Targeting Ewing Sarcoma By ROR1 CAR CXCR2 NK Cells Combined with CamB7-H3 Tri-Specific Killer Engager and Anti-GD2 Antibody

Introduction Patients diagnosed with metastatic Ewing sarcoma (ES) have a dismal outcome. Lines of evidence have shown the importance of NK cell immunity against ES [1]. However, NK cell number and function are low in ES patients, in large part due to the immunosuppressive tumor microenvironment (TME) [2]. Objectives We aim to improve NK therapy against metastatic ES by strategic combinations that simultaneously enhance tumor specific targeting of NK cells via chimeric antigen receptor (CAR), increase NK migration and tumor infiltration by engineering CAR-NK cells to express tumor attracting chemokine receptor CXCR2, enhance NK cell persistence and the immune synapse between cancer and NK cells via a tri-specific killer engager camB7-H3 [3], and improve NK cell ADCC by a GD2 antibody dinutuximab. Methods NK cells were expanded using K562-mbIL-21-41BBL feeder cells and IL-2 [4,5,6]. Expanded NK cells were engineered to co-express ROR1-CAR and CXCR2 using mRNA electroporation. NK cell cytotoxicity was evaluated by luciferase-based cytotoxicity assay. In vitro transwell assays were performed to determine the migratory ability of CAR-NK and CAR-CXCR2-NK cells. An orthotopic mouse model generated by implanting ES cells into the tibia of NSG mice was utilized to evaluate in vivo tumor growth and animal survival. Results We found that compared to mock NK cells, ROR1-CAR-NK cells had significantly enhanced cytotoxicity against ES (A673) cells at various T:E ratios (p