Background. Despite treatment advances, patients with advanced acute myeloid leukemia (AML) have a poor prognosis. While Chimeric Antigen Receptor (CAR) T and NK-cells (CAR-T/NK) targeting CD123 and CD33 have been extensively evaluated in AML, major challenges include their limited efficacy and the risk of myeloablation due to target protein expression on normal hematopoietic cells. Conversely, a brief activation of conventional NK cells with a specific cytokine cocktail (IL-12, IL-15 and IL-18) leads to the generation of cytokine-induced memory-like (CIML or memory-like) NK cells with enhanced anti-AML responses in early phase trials, but AML relapse remains limiting. Novel targeting of CIML NK cells with enhanced efficacy may offer therapeutic benefit.
Mesothelin (MSLN) is an attractive immunotherapeutic target in AML, that is not normally expressed on hematopoietic cells, but has been recently shown to be expressed in a subset of AML patients (14-36% patients), including pediatric/young AML and those with KMT2Ar. We hypothesized that CIML NK cells armed with an MSLN-CAR would demonstrate efficacy in MSLN+ AML. Moreover, while the secondary arming of CAR T cells with IL-12 has been associated with severe toxicity thought mediated primarily by the cis-activation of T cells, we further hypothesized that IL-12 arming of the CAR-NK cells would be safe due to the relatively “benign” secretome of NK cells, while enhancing their potency against AML.
Methods. A MSLN-CAR construct was designed using an scFv targeting the membrane proximal domain of MSLN along with CD8 transmembrane, and 4-1BB/CD3z signaling domains. Single chain IL-12 peptide was created by linking p35 and p40 subunits by an elastin linker separated from the CAR by a P2A sequence. Peripheral blood derived CIML NK cells were transduced using our optimized BaLV protocol; CAR expression was assessed by the HA-tag and IL-12 secretion was confirmed by ELISA. IL12-MSLN-CAR CIML NK cells were characterized for expression of key surface proteins, by RNA-sequencing and by metabolic profiling (Sea Horse XF). Anti-tumor responses including cytotoxicity (7AAD/Annexin) and interferon gamma (IFNγ) responses were assessed by flow cytometry after co-culturing untransduced, MSLN-CAR and IL-12-MSLN-CAR CIML NK cells with a MSLN+ AML cell line NOMO-1. Lastly in vivo efficacy was assessed in IL15 transgenic NOD-SCID g (NSG-Tg(Hu-IL15) mice injected with 1×106 luc+ NOMO-1 cells (tail vein) into the following groups: a) tumor cells alone, b) tumor plus regular MSLN-CAR CIML NK cells (1×106 cells via tail vein), and c) tumor plus IL-12 engineered MSLN-CAR CIML NK cells (1×106 cells via tail vein). The mice were monitored with weekly bioluminescence imaging (BLI) and for survival.
Results. High transduction efficiency was achieved in the CIML NK cells using both MSLN-CAR and IL-12-MSLN-CAR constructs (60-80% range). We observed a significant increase in the expression of several surface proteins including CD25, LFA1, CXCR4 and Sialyl Lewis-X (sLex) in the IL-12 vs regular MSLN-CAR CIML NK cells. Several key genes, including IL12A, IL12B, IFNG, GZMB, IRF1, SOCS3, and ABCA1, were upregulated whereas CISH was downregulated in the IL-12 engineered CAR CIML NK cells. These cells also demonstrated increased metabolic fitness (i.e. increased oxygen consumption rate/OCAR). Compared to the non-IL12 MSLN-CAR, IL12-MSLN CAR CIML NK cells demonstrated dramatically higher in-vitro cytotoxicity (76% vs 35%, p = 0.0067) and IFNγ production (49.46% vs 2.96%, p=0.0004) at 1:1 ET ratio upon 24-hour co-culture with the MSLN+ NOMO-1 cell line. Furthermore, upon co-culture with IL12-MSLN CAR CIML NK cells (but not with MSLN CAR CIML NK cells), NOMO1 cells showed increased expression of MHC class I and II, most likely secondary to the high IFNγ levels secreted by the IL-12 engineered CAR NK cells. In vivo experiments are ongoing, but preliminary results demonstrate significantly enhanced tumor control (BLI/tumor clearance), with the IL12-MSLN CAR NK cells.
Conclusions. This study demonstrates MSLN as a novel CAR target in AML and shows that IL-12 can be safely and feasibly incorporated into MSLN-CAR CIML NK cells, providing a potential therapeutic approach to substantially enhance their anti-tumor responses. Our results support further evaluation of this approach in patients with relapsed/refractory MSLN+ AML with otherwise extremely poor prognosis.
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