to secrete or express certain immunomodulatory molecules. This approach enhances the immunogenicity of the tumour by attracting immune effector cells at the tumour site. We have developed a non-viral vector that targets integrin receptors and can transiently transfect neuroblastoma cells with high efficiency. This system can be easily applied, does not have any DNA size constraints and allows receptor-mediated targeting to cells. In addition, it can be used to deliver combinations of genes carried on different plasmids. We have optimised the lipid and peptide components of the vector as well as the transfection protocol for use in neuroblastoma cells. This vector system has been used to introduce Interleukin-2 and Interleukin-12 genes into neuroblastoma cell lines. The cytokine levels produced are comparable to reported viral systems and the in vitro expression above the potential therapeutic levels lasts for

In the Brown Norway rat Acute Myelocytic Leukemia (BNML) model the graft-versus-host disease (GvHD) and the graft-versusleukemia (GvL) reaction are studied using Wag/Rij rat allogeneic bone marrow and HSV-Tk suicide gene transduced Wag/Rij T cells. Transplantation of T cell containing allogeneic bone marrow generally results in severe GvHD but has a potential GvL effect. The aim is to control the GvHD by selective elimination of the suicide gene transduced donor T cells with the prodrug ganciclovir (GCV). In the rat model optimal conditions are studied to benefit from early GvL and to prevent severe GvHD effects. In the initial experiments Wag/Rij rat T cells were transduced with the SFCMM3 retroviral vector containing the HSV-Tk suicide gene and the truncated nerve growth factor receptor (NGFR). In the presence of polybrene this resulted in a transduction efficiency (TE) of the T cells of between 5 and 15%. After immunoselection

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M.E.M Weijtens, A.M. van Spronsen, A.C.M. Martens, A. Hagenbeek Jordan Laboratory of Hemato-Oncology, Dept. of Hematology, University Medical Center, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands

MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

IMMUNOTHERAPY for NGFR expression, we obtained a population of .95% NGFR1 cells. To acquire enough cells for in vivo experiments the NGFR1 cells are expanded about 20-fold, initially with IL-2 and subsequently with the APD/BSM feeder system. The immuno-selected T cells were up to 700-fold more sensitive to inhibition of in vitro proliferation by GCV than non-transduced rat T cells. While 10 million fresh splenocytes added to an allogeneic BM graft results in lethal GvHD in all rats within 20 days, even up to 80 million transduced, selected and expanded cells added to the BM graft leads to a delayed onset of GvHD which is lethal in only part of the animals. In subsequent experiments the transduction protocol was improved. First, the use of the LZRS retroviral vector and transduction in the presence of retronectint yielded a TE of 30-45%. Culturing of the cells was kept to a minimum and, most importantly, allowing the cells to recover from the MACS separation procedure by culturing them overnight in the presence of IL-2, restored their original potential to induce a lethal GvHD. These observations might have implications for defining the culture conditions for suicide gene transduced T cells to be added to BM grafts for the treatment of acute and chronic leukemia patients within the context of allo-BMT.

745. Genetic Modification of T-lymphocytes: Redirecting Autologous T-cell Specificity to Tumor Cells Farha H. Vasanwala, Tom C. Tsang, David T. Harris Gene Therapy Group, Department of Microbiology and Immunology,University of Arizona, Tucson, Arizona.

Tumor immune escape is associated with a lack of tumor-reactive T lymphocytes. The Tumor Infiltrating Lymphocytes (TIL) therapy postulates the presence of tumor specific T-cells albeit in insufficient numbers for tumor eradication. Thus, there is a need for TIL expansion prior to injection. A modification of TIL therapy would be the genetic modification of existing peripheral T-cells and redirection of their specificity to tumor cells, rather than expansion of TILs to generate sufficient numbers for re-injection. To prove the feasibility of this technique, MO5 (B-16 melanoma transfected with OVA) was used as a tumor model. T-cell receptors were identified and cloned from 2 different hybridomas specific for the MO5 cell line. An alternative to using the full length TCR genes is the use of a single chain T-cell receptor, which would alleviate the need of transfection of two genes into cells and the potential association of the transfected T-cell receptor chains with the endogenous T-cell receptor. A three domain single chain T-cell receptor (3D-scTCR) was created from the tumor specific TCR genes. The Va and the VbCb domains were sewed together with a universal glycine-serine linker using ploymerase chain reaction. To enable signaling through the single chain TCR the transmembrane to intracellular mouse CD3-z gene was ligated to the 39end of the Cb domain in frame with the Cb nucleotide sequence. The full length and single chain T-cell receptors have been cloned into the MMLV retroviral vector for transfection into T-cells. In vitro experiments will support the feasibility of this method of adoptive therapy in in vivo mouse models.

