OS12.6.A Combination therapy of CAR-NK-cells and anti-PD-1 results in high efficacy against advanced-stage glioblastoma in a syngeneic mouse model and induces protective anti-tumor immunity in vivo

Abstract INTRODUCTION Checkpoint inhibitors as well as adoptive cell therapy hold promise for cancer therapy and encouraging treatment responses have already been demonstrated in different cancer indications. Glioblastoma (GB) is the most common and aggressive primary brain tumor. Standard therapy has very limited efficacy in the majority of patients. Analysis of the GB microenvironment (TME) has shown prominent immunosuppressive features, including expression of PD-L1 on tumor cells and increased frequency of FOXP3-positive regulatory T cells. While the surrounding brain is HER2-negative, GB are frequently HER2-positive, suggesting HER2 as a promising target for adoptive immunotherapy. Previous results from mouse glioma models showed efficacy of CAR-NK cells (NK-92/5.28.z) targeted against HER2 as monotherapy with early stage but not with advanced-stage tumors. MATERIALS AND METHODS The murine glioma cell line GL261 was transfected with human HER2. Tumor cells were implanted either subcutaneously or orthotopically into C57BL/6 mice and treated either with HER2-specific NK-92/5.28.z cells alone or in combination with an anti-PD-1 antibody. Effects on tumor growth and survival were determined. Lymphocyte infiltration and immunosuppressive TME were characterized via highplex multi-color flow cytometry (FACS Symphony) and IHC (Phenoptics). Furthermore, gene expression profiles of tumor-infiltrating cells were determined via bulk RNAseq (NanoString). RESULTS Combined treatment with NK-92/5.28.z cells and anti-PD-1 checkpoint blockade resulted in synergistic effects, with tumor regression and long-term survival observed even in advanced-stage tumor bearing mice. Analysis of the TME showed changes in lymphocyte infiltration and increased expression of exhaustion markers in tumor and immune upon combined treatment with NK-92/5.28.z cells and anti-PD-1 antibody resulting in an altered TME. Both, PD-1 and Lag-3 expression are highly upregulated on tumor infiltrating T cells. Total infiltrating lymphocytes show a rather cytotoxic phenotype up combination treatment with NK-92/5.28.z cells and anti-PD-1 antibody CONCLUSION Our data demonstrate that efficacy of NK-92/5.28.z cells can be enhanced by combination with checkpoint blockade, resulting in successful treatment of advanced tumors refractory to NK-92/5.28.z monotherapy. Furthermore, the combination therapy induced a cytotoxic rather than immunosuppressive TME, leading to a primed immune system. To translate the concept of CAR-NK-cell therapy plus checkpoint inhibition to a clinical setting, we are adding a combination therapy cohort to our ongoing phase I clinical study (CAR2BRAIN; NCT03383978).