62 conditional activation of t cells to specifically target c met under hypoxia

62 Conditional Activation of T Cells to Specifically Target c Met under Hypoxia Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S25 CANCER APOPTOSI[.]

CANCER-APOPTOSIS & SUICIDE/CANCER-IMMUNOTHERAPY, ADOPTIVE T CELL THERAPY suppressed cell growth of IFN-lambdas-sensitive carcinoma cells These data collectively suggest that IFN-lambdas is a potential anticancer agent for esophageal carcinoma and useful in combination with chemotherapeutic agents 60 Co-Expression of a Chimeric Antigen Receptor Targeting CD19 (CAR19), Optimized Human IL-15, and iCaspase9 To Enhance the Activity and Safety of Cytotoxic T Lymphocytes Valentina Hoyos,1 Barbara Savoldo,1 Juan F Vera,1 Concetta Quintarelli,1 Helen E Heslop,1 Cliona M Rooney,1 Malcolm K Brenner,1 Gianpietro Dotti.1 Pediatrics, Medicine, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Recurrence of disease remains the largest cause of mortality for many patients transplanted for relapsed or refractory B cell malignancies Modification of primary T cells to express a chimeric antigen receptor (CAR) targeting CD19 represent an attractive strategy to maintain remission after conventional treatment However, their efficacy is limited by poor expansion within the tumor microenvironment The addition of co-stimulatory endodomains, such as CD28, to the CAR may enhance cell expansion in response to the antigen, but cell growth and survival remain suboptimal To further potentiate the expansion and survival of CAR-modified T lymphocytes, we generated a new vector encoding molecules: CAR.19 incorporating the CD28 endodomain, codon optimized hIL15 to enhance cell survival and growth, and an inducible suicide gene based on the expression of Caspase9 (iCasp9) to increase the margin of safety associated with transgenic expression of an autocrine growth factor These three sequences were linked using 2A-like peptide sequences We compared the proliferative capacity, cytotoxic activity and in vivo anti-tumor effects of T lymphocytes expressing either CAR.19-28ζ alone or CAR.19-28ζ, IL15 and the suicide gene T lymphocytes were activated with OKT3/CD28 antibodies and then transduced with retroviral supernatants Phenotypic analysis showed 70±10% and 75±5% transduction efficiency for iCasp9/CAR19-28ζ/ IL15 and CAR19-28ζ T cells, respectively Only the iCasp9/CAR1928ζ /IL15 T cells produced IL15 (>100pg/mL) after stimulation with CD19+ tumor cells T cells kept in culture for weeks stimulated weekly with CD19+ B-CLL cells were co-cultured with CD19+ Daudi cells After 72 hours iCasp9/CAR19-28ζ/IL15 T cells were more efficient eliminating tumor cells compared to CAR19-28ζ T cells (0.7% residual tumor cells vs 14% respectively) Furthermore, labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that the proliferation of the iCasp9/CAR19-28ζ/IL15+ T cells in response to CD19+ tumor cells was greater than that of CAR.19-28ζ control+ T cells Finally, the activation of the suicide gene iCasp9 with a small-molecule dimerizer (CID 50ng/mL) rapidly induced >90% apoptosis of T cells expressing iCasp9/CAR19-28ζ/IL15 Hence all three transgenes were functional To assess the antitumor effects of the modified cells in vivo, we used a xenograft SCID mouse model and an in vivo bioluminescence system CD19+ Daudi cells (1x106) expressing firefly Luciferase (FL) were injected i.p., and on day 4, mice received i.p iCasp9/CAR19-28ζ/IL15+ or CAR1928ζ+ or control T cells (10x106) By day 30 the tumor signal was significantly reduced in mice receiving iCasp9/CAR19-28ζ/IL15+ T cells (ROI2.0x109) In conclusion, our data indicate that a tricistronic vector can effectively be expressed in tumor-redirected human T cells, improving their survival and allowing their destruction should unwanted effects occur Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 61 Effects of Chimeric Antigen Receptor (CAR) Expression on Regulatory T Cells Ibrahim Akalin,1 Serena K Perna,1 Biagio De Angelis,1 Fatma V Okur,1 Cliona M Rooney,1 Helen Heslop,1 Malcolm K Brenner,1 Barbara Savoldo,1 Gianpietro Dotti.