746. Apoptosis versus Necrosis in Prostate Cancer Vaccines

Smruti Desai*, Xiuqin Zhao*, Weitao Song†, Kevin Slawin†, David Spencer* *Department of Immunology, Baylor College of Medicine, Houston, TX 77030 †Department of Urology, Baylor College of Medicine, Houston ,TX 77030. Prostate cancer is the most commonly diagnosed non-skin malignancy in men and the second most common cause of cancerrelated mortality in U.S. males. Conventional treatment modalities for patients with advanced prostate cancer have met with limited success, and recent efforts have refocussed on the development of immune-based strategies as an adjuvant therapy. While several prostate-associated tumor antigens have been idenMOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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tified, a growing body of literature indicates that the mechanism of cell death (i.e. apoptosis versus necrosis) may dramatically influence the efficacy and outcome of antigen presentation. Additional parameters influencing tumor immunogenicity are the type of antigen-presenting cells (APC) involved in phagocytosis and presentation of antigen and the balance between immunomodulatory cytokines. Hence, in the present study, we compared the immunogenicity of prostate tumors killed by apoptotic versus necrotic pathways using the TRAMP murine model. Cell death in TRAMP cells was mediated via apoptosis (g-irradiation or by CID-inducible caspases) or necrosis (heat-shock or homogenization). To further augment the immune response, TRAMP cells were pre-infected with an adenoviral vector co-expressing the TH1 cytokine IL-12 and the costimulatory molecule B7.1. Finally, vaccine efficacy was tested in different anatomical locations by delivering the vaccine subcutaneously (s.c.) or intraperitoneally (i.p). Syngeneic C57BL/6 mice were vaccinated twice at 15-day intervals with different preparations of apoptotic or necrotic TRAMP vaccines and subsequently challenged with viable TRAMP cells. Tumor growth was monitored weekly. A pilot study performed on five animals per group indicated that the tumor volume were considerably smaller in mice vaccinated s.c. with g-irradiated TRAMP cells (328 mm3) as compared to the other groups, which varied between 750-1280 mm3. In contrast, the i.p. groups showed tumor volumes ranging from 719-951 mm3, with the g-irradiated group averaging 719 mm3. Also, the percentage of tumor-bearing mice was greatly decreased in the g-irradiated s.c. group (20%) relative to the other groups (60-80%). Similarly, the i.p. g-irradiated group was 60% tumor-free compared to the other groups (0-20%). Tumor-free animals are presently being tested for antigen-specific CTL activity. It is tempting to speculate that the apoptotic cells delivered by a s.c. route may have been taken up by immature dendritic cells in the periphery, in the milieu of the pro-inflammatory cytokine, IL-12, leading to a more robust TH1-based immune response. In contrast, the apoptotic cells delivered by the i.p route may have been taken up by peritoneal exudate macrophages, which have been observed to suppress immunity in the presence of anti-inflammatory cytokines such as IL-10 and TGF-b. Future experiments will be directed toward testing the immunogenicity of these vaccines in the presence of IL-12 or B7.1 alone or in the complete absence of extra immunomodulatory molecules.

747. Nonviable Tumor Vaccine Cells Inhibit the Generation of Antitumor Immunity A-C Tran, A Lin, F Borellini Cell Genesys, Foster City, CA 94404

Clinical application of cellular tumor vaccines requires the generation, storage, and on-site preparation of large numbers of cells. Due to the multiple steps involved from manufacturing to injection in a patient, some loss of cell viability may be introduced. The effect of non-viable cells in a cellular tumor vaccine on the generation of anti-tumor immunity is unknown. Several possibilities exist: nonviable cells may simply decrease the potency of the vaccine requiring larger cell numbers to be injected; augment potency by acting as a source of necrotic antigen for host antigen presenting cells and possibly providing a “danger” signal to the host; inhibit the generation of anti-tumor response by dilution of live tumor cells or by some other mechanism. To address this question, we studied a well-established cellular tumor vaccine model using B16 melanoma cells engineered by retroviral transduction to express GM-CSF. Dead tumor vaccine cells were mixed with live vaccine cells and injected into syngeneic hosts. The generation of anti-tumor immunity was then assessed by subsequent injections of unmodified B16 cells. Dead tumor vaccine cells clearly decreased the potency of the vaccine. To assess the mechanism of this decreased potency, varying numbers of dead and live vaccine cells were injected. Doubling the dose of a 50% viable vaccine was markedly less effective than a single dose of 100% viable vaccine. Thus, non-viable vaccine cells appear to inhibit the anti-tumor effect of an equivalent number of live vaccine cells. The co-injection of recombinant GM-CSF with

