497 A Recombinant Dimeric Ad3 Fiber Protein Triggers Opening of Epithelial Junctions in Solid Tumors and Improves Trastuzumab/Herceptin and Cetuximab/Erbitux Therapy in Mouse Models Molecular Therapy[.]
CANCER - IMMUNOTHERAPY II Cancer - Immunotherapy II 495 Effective Eradication of Established Ovarian Murine Tumors with MUC16 Targeted T Cells Expressing IL-12 Gene Alena A Chekmasova,1 Samith Sandadi,2 Yan Nikhamin,1 David R Spriggs,1 Renier J Brentjens.1,3 Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY; 2Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY; 3The Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY Despite multimodality therapy with surgery and chemotherapy, most patients with ovarian carcinomas have a poor prognosis For this reason, alternative approaches to treating this disease are urgently needed Autologous T cells may be genetically modified to target tumor antigens through retroviral transduction with chimeric antigen receptors (CARs) We have constructed SFG retroviral vectors encoding first (4H11mz) and second (4H11m28mz) generation CARs as well as IL-12 modified CAR (4H11m28mzIRESmIL12) targeted to the retained extra-cellular domain of MUC16, termed MUC-CD This antigen is over-expressed on most ovarian carcinoma tumor cells Previously we have shown the efficacy of MUC-CD targeted T cells via efficient in vitro cytolytic activity against MUC-CD expressing cell lines and eradication of established MUC-CD+ tumors in SCID-Beige mice In order to mimic the clinical setting and generate the hostile tumor microenvironment, we utilized a syngeneic tumor model using ID8 mouse ovarian tumor cells retrovirally transduced to overexpress MUC-CD antigen The C57BL6 mice were intraperitoneally (i.p.) injected with 1x107 ID8(MUC-CD) tumor cells If left untreated, these mice developed marked ascites and multiple nodular peritoneal tumors by weeks following tumor cell injection In this series of experiments, 5x106 MUC-CD targeted transduced mouse T cells were injected i.p on day and/or post tumor injection We found a statistically enhanced long-term survival of mice treated with 4H11mz+ or 4H11m28mz+ transduced T cells injected day after tumor injection when compared to untreated mice or mice treated with T cells modified to express an irrelevant control CAR (4H11mz compared to 19m28mz with 37% survival up to 120 days, p=0.0036 and 4H11m28mz compared to 19m28mz with 80% survival up to 120 days, p=0.0001) However, treatment of mice with advanced tumors (7 days after i.p injection of ID8(MUCCD) tumor cells) with 4H11m28mz+ T cells demonstrated only 27% long term survival (p=0.005, 4H11m28mz compared to 19m28mz), compared to IL-12 secreting 4H11m28mz T cells treated mice (100% long term survival up to 160 days, p=0.0028) For negative controls, tumor bearing mice were treated with T cells modified to express the irrelevant CD19-targeted 19m28mz or 19m28mzIRESIL12 CARs To assess whether 4H11m28mzIRESmIL12+ T cells eradicate more clinically relevant tumor burdens, we next treated C57BL6 mice 14 days post i.p ID8(MUC-CD) tumor injection At this point, there is evidence of overt disease as assessed by FACS No gross evidence of disease was found on day 120 after the mice were sacrificed Additionally, FACS analysis of peritoneal washes was negative for the presence of MUC-CD+ tumor cells This data supports the further translation of these pre-clinical studies to the clinical setting in the form of a phase I clinical trial in patients with persistent or relapsed ovarian carcinomas Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 496 T Cells Redirected Against HER2 for the Adoptive Immunotherapy of HER2-Positive Malignancies Nabil Ahmed,1,2,3 Vita Salsman,1,2,3 Oumar Diouf,1,2,3 Aidin Ashoori,1,2,3 Claudia Gerken,1,2,3 Lisa Wang,1,2 Peter Anderson,4 Benjamin Musher,1 Shanda Blackmon,3 Teresa Hayes,1 John Hicks,1,2 Hao Liu,1 Winfried Wels,5 Catherine Bollard,1,2,3 Cliona Rooney,1,2,3 Gianpietro Dotti,1,2,3 Adrian Gee,1,2,3 Malcolm Brenner,1,2,3 Helen Heslop,1,2,3 Stephen Gottschalk.1,2,3 Baylor College of Medicine, Houston, TX; 2Texas Children’s Hospital, Houston, TX; 3The Methodist Hospital, Houston, TX; MD Anderson Cancer Center, Houston, TX; 5Georg-Speyer-Haus Chemotherapeutische Forschungsinstitut, Frankfurt, Germany The human epidermal growth factor receptor (HER2) is expressed in a broad range of adult as well as pediatric malignancies The majority of these cancers express HER2 at low levels, rendering HER2 monoclonal antibodies like trastuzumab ineffective Genetic modification of T cells is one attractive approach to overcome this limitation and we have constructed a 2nd generation HER2-specific chimeric antigen receptor (CAR) containing the HER2-specific single chain fragment FRP5, a transmembrane domain, and a CD28.