303 Targeting Ewing sarcoma with anti-IL1RAP chimeric antigen receptor modified natural killer cells

BackgroundMetastatic and recurrent/refractory Ewing sarcoma (ES) has a dismal prognosis,1 largely secondary to therapy resistance within the tumor microenvironment.2 ES have heightened natural killer (NK) cell sensitivity,3 and increased abundance of activated NK cells in ES patient tumors are correlated with extended survival.4 Solid tumors are resistant to NK in large part due to the small number of active NK cells and lack of specific tumor targeting of NK cells.2 Our group has developed a genetically engineered feeder cell to expand peripheral blood mononuclear cells (PBMCs) into NK cells,5 and demonstrated that expanded NK cells engineered to express chimeric antigen receptor (CAR) against various targets including CD20, GD2, ROR1 and MCAM had significantly enhanced cytotoxicity against lymphoma and sarcomas6,7compared to mock NK. IL-1 receptor accessory protein (IL1RAP) is highly expressed on ES cells and minimally expressed in normal tissues as we previously reported.8MethodsHere we developed an anti-IL1RAP CAR NK cell and investigated its efficacy in promoting NK cell cytotoxicity against ES. PBMCs were expanded into NK cells using K562-mbIL21-41BBL artificial antigen presenting cells. The IL1RAP antibody VH domain DNA was codon optimized and synthesized to create the anti-IL1RAP CAR. CAR NK cells were generated by non-viral electroporation of CAR mRNA into expanded NK cells. CAR expression was analyzed by flow cytometry using biotinylated IL1RAP protein. Anti-IL1RAP CAR NK cytotoxicity was evaluated in vitro by luciferase based cytotoxicity assay. CRISPR-Cas9 mediated IL1RAP knockout (KO) in ES cells was utilized to evaluate the specificity of CAR NK targeting.ResultsElectroporation resulted in CAR expression in 70% of expanded NK cells and the CAR expression lasted for at least 6 days (figure 1). We found a significantly increased cytotoxicity of anti-IL1RAP CAR NK cells compared to mock NK cells against ES A673 and SKNMC cells at various effector to target ratios after co-culturing for 4 hours (**p<0.01 and *p<0.05) (figure 2). In the IL1RAP KO A673 cells, we did not observe the significant increase in cytotoxicity with IL1RAP CAR NK compared to mock NK as we did in the wildtype (WT) A673 cells (figure 3), demonstrating the enhanced cytotoxic activity of CAR NK cells is due to specific targeting of IL1RAP.ConclusionsThese findings demonstrate enhanced efficacy of anti-IL1RAP CAR NK cells compared to mock NK cells against ES and support a preclinical evaluation of anti-IL1RAP CAR NK in limiting ES xenograft tumor growth and/or metastasis and prolonging animal survival in vivo.AcknowledgementsThis study is funded by NCI Cancer Moonshot U54 grant (CA232561-01A1).ReferencesLadenstein R, Potschger U, Le Deley MC, Whelan J, Paulussen M, Oberlin O, van den Berg H, Dirksen U, Hjorth L, Michon J, Lewis I, Craft A, Jurgens H: Primary disseminated multifocal Ewing sarcoma: results of the Euro-EWING 99 trial. J Clin Oncol. 2010; 28:3284-3291. http://www.ncbi.nlm.nih.gov/pubmed/20547982 Nayyar G, Chu Y, Cairo MS: Overcoming Resistance to Natural Killer Cell Based Immunotherapies for Solid Tumors. Front Oncol. 2019; 9:51. PMC6378304, https://www.ncbi.nlm.nih.gov/pubmed/30805309 Cho D, Shook DR, Shimasaki N, Chang YH, Fujisaki H, Campana D: Cytotoxicity of activated natural killer cells against pediatric solid tumors. Clin Cancer Res. 2010; 16:3901-3909. 3168562, http://www.ncbi.nlm.nih.gov/pubmed/20542985 Stahl D, Gentles AJ, Thiele R, Gutgemann I: Prognostic profiling of the immune cell microenvironment in Ewing s Sarcoma Family of Tumors. Oncoimmunology. 2019; 8:e1674113. 6844324, http://www.ncbi.nlm.nih.gov/pubmed/31741777 Chu Y, Flower A, Cairo MS: Modification of expanded NK cells with chimeric antigen receptor mRNA for adoptive cellular therapy, in Somanchi S (ed): Methods in Molecular Biology. Natural Killer Cells: Methods and Protocols. New York, Humana Press, Springer, 2016, pp 215-230 Chu Y, Hochberg J, Yahr A, Ayello J, van de Ven C, Barth M, Czuczman M, Cairo MS: Targeting CD20+ Aggressive B-cell Non-Hodgkin Lymphoma by Anti-CD20 CAR mRNA-Modified Expanded Natural Killer Cells In Vitro and in NSG Mice. Cancer Immunol Res. 2015; 3:333-344. http://www.ncbi.nlm.nih.gov/pubmed/25492700 Gardenswartz A, Luo W, Rosenblum JM, Ayello J, and Cairo MS. Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells. Cancer Research 2020;80. Zhang HF, Hughes CS, Li W, He JZ, Surdez D, El-Naggar AM, Cheng H, Prudova A, Delaidelli A, Negri GL, Li X, Orum-Madsen MS, Lizardo MM, Oo HZ, Colborne S, Shyp T, Scopim-Ribeiro R, Hammond CA, Dhez AC, Langman S, Lim JKM, Kung SHY, Li A, Steino A, Daugaard M, Parker SJ, Geltink RIK, Orentas RJ, Xu LY, Morin GB, Delattre O, Dimitrov DS, Sorensen PH. Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma. Cancer Discov. 2021;11(11):2884-2903.Ethics ApprovalThis study was approved by the New York Medical College Institutional Animal Care and Use Committee (protocol number 13911).