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Abstract Chimeric antigen receptor (CAR) based T-cell immunotherapies have transformed the oncology landscape and evolved as part of the standard of care for several hematological malignancies. To generate CAR-T-cell products, autologous patient-derived αβT-cells are engineered with a CAR for tumor cell targeting. However, generating autologous T-cell products fails in a significant number of cases due to the poor quality and quantity of blood-derived T-cells and is associated with manufacturing and logistical complexity as well as high product costs. Therefore, it is crucial to develop strategies for off-the-shelf T-cell products as otherwise many patients are left without this treatment option. Manufacturing allogeneic T-cells from induced pluripotent stem cells (iPSCs) has a great potential to generate cell products with consistently high quality and scalable quantities. In addition, high fidelity gene-editing can be performed in iPSCs to address tumor resistance mechanisms and to prevent exhaustion of the T-cells. 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 ensure stringent monitoring of all process stages. To demonstrate functional activity of iαβT, cytotoxicity and cytokine release assays were performed. We compared the differentiation potential of iPSC lines derived from T-cells (t-iPSCs) to a hematopoietic stem cell-derived iPSC line with an αβTCR knock-in. Hematopoietic progenitor cells were induced from the different iPSC lines and differentiated into iαβT. During the differentiation process, surface markers CD45, CD5 and CD7 were displayed, and cells started to express αβTCRs. Importantly, iαβT derived from the different iPSC lines expressed CD8α and CD8β which is crucial for the function of cytotoxic T-cells. The CD4/CD8 double positive stage is an important step in T-cell development and was observed only when T-cells were generated from t-iPSC, but not from the TCR knock-in cells. Functional analysis of iαβT-cells confirmed their cytotoxic activity and potential to release cytokines after activation. Our scalable process for iαβT differentiation generates CD8-positive T-cells that secrete cytokines and show cytotoxic activity against tumor cells, demonstrating their potential as a promising cell source for TCR-T or CAR-T cancer immunotherapies. Citation Format: Riga Tawo, Michela Mirenda, Olivia Cypris, Philip Hublitz, Nadja Wagner, Michael Epstein, Michael Esquerre, Audrey Holtzinger, Monika Braun, Markus Dangl, Rodrigo Grandy, Daniel Sommermeyer. Development of innovative off-the-shelf cancer immunotherapies with iPSC-derived CD8-positive T-cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 11.
Tawo et al. (Fri,) studied this question.