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Adoptive immunotherapies with genetically modified T lymphocytes are endowed with a means for effectively treating malignant and infectious disorders Transduction of T cells with chimeric antigen receptors (CARs) can redirect the cellular immune response to almost any surface target antigen The function of CAR+ T cells in vivo may, however, be impaired by naturally occurring regulatory T cells (Tregs) Moreover, Tregs themselves might be the inadvertent targets of CAR transfer, resulting in an undesired increase in their presence at the site of activity of CAR-expressing effector T cells To discover the importance of this effect and to devise means to avoid this, we isolated CD4+CD25bright Treg cells from peripheral blood of healthy donors and assessed their inhibitory function using carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay We then transduced peripheral blood polymorphonuclear cells (PBMCs) and Tregs with a chimeric antigen receptor targeting CD19 (CAR-CD19-CD28ζ), since this antigen is present on a high proportion of human B cell malignancies, and is the target for several current trials of T cell immunotherapy using such CAR-CD19 Our results showed that both activated PBMCs and Tregs are transduced and express CAR-CD19 (from 20% to 80%) While CAR-CD19+ T cells showed cytotoxic activity against CD19+ target cells as assessed by 51Cr release assay (71%±34% at E:T ratio 20:1), CAR-CD19+ Tregs lacked effector function (6%±0.1% at E:T ratio 20:1) (p=0.01), but retained inhibitory activity in an MLR [% of proliferation were 68%±18% and 28%±19% when activated T cells were incubated with CD4+CD25- control cells and CAR-CD19+ Tregs, respectively (p=0.001)] More importantly, when CAR-CD19+ Tregs were mixed with CAR-CD19+ T cells (ratio 5:1) they inhibited the anti-tumor effects of these cells (% of remaining CD19+ tumor cells were 49%±21% and 2%±4% when tumor cells were co-cultured with CAR-CD19+ T cells with or without the addition of CAR-CD19+ Tregs) (p=0.02) To discover whether the inhibitory effect on CARCD19+ T cells by CAR-CD19+ Tregs was due to competition for the same epitopes on the CD19 target antigen, we redirected Tregs and T cells with two different CARs (CAR-CD19 and CAR-CD30) We found that inhibition mediated by Tregs was maintained, suggesting that specific regulation rather than epitope/antigen competition was responsible for the observed inhibition Hence, transduction of unseparated peripheral blood T cells transduces both effector T cells and Tregs, and the latter are detrimental to the function of the former Future efforts to generate CAR-modified T cells for human use may need to separate effector T cells from Tregs before transduction or before infusion 62 Conditional Activation of T Cells to Specifically Target c-Met under Hypoxia Sonny O Ang,1 Simon Olivares,1 Drew C Deniger,1 Dean A Lee,1 Richard E Champlin,1 Laurence J Cooper.1 University of Texas MD Anderson Cancer Center, Houston, TX Malignancies localized within chronic and intermittent hypoxic niches, is a negative prognosticator for many aggressive, chemoand radio-resistant solid tumors The decreased oxygen tension, depleted nutrient levels, and low extracellular pH in this tumor microenvironment can limit T-cell viability, cytotoxicity, and thus immunotherapeutic efficacy This makes T-cell mediated elimination of bulky solid tumors difficult However, this also represents a microenvironment that can be exploited by genetic engineering to activate T cells for redirected killing We developed a new approach that S25 CANCER-APOPTOSIS & SUICIDE/CANCER-IMMUNOTHERAPY, ADOPTIVE T CELL THERAPY introduces a molecular sensor of oxygen as a trigger for activation and antigen-dependent cytotoxicity We demonstrate that a novel chimeric antigen receptor (CAR), which uses a scFv specific for c-Met and is capable of activating T cells through chimeric CD28 and/or CD3-ζ signaling endodomain, can be conditionally expressed on T cells in an oxygen-sensitive manner The Sleeping Beauty transposon/ transposase system was used to achieve CAR transgene expression and redirect specificity for c-Met, a receptor highly expressed on many hypoxic tumors By fusing the c-Met-specific CAR to an oxygen dependent degradation domain (ODDD), we stabilized expression of our CAR fusion under hypoxia (1% O2) and demonstrated expected loss of cell-surface expression under normoxia (20% O2) Anticipating the systemic