Overcoming Target Driven Fratricide for T Cell Therapy

Chimeric Antigen Receptor (CAR) T cells expressing the fusion of the NKG2D protein with CD3ζ (NKG2D-CAR T Cells) acquire a specificity for stress-induced ligands expressed on hematological and solid cancers. However, these stress ligands are also transiently expressed by activated T cells implying that NKG2D-based T cells may undergo self-killing (fratricide) during cell manufacturing or during the freeze thaw cycle prior to infusion in patients. To avoid target-driven fratricide and enable the production of NKG2D-CAR T cells for clinical application, two distinct approaches were investigated. The first focused upon the inclusion of a Phosphoinositol-3-Kinase inhibitor (LY294002) into the production process. A second strategy involved the inclusion of antibody blockade of NKG2D itself. Both processes impacted T cell fratricide, albeit at different levels with the antibody process being the most effective in terms of cell yield. While both approaches generated comparable NKG2D-CAR T cells, there were subtle differences, for example in differentiation status, that were fine-tuned through the phasing of the inhibitor and antibody during culture in order to generate a highly potent NKG2D-CAR T cell product. By means of targeted inhibition of NKG2D expression or generic inhibition of enzyme function, target-driven CAR T fratricide can be overcome. These strategies have been incorporated into on-going clinical trials to enable a highly efficient and reproducible manufacturing process for NKG2D-CAR T cells

Efficient shRNA-based knockdown of multiple target genes for cell therapy using a chimeric miRNA cluster platform

Genome engineering technologies are powerful tools in cell-based immunotherapy to optimize or fine-tune cell functionalities. However, their use for multiple gene edits poses relevant biological and technical challenges. Short hairpin RNA (shRNA)-based cell engineering bypasses these criticalities and represents a valid alternative to CRISPR-based gene editing. Here, we describe a microRNA (miRNA)-based multiplex shRNA platform obtained by combining highly efficient miRNA scaffolds into a chimeric cluster, to deliver up to four shRNA-like sequences. Thanks to its limited size, our cassette could be deployed in a one-step process along with all the CAR components, streamlining the generation of engineered CAR T cells. The plug-and-play design of the shRNA platform allowed us to swap each shRNA-derived guide sequence without affecting the system performance. Appropriately choosing the target sequences, we were able to either achieve a functional KO, or fine-tune the expression levels of the target genes, all without the need for gene editing. Through our strategy we achieved easy, safe, efficient, and tunable modulation of multiple target genes simultaneously. This approach allows for the effective introduction of multiple functionally relevant tweaks in the transcriptome of the engineered cells, which may lead to increased performance in challenging environments, e.g., solid tumors

Early signs of clinical activity in Aml patients receiving NKG2D CAR T cell therapy in the absence of pre-conditioning chemotherapy : an alternative strategy to CAR T cell therapy

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Phase 1 studies assessing the safety and clinical activity of multiple doses of a NKG2D-based CAR-T therapy, Cyad-01, in acute myeloid leukemia

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