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50% vaccine dose did not restore efficacy. Our results show clearly that non-viable cells are detrimental to the generation of anti-tumor immunity in a B16 melanoma protection model, and has potentially important implications for clinical application of cellular tumor vaccines. Studies on the mechanism of this effect are underway.

748. Immunogenicity of Enhanced Green Fluorescent Protein (EGFP) in BALB/c Mice: Identification of a H2Kd-restricted CTL Epitope Andrea Gambotto*, Grzegorz Dworacki†, Vito Cicinnati†, Ciprian Almonte‡, Tom Kenniston*, Simon Watkins‡, Albert DeLeo†, Paul Robbins* *Departments of Molecular Genetics and Biochemistry and Surgery †Department of Patology ‡Center for Biologic Imaging

Enhanced green fluorescent protein (EGFP) is a novel marker gene product which is readily detectable using techniques of fluorescence microscopy, flow cytometry, or macroscopic imaging. In the present studies, we show that EGFP is highly immunogenic in BALB/c mice and thus may serve as a useful model tumor antigen in the development of genetic vaccine strategies for the immunotherapy of cancer. A stable transfectant of the transplantable CMS4 sarcoma of BALB/c origin expressing EGFP, CMS4-EGFP-Zeo, was generated. Splenocytes harvested from mice immunized with a recombinant adenovirus expressing EGFP (Ad-EGFP) were restimulated in vitro with CMS4-EGFP. Effector lymphocytes displayed strong cytotoxicity against CMS4-EGFP, but not against mock transfected CMS4-Zeo tumor cells. A number of candidate H2-Kd-binding peptides derived from the EGFP protein, chosen according to an epitope prediction program, were synthesized. These peptides were tested for their ability to bind to H2-Kd molecules and stimulate IFNg-production by splenocytes harvested from Ad-EGFP-immunized mice. Using this methodology, the peptide, HYLSTQSAL (corresponding to EGFP 200-208) which strongly binds to H2-Kd molecules, was identified as a naturally occurring epitope of EGFP. These results should facilitate the use of EGFP as a model tumor antigen in BALB/c mice.

749. Genetic Background Determines Individual Mortality in Response to Systemic Adenoviral Gene Transfer of Interleukin-12

Guillermo Mazzolini, In ˜ igo Narvaiza, Ainhoa Pe´rez-Diez, Cheng Qian, Bruno Sangro, J. Ruiz, Jesu´s Prieto, Ignacio Melero Gene Therapy Unit. Department of Medicine. School of Medicine. University of Navarra. Pamplona. Spain INTRODUCTION: IL-12 has shown remarkable experimental antitumor effects, both upon treatment with recombinant protein or gene transfer. Unfortunately, clinical trials demonstrated unacceptable toxicity of systemic recombinant IL-12 as a result of its ability to potently trigger endogenous IFN-gamma production. MATERIALS AND METHODS:Construction of adenovirus: A Recombinant first generation adenovirus carrying murine IL-12 encompassing IL-12 p35 cDNA, an internal ribosomal entry site (IRES), IL-12 p40 cDNA in a expression cassette under the control of CMV promoter was constructed. Recombinant adenovirus encoding the IL-12 cassette of expression was generated by cotransfection of 293 cells according to standard procedures. Adenovirus carrying LacZ reporter gene under the control of CMV promoter (AdCMVLacZ) was produced similarly. Recombinant adenoviruses were isolated from a single plaque, expanded in 293 cells, and purified by double Cesium chloride ultracentrifugation. A recombinant adenovirus encoding the genes for human IL-12 (AdCMVhIL-12) was generated by similar techniques and will be described in detail elsewhere (C. Qian, et al. manuscript in preparation). Mice and animal experiments: 5 to 8 wk old BALB/c, C57BL/6, DBA/2, F1(BALB/c x C57/BL/6) and B10D2 were housed and treated acording to institutional guidelines and injected with adenovirus, mAbs or Gadolinium chloride iv in ap-

