233 IL12 and IL27 Sequential Gene Therapy for Eliminating Highly Aggressive Tumors Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S91 CANCER IMMUN[.]
CANCER - IMMUNOTHERAPY I irradiated EBV-LCL at 1:1 ratio, both in healthy donors (n=3) and B-CLL patients (n=3) Anti-CD23 CAR expressing T cells from healthy donors secreted 5.5-fold more INF-gamma (3079 pg/ml vs 561pg/mL, p=0.05) and 11-fold more TNF-alpha (187.17 pg/ml vs 16.53 pg/mL, p=0.05), 147-fold more IL-5 (147 pg/ml vs pg/mL, p=0.05) and 13-fold more IL-8 (590 pg/ml vs 43.24pg/mL, p=0.05), compared to non transduced T cells (n=3) In line with these findings, T cells expressing anti-CD23 CAR from B-CLL donors secreted 4.3-fold more INF-gamma (5800.613 pg/ml vs 1340.43 pg/mL, p=0.05) and 16.1-fold more IL-5 (4074.55 pg/ml vs 252.94 pg/mL, p=0.05), compared to non transduced T cells.Altogether these results suggest that for the potentiality to get selective and potent killing of tumor cells, while sparing normal B cells, and for the capability to induce the selective release of immunostimulatory cytokines, CD23targeting through a specific CAR holds great promises for adoptive immunotherapy of B-CLL 231 Expression of Multiple Transgenes in Human T Cells from PiggyBac Transposons Yozo Nakazawa,1 Leslie E Huye,1 Gianpietro Dotti,1 Aaron E Foster,1 Juan F Vera,1 Carl H June,2 Matthew H Wilson,1 Cliona M Rooney.1 Baylor College of Medicine, Texas Children’s Hospital, The Methodist Hospital, Houston, TX; 2University of Pennsylvania, Philadelphia, PA The optimal use of T cells as therapy for cancer will likely require multiple T cell modifications to ensure that T cells home to tumor tissues, recognize tumor cells, proliferate in response to tumor antigen, resist multiple tumor and stroma-derived inhibitory molecules and remain safe The combination of genetic modifications required will be highly tumor-dependent Retroviral vectors readily transduce T cells, but they have limited capacity for multiple gene insertion and co-transduction and are extremely expensive to produce at clinical grade Genetic modification of T cells using transposons is an attractive alternative due to the increased simplicity and decreased cost of production The Sleeping Beauty transposon can stably and persistently express therapeutic genes in primary human T cells, but its use is constrained by inefficient gene delivery (1-3% initial transduction rate) and limited cargo capacity The piggyBac (PB) transposon has higher enzymatic activity and larger cargo capacity in human cell lines and in mice, but its use in human primary cells has not been documented To evaluate the utility of PB for adoptive T cell therapies, we measured its efficiency and capacity in primary human T cells Unstimulated PBMC were transfected with PB transposon (pIR-eGFP) and PB transposase (pCMV-PB) plasmids and then stimulated with OKT3/CD28 mAbs PB transposons mediated ∼20% of stable and long-term GFP transgene expression without selection, representing a 7-20 fold improvement when compared to that previously reported for Sleeping Beauty Pretreatment and maintenance of T cells in the presence of IL-15 (10ng/ml) increased transgene expression up to ∼40% which was stable over a period of weeks Co-transfection of separate transposons, pIR-∆CD19 and pIR-eGFP encoding different marker genes resulted in expression of both proteins in >20% of T cells These double-positive cells were readily enriched by magnetic selection and maintained high expression of both genes (85% coexpression of CD19 and GFP) after expansion Co-transfection with pIR-iCaspase9.2A.∆CD19 and pIR-eGFP also achieved stable expression of three genes (GFP, CD19, and iCaspase9 - an inducible suicide gene) Coculture of gene-transferred T cells with K-562 feeder cells genetically modified to express the co-stimulatory molecules CD80/CD86/41BBL supported ∼300-fold expansion of selected transgenic T cells after 2-weeks of culture in a novel cell bioreactor (GP40, Wilson Wolf Manufacturing Inc.) These results indicate that the PB-transposon might be useful for the multigene expression that may be necessary for routinely successful T cell therapies Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 232 Non-Integrating Lentiviral Vectors for Vaccination Katarzyna Karwacz,1 Luis Apolonia,2 David Escors,1 Adrian Thrasher,2 Mary Collins.1 Infection and Immunity, University College London, London, United Kingdom; 2Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom Our lab has previously demonstrated that vaccination with lentiviral vectors generates potent CD8+T cell responses in vivo and protects mice from tumour challenge However, vector integration into the host