ζ costimualtory/signaling domain (HER2-CAR) T cells expressing HER2-CARs (HER2-CAR T cells) had potent antitumor activity in preclinical animal models Based on these preclinical findings we developed three Phase I/II clinical studies for patients with lung cancer, osteosarcoma, and glioblastoma multiforme Due to the adverse event reported after the infusion of 1010 HER2-CAR T cells after nonmyeloablative conditioning in a patient with colon cancer, we designed our trials without lymphodepleting chemotherapy starting at T-cell doses of 104 cells/m2 or 106 cells/m2 Our clinical studies for patients with osteosarcoma and lung cancer have recently opened for accrual and we have infused so far patients with HER2-CAR T cells Patients received cell doses between 104/m2 to 1x105/m2cells Infusions were well tolerated without systemic side effects, and no increase of proinflammatory cytokines (IL-2, IFN-γ, GM-CSF, TNF-α) in the patients’ plasma was observed post infusion HER2CAR T cells were detectable for up to weeks post infusion and localized to sites of disease In conclusion, HER2-CAR T cells have shown promising antitumor activity in preclinical animal models We are currently evaluating the safety and efficacy of HER2-CAR T cells in three Phase I/II clinical studies Initial safety data is encouraging, warranting further active exploration of HER2-CAR T-cell based therapies for cancer 497 A Recombinant Dimeric Ad3 Fiber Protein Triggers Opening of Epithelial Junctions in Solid Tumors and Improves Trastuzumab/Herceptin and Cetuximab/Erbitux Therapy in Mouse Models Ines Beyer,1 Hongjie Wang,1 ZongYi Li,1 Roma Yumul,1 Andre Lieber.1 Division of Medical Genetics, University of Washington, Seattle, WA The effectiveness of cancer therapeutics is decreased by the blocking of intratumoral diffusion and/or physical masking of target receptors on malignant cells In immunohistochemical studies of tumor sections from breast cancer or non-small cell lung cancer xenografts, we observed colocalization of epithelial junction protein with Her2/ neu or with EGFR - tumor-associated antigens that are the targets for monoclonal antibodies i.e trastuzumab (Herceptin) and cetuximab (Erbitux), respectively In epithelial cells, adenovirus serotype binding to the junction protein desmoglein (DSG2) triggers events reminiscent of epithelial-to-mesenchymal transition, leading to the transient opening of intercellular junctions This opening improves access to receptors, e.g Her2/neu and EGFR1, that are trapped S191 CANCER - IMMUNOTHERAPY II in these intercellular junctions In addition to complete virions, penton recombinant dodecahedral particles (PtDds) and dimeric Ad3 fiber domains (Ad3K-K), can trigger DSG2-mediated opening of intercellular junctions In in vitro studies we show a significant improvement of trastuzumab dependent cell killing of the Her2/neu positive breast cancer cell line BT474M1 subsequent to treatment with the dimeric Ad3K fiber We also tested whether the application of Ad3K-K would result in the improvement of trastuzumab or cetuximab therapy in vivo In mouse models with xenograft breast cancer tumors (BT474-M1, HCC 1954) and xenograft lung cancer tumors (A549), Ad3K-K improved the efficiency of the monoclonal antibody used and enabled control of tumor growth in subcutaneous tumor models We are currently testing a combination of Ad3K-K and cetuximab therapy in an orthotropic lung metastasis model, where we expect to see a benefit in the overall survival and control of tumor growth in the group that received the co-therapy Our results have potential implication for cancer therapy with T-cells or encapsulated chemotherapy drugs 498 Genetic Editing of T Lymphocyte Specificity for Safe and Effective Adoptive Immunotherapy Pietro Genovese,1,2 Elena Provasi,2,3 Angelo Lombardo,1,2 Zulma Magnani,3 Pei-Qi Liu,4 Andreas Reik,4 Victoria Chu,4 David E Paschon,4 Lei Zhang,4 Jurgen Kuball,5 Attilio Bondanza,3 Giulia Casorati,3 Fabio Ciceri,3 Claudio Bordignon,3 Philip D Greenberg,5 Michael C Holmes,4 Philip D Gregory,4 Luigi Naldini,1,2 Chiara Bonini.2,3 HSR-Telethon Institute for Gene Therapy, Milan, Italy; 2Vita Salute San Raffaele University, Milan, Italy; 3San Raffaele Scientific Institute, Milan, Italy; 4Sangamo BioSciences, Richmond; Fred Hutchinson Cancer Research Center, Seattle Transfer of a T cell receptor (TCR) from a high-avidity tumorspecific T cell (CTL) to polyclonal T cells may overcome the challenges of expanding rare tumor-specific CTLs under conditions that preserve function and prevent exhaustion Unfortunately, the full potential of this approach is limited by the co-expression in the same lymphocyte of the endogenous and exogenous TCR chains, resulting in competition for surface expression and acquisition of novel unpredicted specificities due to TCR mispairing between the chains, which might lead to dangerous autoreactivity Thus, we developed two sets of Zinc Finger Nucleases (ZFNs) to target double-strand breaks to the constant regions of the TCR α (TRAC) and β (TRBC1 and TRBC2) genes and disrupt their coding potential by the induced Non Homologous End Joining (NHEJ) repair process We used a stimulation protocol based on anti-CD3/CD28 antibodyconjugated beads and culture with low doses of IL-7/IL-15 to preserve cells with an early T cell differentiation phenotype Upon stimulation, delivery of either ZFN set by Adenoviral vectors (Ad5/F35) resulted in functional inactivation of the target genes in primary T cells (>45%), as indicated by the generation of cells that lack surface expression of the