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propriate vehicles. Cytokines: IFNgamma and IL-12 concentrations were assessed by comercial ELISA kits. RESULTS AND CONCLUSIONS:We report that systemic administration of a recombinant adenovirus encoding IL-12 (AdCMVmIL-12) has a dramatically different survival outcome in distinct mouse strains over a wide range of doses. At 2.5 x 109 pfu, systemic AdCMVmIL-12 killed all C57BL/6 mice but spared all BALB/c mice. IFN-gamma levels in sequential serum samples of such mice showed a much higher IFN-gamma production in C57BL/6 than in identically treated BALB/c. Moreover, C57BL/6 splenocytes stimulated in vitro with rIL-12 showed a much higher production of IFN-gamma than BALB/c splenocytes. F1 (C57BL/6 x BALB/c) mice showed no mortality and BALB/c-like serum concentrations of IFN-gamma in response to AdCMVmIL-12 injection. In contrast, DBA/2 which share H2d haplotype with BALB/c show both high sensitivity and high IFNg production, indicating that phenotype was not clearly linked to this locus, as also confirmed with B10.D2 mice. C57BL/6 mice were protected by administration of blocking anti IFN-gamma mAbs and also by simultaneous depletion of T-cells (both CD41 and CD81), NK-cells, and macrophages, but not by depleting each population separately. Human PBMCs from healthy donors showed great dispersion in their production of IFN-gamma upon in vitro stimulation with rhIL-12 or in vitro infection with AdCMVhIL-12. Such a result suggests that patients might also show individual variability in toxicity caused by IL-12-gene transfer.

750. Recombinant Adenovirus Encoding Human Papillomavirus Type 16 E5 Gene as a Vaccine to Tumor

Show-Li Chen†, Dai-Wei Liu†‡, Chang-Shun Hsieh†, Yeou-Ping Tsao§ † Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, ROC ‡Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, ROC §Department of Ophthalmology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan, ROC HPV-16 is the etiologic agents of cervical cancer, and encode three transforming oncogenes-such as E5, E6 and E7. HPV gene products are thus unique tumor antigens and served as ideal materials to be used as tumor vaccine. During the viral infection, E5 is expressed in earlier stage of neoplastic transformation of cervical epithelium, immune response arose may have a higher chance of eradicate tumor cells. Hence, we evaluate HPV-16 E5 protein as a tumor vaccine to cervical cancer. First, we generated recombinant adenovirus encoding E5 protein, and established a syngenic animal model which can express E5 protein. The efficacy of vaccination by adenovirus encoding E5 gene in the syngenic animal model was evaluated. The results reveal that E5 vaccination could protect tumor growth through cytotoxic T lymphocyte and induce CD8-dependent but CD4-independent CTL activity in vitro. Moreover, studies by using knock out mice with distinct T cell deficiencies confirm that CTL-induced tumor protection is CD8-dependent but CD4independent. It could be a potential therapeutic treatment for cervical cancer containing HPV-16 E5.

751. Electrically Enhanced Delivery of IL-12 for the Treatment of Murine Melanoma

Lee Lucas*, Loree Heller†, Carlos Pottinger†, Richard Heller*†‡ *Medical Microbiology/IBS, University of South Florida, Tampa, FL 33612 †Center for Molecular Delivery, University of South Florida, Tampa, Florida 33612 ‡Department of Surgery, University of South Florida, Tampa, FL 33612 The lack of an effective therapy that could be relied upon for a consistent therapeutic effect in the treatment of advanced melanoma makes it an ideal candidate for the development of novel MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

