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558 Genetically Modified Human T Cells Targeted to Retained Epitopes of the MUC16 Antigen Are Functional and Mediate Lysis of Ovarian Tumor Cell Lines Molecular Therapy Volume 17, Supplement 1, May 20[.]
CANCER - IMMUNOTHERAPY II hybridization and chromosome analysis PB transposition of human T cells to redirect specificity to a desired target such as CD19 is a new approach with therapeutic implications for generating T-cell therapy for B-lineage malignancies be optimized These findings are key to gaining information critical for improving the efficacy of genetically engineered therapeutic cellbased vaccines for the treatment of cancer, and potentially, chronic infectious diseases 557 Protection Against Tumorigenesis by 3-Methylcholanthrene in Mice by IL-15 Gene Transfer Xianghui He,1 Yilin Xu,1 Shige Yu,1 Weidong Li,1 Liwei Zhu.1 Department of Surgery, Tianjin General Surgery Institute, Tianjin Medical University General Hospital, Tianjin, China 556 Tracking the In Vivo Migration of Dendritic Cell-Based Vaccines Transduced with Viral Vectors by Magnetic Resonance Imaging Sonali DeChickera,1 John Barrett,1 Christy Willert,1 Jonatan Snir,2 Roja Rohani,2 Paula Foster,2 Gregory A Dekaban.1 BioTherapeutics Research Group, Robarts Research Institute, London, ON, Canada; 2Imaging Research Laboratories, Robarts Research Institute, London, ON, Canada Despite recent therapeutic advances, including the introduction of novel cytostatic drugs and therapeutic antibodies, many cancer patients continue to experience recurrent or metastatic disease Current treatment options, particularly for those patients with metastatic breast, prostate or skin cancers, are complex and have limited curative potential Recent clinical trials however, have shown that cell-based therapeutic vaccines may be used to generate anti-tumor immune responses Dendritic cells (DC) have proven to be the most efficacious cellular component for therapeutic vaccines, serving as both the adjuvant and antigen (Ag)-delivery vehicle A noninvasive, non-radioactive method to determine the fate of DC-based vaccines administered to human subjects is not readily available In this study we demonstrate that in vitro-generated mouse DC can be readily labeled with superparamagnetic iron oxide nanoparticles, Feridex, without altering cell morphology, or their phenotypic and functional maturation Feridex-labeling enables the detection of DC in vivo following their migration to draining lymph nodes using a 1.5 or Tesla clinical magnetic resonance scanner We report a semi-quantitative approach for analysis of MR images and show that the Feridex-induced signal void volume, and fractional signal loss, correlates with the delivery and migration of graded, small numbers of in vitro-generated DC The analysis of DC migration also revealed that Feridex-treated DC not migrate as efficiently as untreated DC to the draining LN This correlated with decreased endocytic activity exhibited by Feridex-treated DC as well as a reduction in antigen specific T cell proliferation in draining lymph nodes following injection of Feridex labelled DC Thus the effect of Feridex on DC migration and function must be more carefully assessed before clinical application Furthermore, the order and timing of antigen loading and/or introduction of immunomodulatory genes through the use of viral vectors in the presence of Feridex must also Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy Interleukin-15 (IL-15) plays a key role in regulating both innate and adaptive immune responses It promotes the survival, proliferation, activation and maintenance of natural killer (NK) cells and CD8+ T cells, also stimulates the function of neutrophils, macrophages and dendritic cells We previously reported an amplified IL-15 expression plasmid vector pHi2-spIL15-CMV-tat and carcinoembryonic antigen (CEA)-positive tumor specific amplified IL-15 expression plasmid vector pHi2-spIL15-CEA-tat Significant amount of IL-15 expression was achieved in vitro Injection of IL-15 expression plasmids into the abdomen increased the survival of mice inoculated intraperitoneally with CT-20 tumor We further investigated the therapeutic effect of IL-15 plasmid in vivo IL-15 expression plasmid was injected intravenously into Balb/c mice and increased serum IL-15 was detected The amount of T cells and NK cells in the peripheral blood and spleen of transfected mice were increased Importantly, methylcholanthrene (MC) knot was implanted into the glandular stomach of mice to induce gastric tumor and decreased tumorigenesis was observed in the IL-15 expression plasmid transfected mice The ratio of CD4+CD25+ regulatory T cells decreased in the IL-15 plasmid transfected mice compared to control group In conclusion, IL-15 gene transfer could protect mice against methylcholanthrene induced primary tumor formation 558 Genetically Modified Human