T cell-based immunotherapies are at the forefront of an emerging wave of medical discovery focused on harnessing the power of the immune system to treat cancer and other immune disorders. The most dramatic clinical outcome in this new era of cancer treatment has been seen in clinical trials evaluating autologous chimeric antigen receptor (CAR) therapy for the treatment of refractory B cell acute lymphoblastic leukemia where complete responses have reproducibly occurred in 80-90% of patients. While patient-specific CAR therapy hold great promise as a potentially curative therapeutic option, several obstacles hamper its range of application including the challenge of controlling the T cell product consistency, the inherent variability of manipulating a heterogenous cellular population and the inability to create the quantity of CAR T cells needed to support a wide patient base.
Human induced pluripotent stem cells (hiPSC)-derived T cells represent a practical and sustainable source of highly defined lymphocytes for cancer immunotherapy. We previously demonstrated a cellular reprogramming platform supporting the efficient derivation of naïve state, self-renewing, clonal hiPSC lines that can be effectively engineered with multi-gene and multi-loci targeting strategies. In addition, guided by a small molecule stage-specific differentiation protocol to derive definitive hemogenic endothelium, we have previously developed a highly efficient and scalable hiPSC differentiation system that delivers a consistent and reproducible source of cellular material for further hematopoietic specification. We have also previously demonstrated that hiPSCs reprogrammed from T cells encoding a rearranged endogenous αβTCR (TiPSCs) can generate effector T lymphocytes that, when engineered with a CD19-specific CAR, confer antigen-specific cytotoxic activity against CD19-expressing tumors in vivo. However, the derived T cells appeared skewed both phenotypically and functionally towards an innate γδ T cell subset expressing the CD8αα co-receptor, and were unable to induce complete tumor regression as seen with primary CDαβ CAR T cells. Therefore, to capitalize on the therapeutic potential of TiPSC-derived T cells, it is essential to generate CD8αβ-expressing CTLs.
Through specific modulation of the Notch and T cell receptor (TCR) signaling pathways, we have now developed a directed TiPSC differentiation protocol that recapitulates the developmental stages of T cell commitment and have generated CD4-CD8αβ+ single positive T lymphocytes as defined by phenotype and gene expression profile analysis. In vitro assays validate the functionality of these TiPSC-derived T lymphocytes as demonstrated by proliferation in response to CD3/CD28 stimulation (>90% entry into cell cycle), upregulation of activation markers CD25 and CD69 (>50%, >90% respectively), production of the T cell effector cytokines IL2, IFNγ and TNFα (>70%, >30%, >90% respectively) and the release of the cytolytic proteins Granzyme B and Perforin upon target cell engagement. Utilizing our directed TiPSC differentiation platform, a single TiPSC clone produces a clonal and expandable CD4-CD8αβ+ CTL population, wherein greater than 5x106 CD4-CD8αβ+ T cells are derived from the single input TiPSC. Furthermore, we demonstrate that CAR-TiPSC can also differentiate efficiently into CD4-CD8αβ+ T cells using our directed differentiation protocol and these CD4-CD8αβ+ CAR T cells target tumor in an antigen specific manner, 70% lysis of CD19+ Raji vs. 5% CD19- Raji, 77% lysis of CD19+ NALM6 vs. 10% CD19- NALM6. Further in vitro and in vivo studies are ongoing and will be discussed. In summary, the advances presented here demonstrate the combination of engineered hiPSC clones, T cell differentiation and CAR-T technologies to produce a renewable source of off-the-shelf engineered T cells for cancer immunotherapies.
Clarke: Fate Therapeutics Inc.: Employment, Equity Ownership. Chang: Fate Therapeutics Inc.: Employment, Equity Ownership. Sasaki: Fate Therapeutics Inc.: Employment, Equity Ownership. Lee: Fate Therapeutics Inc.: Employment, Equity Ownership. Lan: Fate Therapeutics Inc.: Employment, Equity Ownership. Valamehr: Fate Therapeutics: Employment, Equity Ownership.
Asterisk with author names denotes non-ASH members.