CELL INTELLIGENCE · 肿瘤细胞治疗研究

BACKGROUND: The prognosis of patients with metastatic/relapsed/refractory Ewing sarcoma (ES) is dismal. Natural killer (NK) cells are highly cytotoxic to ES but limited by resistance within the ES tumor microenvironment (TME). Here we sought to overcome ES resistance to NK cells by a combinatorial immunotherapy approach simultaneously enabling NK tumor-specific-targeting via chimeric antigen receptor (CAR) against a novel ES target interleukin-1 receptor accessory protein (IL1RAP), circumventing transforming growth factor beta (TGF )-mediated NK immunosuppression by TGF 1-imprinting, increasing NK cell antibody-dependent cellular cytotoxicity (ADCC) via an anti-GD2 antibody dinutuximab, and improving NK cell persistence and ADCC by an IL-15 agonist, NKTR-255. METHODS: Peripheral blood mononuclear cells were expanded into NK and TGF 1-imprinted-NK (imNK) cells using antigen-presenting feeder cells co-expressing IL-21 and 4-1 BBL. Anti-IL1RAP-CAR messenger RNA was electroporated into NK or imNK cells. In vitro cytotoxicity assays were performed to investigate the efficacy of anti-IL1RAP-CAR-NK/imNK cell alone or combined with NKTR-255 and/or dinutuximab against ES cells. Xenograft mouse models of ES were used to investigate the antitumor efficacy of the combinatorial CAR-NK/imNK cell therapy against ES in vivo. Single-cell RNA sequencing and mass cytometry analyses of cells from xenograft tumors were performed to identify mechanisms of response/resistance to this combinatorial immunotherapy. RESULTS: We found that anti-IL1RAP-CAR-NK cells significantly and specifically enhanced NK cytotoxicity in vitro and decreased tumor growth and lung metastasis in vivo against IL1RAP+ES. TGF 1-imprinting significantly enhanced in vitro cytotoxicity and tumor infiltration of CAR-NK cells, leading to significantly reduced tumor growth and improved animal survival in the ES orthotopic mouse model. Compared with single agent or double combinations, the triple combination of imprinted-CAR-NK (CAR-imNK) cells and NKTR-255 with dinutuximab had superior antitumor efficacy against IL1RAP+GD2+ ES. Mechanistic studies on single cells from the xenograft tumors revealed increased apoptosis of ES cells, upregulated expression of ligands to NK inhibitory receptors on ES cells, and enhanced mouse macrophage migration in the ES TME in response to the CAR-imNK+NKTR-255+dinutuximab therapy. CONCLUSIONS: Our preclinical data demonstrate that combinatorial innate immunotherapy leveraging tumor-targeting TGF 1-imprinted IL1RAP-CAR-NK cells combined with an IL-15 agonist and an anti-GD2 antibody is a promising novel therapeutic strategy for targeting metastatic/relapsed/refractory ES.