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

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. 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. 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. 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<0.05) (Fig 1A). While the in vitro cytotoxicity of CAR-NK and CAR-CXCR2-NK cells was very similar (Fig 1A), CXCR2-CAR-NK cells showed significantly enhanced migration towards the conditioned media of ES cells (p<0.05 and p<0.01) (Fig 1B). Both dinutuximab (Fig 1C) and camB7-H3 (Fig 1D) significantly enhanced in vitro cytotoxicity of CAR-CXCR2 NK cells against ES A673 cells (p<0.05 and p<0.01). Furthermore, CAR-CXCR2-NK cells combined with camB7-H3 and dinutuximab had significantly enhanced anti-tumor effects against ES in the orthotopic xenograft mouse model, as evidenced by significantly reduced tumor growth (p<0.05 and p<0.01) (Fig 1E) and prolonged animal survival (p<0.05 and p<0.01) compared to controls (Fig 1F). Our data demonstrated enhanced anti-tumor efficacy of ROR1-CAR-CXCR2-NK cells combined with TriKE and anti-GD2 against ES in vitro and in vivo, suggesting a potential novel effective immunotherapy for patients with high-risk ES. Supported by U54 CA232561-01A1 and ALSF reach grant. Dinutuximab is generously provided by United Therapeutics.