IMMU-50. SYSTEMIC ADOPTIVE TRANSFER IMMUNOTHERAPY WITH TCR-TRANSDUCED T-CELLS TARGETING NY-ESO-1 FOR MENINGIOMA

INTRODUCTION The lack of immunotherapeutic antigen targets selectively expressed on meningiomas have historically hindered its immunotherapy development. However, recent literature showed that in meningiomas, NY-ESO-1 is the most frequently expressed Cancer-Testis(CT)-antigen, which is a group of proteins silent in somatic cells but reactivated in cancers. NY-ESO-1 also has higher expression in higher-grade meningiomas. Decitabine is known to upregulate CT-antigens and augment immunotherapy. To evaluate systemic adoptive transfer for meningiomas, we tested the efficacy of NY-ESO-1 T-cell-receptor(TCR)-transduced T-cells in vitro and in vivo, and the role of decitabine in meningioma immunotherapy. METHODS NY-ESO-1 TCR-Transduced T-cells were generated by double-transfection with supernatants from PG-13 retroviral packaging cell line encoding HLA-A*0201–restricted NY-ESO-1(157 – 165)-specific TCR. In vitro cytolysis was measured using the xCelligence Real-Time Cell Analyzer System. We utilized human meningioma cells SF1335(Grade I, HLA-A2.1 positive) and CH157(Grade III, HLA-A2.1 negative) in vitro and in vivo. RESULTS CH157 and SF1335 have high and low NY-ESO-1 expression, respectively. Decitabine upregulated NY-ESO-1 mRNA expression in SF1335 by 6-fold after 2 days and by 100-fold after 9 days, with similar effect on protein expression. Co-culturing CH157, CH157-HLA-A2.1(CH157 transduced with HLA-A2.1 vector), SF1335, and decitabine-pretreated-SF1335 cells individually with NY-ESO-1 TCR transduced T-cells at a ratio of 1:1 resulted in 0%, 65%, 20% and 40% cytolysis at 10hours, respectively. Systemic(intravenous) adoptive transfer of TCR-transduced T-cells significantly increased overall survival in NSG mice with intracranial xenografts of CH157-HLA-A2.1 (p=0.04) and SF1335(not treated with decitabine) (p=0.06). CONCLUSIONS NY-ESO-1 TCR-transduced T-cells induce NY-ESO-1 and HLA-A2.1-specific cytolysis in meningioma in vitro, and systemic adoptive transfer confers a statistically significant survival benefit in vivo for meningiomas with high NY-ESO-1 expression. Decitabine upregulates NY-ESO-1 expression and increases tumor cytolysis by TCR-transduced T-cells in meningiomas. Targeting NY-ESO-1 may be a clinically feasible immunotherapeutic strategy to treat aggressive meningiomas.

High Titer Lentivector from a Stable Lenti-Producer Efficiently Corrects CD34+ Hematopoietic Stem Cells from Patients with p47phox-Deficient Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to mutations in the genes for the NADPH subunits (the genes for p47phox, p22phox, p67phox, p40phox autosomal chronic granulomatous disease), or gp91phox (X-linked chronic granulomatous disease). This results in a failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs), the major defect in chronic granulomatous disease causing recurrent infections and granulomatous complications. Hematopoietic stem cell transplantation (HSCT) with a suitable donor is potentially curative. However, in the absence of HLA-matched donor, gene therapy using autologous gene-corrected HSC offers potential for significant clinical benefit. To date, despite myeloid conditioning, gene therapy for CGD patients using gamma-retroviral vectors have achieved either minimal long-term gene marking and engraftment, or has been associated with insertional mutagenesis. In contrast, lentivector-mediated gene therapy has successfully treated patients with Wiskott-Aldrich syndrome and Metachromatic Leukodystrophy without any dysregulated clonal expansion. We used a lentivector construct which incorporates an MND internal promoter, a modified self-inactivating MoMuLV LTR U3 region with myeloproliferative sarcoma virus enhancer, and a 650bp single chicken b-globin insulator encoding codon-optimized p47phox gene. Mutations in p47phox accounts for the majority of AR-CGD. The production of large-scale, consistently-high-titer lentivector using a transient 4-plasmid transfection system however, is labor- and cost-prohibitive. To address this, we applied concatemeric array transfection of pCL20cW650 MND-p47-OPT into a stable packaging cell line (GPRTG) for HIV-based lentiviral vectors to create a stable producer of VSV-G pseudotyped pCL20cW650 MND-p47OP. The concatemer array of HIV lentiviral vector construct and bleomycin selectable gene cassette showed 10 copies of lentiviral vector in a stable producer line, capable of producing vector at 10^7 IU/ml. Hematopoietic CD34+ stem cells from p47phox- CGD were transduced with pCL20cW650 MND-p47-OPT vector (MOI 10) with 2 overnight transductions following 24 hours pre-activation with SCF, FLT-3L and TPO (100ng/ml). Following three weeks in vitro culture, non-transduced or transduced p47 CGD HSC versus normal HSC were 0%, 42% and 20% p47phox positive, respectively. To determine functional correction, PMA stimulated oxidant production was measured using the dihydrorhodamine assay, confirmation similar levels of oxidant generation in transduced patient cells compared with normal controls. More than 90% of CFU were vector positive, indicating a high level of gene marking. Transduced and control naïve p47phox-patient CD34+ HSC were transplanted into 20 immunodeficient Nodscid-gc deficient (NSG) mice, and at 13 weeks post-transplant the CD13+ human neutrophils arising in mouse bone marrow were assessed for p47phox expression. Over 40% CD13+ neutrophils expressed p47phox protein from NSG mice transplanted with transduced p47-patient CD34 HSC, compared with 74% or 0% in mice transplanted with normal CD34 or p47 patient naive CD34 cells respectively. Detailed histopathology of each transplanted mice confirmed the absence of vector insertion-related myeloid tumors, and deep sequencing of bone marrow CD45+ human cells from each mouse also demonstrated polyclonal distribution of vector integration sites. In conclusion, we provide preclinical data demonstrating the efficacy and safety of high titer VSVg-pseudotyped lentivector (CL20cW650 MND-p47-OPT) generated by our stable GPTRG p47 lenti-producer for correction of p47phox-deficient human CD34 HSC. Disclosures No relevant conflicts of interest to declare.