transducing T cells with a self-inactivating lentiviral vector resulted in reduced levels of 4-1BB.zeta CD19 CAR expression allowed excellent expansion, similar ...
Cancer-Targeted Gene and Cell Therapy I transducing T cells with a self-inactivating lentiviral vector resulted in reduced levels of 4-1BB.zeta CD19 CAR expression allowed excellent expansion, similar to that of T cells with 28.zeta CD19 CAR. We also observed improved cytotoxicity of T cells with lower expression of 4-1BB.zeta CAR in co-culture assays with CD19+ cell lines Daudi (5-fold reduction in remaining tumor cells with lentiCAR and 22-fold for IRES-CAR) and Raji (18-fold reduction for both lenti- and IRES-CAR constructs) and sustained performance in sequential killing assays. These studies reveal a mechanism that limits expansion and function of T cells expressing high levels of 4-1BB.zeta CD19 CAR and provide a basis for rational choice of expression systems and design of chimeric molecules that incorporate the 4-1BB signaling domain.
211. Combination of Forced Transduction of P53 and an Agent That Blocks MDM2-p53 Interactions Produces Synergistic Cytotoxicity on Mesothelioma Defective of the INK4A/ARF Region
Masatoshi Tagawa1, Takao Morinaga1, Zhihan Li1, Thao T. T. Nguyen1, Boya Zhong1, Shuji Kubo2, Ikuo Sekine3, Yuji Tada4, Koichiro Tatsumi4, Hideaki Shimada5, Kenzo Hiroshima6 1 Division of Pathology and Cell Therapy, Chiba Cancer Center Res Inst, Chiba, Japan, 2Department of Genetics, Hyogo College of Medicine, Nishinomiya, Japan, 3Department of Medical Oncology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan, 4Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan, 5Department of Surgery, School of Medicine, Toho University, Tokyo, Japan, 6Department of Pathology, Tokyo Women’s Medical University Yachiyo Medical Center, Yachiyo, Japan A majority of malignant mesothelioma specimens possesses the wild-type p53 gene with a homologous deletion in the INK4A/ ARF locus which encodes the p14ARF and the p16INK4A genes. Consequently, the p53 downstream pathways are functionally inactivated since the p14 defect augments activities of MDM2 which mediates p53 degradation through the ubiquitination processes. Furthermore, a loss of p16 induces pRb phosphorylation and uninhibited cell cycle progression through up-regulated CKD activities. We then examined possible anti-tumor effects of MDM2 inhibitors, Nutlin-3a and RITA, on human mesothelioma cells in combination of adenoviruses expressing the p53 gene (Ad-p53). Nutlin-3a, inhibiting the MDM2-p53 interactions, increased p53 phosphorylation levels and suppressed viability of mesothelioma cells in a p53-dependent manner. RITA, although blocked the MDM2-p53 interactions and phosphorylated p53, inhibited viability of mesothelioma cells independently of the p53 geneotype. Transduction of mesothelioma cells with Ad-p53 induced apoptotic cell death irrespective of the genotype. Moreover p21 induced by Ad-p53 dephosphorylated pRb and subsequently inhibited cell cycle progression. A combinatory use of Ad-p53 and Nutlin-3a or RITA, produced synergistic cytotoxicity of mesothelioma irrespective of the p53 geneotype. In addition, we demonstrated that the combination produced anti-tumor effects in an orthotopic animal model, mesothelioma developed in the pleural cavity in nude mice. In contrast, heat shock protein 90 inhibitors, blocking functions of MDM4, a homologous gene product of MDM2 without ubiquitination activities, did not produce the synergistic cytotoxicity with Ad-p53. These data collectively showed that MDM2 inhibitors enhanced expression levels of endogenous and exogenously transduced p53 through increased stability of the gene produce, and consequently activated the p53-mediated apoptosis. The p53-directed therapy is thus a possible therapeutic strategy for mesothelioma with the INK4A/ARF deletion. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
212. Lentiviral Insertional Mutagenesis Helps to Uncover the Mechanisms of Resistance to AZD9291 and CO-1686 in EGFR-Mutant Lung Adenocarcinoma Valentina Pirazzoli, Giulio Spinozzi, Erika Tenderini, Fabrizio Benedicenti, Andrea Calabria, Eugenio Montini HSR-TIGET, Milano, Italy
