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Long-term follow-up of adoptive transfer of autologous T cells expressing a chimeric antigen receptor (CAR) directed to CD19 antigen has demonstrated encouraging, durable clinical outcome in various B cell malignancies. However, to make such CAR-T cells available to a broader base and to reach a more diverse patient population, challenges associated with product consistency, cost of manufacture, precision genetic engineering and on-demand availability still need to be addressed. FT819 is a first-of-kind off-the-shelf CAR-T cell product candidate derived from a renewable master pluripotent cell line. FT819 comprises precise genetic engineering of multiple targeting events at the single cell level and is produced using a clonally-derived master cell bank (MCB) that serves as the starting material to support consistent and reproducible clinical manufacturing. The engineered features of FT819 include the targeted integration of a novel CD19 1XX-CAR into the T-cell receptor α constant (TRAC) locus to provide antigen specificity, enhanced efficacy and temporally-regulated CAR expression driven by an endogenous (TCR) promoter. Such features are designed to also eliminate the possibility of graft versus host disease (GvHD) by nullifying the TCR. To develop the MCB for FT819, αβ T cells were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently engineered to direct CD19 1XX-CAR into the TRAC locus with knockout of the TCR. To generate clonal lines, engineered iPSCs were sorted by flow cytometry for various markers and single cells were seeded into individual wells of feeder-free 96-well plates. Engineered iPSC clones were screened for integration of CAR into the TRAC locus by amplifying the genomic DNA flanking the homologous recombination site and confirmed by a SNP phasing assay. Clones were further screened for random integration of donor template by quantitative PCR and the CAR copy number was confirmed by droplet digital PCR. Genome stability of each clone was also confirmed by karyotype analysis. Overall, the described screening initiative surveyed 774 clones to select the ideal MCB for FT819. Utilizing our stage-specific T cell differentiation and expansion protocol, we demonstrated that T cells derived from the FT819 (FT819-iTs) expanded greater than 100,000-fold during the clinical manufacturing process and the cells expressed greater than 95% T lymphocyte markers such as CD45, CD7, intracellular CD3, and TRAC-regulated CAR. Further modifications to the T cell differentiation protocol resulted in enhanced expression of CD8 αβ from less than 25% to greater than 70% of the total population. In addition, expression of CD2, CD5, and CD27 was increased by approximately 5- to 20-fold. In vitro functional studies showed that FT819-iTs possess antigen specificity as confirmed by cytokine release and cytotoxic T lymphocytes (CTL) assays. Upon stimulation with a wild type acute lymphoblastic leukemia line, Nalm-6, FT819-iTs expressed 30% CD107a/b compared to 2% when stimulated by Nalm-6 CD19KO. In an in vitro CTL assay, greater than 80% of Nalm-6 WT cells were lysed with effector to target (E:T) ratio at 10:1 as compared to Nalm-6-CD19KO, which showed less than 10% lysis at the same E:T ratio. Finally, in an in vivo tumor model, FT819-iTs generated from our original and modified T cell differentiation protocols showed similar tumor burden control and prolonged survival rate when compared to primary CAR-T cells (days of survival >80days, p>0.1). In a more stringent in vivo model, FT819-iTs generated from the modified differentiation protocol demonstrated higher anti-tumor response and better animal survival rate compared to iTs from the original T cell differentiation protocol (Day 30 p Disclosures Chang: Fate Therapeutics: Employment. Van Der Stegen:Memorial Sloan Kettering Cancer Center: Employment. Mili:Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Lai:Fate Therapeutics: Employment. Witty:Fate Therapeutics: Employment. Lindenbergh:Memorial Sloan Kettering Cancer Center: Employment. Yang:Fate Therapeutics: Employment. Husain:Fate Therapeutics: Employment. Shaked:Fate Therapeutics: Employment. Groff:FATE THERAPEUTICS: Employment. Stokely:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Chu:Fate Therapeutics: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. ORourke:Fate Therapeutics: Employment. Gutierrez:Fate Therapeutics: Employment. Riviere:Juno Therapeutics: Consultancy, Equity Ownership, Research Funding; Fate Therapeutics: Consultancy; Memorial Sloan Kettering Cancer Center: Employment. Sadelain:Memorial Sloan Kettering Cancer Center: Employment; Fate Therapeutics: Consultancy, Patents & Royalties; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