IMMUNOTHERAPY therapeutic approaches. One such approach is the development of a genetic immunotherapy protocol. Murine melanoma has been treated in pre-clinical trials with cytokines such as recombinant IL-12. IL-12 is known for its anti-angiogenic properties as well as its T cell and NK cell stimulation effects. Due to the possibility of adverse side effects of recombinant IL-12, a treatment scenario involving delivery of IL-12 in the form of plasmid DNA may be more tolerable. Recent studies have shown that expression of plasmid DNA can be increased in many tissues with the addition of in vivo electroporation. Therefore, increasing the expression of plasmid DNA encoding IL-12 with the use of in vivo electroporation may yield a therapeutic effect when treating melanoma. In these studies, mice bearing B16F10 melanoma tumors were treated twice either intra-tumorly (IT) or intra-muscularly (IM) with a plasmid encoding IL-12 followed by electrical pulses. Mice receiving IT treatment had a longer survival time despite lower serum IFN-g levels compared to IM treatment. This observation suggests a role of IL-12 at the tumor site independent of systemic levels of IFN-g. In one experiment two out of four mice remained tumor free through Day 158 at which point they were challenged with another dose of B16F10 cells in the opposite flank. By Day 208, one mouse developed a small tumor while the second mouse remained tumor free until it was euthanized on Day 230. This result suggests formation of a possible immune memory response. These studies reveal that delivery of a plasmid coding for IL-12 with in vivo electroporation can result in tumor regression, with the possible formation of an immune memory response. Work is being continued to elucidate the effective electroporation parameters for these treatment scenarios and to study the role of IL-12 at the tumor site. Future studies for this project will examine the use of these protocols as a potential treatment for metastatic disease.

752. Retroviral Gene Transfer of GM-CSF Gene into Cytokine-dependent Growth TF1-cells and Reinjection into SCID Mice: A Hematological Tumor Animal Model

M Imbert*, M Di Ianni‡, A Tabilio‡, N Maroc*, P Mannoni*, C Bagnis* *Institut Paoli-Calmettes. Centre de The´rapie Ge´nique. BP156. 13273, Marseille, CDX09, France †University of Perugia ‡University of Perugia - Instituto di Ematologia, Italy TF-1 is a CD341 erythroleukemia derived cell line which requires a cytokine stimulus to proliferate such as GM-CSF, IL3 or Erythopoietin. High in vitro transducibility of this cell line using retroviral or lentiviral vectors argued for the use of TF1 to design a human hematological tumor animal model but numerous experiments clearly showed absence of tumorigenicity in classical immunocompromised murine models. Assuming autocrine production of GM-CFS by TF1 cells can favor growth independent proliferation and subsequent tumorigenicity, TF1 cells were transduced with a retroviral vector coexpressing NeoR and the human GM-CSF gene. G418 resistant TF1 cells were found able to proliferate in vitro without addition of GM-CSF in the culture medium. Southern-blot assay and analysis of exogenous GM-CSF transcripts confirmed presence and transcription of the transgene in selected cell populations, respectiveley. Surprisingly no significant production of GM-CSF was detected by ELISA in these genetically modified cell population suggesting low amount of GM-CSF is able to promote proliferation of TF1 cells. When reinjected into SCID mice, either intraperitoneally or intravenously, tumor masses were detected in the testis. Cells derived from tumor masses still exhibit TF1 cell behavior, cytokine independent growth and G418 resistance. Comparison of the transcription profile of native TF1 cells, retrovirally transduced cells prior to reinjection, intravenous and intraperitoneal reinfused cells among 250 genes using a macro array procedure did not show any significant difference. Altogether, these data suggest that, in the testis only, local expression of GM-CSF by trapped MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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genetically modified TF1 cells allows GM-CSF/TF1 cells to proliferate and generate a tumor mass without any further tumorigenic genetic alteration. This animal model could be of interest to design cancer gene therapy approaches aimed at interfering with hematological tumor growth first events.

753. Evidence for Protective Immune Stimulation from a(1,3) Galactosyl Epitopes in Mice R. C. Unfer*, D. J. Hellrung†, C. J. Link, Jr.*† *Human Gene Therapy Research Institute, John Stoddard Cancer Center, Iowa Methodist Medical Center, Des Moines, IA †Iowa State University, Ames, IA

The a(1,3) galactosyl epitope (agal) is recognized by the human immune system during complement-mediated hyperacute xenograft rejection. The future goal of in vivo delivery of the aagal gene into agal-negative tumor cells, it’s expression, and their subsequent killing is dependent upon demonstration of a protective immunity generated by exposure to agal. Previous in vitro work by our Institute and others has demonstrated cytolysis of agal transduced tumor cells by human serum. We designed two experiments to determine whether immune priming with agal would provide any evidence for protection from tumor cell growth. In the first part of this study, C57BL/6 a(1,3) galactosyltransferase knockout mice (agal-negative) immunized with agalrich rabbit RBC, and two non-immunized control groups of C57BL/6 a(1,3) KO mice, and wild-type C57BL/6 mice were implanted with dilutions of agal-positive MC 38 colonic adenocarcinoma cells. Mice were monitored for tumor growth and morbidity. Results indicate a protective immunity was generated by prior exposure to the agal epitope in immunized C57BL/6 a(1,3) KO mice signified by lower morbidity and increase survival. In the second part of this study, C57BL/6 a(1,3) galactosyltransferase KO mice were immunized with rabbit RBC, implanted with 105 cells of the agal-negative B16.F10 tumor cell line and divided into three groups. Group A received only RBC and tumor cells, while groups B and C were implanted with 107 agal-positive vector-producing cells (VPC). Group 3 mice were also treated with GCV (10mg/ml). Increased survival was observed among group C mice indicating a protective effect from both exposure to agal-positive VPC and GCV. These data suggest that innate immunity in humans might be directed against a(1,3) galactosyltransferase expressing tumors.