The discovery of cancer-driver mutations, accounting for the growth and spreading of cancer cells, led to the development of anticancer targeted therapies, which hit in a specific manner cell pathways directly involved in tumor progression. This new class of therapeutic agents has been shown to be more effective and less toxic than conventional chemotherapy in advanced forms of cancer. However, the inevitable development of acquired resistance has limited their success. An example of this concept is the case of Epidermal Growth Factor Receptor (EGFR)-mutant lung cancer. The discovery of EGFR mutations that confer sensitivity to the Tyrosine Kinase Inhibitors (TKI) erlotinib and gefitinib have underlined the importance of defining molecular subgroups to design more efficacious targeted therapies. Unfortunately, on average ~1 year after starting treatment, resistance to these agents, caused by EGFR secondary mutation T790M, occurs at high frequency. This observation suggests that strategies to delay or prevent the emergence of this resistance mechanism would prolong the lives of many lung cancer patients with EGFR mutations. AZD9291 and CO-1686 are two novel thirdgeneration EGFR TKIs designed to irreversibly and specifically target both the initial activating EGFR mutations and the resistance T790M. Phase I/II studies show compelling clinical activity of these compounds. Nevertheless the observed progression-free survival is about 12 months and the reasons of the relapse are under evaluation. We took advantage of a lentiviral vector (LV) -based insertional mutagenesis platform, developed by our lab, to screen genes that confer resistance to CO-1686. To this aim T790M+ (PC9BRc1) and T790M- (PC9) human lung adenocarcinoma cells were transduced with a genotoxic LV, harboring the Spleen Focus Forming Virus enhancer/promoter in the Long Terminal Repeats (LV-SF-LTR) or a non-genotoxic LV with self-inactivating LTR. After 2 weeks, transduced cells were divided in three groups receiving Erlotinib, CO-1686 or DMSO as control. In our rationale, the integration of the genotoxic LV in the cellular genome can potentially deregulate the expression of neighboring genes that contribute to confer resistance to these TKIs. Therefore, exposure to the treatment would result in the selection and expansion of the cell clones harboring those specific traceable mutations. Drug-resistant colonies were obtained after 4 weeks of erlotinib and 6 weeks of CO-1686 treatment. While results on PC9BRc1 are still pending, sequencing analysis performed on 100,000 LV integration sites retrieved by PCR-based technologies on PC9 drug-resistant colonies identified SOS1 as the principal LV-induced gene deregulation responsible for the resistance to both Erlotinib and CO-1686. A similar experiment aimed at identifying the AZD9291-induced resistance in lung cancer cells is on-going. The identification of biomarkers of resistance will allow the development of new therapies to overcome resistance, improving the life expectancy of lung adenocarcinoma patients.
213. Detection of Integration Sites of CD19 Chimeric Antigen Receptor Gene in a Non-Viral Gene Transfer System Motoharu Hamada Pediatrics, Nagoya University, Nagoya, Japan
BACKGROUND: Chimeric antigen receptor (CAR)-modified T cells targeting CD19 have exhibited marked activity in hematological malignancies, such as acute lymphoblastic leukemia, chronic S83
Cancer-Targeted Gene and Cell Therapy I lymphocytic leukemia, and B-cell lymphomas. Many of these results have been obtained using CD19-CAR T cells established by retro- or lentiviral vectors. We developed a non-viral gene-transfer method using the piggyBac transposon system wherein the CD19-CAR gene was integrated into the genome by cut paste mechanism of the transposase. In this system, the expression of CAR was permanent, and the in vitro and in vivo cytotoxic activity on several cancer cell lines was confirmed. In the process of such gene transfer, however, insertional mutagenesis and subsequent activation of proto-oncogenes are a concern. In this report, we aimed to detect the integration sites of CAR genes to evaluate the safety of our CD19-CAR T cells. METHOD: CD19-CAR T cells were produced by transfecting two plasmids containing piggyBac and a CD19-CAR gene by electroporation. We detected the integration sites of CAR genes by inverse polymerase chain reaction and subsequent next-generation sequencing using MiSeq. An analytic pipeline was developed to identify and to classify the integration sites. Proto-oncogenes were defined according to the Cancer Gene Census of the Catalogue of Somatic Mutations in Cancer database. RESULTS: We detected 181 independent integration sites of which five were in exons (CASP10, F2R, CYP51A1-AS1, ULBP2, and SLC24A2) and 113 in introns. The integration sites demonstrated no preference for specific sites. Five integration sites (2.8%) were in the introns of known proto-oncogenes (CDK6, MAML2, RAD51B, RUNX1, and EP300). This percentage of integrations into proto-oncogenes is comparable with that of random integration (2.4%) and lower than that of retro- and lentiviral vectors (6.3-10.4%) according to previous report using marker genes (Galvan et al. J Immunother 2009). CONCLUSION: We confirmed that our piggyBac-mediated transduction system randomly transduced the CAR gene into the genomic DNA. Therefore, this system is considered safer than viral vector system in terms of genotoxicity for CAR transduction into human T cells; however, the possibility of leukemic transformation caused by insertional mutagenesis should be carefully monitored by in vivo clonal analysis.
