521 Multiplex Genome Editing of TCR°/CD52 Genes as a Platform for “Off the Shelfâ€? Adoptive T Cell Immunotherapies Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Soci[.]
CANCER-IMMUNOTHERAPY I IL-12 and Decorin (hDCN) (RdB/IL12/hDCN) as suitable therapeutic adjuvant to overcome immune suppression by down-regulation of TGF-β through hDCN Intratumoral administration of RdB/IL12/ hDCN promoted enhanced antitumor effects compared with an oncolytic adenovirus expressing IL-12 or hDCN alone (RdB/IL12 or RdB/hDCN, respectively) We showed markedly elevated levels of interferon-γ (IFN-γ), tumor necrosis factor-a (TNF-a) and IFN-γ secreting cells in RdB/IL12/hDCN-treated tumors Moreover, Treg in draining lymph nodes (DLNs) dramatically decreased in mice treated with RdB/IL12/hDCN, suggesting an association with the down-regulation of TGF-β in tumor treated with RdB/IL12/hDCN Consistent with these data, tumor tissue injected with RdB/IL12/ hDCN showed increased infiltration of CD4+ T and CD8+ T cells infiltration Furthermore, hDCN enhanced viral distribution and tumor penetration, leading to improved virus-mediated cancer gene therapy by overcoming the extracellular matrix barrier within tumor masses Together, these results provide new insight into the ability of hDCN to eliminate immune suppression and demonstrate that adenovirus co-expressing IL-12 and hDCN is a promising therapeutic tool for cancer treatment than adenovirus expressing IL-12 alone to mice boosted with control aAPC/CD40L/OX40L (p=0.002) Forty-eight percent were tumor free, while only 8% of mice boosted with control aAPC/CD40L/OX40L cleared the tumor (p≤0.037) Although K562-derived vaccines have been extensively used in clinical trials, to further increase the safety profile of this approach we also incorporated the inducible caspase9 safety switch We found that its activation effectively eliminates aAPC which had purposely been engrafted in NSG mice to mimic an unforeseen circumstance of aAPC accidentally inoculated without prior irradiation In conclusion, we have identified a strategy based on an “off the shelf” vaccine that can be used to stimulate CAR-redirected virus-specific CTLs in vivo ensuring their longer persistence, which results in an improved antitumor activity 520 A PD1-CD28 “Switch Receptor” Is Able to Augment Mesothelin-Directed Chimeric Antigen Receptor T Cell Therapy in a Resistant In Vivo Model of Human Tumor 519 In Vivo Boosting of CAR-Redirected Virus Specific CTLs by Artificial Antigen Presenting Cells Edmund K Moon,1 Raghuveer Ranganathan,1 Jing Sun,1 Xiaojun Liu,2 Carl H June,2 Steven M Albelda,1 Yangbing Zhao.2 Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 2Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA Virus-specific cytotoxic T lymphocytes (CTLs) redirected with a GD2-specific chimeric antigen receptor (GD2.CAR) induces objective clinical responses in patients with relapsed/refractory neuroblastoma (NB) [Pule et al Nat Med 2006] However the in vivo persistence of these cells remains limited Using Cytomegalovirus-(CMV)-specific CTLs as a model, we sought to boost GD2.CAR-CMV-CTLs in vivo by using artificial antigen presenting cells (aAPC) based on the K562 cell line, encoding the pp65-CMV antigen and CD40L/OX40L costimulatory molecules The rational of this vaccine design relies on the in vivo pp65-antigen-cross presentation to professional dendritic cells (DC) by apoptotic/necrotic bodies originating from irradiated pp65-modified/K562 CD40L induces DC maturation, while OX40L is mediating CD4+ T-cell activation As shown in Table 1, we found that gene modified aAPC reproducibly reactivated CMV-CTLs from 11 CMV+ donors both ex vivo and in NSG mice grafted with PBMC from CMV+ donors and boosted in vivo with irradiated aAPCs Of note the co-expression of both OX40L and CD40L (aAPC/CD40L/ OX40L/pp65) showed the highest activation of CMV-CTLs in vivo (*p=0.048) Adoptive Cell therapy (ACT) using genetically-modified T cells has shown great promise in diseases such as melanoma and hematologic malignancies Our group has recently applied this paradigm-changing technology to malignant pleural mesothelioma (MPM) by creating T cells that express a chimeric antigen receptor (CAR) that targets the highly expressed tumor antigen, mesothelin (mesoCAR T cells) Previously published work showed the mesoCAR T cells could reduce the size of human MPM cell line (M108) flank tumors in mice Based on this data, a Phase I trial in MPM patients using mesoCAR T cells has begun One limitation of ACT using tumor-infiltrating lymphocytes (TILs) using their native T cell receptors has been functional inhibition This important phenomenon has not yet been fully explored in CAR signaling Employing a novel resistant in-vivo model of tumor-induced mesoCAR T cell hypofunction, we have demonstrated upregulation of PD1 on tumor-infiltrating mesoCAR T cells and of PDL1 on tumors infiltrated by those cells To block this inhibition, we have created a PD1-CD28 “switch receptor” in which the extracellular domain of PD1 was linked to the cytoplasmic domain of CD28 and expressed along with a mesoCAR receptor Genetically modifying mesoCAR T cells with a PD1-CD28 switch receptor led to increased pro-inflammatory cytokine secretion, infiltrating T cell proliferation, tumor control, and resistance to tumor-induced hypofunction Ignazio Caruana,1 Gerrit Weber,1 Barbara Savoldo,1 Gianpietro Dotti.