754. Expression and Release of Stable and Active Forms of Murine Granulocytic Colony Stimulating Factor (mGM-CSF) Targeted to Different Subcellular Compartments

HJ Arteaga*†‡, AM Jama‡, B Christensson†, G Gahrton*, CIE Smith†‡, MS Dilber* *Department of Medicine and †Department of Clinical Immunology, Microbiology, Pathology and Infectious Diseases at Huddinge University Hospital. Karolinska Institute, Sweden. ‡Center for Biotechnology, Department of Biosciences at Novum. Karolinska Institute, Sweden. Cells modified by gene transfer technology can be used as delivery source of products with pharmacological activity. Usually, these products are released as a secreted substance. However, recombinant proteins could also be targeted to specific intracellular locations, stored there and released after cell lysis. Alternatively, for therapeutic purposes, secreted proteins such as cytokines, when relocated to the plasma membrane could, more efficiently, interact with appropriate receptors expressed by other cells. Cytokines have been used for a long time as immunomodulators. However, one of the main drawbacks of systemically applied cytokines is their high toxicity. In addition, cytokines work in a paracrine form and frequently after cell to cell interaction. Therefore, a very restricted release of cytokines - in time and localization area - could be desired for most of their therapeutic

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DNA VIRUS VECTORS applications. The murine granulocytic colony-stimulating factor (mGM-CSF) is one of the most promising cytokine candidates for cancer immunotherapy and as adjuvant of DNA vaccines. With the aim of improving the administration and release of cytokines in a very localized area, we have designed vectors expressing the mGM-CSF cDNA with different localization signals. Using this strategy it was shown that: cytokines can be targeted to different subcellular compartments, expressed there as stable active proteins, stored inside the cells and released after cell lysis. Moreover, a plasma membrane targeted form of mGM-CSF displayed substantial amount of biological activity. These vectors could have potential applications in immunotherapy for tumors and DNA vaccination protocols.

755. Use of Dendritic Cells as Cancer Vaccines Targeted against Mutant K-Ras Oncogenes

Kristen Radford, Susanna Pasquini, Richard Aspinall, Nicholas R. Lemoine Imperial Cancer Research Fund Molecular Oncology Unit , Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, United Kingdom. The aim of this study is to use this approach to generate new treatment strategies for patients with pancreatic cancer. Codon 12 mutations of the k-ras oncogene occur in over 90% of pancreatic carcinomas. There is evidence that endogenously processed mutant ras peptides in animal models and peptide vaccinations are able to induce CTL reactivity against ras oncogene-expressing tumor cells in mice and in cancer patients. Previous studies in our laboratory using naked DNA vaccination of mutant ras genes have indicated a potential use of these genes as antigens in cancer immunotherapy. Dendritic cells engineered to express tumour antigen represent a powerful method for inducing a more effective anti-tumor response. We are investigating the use of different antigen delivery systems such bacteria, adenovirus and SV40 viral vectors to transfect dendritic cells ex vivo with mutant ras genes. The efficacy of this vaccination approach is being evaluated in animal models to protect against tumor challenge and ultimately explore the possibility of the use of this therapeutic approach in clinical trials with patients affected with malignancies showing a high frequency of k-ras mutations, such as pancreatic and colorectal cancer.

