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209 TCR Gene Editing in a Single Step of T Cell Activation To Redirect T Cell Specificity and Prevent GvHD Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene[.]
Cancer – Immunotherapy, Cancer Vaccines I in vitro Furthermore, to assess the anti-tumor effect of anti-FSHR T-cells, we established human OvCa xenografts in NSG mice by subcutaneous flank injection of CaOV3 cancer cell line followed by two intravenous redirected T-cell administrations As shown by caliper measurements, FSHR-redirected T-cells elicited anti-tumor potential with suppressed outgrowth of established tumor Tumor growth was statistically reduced in comparison to control, GFP engineered T-cell treated group or PBS treated animals These data suggest that systemic delivery of anti-FSHR T-cells to mice bearing FSHR-expressing tumors may result in tumor trafficking, activation, and lysis at location of tumor In summary, we have successfully exploited for the first time a novel therapeutic antigen, the FSHR protein, expressed on tumor cell surface, to deliver T-cell based immune-therapy of cancer Given its highly restricted expression in cancer, cancer-associated vessels and gonadal tissues, the rationale for FSHR targeting is strong and supports further exploration of FSHR-targeted immune therapy approaches for cancer patients 207 Optimal Activation of Both CD28 and 4-1BB in CAR-Targeted T Cells Results in Enhanced Tumor Eradication Zeguo Zhao,1 Sjoukje J.C van der Stegen,1 Maud Condomines,1 Fabiana Perna,1 Gertrude Gunset,1 Jason Plotkin,1 Michel Sadelain.1,2 Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY; 2Immunology Program, Sloan Kettering Institute, New York, NY Chimeric Antigen Receptors (CARs) are a novel drug class, consisting of engineered receptors for antigen that redirect and reprogram T-cell function We have shown in both xenogeneic tumor models as well as in leukemia patients, that CD19-targeted T-cells are capable of inducing complete remission of relapsed or refractory B cell malignancies The inclusion of appropriate co-stimulation is essential for the potency, quality and durability of the CAR T cell response Here we show that the co-stimulatory domain incorporated within the CAR dictates the kinetics of in vivo anti-tumor response and T-cell accumulation Additionally, expression of co-stimulatory ligands complementary to the second-generation CAR provides an optimal balance of cytolytic activity and T-cell accumulation resulting in superior anti-tumor function The combination of 19-28z CAR with 4-1BBL was associated with activation of the endogenous Interferon-β (IFNβ) pathway in an IRF7-dependent manner This enabled very small T-cell doses to eradicate systemic established leukemia in a xenogeneic model The unexpected role of the IRF7/IFNβ pathway provides insight into the synergy behind co-stimulatory molecules in T cell activation and a novel mechanism through which engineered T cells can increase tumor control 208 Single-Cell Gene Expression Profiles of Genetically Modified T Cells for Adoptive Immunotherapy Sonny O Ang,1 Laurence J.N Cooper.1 Pediatrics, University of Texas MD Anderson Cancer Center, Houston, TX System-level characterization of cell states requires detailed portrayal of cellular components such as gene transcripts or proteins Here, we exploit a robust single-cell platform technology to characterize T cells expanded ex vivo intended for human application Coupled with advanced custom analytics, we identify “drivers” (relatively few, but vital) genes which orchestrate T-cell activation and clonal expansion programming, versus “passengers” (relatively many, less critical) or resultant/downstream transcripts S82 Detailed analyses reveal that there is considerable variability between individual activated T cells, implying that cell state may undergo multiple stepwise transitions in a stochastic manner Compared to interrogation of bulk T-cell populations (e.g., analyzed using RNASeq, microarrays, and Northern blotting), where heterogeneity in expression signals is attenuated by temporal and cell-cycle averaging, the genetic heterogeneity between cells in isogenic populations may be a reflection of fundamental phenomena such as transcriptional bursting Our model describes cell state oscillation in tandem with transcriptional repressive (closed chromatin) and permissive conformations (open chromatin), versus simpler probabilistic models of transcription T-cell variability can significantly impact upon individual cell behavior within seemly isogenic populations, and may be essential in molding survival and cytotoxicity within certain contexts, such as the rapidly changing and stressful tumor microenvironments We find that lactate dehydrogenase A (LDHA) is a leading indicator for the metabolic adaptation during T-cell activation and together with other energetics related genes such pyruvate kinase isoform (PKM2), can be harnessed to adjust our culture conditions during ex vivo expansion These cellular fingerprints suggest useful ways for tuning the balance between oxidative and non-oxidative glucose metabolism to enhance the killing and thus therapeutic potential of our T cells Our framework to harness catalogs of gene expression data into clinically-applicable insights can be adapted for meaningful characterization of other therapeutically-relevant source populations such as hematopoietic stem cells and reprogrammed NK cells This is being reduced to practice by understanding the heterogeneity within and between genetically modified T-cell products generated for clinical use 209 TCR Gene Editing in a Single Step of T Cell Activation To Redirect T Cell Specificity and Prevent GvHD Pietro Genovese,1 Sara Mastaglio,2 Zulma Magnani,2 Elisa Landoni,2,3 Barbara Camisa,2 Giulia Schiroli,1,3 Elena Provasi,1 Angelo Lombardo,1,3 Andreas Reik,4 Nicoletta Cieri,1 Maurilio Ponzoni,2 Fabio Ciceri,2 Claudio Bordignon,2 Michael C Holmes,4 Philip D Gregory,4 Luigi Naldini,1,3 Chiara Bonini.2 TIGET, Milan, Italy; 2San Raffaele Scientific Institute, Milan, Italy; 3San Raffaele University, Milan, Italy; 4Sangamo BioSciences, Richmond Transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach for cancer adoptive immunotherapy Yet, TCR gene transfer into mature T cells results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities Thus, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to α and β chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes (Provasi et al, Nat Med 2012) While successful, the complete editing requires multiple manipulation steps involving repeated cell activation cycles and transductions To reduce the duration and complexity of cell product generation, we recently developed a ‘single TCR editing’ (SE) procedure, based on the disruption of the endogenous TCR α chain only followed by the transfer of the tumor specific TCR genes This SE method generates redirected T cells fully devoid of their natural TCR repertoire in a single round of cell activation We validated the SE protocol exploiting an HLA-A2 restricted TCR specific for NY-ESO-1 (expressed by a considerable proportion of high risk multiple myeloma) The SE strategy allowed rapid production of high numbers of tumor specific T cells enriched for an early differentiation phenotype When fucntionaly tested (co-culture, γ-IFN and 51Cr release) against the Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy Cancer – Immunotherapy, Cancer Vaccines I U266 myeloma cell line (NY-ESO-1+HLA-A2+), all NY-ESO-1 redirected T cells showed a strikingly high killing activity However, when we assess the alloreactive potential of the different redirected T cells in mixed lymphocyte reactions, we observed that the allogeneic lysis by SE T cells was markedly lower (p=0.05) than that of conventional TCR transfer cells (TR) These results were validated in NSG mice, where the genetically modified T cells were infused after the engraftment of the U266 myeloma All animals treated with tumor specific T cells were completely myeloma-free at the time of sacrifice, demonstrating the powerful anti-tumor potential of the NY-ESO-1 redirected T cells However, the overall survival of mice treated with TR vs SE cells was 26% vs 100% respectively (p