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX (SFU/105 cells) In vitro IFNγ aAPC/CD40L/OX40L 92±19 In vivo IFNγ 27±6 aAPC/pp65 292±56 57±24 aAPC/CD40L/pp65 502±104 41±14 aAPC/OX40L/pp65 357±40 35±17 aAPC/CD40L/OX40L/pp65 477±91 101±21* Using the same NSG model, we also found that when CMV-CTLs were genetically modified to express the GD2.CAR, vaccinations in vivo with aAPC/CD40L/OX40L/pp65 also increases the frequency of IFNg+ T cells responding to GD2 (71±24 IFNg+SFU/105 cells) and pp65 antigen (85±16), compared to mice vaccinated with the control aAPC/CD40L/OX40L (23±8, p=0.048, and 41±11, p=0.035, for GD2 and pp65, respectively) This boosting ultimately promoted better antitumor effects Indeed, NSG mice engrafted with GD2+ tumor cells, receiving GD2.CAR+CMV-CTLs and boosted in vivo with aAPC/CD40L/OX40L/pp65 had better tumor reduction compared Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy 521 Multiplex Genome Editing of TCRα/CD52 Genes as a Platform for “Off the Shelf” Adoptive T-Cell Immunotherapies Laurent Poirot,1 Brian Philip,2 Cécile Schiffer-Mannioui,1 Sophie Derniame,1 Agnes Gouble,1 Sylvain Arnould,1 Julianne Smith,1 Martin Pule,2 Andrew Scharenberg.1 Cellectis Therapeutics, Paris, France; 2Department of Hematology, UCL Cancer Institute - University College of London, London, United Kingdom Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has emerged as a powerful approach to treating cancer However, a limitation of this approach is that CAR T-cells must be generated on a bespoke basis To permit adoptive T cell therapy using allogeneic T cells, we have developed a platform for the large scale production of “off-the-shelf” CAR T-cells from unrelated 3rd party donor T-cells We overcome the S201 CANCER-IMMUNOTHERAPY I key barriers to the adoptive transfer of 3rd party CAR T-cells via application of Transcription Activator-Like Effector Nucleases (TALEN) gene editing technology – we eliminate the potential for T-cells bearing alloreactive TCR’s to mediate GvHD by disrupting the TCRalpha constant (TRAC) gene, and we exploit the requirement for preparative lymphodepletion for engraftment of allogeneic CAR T-cells through disruption of the CD52 gene Simultaneous editing of the TRAC and CD52 genes results in TCR/CD52-deficient T-cells, which can be administered with concomitant alemtuzumab-mediated lymphodepletion/ immunosuppression to both promote engraftment and suppress rejection In order to achieve simultaneous inactivation of the TRAC and CD52 genes in primary human T-cells, we developed and optimized a method for electroporation of TALEN mRNAs into T-cells that consistently achieves 40% double knock out rates This TALEN electroporation step was incorporated following transduction of a CAR gene cassette into a two-week, large scale, process for GMP manufacturing of TCR/CD52-deficient CAR T-cells We provide proof of concept for the general applicability of this approach by manufacturing a TCR/CD52-deficient universal CD19 CAR T-cell (UCART19), and demonstrating that this product does not mediate alloreactivity, and can be selectively engrafted in the presence of alemtuzumab while maintaining potency equivalent to standard CD19 CAR T-cells in an orthotopic CD19+ lymphoma model of somatic knockout of the endogenous TCR genes (by transient exposure to alpha and/or beta chain specific Zinc Finger Nucleases - ZFNs) and introduction of tumor-specific TCR genes by lentiviral vectors (Provasi, Genovese et al Nat Med 2012) While the complete editing (CE) procedure requires multiple manipulation steps, ‘single TCR editing’ (SE), based on the disruption of a single endogenous TCR chain, followed by gene transfer of a tumor specific TCR, enables the generation of redirected T cells devoid of their natural TCR repertoire with a single round of T cell activation, improving the feasibility of its clinical translation By completely abrogating the endogenous repertoire, single TCR editing overcomes the risk of Graft versus Host Disease (GvHD) after allogeneic hematopoietic stem cell transplantation We