Background T-cell immunotherapy with chimeric antigen receptors (CARs) has evolved as part of the standard of care for several hematological malignancies and has transformed the oncology landscape. CAR-T-cell products are traditionally generated from autologous, patient-derived αβT-cells engineered with a CAR for tumor cell targeting. Generating autologous T-cell products for each patient is associated with manufacturing and logistical complexity and high product costs. Furthermore, manufacturing fails in a significant number of cases due to the poor quality and quantity of blood-derived T-cells and restrictions apply in terms of throughput to produce autologous cell products. Consequently, many patients are left without this treatment option, so it is crucial to develop strategies for off-the-shelf T-cell products. Immune cells derived from induced pluripotent stem cells (iPSCs) offer the opportunity to manufacture allogeneic T-cell products with consistently high quality and scalable quantities. Use of iPSCs as a starting material makes it easier to introduce several genetic modifications (e.g. to enhance both cell persistence and tumor infiltration) addressing tumor resistance mechanisms for both liquid and solid tumors.
Methods Using a validated GMP iPSC line modified with an NY-ESO-1-specific T-cell receptor (TCR) knock-in, we have established a feeder-free differentiation protocol that enables robust production of iPSC-derived αβT-cells (iαβT). Flow cytometry and single cell transcriptome analysis ensured a stringent monitoring of all process stages. To demonstrate functional activity of our iαβT, we performed cytotoxicity and cytokine release assays against tumor cell lines presenting the NY-ESO-1 antigen.
Results Successful knock-in of the NY-ESO-1-TCR-transgene cassette into the iPSC line was confirmed by marker-gene expression. Hematopoietic progenitor cells were induced from knock-in-enriched iPSCs and differentiated into iαβT. During the differentiation process, the T-cell markers CD45, CD5 and CD7 were displayed and cells started to express the TCR. After activation of iαβT, the fraction of NY-ESO-1-TCR-positive cells increased to over 95%. Importantly, iαβT expressed CD8α and CD8β which is crucial for the function of cytotoxic T-cells. Transcriptome analysis validated the efficient differentiation from pluripotent cells towards cells with a T-cell-specific gene expression profile. Co-culture experiments with NY-ESO-1 antigen presenting tumor cell lines confirmed cytotoxic activity of iαβT and their potential to release cytokines.
Conclusions Our scalable iαβT differentiation process enables us to generate CD8+ T-cells that secrete cytokines and show cytotoxic activity, indicating their potential as a promising cell source for TCR-T or CAR-T cancer immunotherapies.
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