CD3/TCR complex (CD3neg) Once sorted, CD3neg cells proved stable in culture for more than 50 days, maintaining an early T cell phenotype (CD62L+ CD28+ CD27+ IL7Ra+), and were permissive to further lentiviral vector (LV) transduction For complete editing of T cell specificity, we selected a codon-optimized, cystein-modified TCR specific for the Wilm’s Tumor Antigen (WT1), involved in oncogenic transformation in several tumors, and developed a strategy for sequential disruption of each endogenous TCR chain followed by LV transfer of the respective WT1-specific chain Using this approach we obtained a population of TCR-edited lymphocytes, carrying only the tumor-specific TCR that, in the absence of competition, was robustly expressed at physiological levels Genetic editing did not affect the proliferative potential and the clonability of engineered cells nor their capacity to differentiate into effector and effector memory T S192 cells TCR-edited lymphocytes were superior to conventional TCRtransferred cells in WT1 pentamer binding, specific recognition and lysis of WT1pos targets, including primary acute myeloid leukemia blasts Importantly, these cells showed sharply reduced non-specific reactivity, including alloreactivity, demonstrating the advantage of preventing TCR mispairing Overall, our results demonstrate that full genetic editing of T cell specificity is feasible, and allows the rapid generation of effective and safe T cells for adoptive immunotherapy 499 Primary Human CD8+ T Cells Engineered To Express a PD1-CD28 Chimeric Receptor Are Co-Stimulated through the Exploitation of Tumor Expressed PD-L1 Megan E Prosser,1 Christine E Brown,1 Michael C Jensen.2 Department of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute of the City of Hope, Duarte, CA; 2Center for Immunity and Immunotherapies, Seattle Childrens Research Institute, Seattle, WA Adoptive immunotherapy is a promising therapeutic approach for the treatment of malignancies However, several challenges must be addressed to enhance therapeutic efficacy, including the loss of effector function of adoptively transferred cells in the tumor microenvironment Tumors elicit multiple mechanisms for immunosuppression of transferred cells including deficiency of costimulation and expression of inhibitory ligands T cell activation requires both antigenic stimulation through the T cell receptor as well as a costimulatory second signal This secondary costimulatory signal is commonly deficient within the tumor microenvironment resulting in T cell anergy In addition, tumors often express the inhibitory ligand Programmed Death Ligand (PD-L1) which interacts with Programmed Death (PD-1) expressed on the T cell surface, resulting in T cell exhaustion, characterized inhibition of TcR signaling, and enhanced apoptosis We hypothesize that the development of a PD1-CD28 chimera could exploit the PD-L1 tumor derived inhibitory mechanism to result in tumor-induced costimulation and maintenance of effector function of adoptively transferred CD8+ T cells, thereby addressing the two aforementioned immunosuppressive mechanisms Preliminary assessment of this PD1-CD28 chimera in transformed cells reveals that fusion of the PD1 extracellular domain to the intracellular signaling domain of CD28 does not alter PD-L1 binding to the extracellular PD-1 moiety Furthermore, the chimera results in increased phosphorylation of ERK and AKT suggesting increased signaling through CD28- and TCR- signaling pathways in transformed cells upon ligand binding In addition, this PD1-CD28 chimera results in increased costimulatory cytokine production in both transformed T cell lines as well as primary human CD8+ T cells The PD1-CD28 chimera also provides a proliferative advantage, as well as increased accumulation of cytotoxic granules for CD8+ T cells This work suggests that genetically engineering T cells to express the PD1CD28 chimera may exploit an intrinsic tumor immunosuppressive mechanism to result in costimulation and maintenance of effector function of adoptively transferred cells 500 CD56-Specific T Cells Can Distinguish between Allogeneic and Autologous CD56+ Targets Denise L Kellar,1 Sonny Ang,1 Simon Olivares,1 Helen Huls,1 Laurence J N Cooper.1 Division of Pediatrics, UT MD Anderson Cancer Center, Houston, TX Some candidate tumor-associated antigens (TAAs) are also expressed on T cells limiting the use of targeted T-cell therapy CD56 is an attractive TAA with expression on many malignancies, yet CD56 upregulation on a subset of activated T cells could lead to Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... the chimera results in increased phosphorylation of ERK and AKT suggesting increased signaling through CD28- and TCR- signaling pathways in transformed cells upon ligand binding In addition,...CANCER - IMMUNOTHERAPY II in these intercellular junctions In addition to complete virions, penton recombinant dodecahedral particles (PtDds) and dimeric Ad3 fiber domains (Ad3K-K), can trigger... tumor models We are currently testing a combination of Ad3K-K and cetuximab therapy in an orthotropic lung metastasis model, where we expect to see a benefit in the overall survival and control of