The advent of off-the-shelf chimeric antigen receptor (CAR) T cell therapeutics is widely recognized to be a major potential advancement for the treatment of cancer. Several obstacles currently hamper the broad use of CAR T cells, including the inherent variability and cost of manufacturing of autologous cellular populations, the absolute requirement for precise genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity. Here we present pre-clinical data for FT819, a first-of-kind off-the-shelf human induced pluripotent stem cell (hiPSC)-derived CAR T cell product. FT819 is defined by the precise genetic engineering of multiple targeting events at the single cell level to create a clonal master iPSC line. The engineered features include the targeted integration of a novel, modified CD19 CAR into the T cell receptor α (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease (GvHD), and the expression of a high-affinity, non-cleavable form of CD16 (hnCD16) to deliver an adjustable system to address tumor antigen escape. Through a proprietary cellular reprogramming platform, peripheral blood derived T cells are converted to hiPSCs, engineered to contain the modified CD19 CAR targeted into the TRAC locus and hnCD16, and clonally selected to create a master hiPSC line (TRAC-TiPSC, FT819). Molecular characterization of the TRAC-TiPSC master cell line by 5' junction, 3' junction and internal sequence PCR confirmed homology directed repair and bi-allelic targeting of the CD19 CAR into the TRAC locus. The origin of the clonal master cell bank was confirmed to be a TCRαβ T cell by PCR-mediated detection of TCRδ locus deletion and methyl-seq analysis of the TCRα locus. Flow cytometric analysis demonstrated the maintenance of a uniform population of hiPSCs (>95% SSEA4/TRA-1-81/OCT4/NANOG) and expression of hnCD16 transgene (>95% CD16). Utilizing our stage-specific T cell differentiation protocol, we demonstrate that the TRAC-TiPSCs yield TRAC-iT cells with uniform expression of the CAR (>95%), complete elimination of TCR surface expression and clinically enabling expansion through the manufacturing process (>50,000 fold). To confirm the lack of alloreactivity conferred by the deletion of endogenous TCR expression, mixed lymphocyte reactions were performed using TRAC-iT, primary TCR+ T cells and primary TCR+CAR+ T cells as responders and HLA-mismatched peripheral blood mononuclear cells (PBMCs) as targets. In comparison to primary T cells and primary CAR-T cells, TRAC-iT did not respond and proliferate in response to TCR stimulation or HLA-mismatched PBMCs indicating that the risk of GvHD was alleviated. In vitro functional studies established that TRAC-iT possess a potent cytotoxic T lymphocyte response to CD19 antigen challenge in a similar manner to peripheral blood CAR T cells as demonstrated by expression of markers indicative of degranulation (CD107a/b, Granzyme B), T cell activation (CD69, CD25), and production of INFγ, TNFα and IL2. Importantly, TRAC-iT targeted tumor in an antigen specific manner as verified by lysis of CD19+, but not CD19-, tumor cell lines as seen by in vitro cytolytic assays (50% killing E:T; TRAC-iT = 1:8, primary CAR-T = 1:4). In vivo studies demonstrated that TRAC-iT cells effectively control tumor progression in a mouse model of acute lymphoblastic leukemia Nalm6 (TRAC-iT versus no treatment, p Disclosures Clarke: Fate Therapeutics Inc.: Employment. Chang:Fate Therapeutics Inc.: Employment. Husain:Fate Therapeutics Inc.: Employment. Lai:Fate Therapeutics Inc.: Employment. Peralta:Fate Therapeutics Inc.: Employment. Stokely:Fate Therapeutics Inc.: Employment. Abujarour:Fate Therapeutics Inc.: Employment. Dinella:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment. Pribadi:Fate Therapeutics Inc.: Employment. Chu:Fate Therapeutics Inc.: Employment. Truong:Fate Therapeutics Inc.: Employment. Sabouri-Ghomi:Fate Therapeutics Inc.: Employment. Meza:Fate Therapeutics Inc.: Employment. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Sadelain:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.