214. Specific Targeting of IL-13 Receptor alpha 2 Expressing Breast Cancer Cells by Paramyxovirus-Pseudotyped Lentiviral Vectors Displaying IL-13
Anna R. Kwilas, Wu Ou, Michael P. Marino, Takele Argaw, Akiko Suzuki, Bharat Joshi, Syed R. Husain, Raj K. Puri, Jakob Reiser Division of Cellular and Gene Therapies, CBER, FDA, Silver Spring, MD The ability to selectively target specific cell types is highly desirable for the in vivo application of viral vectors for the treatment of multiple diseases including cancer. Lentiviral vectors (LV) have the advantage of being easily pseudotyped allowing their tropism to be altered. LV can also be engineered to deliver therapeutic or cytotoxic genes directly to cancer cells. IL-13 receptor alpha 2 (IL13Rα2) is overexpressed in many different tumor types, including glioma, sarcoma, kidney, breast and ovarian cancer, making it an attractive target for tumor therapy. We have previously shown that firefly luciferase-expressing LV pseudotyped with a truncated fusion (F) protein derived from measles virus (MV) and a tail-truncated, receptor-blind MV hemagglutinin (H) protein bearing IL-13 at the C-terminus were capable of transducing IL-13Rα2-positive U251 glioma cells when administered intratumorally. In addition, we have shown the ability of LV to be pseudotyped using the F and H proteins from the Tupaia paramyxovirus (TPMV). TPMV does not infect human cells, potentially making it a safer choice for pseudotyping LV for in vivo delivery. Similar to the MV H protein, the TPMV H protein can also display IL-13 and bind specifically to IL-13Rα2positive tumor cells. We plan to confirm and extend these findings by examining the targeting of LV-MV-IL-13 and LV-TPMV-IL-13 S84
in metastatic orthotopic models of breast cancer by intratumoral and intravenous administration. In addition, LV expressing HSV1 thymidine kinase (TK) were constructed and are being evaluated for antitumor efficacy.
215. The Use of miR-122 and Its Target Sequence in Adeno-Associated Virus-Mediated Cytotoxic Gene Therapy
Daniel Zhang1, Jun Li2, Zifei Yin2, Georgy Aslanidi1, Arun Srivastava3, Changquan Ling2, Chen Ling1 1 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL, 2Department of Traditional Chinese Medicine, Second Military Medical University, Shanghai, China, 3Powell Gene Therapy Center, University of Florida, Gainesville, FL There are two challenges in adeno-associated virus (AAV)mediated cytotoxic gene therapy: i) overexpression of the cytotoxic transgene in the producer cell lines, such as HEK293, hampers efficient AAV vector production; and ii) most, if not all, AAV serotype vectors possess hepatic tropism since liver is the major tissue for cellular metabolism. These two challenges can be partially resolved by tissue specific promoters. However, in the cases that a specific promoter is not available, constitutively active promoters, such as the chicken β-actin (CBA) promoter, have to be used. For example, there is no well-studied specific promoter for hepatic satellite cells, which are the major cell type involved in liver fibrosis. In addition, most tissue-specific are weak promoters and are often too large to be packaged into AAV vectors. To address the above problems simultaneously, we explored the use of miR122 and its target sequence for the conditional regulation of transgene expression not only in the producer cells during AAV production, but also in the liver after systemic delivery. We first established a HEK293 cell line that overexpresses miR122. The functional miR122 expression was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) as well as Western blot assays against cellular proteins that are known to be regulated by miR122, such as c-Met. The expression of miR122 showed little effect on HEK293 cell growth and AAV protein expression. Next, we generated rAAV plasmids harboring suicide genes under the control of CBA promoter and a miR122 target (miR122T) sequence in the 3’-UTR. Two strong cytotoxic genes were selected, which encodes trichosanthin (a type 1 ribosome-inactivating protein) and diphtheria toxin (an exotoxin causing diphtheria). The presence of miR122T sequence had little effect on the protein functions in miR122-free cells, but significantly reduced the protein expression in the cells overexpressing miR122. Consequently, the AAV8 vectors carrying suicide genes and miR122T sequence yielded significantly increased production, up to 75-fold, in the HEK293-miR122 cells, when compared to that in the parental HEK293 cells (Table 1). Finally, we showed that AAV8 vectors that were produced from the HEK293-miR122 cells preserved the same tropisms and full bioactivity in vivo. Most importantly, the AAV8 vectors carrying a miR122T sequence mediated little transgene expression in normal liver. Taken together, we conclude that the use of HEK293-miR122 cells and a miR122T sequence should be applied to attenuate the transgene cytotoxicity during AAV vector production and infection of normal liver tissues.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
