305. αγ-T Cell Receptor-Based Gene Therapy Targeting the Common Tumor-Antigen Survivin for Therapy of Hematological Malignancies

GENE THERAPY FOR HEMATOLOGIC DISEASES: FROM MODELS TO CLINICAL RESULTS EVs into microglia by fluorescence microscopy. Comparison of the miRNA levels in GBM-derived EVs revealed that a number of different miRNAs were specifically enriched in EVs as compared to the cells of origin. Based on array data miRNAs with elevated levels in EVs were chosen, including miR451, which showed the highest enrichment in EVs. The level of miR451 in microglia was monitored after exposure to GBM-EVs and showed up to 15-fold increased levels after 24 and 48 hours of exposure as compared to untreated microglia. We are currently assessing changes in levels of mRNAs targeted by miR451 in microglia. This study supports a role for EVs in intercellular communication and provides a platform for designing a therapeutic delivery system of miRNAs. We have shown that EVs shed by GBM cells contain a unique repertoire of miRNAs and are efficiently taken up by microglia. Furthermore, analysis of miR451 showed that the content of the EVs could be transferred from the GBM cells to the microglia. Future experiments will focus on the change in phenotype and the physiologic effect of the EV-mediated transfer of miR451 in microglia, which should give us insight into how GBMderived EVs can alter the tumor microenvironment.

305. αβ-T Cell Receptor-Based Gene Therapy Targeting the Common Tumor-Antigen Survivin for Therapy of Hematological Malignancies

Caroline Arber,1 Harshal Abhyankar,1 Helen E. Heslop,1 Gianpietro Dotti,1 Barbara Savoldo.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX. Genetically engineered T cells redirected to tumor-associated antigens (TAAs) have produced complete responses in patients with selected malignancies. We have sought to broaden this approach by targeting survivin, over-expressed in virtually every human cancer, while low/absent in healthy adult tissues. However, the potential clinical impact of this targeting has previously been challenged by evidence that ectopic expression of a survivin-TCR, generated in an HLA-mismatched culture system and with remarkably high avidity, had fratricide effects on normal cells. To overcome this limitation, we isolated a new T-cell clone targeting the known HLA-A*02-restricted epitope (survivin95-104, ELT) and its variant survivin96-10497M (LML) starting from autologous cultures, using our previously validated culture system. In 51Cr-release assays, this T-cell clone, with nanomolar avidity, displayed specific killing against the survivin+HLA-A*02+ leukemia cells BV173 (39±16% specific lysis, E:T 40:1) and multiple myeloma cells U266 (20±7%) but not the HLA-A*02– HL-60 cells (2±2%). Furthermore, colony formation of primary myeloid leukemias was inhibited (>50% reduction) while that of healthy bone marrow (BM) was normal. The TCR - and -chains were thus cloned in an optimized retroviral vector and transduction of CD8+ cells resulted in efficient expression of the transgenic TCR (89±4%, n=6). Two stimulations with LML-pulsed artificial antigen presenting cells and IL2 resulted in an enrichment of LML-tetramer+ cells (97±1%) and similar fold T-cell expansion regardless of TCR expression by HLA-A2+ or HLA-A2– T cells (25±5 vs 26±5 fold, n=5/each). Furthermore, fratricide was not detected in 51Cr-release assays against HLA-A2+ activated T cells by TCR+ T cells from HLA-A2+ (1±2%, E:T 20:1) or HLA-A2– donors (6±3%, n=7/each) and killing only occurred when targets were pulsed with survivin peptides (46±12% vs 55±7% for LML, 68±14% vs 62±16% for ELT). As compared to non-transduced (NT) T cells, survivin-TCR+ T cells showed significant lysis of BV173 (46±14% vs 8±6%, E:T 20:1, n=12, p<0.001) and U266 (27±12% vs 14±6%, p=0.003) but not HL-60 (14±7 vs 14±6 %, p=NS). Blocking of targets with HLA class I antibody confirmed the HLA-restriction of TCR transgenic cells. Importantly, transgenic cells recapitulated the function of the original clone by inhibiting colony formation (range 32-78% reduction, n=5) of primary myeloid leukemias while preserving Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