exploited a HLA-A2 restricted TCR specific for NY-ESO-1, expressed by several solid tumors and hematological malignancies, to compare safety and efficacy of unedited TCR transferred (TR), SE and CE T cells Genetically modified T cells were tested in vitro for TCR expression, differentiation phenotype and killing ability, and in vivo in NSG mice engrafted with the U266 (HLA-A2+ and NY-ESO-1+) myeloma cell line Gene editing ensured the preservation of an early differentiated T cell phenotype, enriched in central memory and stem memory T cells Upon lentiviral transfer of the NY-ESO-1-specific TCR, we observed significantly higher levels of the tumor-specific TCR expression, measured as dextramer binding capacity, in edited versus transferred T cells Edited T cells were more efficient than transferred T cells in killing NY-ESO-1+ myeloma cell lines Importantly, SE and CE T cells displayed no reactivity against NY-ESO-1- targets In NSG mice, NY-ESO-1 redirected SE and CE T cells completely eliminated the NY-ESO1+ HLA-A2+, WT1- U266 myeloma line (19 out of 19 mice), that accumulated in the bone marrow in the presence of WT1-redirected CE T cells (5 out of mice) Importantly, mice infused with CE or SE T cells did not develop xenogeneic GvHD In conclusion, the TCR single editing approach allows the rapid generation of large numbers of tumor specific T cells with high anti-tumor efficacy and, being devoid of off-target reactivity, unable to cause GvHD 523 Clinical Application of TCR GeneTransduced Lymphocytes for Patients With Epitherial Cancer and Other Types of Malignancy: In Vivo Persistence of Adoptively Transferred TCR Gene-Transduced Lymphocytes With Anti-Tumor Reactivity in Patients We propose that this approach may be used as a platform for large scale manufacturing of allogeneic CAR T-cell products directed against arbitrary targets for administration as “off the shelf” immunopharmaceuticals 522 Single Edited T Cells Redirected Towards NY-ESO-1 Ensure Tumor Rejection Without Inducing Xenogeneic GvHD Sara Mastaglio,1 Pietro Genovese,1 Zulma Magnani,1 Barbara Camisa,1 Elisa Landoni,1 Elena Provasi,1 Angelo Lombardo,1 Andreas Reik,2 Nicoletta Cieri,1 Maurilio Ponzoni,1 Fabio Ciceri,1 Claudio Bordignon,3 Michael C Holmes,2 Philip D Gregory,2 Luigi Naldini,1 Chiara Bonini.1 San Raffaele Scientific Institute, Milan, Italy; 2Sangamo BioSciences Inc, Richmond; 3MolMed SpA, Milan, Italy T cell receptor (TCR) gene transfer has yielded promising clinical results in cancer patients To permanently remove the expression of the endogenous TCR and the risk of TCR chain mispairing, our group developed a TCR gene editing approach, based on the combination S202 Hiroaki Ikeda,1 Shinichi Kageyama,1 Naoko Imai,1 Yoshihiro Miyahara,1 Mikiya Ishihara,1 Naoyuki Katayama,2 Hirofumi Yoshioka,3 Daisuke Tomura,3 Ikuei Nukaya,3 Junichi Mineno,3 Kazuto Takesako,3 Hiroshi Shiku.3 Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Tsu, Japan; 2Hematology and Oncology, Mie University Graduate School of Medicine, Tsu, Japan; 3Center for Cell and Gene Therapy, Takara Bio Inc., Otsu, Japan Engineering the antigen receptor gene in patients’ lymphocytes is one promising strategy to create antigen-specific lymphocytes without senescent phenotypes The strategy provides an opportunity to extend the application of adoptive T cell therapy for cancer patients However, this concept has not been well verified in patients with epithelial cancer or hematological malignancy We completed a phase I clinical trial of TCR gene therapy targeting MAGE-A4 to treat esophageal cancer patients without lymphodepleting pre-conditioning The trial was designed as a cell-dose escalation consisting of three cohorts, 2x108, 1x109 and 5x109 cells/ patient The treatment was tolerable with no adverse events associated with transferred cells In all patients, the transferred lymphocytes were detected in their peripheral blood in a dose-dependent manner during the first 14 days The infused cells persisted more than months after the transfer and sustained the reactivity to the antigen-expressing Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ... platform for large scale manufacturing of allogeneic CAR T- cell products directed against arbitrary targets for administration as “off the shelf” immunopharmaceuticals 522 Single Edited T Cells... completed a phase I clinical trial of TCR gene therapy targeting MAGE -A4 to treat esophageal cancer patients without lymphodepleting pre-conditioning The trial was designed as a cell- dose escalation consisting... eliminate the potential for T- cells bearing alloreactive TCR’s to mediate GvHD by disrupting the TCRalpha constant (TRAC) gene, and we exploit the requirement for preparative lymphodepletion for