CAR-NK cell therapy combined with checkpoint inhibition induces an NKT cell response in glioblastoma

Abstract

Glioblastoma is the most aggressive primary brain tumor with limited efficacy of established therapies, and a pronounced immunosuppressive tumor microenvironment. Targeting HER2 with local immunotherapy allows for high tumor specificity in the brain with physiologically very low expression. Monotherapy with CAR-NK cells targeted against HER2 has previously shown efficacy in medium-sized GL261/HER2 tumors. Advanced GL261/HER2 tumors were treated by local CAR-NK cell injection combined with systemic anti-PD-1 checkpoint blockade. Tumor growth and survival were monitored. In-depth characterization of the microenvironment was performed by multiplex immune fluorescence, spectral flow cytometry and RNAseq. Untreated GL261/HER2 tumors were characterized by local immunosuppression and high PD-L1 expression. Combined treatment with NK-92/5.28.z and systemic anti-PD-1 induced robust anti-tumor response and long-term survival. Multiplex immunofluorescence and spectral flow cytometry showed increased CD4+ T cell infiltration in mice treated with CAR-NK cell and anti-PD-1 combination therapy. A cluster of T cells specifically emerging in the combination therapy group expressed markers of NKT cells, which was further verified by immunofluorescence staining. The combination therapy reverted the immunosuppressive tumor microenvironment with increased T and NKT cell infiltration. This resulted in successful treatment of advanced orthotopic tumors refractory to CAR-NK cell monotherapy.