Genetically Enhanced Pluripotent Stem Cell-Derived T Lymphocytes for Off-the-Shelf Cellular Immunotherapy

Cellular immunotherapies are poised to transform the treatment of cancer and immunological disorders. In the most promising setting to date, genetic modification to the T lymphocytes in the form of chimeric antigen receptors (CAR) has dramatically increased therapeutic efficacy with reported initial complete remission rates in acute lymphoblastic leukemia ranging between 80-100%. However, pressing challenges remain to be solved to ensure that engineered T-cell immunotherapies can be cost-effectively and consistently manufactured, and safely and reliably delivered at the scale necessary to support wide patient base commercialization.

Human induced pluripotent stem cell (hiPSC) derived T lymphocytes represent a unique, renewable source of genetically engineered T cells for "off-the-shelf" immunotherapy. Through the precise genetic engineering at the hiPSC stage, clonal and uniform populations of modified cell lines can be banked and reliably tapped into on demand to generate highly efficacious T cells for therapeutic applications. Although great progress has been made, several challenges need to be addressed including the ability to enhance effector function through genome-engineering of persistence, targeting, histocompatibility and controlled safety mechanisms at the hiPSC juncture while retaining the capacity to efficiently and reproducibly generate the intricate stages of lymphocyte development in an accurate and scalable process. We have previously demonstrated that our proprietary reprogramming platform supports efficient and rapid derivation of clonal hiPSC lines with properties indicative of the naïve state of pluripotency. In addition to maintaining a homogeneous population of hiPSCs, our platform enables efficient multi-gene and multi-loci targeted engineering at a single cell level resulting in clonal population of pluripotent cell lines with desired genetic attributes.

Here we will provide an update on our "off-the-shelf" T-cell immunotherapy preclinical program where engineered hiPSC lines are uniquely used as the renewable starting material. We will also highlight our novel differentiation platform to derive definitive hematopoietic progenitor cells termed hemogenic endothelium (HE); a well-defined, small molecule-driven, staged process that is currently being translated into cGMP (current good manufacturing practice) settings. The highly efficient differentiation system (on average >65% hiPSC to CD34 conversion) delivers approximately 100 CD34+ HE cells per each input hiPSC, representing a highly scalable process that is further expanded during lymphocyte differentiation and maturation. To validate that the iCD34+ HE is definitive in nature we demonstrate that during further hematopoietic differentiation the emerging CD43+ hematopoietic cells exhibit Notch dependency and high expression of key genes such as MYB and the HOXA cluster, found only in definitive hematopoietic progenitors. The hiPSC-derived HE exhibits multi-lineage potential and can be successfully cryopreserved and banked, serving as a highly-stable cell bank for subsequent therapeutic use. Through genetic modifications at the single cell hiPSC stage, we confer antigen-specificity via the expression of temporally inducible CARs as premature expression of CAR proteins during in vitro differentiation has been found to skew development towards innate-lymphoid like lineages. Utilizing our stage-specific hematopoietic differentiation platform we have identified the optimal developmental window to induce the expression of CAR proteins to maintain optimal differentiation towards functional effector lymphocytes. The hiPSC-derived engineered T lymphocytes are currently under preclinical investigation for in vitro and in vivo effector function including thymic rejuvenation, T cell repertoire repopulation, target specific recognition and enhanced killing potential. Preliminary data suggests that hiPSC-derived lymphocytes are functional and can home to their respective niche to support initial repopulation in vivo. Our study continues to support that naïve hiPSCs are an ideal renewal source for "off-the-shelf" hematopoietic cell-based immunotherapies and represent a potentially exponential advancement in adoptive T cell therapy.