Ewing sarcoma and osteosarcoma are the two most common pediatric malignant bone tumors. Patients who present with metastatic disease or relapse have a dismal average 5-year survival of only 20%. Novel therapeutic approaches are desperately needed. The melanoma cell adhesion molecule (MCAM/CD146/MUC18) is highly expressed in sarcoma and metastatic pediatric tumors. MCAM depletion inhibits Ewing sarcoma cell migration in vitro and tumor metastasis in vivo. High MCAM levels in patient tumors are associated with poor survival in Ewing sarcoma. MCAM therefore constitutes a unique and novel target for immunotherapy against MCAM+ sarcomas. We previously demonstrated that natural killer (NK) cell engineered to express chimeric antigen receptor (CAR) against CD20 significantly reduced tumor burden, prevented tumor dissemination, and extended survival of treated mice compared to mock NK treatment in humanized Burkitt lymphoma xenograft mouse model. We hypothesize that anti-MCAM CAR NK cell will enhance the inherent NK-sarcoma cytotoxicity by better targeting sarcoma cells. Here we develop an anti-MCAM CAR NK cell and investigate its efficacy in promoting NK cell cytotoxicity against Ewing sarcoma and osteosarcoma. MCAM expression in sarcoma cell lines and tumor samples were validated by flow cytometry and western blotting. Peripheral blood mononuclear cells (PBMCs) are expanded into NK cells ex vivo using K562-mbIL21-41BBL artificial antigen presenting cells (aAPC). Anti-MCAM CAR NK cells are generated by non-viral electroporation of anti-MCAM CAR mRNA into expanded NK cells. Anti-MCAM CAR NK cytotoxicity is evaluated in vitro by luciferase based cytotoxicity assay. We detected various levels of MCAM expression in Ewing sarcoma and osteosarcoma cell lines and tumors. In addition, we noticed an interesting patchy expression pattern of MCAM in the Ewing sarcoma tumor sample. Importantly, we found a significantly increased cytotoxicity of anti-MCAM CAR NK cells compared to mock NK cells (59.9±10 vs 13.3±3.7 at E:T ratio of 5:1, p=0.006; and 65.6±7 vs 13.7±3.4 at 10:1, p=0.004) against Ewing sarcoma TC71 cells after co-culturing for 4 hours. A similar result was observed at the time point of 48 hours (91.5±8 vs 59±0.5 at E:T of 5:1, p=0.018). Anti-MCAM CAR NK cells also showed significantly enhanced cytotoxicity against U2OS osteosarcoma cells compared to mock NK cells (32.6±23.2 vs 0.5±19.5 at E:T of 0.5:1, p=0.004; 47.1±27.4 vs 10.2±24 at E:T of 1:1, p=0.001) at the time point of 48 hours. In summary, these findings demonstrated enhanced efficacy of anti-MCAM CAR NK cells compared to mock NK cells against Ewing sarcoma and osteosarcoma cells and support a further preclinical investigation whether targeting MCAM by CAR NK will limit sarcoma growth and/or metastasis and increase survival in vivo.
Citation Format: Aliza Gardenswartz, Wen Luo, Jeremy M. Rosenblum, Janet Ayello, Mitchell S. Cairo. Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6594.