genome risks inducing insertional mutagenesis Therefore, we tested efficiency of non-integrating lentivectors expressing ovalbumin and co-expressing ovalbumin with dendritic cell activators in vaccination protocols For this purpose, we have generated lentivectors with mutations in the pol gene and attachment sites (DNW/2∆att) Vectors expressing DC activators MKK6 and vFLIP (along with OVA) carried a triple mutation in the integrase (MKK6/DNW and vFLIP/DNW) We detected transgene expression from nonintegrating vectors in draining lymph nodes 24 and 72 hours after subcutaneous injection in mice We showed that all nonintegrating vectors used generate CD8+T cell specific responses in vaccinated mice, which, at a dose of 250ng RT activity, are comparable to responses generated by vaccination with 10ng RT activity of integrating vector We have tested those vectors for tumour therapy in mice that had been previously injected with EG7.OVA mouse tumoral cell line Immunization with 150ng RT of DNW/2∆att, integrating, vFLIP/DNW and MKK6/DNW lentivectors resulted in 10, 30, 55 and 60% of surviving mice respectively Next, we measured the number of specific CD8+T cells over a period of 50 days and found that MKK6/DNW and vFLIP/DNW rise a high specific response in a shorter time which might explain higher percentage of surviving mice in these groups Finally, we compared persistence of antigen presentation in mice immunized with integrating and nonintegrating lentivectors (also those expressing DC activators) We found that regardless of the vector used for vaccination, the antigen is presented in all mice for at least 30 days after vaccination We have shown that nonintegrating lentivectors are effective tools for tumour therapy and they should be considered an important alternative to integrating vectors 233 IL12 and IL27 Sequential Gene Therapy for Eliminating Highly Aggressive Tumors Shiguo Zhu,1 Boyu Zhang,1 Jeffry Cutrera,1 Shulin Li.1 CBS, Louisiana State University, Baton Rouge, LA Eradication of residual malignancy and metastatic tumors via systemic approach is the key for successfully treating cancer and increasing the cancer patient survival Systemic administration of IL12 protein in an acute large dose is effective but toxic Systemic administration of IL12 gene by persistently expressing a low level of IL12 protein may reduce the systemic toxicity, but only eradicates IL12 sensitive tumors Here, we discovered that sequential administration of IL12 and IL27 encoding DNA, referred to as sequential IL12IL27 gene therapy, not only eradicated IL12 sensitive tumors from 100% of mice but also eradicated the highly malignant 4T1 tumors from 33% of treated mice in multiple independent experiments This IL12-IL27 sequential gene therapy is not only superior to IL12-IL12 sequential gene therapy for eliminating tumors, but also for inducing CTL activity, increasing T cell infiltration into tumors, and yielding a large number of tumor-specific IFNg positive CD8 T cells More importantly, the IL12-IL27 sequential gene therapy yielded a strong anti-tumor immune memory compared to IL12-IL12 gene therapy Both reversal of the administration sequence and co-administration of IL12 and IL27 impaired the tumor eradication in 4T1 tumor bearing mice This IL12-IL27 sequential gene therapy, via sequential administration of IL12 and IL27 encoding plasmid DNA into tumorS91 CANCER - IMMUNOTHERAPY I bearing mice through intramuscular electroporation, provides a simple but effective approach for eliminating inaccessible residual tumors 234 In Depth Analysis of Sleeping Beauty Transposition for Therapeutic Gene Therapy Using CD19-Specificic T Cells Sourindra N Maiti,1 Branden Moriarity,2 Pullavathi Rao,3 Yi Jue Zhao,3 Margaret Dawson,1 Simon Olivares,1 Matthew Figliola,1 Ling Zhang,1 Helen Huls,1 Pallavi Manuri,1 Harjeet Singh,1 Zeming Jin,1 David A Largaespada,2 Perry Hackett,2 Partow Kebriaei,4 Richard Champlin,4 Laurence J N Cooper.1 Pediatrics, Box 907, M.D Anderson Cancer Center, Houston, TX; Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 3Pediatrics, Baylor College of Medicine, Houston, Tx; 4Stem Cell Transplantation, M.D Anderson Cancer Center, Houston, TX Genetic modification of primary human T cells to redirect their specificity towards desired antigen(s) expressed on tumor cells can be undertaken to produce clinical grade T-cells with therapeutic potential We and others have reported that the non-viral Sleeping Beauty (SB) transposon/transposase system can mediate genomic integration and long term expression of introduced transgenes in T cells Using this SB system we have introduced a chimeric antigen receptor (CAR) to render primary T cells