(F1220) ADVANCING ALLOGENEIC CAR-T CELL THERAPY: OVERCOMING CHALLENGES IN AUTOLOGOUS GRAFTING AND LENTIVIRAL TRANSDUCTION THROUGH IPSC AND CRISPR TECHNOLOGIES FOR T-CELL MALIGNANCIES
Abstract: Recently discovered adoptive cell therapies such as CAR-T cells have shown great success in treating hematological malignancies, although several factors prevent such therapies from reaching their full potential. Parallelly, advances in stem cell differentiation and engineering strategies hold the potential to overcome the current limitations of CAR-T cells in especially challenging scenarios such as T cell-derived malignancies. This project addresses key challenges in CAR-T therapy development for T cell malignancies such as difficult T cell isolation, fratricide, and poor phenotypes. We will achieve this by overcoming autologous grafting limitations by using iPSCs to create allogeneic CAR-T cells and optimizing CAR-T cell production by introducing strategic gene modifications on the stem cell stage resulting in an improved cell product.
Our results show that individual KOs of potential T cell targets CD1a, CD2, CD3, CD5, CD7, CD30, and CD37 in T cells do not result in collateral loss of other markers and maintain proper activation upon stimulation. Moreover, lentiviraly-transduced CD3/7-targeting CAR-T cells with CD3/7 KO exhibit a favorable phenotype and excellent cytotoxic potential, as confirmed by flow cytometry and Incucyte assays. While CRISPR-based CAR delivery into T cells has been successful, further optimization is ongoing to improve efficiency. Lentiviral delivery of the CAR and clone selection in iPSCs have also been successful, yielding pure cultures of CAR-expressing stem cells ready for differentiation into CAR-T cells. Additionally, iPSCs from the CiRA Center (Kyoto), lacking human leukocyte antigen (HLA), are engineered to carry the CAR, target KO, and additional deletions (e.g., PD-1), ready to start the allogeneic T cell differentiation protocol.
This work lays the foundation for allogeneic CAR-T cell products with enhanced safety for T cell malignancies. At the same time, iPSC technology promises to overcome autologous grafting challenges, addressing clinical and economic obstacles in CAR-T therapy.
