Targeting Ewing Sarcoma By IL1RAP CAR Modified Ex-Vivo Expanded TGFβ Imprinted NK Cells in Combination with IL-15 Agonist and Anti-GD2 Antibody

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Objectives Metastatic Ewing sarcoma (ES) has a dismal prognosis, largely due to therapy resistance within the tumor microenvironment (TME). NK cell activity in solid tumors can be improved by enhancing tumor specific targeting of NK cells, increasing NK cell function and persistence and overcoming TGFβ-mediated immune suppression [1]. Interleukin-1 receptor accessory protein (IL1RAP) is a novel immunotherapy target highly expressed on ES cells [2]. Addition of TGFβ during NK expansion epigenetically reprograms NK cells (TGFβi-NK) to enhance tumor homing, lytic activity, and cytokine productions [3]. Dinutuximab is an FDA approved mAb against GD2, which is overexpressed in ES. NKTR-255 is a recombinant hIL-15 agonist that boosts NK cell numbers, persistence and ADCC . Here we develop an IL1RAP CAR engineered TGFβ imprinted NK cell in combination with NKTR-255 and dinutuximab to overcome the ES TME resistance. Methods PBMCs were expanded into NK or TGFβi-NK cells using feeder cells without or with TGFβ-imprinting, respectively. The IL1RAP CAR NK cells were generated by electroporation of CAR mRNA into expanded NK cells. NK cell cytotoxicity was evaluated by luciferase based cytotoxicity assay . IFN-γ and perforin secretion were analyzed by ELISA . An orthotopic mouse model generated by implanting ES cells into the tibia of NSG mice was utilized for evaluating in vivo tumor growth and animal survival. Results We found a significantly increased cytotoxicity of IL1RAP CAR NK cells compared to mock NK cells against ES cells at various target to effector ratios (T:E=20:1, 10:1, 5:1, 2:1, 1:1) (p<0.01 and p<0.05) which is specific to IL1RAP+ ES cells but not IL1RAP KO cells (Fig 1A). IL1RAP CAR NK cells secreted significantly higher levels of IFN-γ and perforin than mock NK cells (p<0.01 and p<0.05). IL1RAP CAR expression in TGFβi-NK cells further enhanced NK cell cytotoxicity in vitro against ES cells (Fig 1B). Furthermore, we found that, compared to IL1RAP CAR NK alone or CAR NK combined with dinutuximab or CAR NK with NKTR-255, CAR NK cells combined with NKTR-255 and dinutuximab had significantly enhanced cytotoxicity against ES cells in vitro (p<0.01 and p<0.05) (Fig 1C), and had significantly enhanced anti-tumor effects against ES xenograft tumors in orthotopic mouse model of ES, as evidenced by significantly reduced tumor growth (p<0.01 and p<0.05) (Fig 1D) and prolonged animal survival (p<0.001, p<0.01 and p<0.05) (Fig 1E). Conclusions Our data demonstrated enhanced anti-tumor efficacy of IL1RAP CAR NK/TGFβi-NK cells alone and combined with NKTR-255 and dinutuximab against ES in vitro and in vivo. These CAR engineered TGFβi NK cells in combination with NK function and persistence enhancing immune modulators constitute potential novel effective immunotherapies for patients with high-risk ES. This study was supported by the NCI Cancer Moonshot U54 Grant (CA232561-01A1).

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