T Cells Targeted to Retained Epitopes of the MUC16 Antigen Are Functional and Mediate Lysis of Ovarian Tumor Cell Lines Alena A Chekmasova,1 Yan Nikhamin,1 Dharmarao Thapi,1 David R Spriggs,1 Renier J Brentjens.1,2 Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY; 2The Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY Ovarian carcinoma remains the cancer with the highest mortality rate among gynecologic malignancies Epithelial ovarian cancer is an immunologic tumor capable of stimulating an antitumor immune response, as evidenced by the fact that patient prognosis is linked both to the degree tumor infiltration by cytotoxic host T cells, as well as the number of suppressive CD4+ CD25HI regulatory T cells present within the tumor microenvironment T cells may be genetically modified to express chimeric antigen receptors (CARs) targeted to specific tumor antigens The ovarian cancer antigen CA125 (MUC16) is a high-molecular weight glycoprotein that is expressed by 80% of ovarian tumors and is therefore attractive target for T cell therapy However, most of the extracellular portion of MUC16 is secreted, limiting enthusiasm for this target antigen To address this limitation we have generated MAbs targeted to the retained extracellular portion of the MUC16 antigen Using the 4H11 and 4A5 hybridomas we have generated a series of CARs targeted to the retained extracellular domain of MUC16 (MUC-CD) The resulting CAR genes, termed 4H11z and 4A5z, were subcloned into the MMLV retroviral vector SFG Human T cells isolated from PBMC obtained from healthy donors were retrovirally transduced, and expression of the 4H11z and 4A5z CARs was verified by FACS and Western blot S213 CANCER - IMMUNOTHERAPY II analyses To assess CAR function we monitored the proliferation, cytokine secretion (IL-2 and IFNγ), and cytotoxicity of transduced T cells following co-culture on NIH-3T3 murine fibroblasts modified to express MUC-CD and the co-stimulatory ligand B7.1 (3T3(MUC/7.1)) or ovarian cell lines OV-CAR3 and SK-OV3 genetically engineered to over-express MUC16-CD 4H11z+ T cells demonstrated greater proliferative and cytotoxic effects on AAPCs and ovarian tumors when compared to 4A5z modified T cells For this reason 4H11z will be further developed into second generation CARs with co-stimulatory capacity and studied in previously established xenotransplant and syngeneic mouse tumor models Ultimately we hope to translate this targeted adoptive T cell approach to the clinical setting in patients with relapsed, chemotherapy-refractory ovarian cancer 559 4-1BB and CD28 Signaling Plays a Synergistic Role in Redirecting Umbilical Cord Blood T Cells Against B-Cell Malignancies Syam Tammana,1 Xin Huang,1 Michael Milone,2 Linan Ma,3 Bruce L Levine,2 Carl H June,2 John E Wagner,1 Xianzheng Zhou.1 Pediatrics, University of Minnesota, Minneapolis, MN; Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 3Biostatistics and Informatics Core, University of Minnesota, Minneapolis, MN The role of co-stimulatory molecules in umbilical cord blood (UCB) T cell activation and effector functions remains elusive To investigate the effect of co-stimulatory molecules (CD28 and 4-1BB) on UCB T cells, we transduced UCB T cells with lentiviral vectors expressing GFP and a single chain chimeric antigen receptor (CAR) for CD19 containing an intracellular domain of CD3 ζ chain and either a 4-1BB (UCB-19BB) or a CD28 intracellular domain (UCB-28) or both (UCB-28BB) or neither (UCB-19) We found that UCB-19BB and UCB-28BB T cells exhibited more cytotoxicity to CD19+ leukemia and lymphoma cell lines than UCB-19 and UCB-28 although differences in secretion of IL-2 and INF-γ by these T cells was not evident In vivo adoptive transfer of these UCB T cells into intraperitoneal tumor-bearing mice demonstrated that UCB19BB and 28BB T cells mounted the most potent anti-tumor response The mice, adoptively transferred with UCB-28BB cells survived longer than the mice with UCB-19BB In addition, UCB-28BB T cells also mounted more robust anti-tumor responses than UCB-19BB in a systemic human lymphoma model Our data suggest a synergistic role of 4-1BB and CD28 co-stimulation in engineering anti-leukemia UCB effector cells and implicate in design of redirected UCB T cell Phase I trial for refractory leukemia and lymphoma 560 The CE7R Chimeric Antigen Receptor Re-Directs T Cell Specificity to a Broad Range of L1-CAM Expressing Human Malignancies Michael Stastny, Christine Brown, Hao Hong, Wen-Chung Chang, Michael C Jensen Division of Cancer Immunotherapeutics & Tumor Immunology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA L1-CAM, initially identified as a neural cell adhesion molecule, is commonly expressed on metastatic neuroblastoma, and has also been more recently associated with progression and metastasis in several common human malignancies such as colorectal, lung, and ovarian cancer Therefore L1CAM has been identified