capable of targeting B-lineage-specific antigen CD19 expressed on malignant B cells For clinical infusion, CD19-specific CD4+ and CD8+ T cells expressing CAR and markers for central memory were obtained by CAR-dependent propagation on irradiated clinical-grade CD19+ artificial antigen presenting cells (aAPC) derived from K562 and modified to stably express co-stimulatory molecules We now report an in depth study of these genetically modified T cells beyond their ability to selectively lyse CD19+ tumor targets An analysis of TCR Vbeta expression before and after electroporation/propagation demonstrated an outgrowth of a polyclonal population of T cells which is consistent with efficient gene transfer High throughput comprehensive global pyrosequencing of transposon-chromosome junctions confirmed that stable gene expression was due to SB-mediated transposition Fluorescent in situ hybridization (FISH) and real time genomic Q-PCR analyses using CAR-specific probes and primers demonstrated that transposon integrated on average once per T-cell genome Measurements of telomere length by quantitative-FISH, as a function of aging, demonstrated that in vitro propagated T cells maintained a telomere length equivalent to unmodified T cells In summary, these data reveal that CD19-specific T cells can be stably and efficiently modified by electro-transfer of SB DNA transposon/transposase resulting in cells containing a single integrant which can be propagated to clinicallymeaningful numbers on aAPC without replicative senescence (Figure) These data were used to successfully achieve institutional and federal regulatory approval and are the foundation for a clinical trial for the adoptive transfer of autologous CD19-specific T cells in patients with high-risk B-lymphoid malignancies after autologous hematopoietic stem-cell transplantation S92 235 IFN-α-Producing Plasmacytoid Dendritic Cells Mediate Brain Tumor Regression Induced by Adenoviral Vectors Expressing HSV1-TK and Flt3L Marianela Candolfi,1 Kader Yagiz,1 Gwendalyn D King,1 Patricia Lin,2 AKM G Muhammad,1 James F Curtin,1 Chunyan Liu,1 Kurt M Kroeger,1 Pedro R Lowenstein,1 Maria G Castro.1 Gene Therapy Research Institute, Cedars Sinai Medical CenterUCLA, Los Angeles, CA; 2Flow Cytometry Core Facility, Cedars Sinai Medical Center-UCLA, Los Angeles, CA The immunosuppressive nature of the tumor microenvironment has been postulated as the main obstacle to induce effective antitumor immune responses This scenario worsens when the tumor is located in an immune-privilege organ, such as the brain We therefore aimed to modify the tumor microenvironment of intracranial glioblastomas (GBM) using adenoviral vectors (Ads) expressing HSV1-TK (Ad-TK) and Flt3L (Ad-Flt3L) This approach combines the cytotoxic effect of TK + gancyclovir, which leads to release of inflammatory molecules and tumor antigens due to tumor cell death, with the immune-stimulatory effect of Flt3L, which recruits antigen presenting cells (APCs) into the tumor microenvironment This combined immunotherapy leads to 70% long term survival in rats bearing large intracranial GBM and this effect depends on CD4+ and CD8+ T cells We now characterized the APCs involved in the anti-tumor immune response induced by Ad-TK/GCV+Ad-Flt3L Rats bearing intracranial CNS-1 GBM tumors were injected with Ad-TK/GCV+Ad-Flt3L into the tumor mass After and 12 days, we harvested the tumor infiltrating immune cells and characterized them by flow cytometry We found plasmacytoid dendritic cells (pDCs), B cells, conventional DCs, and macrophages infiltrating the tumor mass Tumor pDCs were capable of in vitro phagocytosis of PKH67labeled CNS-1 cell debris, and 1µm FluoSpheres Incubation of tumor pDCs with the TLR-9 agonist ODN Cpg2216 (50 µg/ml) stimulated release of IFN-α, TNF-α and IL-6, as well as upregulation of MHCII expression To mimic the therapeutic effect of Ad-Flt3 we injected bone marrow-derived pDCs, or an Ad expressing IFN-α (Ad-INF-α), into the tumor mass pDCs were purified from the bone marrow of Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ...CANCER - IMMUNOTHERAPY I bearing mice through intramuscular electroporation, provides a simple but effective approach for eliminating inaccessible residual tumors 234 In Depth Analysis... 234 In Depth Analysis of Sleeping Beauty Transposition for Therapeutic Gene Therapy Using CD19-Specificic T Cells Sourindra N Maiti,1 Branden Moriarity,2 Pullavathi Rao,3 Yi Jue Zhao,3 Margaret... B-lineage-specific antigen CD19 expressed on malignant B cells For clinical infusion, CD19-specific CD4+ and CD8+ T cells expressing CAR and markers for central memory were obtained by CAR-dependent propagation