circulation of infused T cells in vivo, our oxygen-sensitive CAR is designed to deactivate upon exiting from hypoxic tumor microenvironment to minimize deleterious off-target T-cell mediated effects Importantly, we demonstrate CAR-dependent cytotoxicity of c-Met+ cells by the reprogrammed T cells in an oxygen dependent manner (Figure) This approach to gene therapy of T cells has the advantage of transforming hypoxia from an adverse factor to a triggering mechanism thereby minimizing CAR-mediated potential off-target killing of normoxic tissues In summary, we have developed an approach to conditional activation of T cells through an introduced CAR for the targeting of hypoxic solid tumors Figure: (i) Schematic showing fusion of CAR to ODDD and degradation by ubiquitination under normoxia, but not hypoxia (ii) Selective T-cell killing of c-Met+ tumor under hypoxia 63 Immunotherapy of Metastatic Melanoma Using Genetically Engineered GD2-Specific T Cells Eric S Yvon,1 Barbara Savoldo,1 Valentina Hoyos,1 Aurélie Dutour,2 Catherine M Bollard,1 Gianpietro Dotti,1 Malcolm K Brenner.1 Center for Cell & Gene Therapy, Baylor College of Medicine, Houston, TX; 2INSERM U590, Centre Léon-Bérard, Lyon, France effector cells that bypass tumor immune escape mechanisms that are due to abnormalities in protein-antigen processing and presentation Moreover, these transgenic receptors can be directed to tumor associated antigens that are not protein derived We have generated chimeric T cells specific for the ganglioside GD2, which is expressed on a high proportion of melanoma cells We joined an extracellular antigen-binding domain derived from the GD2-specific antibody sc14 G2a to cytoplasmic signaling domains derived from the TCR z-chain, with the endodomains of the co-stimulatory molecules CD28 and OX40 In this study, we used three melanoma cell lines expressing different level of GD2 (CLB = low, SENMA = medium and P1143 = high) In a standard hours chromium release assay, the CAR-GD2 T lymphocytes were also able to kill GD2+ melanoma cells in vitro in an antigen-dependant manner (Figure) In addition, and upon co-incubation with GD2-expressing melanoma cells, these CAR-GD2 T lymphocytes secrete cytokines, producing 5208 to 9221 pg/ml/1x106 cells/24hrs of IFN-g; 77 to 333 pg/ml/1x106 cells/24hrs of TNFα, and from 83 to 232 pg/ml/1x106 cells/24hrs of IL5 Most critically, these CAR-GD2 T lymphocytes secreted from 20 to 169 pg/ml/1x106 cells/24hrs of IL2, and as a consequence, CFSE labeling showed the CAR-GD2 T lymphocytes proliferated extensively upon engagement of their chimeric receptor by GD2+ tumor cells Overall, cytokine secretion matched the pattern and level obtained following engagement of the native CD3 receptor by cognate antigen and co-stimulatory molecules.The CAR-GD2 T lymphocytes were also functional in vivo in a metastatic xenograft model (Figure 2) The melanoma cell line P1143 was labeled with FireFlyLuciferase Mice injected with CAR-GD2 T lymphocytes had a significant survival advantage over control animals with 80% survival at day 100 versus maximum survival of 68 ± days or 72 ± 12 days for the groups receiving tumor alone or non transduced T lymphocytes respectively (p=0.006) (Figure 3) Hence the ganglioside antigen GD2, expressed on the majority of primary melanoma cell lines can be targeted in vitro and in vivo by gene modified T cells expressing a compound CAR–co-stimulatory molecule directed to this antigen This approach may be of value in humans with advanced melanoma Genetic engineering of human T lymphocytes to express tumordirected chimeric antigen receptors (CAR) can produce anti-tumor S26 Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ... hypoxic tumor microenvironment to minimize deleterious off -target T- cell mediated effects Importantly, we demonstrate CAR-dependent cytotoxicity of c- Met+ cells by the reprogrammed T cells in... demonstrate that a novel chimeric antigen receptor (CAR), which uses a scFv specific for c- Met and is capable of activating T cells through chimeric CD28 and/or CD3-ζ signaling endodomain, can be conditionally...CANCER-APOPTOSIS & SUICIDE/CANCER-IMMUNOTHERAPY, ADOPTIVE T CELL THERAPY introduces a molecular sensor of oxygen as a trigger for activation and antigen-dependent cytotoxicity We demonstrate