Modulation of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma

Background \u0026 Aim Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the highly immunosuppressive tumor microenvironment. These adoptively transferred CAR T cells are susceptible to inhibition via the engagement of inhibitory receptors on their surface including PD1, CTLA4, LAG3 and BTLA. The recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of these co-inhibitory receptors may represent a common mechanism of T cell inhibition. Upon its recruitment and activation, SHP2 dephosphorylates key stimulatory signaling molecules, thus limiting T cell activation. We hypothesized that disrupting SHP2 expression will simultaneously offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Methods, Results \u0026 Conclusion CRISPR/Cas9 technology was used to knockout (KO) SHP2 from human T cells. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 signaling endodomain) targeting HER2. The phenotype of wild-type and edited CAR T cells was investigated using mass cytometry and confirmed with flow cytometry. Their function was tested in vitro using xcelligence (an electrical impedance-based long-term cytotoxicity assay) and in vivo in an orthotopic xenograft mouse model of GBM. Efficient SHP2 KO in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect T cell expansion, proliferation or phenotypic profile. However, SHP2KO CAR T cells eliminated LN229 cells (a GBM cell line) in vitro more efficiently compared to wild-type CAR T cells. In vivo, SHP2KO CAR T cells showed better control of engrafted LN229 tumor cells and improved the survival of treated mice in comparison to wild-type CAR T cells. In conclusion, these results suggest that SHP2 may limit the function of CAR T cells and that SHP2 deletion may improve their anti-GBM activity.