IL1RAP, CAR, TGFβi, CXCR2 Modified NK Cells Enhances In Vitro Efficacy in Ewing Sarcoma

Wen Luo, PhD

Modified natural killer (NK) cells (IL1RAP CAR NK, TGFβi-NK, IL1RAP CAR CXCR2 NK) demonstrated enhanced antitumor activity alone and combined with NKTR-255 and dinutuximab against Ewing Sarcoma (ES) tumor cells in vitro, providing rationale for further preclinical study of these cells.

The preclinical data were presented by Wen Luo, PhD, assistant professor, Pediatrics and Pathology, New York Medical College, at the 2024 Tandem Meetings |Transplantation & Cellular Therapy Meetings of ASTCT and CIBMTR, held in San Antonio, Texas, February 21-24, 2024. 

“ES is a malignant pediatric bone associated sarcoma and it is characterized by fusion oncogene in EWS/FLI. Patients with localized ES have around 75% 5-year overall survival (OS) but for patients with metastatic disease, they only have a dismal outcome of around 30% 5-year OS, and patients with relapsed/refractory disease have only 5% 6-month event-free survival. Current treatment including surgery, radiation, and chemotherapy have failed to improve patient outcomes for decades, so novel therapeutics strategies are urgently needed,” Luo said during her presentation.

Lup and colleagues investigated several strategies to enhance efficacy and persistence of NK cells in models of ES, as it has been reported that an increased abundance of activated NK cells in ES tumors correlates with extended OS. ES cells are highly sensitive to expanded NK cells in vitro and in vivo, however, there are challenges to overcome with the tumor microenvironment.

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“Ewing sarcoma cells express high levels of ligands to NK activating receptors and low levels to inhibitory receptors. This renders ES cells highly sensitive to NK cells and suggests potential therapeutic efficacy of NK cells against ES,” Luo said.

The investigators developed a strategy to yield a thousand-fold expansion of NK cells, which were epigenetically modified by adding TGFβiinto stimulation media to enhance NK specific lysis of tumors cells. TGFβi is a major mechanism of NK cell resistance. They explored IL-15 agonists, using NKTR-255 to enhance response to ADCC-mediated therapy. This combination reduced lung metastases and extended animal survival in an orthotopic mouse model of ES.

They modified ex vivo expanded NK cells to express a CAR against IL1RAP, a novel upregulated by EWS/FLI that promotes anoikis resistance and tumor metastases in ES. To overcome poor NK cell infiltration into ES tumors, the researchers engineered NK cells armored with CXCR2, an IL-8 receptor, to facilitate the NK cell migration and infiltration

Luo and colleagues combined these above approaches and evaluated the modified NK cells in xenograft models. Compared to mock NK cells against ES cells, IL1RAP CAR NK cells displayed increased cytotoxicity at various effector to target ratios (P <.01 and P <.05) and secreted significantly higher levels of IFN-γ and perforin (P <.01 and P<.05). IL1RAP was identified as the driver in enhancing cytotoxicity as no significant increase was seen IL1RAP knockout cells.¹

Looking at IL1RAP CAR expression in TGFβi-NK cells, further enhanced toxicity was seen in vitro against ES cells and TGFβi-NK cells significantly slowed down ES tumor growth in vivo (P <.05). Lastly, combining NKTR-255 and dinutuximab with the modified NK cells enhanced this activity even further, and armoring the cells with CXCR2 demonstrated significantly enhanced migration toward IL-8 conditioned media of ES cells (P <.01).¹

“We are actively investigating the in vivo antitumor efficacy of these NK cell combinatorial therapies in ES. If the results are promising, we will perform GMP-grade manufacturing and preclinical evaluations to apply for an IND for a clinical trial to test safety and efficacy of NK cell combinatorial therapy in ES patients,” Luo concluded. “If ES sarcoma is still resistant to the proposed therapies, we will identify the resistant mechanisms as well as targets for novel combinatorial therapy.”

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REFERENCE

1. Luo W, Zhang HF, Li Wei, et al. Targeting Ewing Sarcoma By Genetically and Epigenetically Modified Ex-Vivo Expanded NK Cells in Combination with NKTR-255 and Dinutuximab. Presented at: 2024 Tandem Meetings, February 21-24, San Antonio, Texas. Abstract #107.