as a potential therapeutic target for cancer therapy in many studies Our group has developed a chimeric antigen receptor (CAR), designated CE7R, composed of a single chain antibody extracellular domain derived from the L1-CAM-specific murine CE7 hybridoma and the cytoplasmic tail of huCD3-zeta chain that when expressed by S214 human cytotoxic T lymphocytes (CTLs) redirects their specificity to L1-CAM positive tumor cells We have previously reported on these CE7R expressing CD8+ CTL clones and their ability to specifically recognize and kill human neuroblastoma cells Moreover, in a first-inhuman clinical trial we showed that infusion of up to 109 autologous cloned CE7R+ CD8+ CTLs to children with advanced refractory neuroblastoma was safe and not associated with clinically detectable toxicities to non-malignant L1-CAM expressing tissues such as brain, adrenal medulla, and sympathetic ganglia Utilizing the murine CE7 monoclonal antibody, here we have found a broad range of human tumors cells lines express L1CAM, including not only neuroblastoma lines Be-2, 10HTB, and 11HTB as expected, but also the glioma line U251T, the ovarian cancer line OVCAR3, the renal carcinoma line Caki-1, and small cell lung cancer (SCLC) cell lines H82 and H209 We also explored the ability of CE7R+ CTLs to specifically lyse L1-CAM positive tumor cells by chromium release assays Our data revealed that the newly identified L1-CAM+ tumor lines (including U251T, OVCAR3, Caki-1, H82 and H209) were indeed sensitive to re-directed killing by the genetically engineered T cells Moreover, pre-incubation of tumors with CE7 antibody inhibited the CE7R+ CTL-mediated killing, demonstrating the antigenic specificity of these cells.Thus, overall, these data suggest that adoptive transfer of CE7R+ autologous CTL could have clinical utility in the treatment of L1-CAM positive tumors such as glioma, ovarian carcinoma, renal carcinoma and SCLC We are currently extending these results toward the development of L1-CAM targeted immunotherapy against ovarian cancer 561 IL-12 Enhances the Efficacy of Oncolytic Adenoviral Therapy in an Immunocompetent Preclinical Model of Prostate Cancer Vladimir V Ternovoi,1 Yingshu Zhang,1 Kenneth N Barton,1 Svend O Freytag.1 Radiation Oncology, Henry Ford Health System, Detroit, MI Introduction: For the past 15 years our translational research program has been developing a multi-modal gene therapy-based approach for the treatment of cancer Our approach utilizes a replication-competent, oncolytic adenovirus armed with a pair of therapeutic suicide genes The combined effects of the oncolytic adenoviral and suicide gene therapies result in significant tumor cell destruction and release of tumor antigens The innate immune recognition of adenovirus induces the robust immune response, which may promote efficient cross-presentation of tumor antigens through DC activation, increased activity of NK cells and macrophages, priming of T cells, and increased T cell survival Nevertheless, the immune-suppressive tumor environment is known to impede the development of durable anti-tumor immune responses Introduction of immunostimulatory cytokines in the context of replication-competent adenovirus-mediated suicide gene therapy might re-activate tumor resident immune effector cells and provide the stimulus necessary for the recruitment, differentiation, maturation and programming of immune cells Objective: To test the hypothesis that interleukin 12 (IL-12) immunotherapy will improve the efficacy of replicationcompetent adenovirus-mediated suicide gene therapy and result in the development of anti-tumor immunity in a syngeneic mouse model of prostate cancer Results: We generated a replication-competent adenovirus (Ad5-yCD/mutTK SR39rep-IL12) expressing a yeast cytosine deaminase (yCD)/mutant SR39 HSV-1 thymidine kinase (TKSR39) fusion gene and a single-chain murine interleukin 12 gene Ad5-yCD/mutTKSR39rep-IL12 expressed the expected IL-12 protein product and IL-12 expression did not impede adenovirus replication Ad5-yCD/mutTKSR39rep-IL12 anti-tumor activity was evaluated in the immune-competent TRAMP-C2 tumor model Relative to an oncolytic adenovirus lacking IL-12 (Ad5-yCD/mutTKSR39rep), intratumoral injection of Ad5-yCD/mutTKSR39rep-IL12 resulted in a Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ... IL-2 and INF-γ by these T cells was not evident In vivo adoptive transfer of these UCB T cells into intraperitoneal tumor- bearing mice demonstrated that UCB19BB and 28BB T cells mounted the most... demonstrating the antigenic specificity of these cells. Thus, overall, these data suggest that adoptive transfer of CE7R+ autologous CTL could have clinical utility in the treatment of L1-CAM positive... might re-activate tumor resident immune effector cells and provide the stimulus necessary for the recruitment, differentiation, maturation and programming of immune cells Objective: To test the