756. Production of Allogeneic Prostate Cancer Vaccine (GVAX®): Characterization and Bioequivalence Studies Ken Ho, Andy Lin, Richard Lazar, Johanna Sy, Annie-Chen Tran, Flavia Borellini Cell Genesys, Inc. Foster City Ca 94404

The allogeneic GVAXt cancer vaccine is composed of two prostate cell lines (PC3 and LNCaP) that have been genetically modified by retroviral transduction to secrete GM-CSF. After production, the cells are lethally irradiated and frozen. Manufacture of GVAXt for clinical use was first carried out in 1997 using flat-stock culture vessels and FBS-containing medium. Subsequently, several changes were introduced in the manufacturing process including adaptation of vaccine cells to serum-free suspension culture in the absence of serum. These changes were introduced to make the production of GVAXt a scaleable process and to eliminate the risks of introducing adventitious agents with the use of FBS. To assess the effect of these modifications, GVAXt cancer vaccine lots manufactured by different processes were extensively characterized and compared with regard to mRNA and protein expression, viability, GM-CSF production and activity, immunogenicity in mice, and reactivity to serum from GVAXt vaccine recipients. The data collected in this study indicate that, within the limitation of the available technologies, no significant differences are observable between lots of GVAXt produced by the different manufacturing processes. This study represents an approach

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towards addressing the characterization of complex biological therapeutics.

DNA VIRUS VECTORS 757. Extending the Packaging of Recombinant Adenoassociated Virus

Ruian XU*†, Hsu Ma*†, Patricia Lawlor*, Mihail Mastakov*, Paola Leone†, Kai-Luk Choi*, Qi Chen*, Matthew During*† *Gene Therapy Center, University of Auckland, New Zealand †CNS Gene Therapy Center, Jefferson Medical College, Philadelphia, Pennsylvania, USA Recombinant adeno-associated viruses (rAAV) are among the most promising vectors for gene transfer in humans. However, the parvovirus has restricted packaging capacity that has prevented its wider application in gene therapy. We wished to determine whether recombinant AAV could be similarly overstuffed to generate vectors with genomes greater than 120% of wild-type. Specifically, we wished to determine whether an expression cassette of ;5.8 kb inclusive of the AAV ITRs can be packaged into an AAV-2 capsid efficiently and without loss of infectivity and transduction titer. Four viral vectors, AAV/PPE-luc (107% of wild type AAV in length), AAV/PPE-luc-wpre (123% of wild type AAV in length), AAV/GFAP-luc (98% of wild type AAV in length) and AAV/ GFAP-luc-wpre (117% of wild type AAV in length), were generated by a helper-free two plasmid transfection method (Grimm et al., 1998). The expression cassette on all constructs was flanked by AAV-2 145 bp ITRs. The two promoters investigated were the neuronal promoter, a 2.7 kb rat preproenkephalin (PPE) fragment, and an astrocytic 2.2 kb human glial fibrillary acidic protein (GFAP) promoter. Each promoter was used to direct expression of a firefly luciferase cDNA, with the 39 component of the expression cassette containing either a 400 bp bovine growth hormone polyadenylation signal (BGH) alone, or a 650 bp Woodchuck Hepatitis Virus Post Regulatory Element (WPRE) immediately 59 to the BGH polyA. For quantitative gene expression analysis, rAAV viral vectors were extracted with 1,2,2-trichlorotrifluoroethane, and then ultracentrifuged in CsCl at 80,000 rpm for 24 hours twice in order to concentrate and remove any empty particles. The rAAV titer was based on a sandwich ELISA technique (kit provided by Progen, Germany). Quantitative transgene expression was determined by a luciferase assay. Luciferase gene expression was further confirmed by immunohistochemistry. For immunohistochemical analysis, viral vectors were purified by a Biocad Sprint high-performance liquid chromatography (HPLC) system with a POROS HE heparin column. The vector titer was determined by an ABI 7700. Western blotting showed that all four vectors examined contained the viral structural proteins, VP1, VP2 and VP3 in appropriate ratios. Southern blotting revealed that the rAAV genome was present fully intact in each of the vectors respectively, including the 5.75 kb PPE construct. In contrast to previous reports which suggested that rAAV containing .5.2kb genomes lose transductional efficiency, enzyme assays showed significant enhancement of expression from our WPRE plus oversized vectors. Ongoing studies are attempting to define the absolute limit for efficient packaging as well as determining whether variability in titers obtained from over-sized genomes is sequence dependent. The versatility of rAAV is strongly compromised by its small size. The standard dogma of 110% above wild-type can be challenged, and for a given sequence, packaging can be extended to vector genomes of greater than 120% of wild-type, thus increasing the potential applications of the vector and inclusion of a greater range of cDNAs, promoters and postregulatory elements. MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy