NK cells are an emerging cell therapy for cancer, however, the optimal approaches to maximize NK cell anti-tumor attack are unclear. NK cells exhibit memory-like (ML) properties following combined cytokine (IL-12/15/18) pre-activation, evidenced by enhanced responses to cancer cells upon re-stimulation weeks later. A first-in-human clinical trial for acute myeloid leukemia (AML) (Romee R et al., Sci Transl Med, 2016) revealed that 7 of 11 (54%) evaluable patients responded to ML NK cell therapy. To inform key aspects of response, we used mass cytometry to track ML NK cell diversity, checkpoints, and effector functions in AML patients treated with ML NK cells. Multidimensional analyses (viSNE) of patient samples collected 7 days after NK transfer accurately identified in vivo-differentiated ML NK cells that were distinct from conventional NK (cNK) cells: CD56hiCD11bloCD62L+ NKG2AhiNKp30hi Ki-67+ (cNK: 3%±0.5% vs. ML: 87%±5%, mean±SEM within ML gate, P<0.05, N=10). In a second clinical trial of MHC-haploidentical hematopoietic transplantation (HCT), augmented with same-donor IL-12/15/18 activated NK cells (NCT02782546), mass cytometry identified marked ML NK cell expansion in vivo in this immune-compatible environment. In the first two patients treated, ML NK cells expanded (>1000-fold expansion in vivo, peak >2000 cells/uL blood), persisted >= 60 days, and were distinct from immature CD56brightKIR-CD16- NK cells developing from the graft. These ML NK cells exhibited potent anti-leukemic functional responses at day +28. Utilizing the phase 1 study cohort, Citrus analysis identified increased NKG2A expression as significantly correlated with treatment failure [median NKG2A = 89±25 (treatment failure); 8±3 (clinical response); p=0.007, FDR<0.1]. NKG2A is an inhibitory receptor that binds to non-classical MHC HLA-E expressed on AML. We hypothesized that NKG2A/HLA-E interactions in vivo represent a key checkpoint on ML NK cell responses. Consistent with this idea, HLA-Ehi AML blasts resulted in reduced ML NK cells responses (P<0.05) in vitro. ML NK cells also triggered with HLA-E+ K562-AML in the presence of control or anti-NKG2A blocking antibodies. Increased functional responses including IFN-γ (p=0.02) and TNF (p=0.05) production by NKG2A-blocked ML NK cells were detected, compared to isotype-treated ML NK cells. Similar results were observed with HLA-E+ primary AML blasts as targets, showing that ML NK cells treated with NKG2A blockade produced significantly more IFN-γ (p=0.001). Thus, mass cytometry identified that in vivo-differentiated ML NK cells are distinct from cNK cells, and exhibit marked expansion and persistence in an immune-compatible environment. NKG2A was identified as a key ML NK cell checkpoint in vivo, and blockade of NKG2A signals may enhance the clinical efficacy of ML NK cell therapy for AML patients.

Citation Format: Melissa M. Berrien-Elliott, Julia A. Wagner, Rizwan Romee, Michelle Becker-Hapak, Timothy Schappe, Carly Neal, Ethan McClain, John DiPersio, Peter Westervelt, Amanda F. Cashen, Todd A. Fehniger. Mass cytometry identifies the expansion, persistence, and immune checkpoints of adoptively transferred memory-like NK cells in patients with leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5704.