Harnessing iPSC-Derived γδ CAR-T Cells: A Breakthrough in Cancer Immunotherapy

A novel approach to cancer treatment is presented in this study, utilizing allogenic CAR-T cell therapies to overcome limitations associated with autologous cell therapies. The primary concern addressed is the risk of graft versus host disease (GvHD), which is mitigated by employing a subset of γδ CAR-T cells that target invariant antigens, thus avoiding the hypervariability of MHC molecules.

The methodology involves the generation of induced pluripotent stem cells (iPSCs) from T cells, specifically γδ T cells, through a reprogramming process. These T cell-derived iPSCs (TiPSCs) are then genetically modified using CRISPR to incorporate a CD19 CAR transgene. The TiPSCs undergo a two-stage differentiation: first into CD34-expressing hematopoietic progenitor cells (HPCs), followed by a differentiation process yielding uniform γδ CAR-iT cells. The purity and identity of these cells are evaluated using flow cytometry, and their response to growth factors is determined through intracellular staining and mRNA analysis.

The study demonstrates that γδ CAR-iT cells are capable of responding to IL-2 and IL-15, with notable differences in STAT3 and STAT5 phosphorylation levels. The cytokine-induced gene expression is generally more pronounced in cells treated with IL-15. Furthermore, these cells exhibit a reduced release of inflammatory cytokines such as IFN-γ compared to conventional ab CAR-T cells. In cytotoxicity assays, γδ CAR-iT cells demonstrate sustained killing of tumor cells and, in an in vivo model using immunodeficient mice, significantly reduce tumor burden with a high rate of tumor growth inhibition.

To enhance the clinical application, the study also explores methods for the isolation, expansion, and reprogramming of human γδ T cells. A universal activation protocol is established, leading to robust expansion of γδ T cells from various donors. The reprogramming of these cells results in the creation of new γδ TiPSC lines, which are confirmed to possess the rearranged γδ TCR locus and exhibit killing activity in vitro.

In conclusion, the γδ CAR-iT cells offer a promising off-the-shelf solution for cancer treatment, with the advantage of being universally applicable and devoid of GvHD risks. They are easily manufactured, and the study outlines a comprehensive process for reprogramming, genetic modification, and differentiation. The cells' potent tumor-killing capabilities and reduced inflammatory cytokine release suggest a potential reduction in cytokine-mediated toxicities, paving the way for safer and more accessible allogenic cell therapies for various types of cancer.