Effective Targeting of Multiple Tumours by Combining CRISPR-based Genome Editing and CAR/TCR-engineering in Human T Cells

Cancer immunotherapy culminated in the last years FDA-approval of the CAR-T cells directed against haematological malignancies. However, in the field of the solid neoplasm, canonical scFv-based CARs were found to be less effective. Beyond the immunosuppressive nature of solid tumour microenvironment, this lack of functionality was also attributed to the xenogeneic origin of the scFv-based CAR and their potential immunogenicity. In the current work, we have designed an ‘autologous’ CAR, based on the extracellular domain of the human NCR2/NKp44 receptor. The latter belongs to the NCR family which are receptors derived from NK cells and which was shown to mediate the recognition of cancer-associated ligands. By combining NK tumour recognition pattern with T cell effectiveness, we showed that an optimized NCR2-CAR, namely s4428z, confer T cells with the specific recognition and killing of solid tumor cell lines from different histologies.

Beyond specificity, signals conveyed by immune checkpoints may dampen engineered T-cell function such as those mediated by the CD155/TIGIT immuno-inhibitory pathway. Herein, we explored the benefit of CRISPR-Cas9 technology to knock out the TIGIT receptor in human T-cells and its impact on T-cell response. Finally, we also apply CRISPR-Cas9 technology to knock-out TIGIT and its related receptors in NY-ESO1-specific TCR, namely 1G4 to improve the T cells anti-tumour function.

Overall we demonstrate the feasability of the combination of CRISPR-Cas9 approach in T cells engineered to express specific anti-tumour receptors. Moreover, we show that knock-out of receptors beloging to the CD155/TIGIT pathway in engineered T cells is beneficial to the anti-tumour responce.