Pluripotent Cell-Derived Off-the-Shelf TCR-Less CAR-Targeted Cytotoxic T Cell Therapeutic for the Allogeneic Treatment of B Cell Malignancies

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<0.0001). To validate the capability of TRAC-iT to simultaneously target multiple antigens, TRAC-iT was co-cultured with mixtures of CD19+CD20+ and CD19-CD20+ tumor cells in the presence of anti-CD20 monoclonal antibody, Rituxan. In vitro cytolytic assays demonstrate that only TRAC-iT cells can effectively identify and eliminate CD19-CD20+ tumor cells when combined with Rituxan. Importantly, the antibody-dependent cellular-cytotoxicity did not appear to interfere with CAR function as TRAC-iT maintained its directed cytotoxic capacity. Collectively, these preclinical studies suggest that FT819 is a consistent and uniform off-the-shelf product than can be effectively and safely used in the treatment of B cell malignancies in the allogeneic setting.