normal clonogenic capacity of healthy BM or cord blood (n=5). When tested in vivo in a xenograft model of established systemic acute leukemia (FFLuc+BV173) using bioluminescent imaging, leukemia progression was significantly slower in mice treated with survivin-TCR+ versus NT T cells (14 vs 160-fold 28 days after T-cell therapy). This translated in an improved overall survival (n=12/group, p=0.038), although all mice eventually succumbed to the aggressive disease. In conclusion, we have generated a survivinTCR that can be efficiently expressed in polyclonal T cells and provides antitumor activity in vitro and in vivo without affecting the survival of T cells or normal hematopoietic progenitors.

Gene Therapy for Hematologic Diseases: From Models to Clinical Results 306. Lentiviral Vector-Based Gene Therapy for Wiskott-Aldrich Syndrome: Preliminary Results from the French Center

Salima Hacein-Bey-Abina,1,2 Johanna Blondeau,1 Laure Caccavelli,1 Sabine Charrier,3 Capucine Picard,2,4 Liliane DalCortivo,1 Guillem Cross,5 Stephane Blanche,2,5 Frederic Bushman,6 Nirav Malani,6 Alain Fischer,2,5,7 Adrian Thrasher,8 Anne Galy,3 Marina Cavazzana-Calvo.1,2 1 Biotherapy Dpt and Clinical Investigation Center, Univ. Hospital Necker Enfants Malades, APHP, Paris, France; 2Faculte de Medecine, Universite Paris Descartes, Paris, France; 3Research Unit UMR 951, Genethon, Evry, France; 4Centre d’Etudes des Deficits Immunitaire, Univ. Hospital Necker Enfants Malades, APHP, Paris, France; 5Pediatric Immunology & Hematology Unit, Univ. Hospital Necker Enfants Malades, APHP, Paris, France; 6 Dept. of Microbiology, Univ. of Pennsylvania School of Medicine, Philadelphia, PA; 7research Unit U768, INSERM, Paris, France; 8 Institute of Child Health, Univ. College of London, London, United Kingdom. The Wiskott Aldrich Syndrome (WAS) is a complex primary immunodeficiency disorder characterized by thrombocytopenia, recurrent infections, eczema, autoimmunity and lymphomas. We developed a phase I/II clinical protocol based on ex vivo gene transfer in hematopoietic progenitor/stem cells with a VSVg pseudotyped, 3rd generation SIN lentiviral vector (LV) physiologically expressing WAS cDNA from WAS gene promoter. Genethon sponsors several trials with this vector and preliminary results from the French center are following. Four score 5 WAS patients without HLA-identical donor underwent gene therapy. These patients received a myelosuppressive conditioning with busulfan, fludarabin associated or not with antiCD20 and/or alemtumuzab in case of autoimmunity. The first two patients 15.5 and 10 years-aged received transduced mobilized peripheral stem cells. The two others 10 months and 3 years-aged received transduced autologous bone marrow CD34+ cells. The median infused dose of CD34+ cells/kg of body weight was 9x106; [6.8- 11x106] with a mean vector copy number/cell (VCN) in CD34+ cells of 1.5 +/-1. After engraftment, WASP-positive leukocyte subpopulations’ frequency in PBMC increased mainly in lymphoid cell population consistent with expected WASP expression. At the last follow-up (FU) [18 to 6 months]: -Patients’WASP-positive cell subsets’ levels (P1 to P4) were respectively -WASP+T cells:, 75%, 28%, 54% and 29%; WASP+B cells: 27%, 11%, 57% and 14% and WASP+NK cells:58%, 52%, 64% and 78% respectively. - mean VCN was 0.39+/- 0.2 (range: 0.12 to 0.6) in PBMC and 0.17 +/- 0.11(range: 0.11 to 0.3) in granulocytes. WASP expression was also significantly observed in platelets in 2 patients. Today, P1 is independent from platelet transfusions, all patients improved their clinical signs, except P 2 who died 5 months post-treatment from opportunistic viral infections resistant to all conventional antiviral drugs and affecting S117