Memory-like NK Research Articles

Memory-like differentiation enhances NK cell responses against colorectal cancer

ABSTRACT Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.

Combined IL-12, IL-15 and IL-18 activation induces epigenetic changes in human NK cells during the memory-like state transition

Abstract NK cells exhibit memory properties after activation with haptens, viral infection, or cytokines with each stimulus having distinct biology. Memory-like (ML) NK cells differentiate over one week after brief activation with combined IL-12/15/18, and have augmented responses upon restimulation by cytokines, activating receptor, or tumor targets. While much is known about their functional characteristics, there are large gaps in our understanding of the molecular mechanisms involved in programming conventional (c)NK cells into ML NK cells. As ML NK cells have enhanced ability to produce IFN-γ after multiple cell divisions, this suggests that the IFNG gene, and others, may be epigenetically poised. Therefore, we hypothesize that activation with IL-12/15/18 triggers epigenetic remodeling of cNK cells to ML NK cells that drives enhanced functionality. To test this, we performed ATAC-seq on baseline cNK cells, IL-12/15/18 activated NK cells, and in vitro differentiated cNK and ML NK cells after one week. Activation with IL-12/15/18 induces dramatic early epigenetic remodeling with >10,000 differentially accessible regions (DARs) compared to cNK cells. Moreover, ML differentiation generates epigenetically distinct ML NK cells (>1,000 DARs) compared to in vitro differentiated cNK cells, including increased IFNG locus accessibility, suggesting epigenetic mechanisms regulate their enhanced functionality. Also, ML NK cells are markedly different from IL-12/15/18 activated NK cells (>15,000 DARs) suggesting not all epigenetic changes are immediately induced, and time is required for full implementation of the memory-like state. Together, this supports that ML NK cells rely on epigenetic programming for enhanced function. Supported by grants from NIH (P50CA171963, R01CA205239, P30CA91842, 1F31GM146361-01).

CD8α identifies human NK cells with altered proliferation, metabolism, and IL-15 signaling

Abstract NK cells are cytotoxic innate lymphoid cells that can differentiate into memory-like (ML) NK cells following IL-12/15/18 stimulation. Previous work identified that CD8α expression on donor ML NK cells was negatively associated with outcome for cellular therapy of AML. However, the role of CD8α on human NK cells remains poorly studied, and murine NK cells are CD8α-precluding study in mice. We found that CD8α-NK cells had greater proliferation and survival in vitro and in vivo. Interestingly, we observed that CD8α expression was dynamic, and a subset of CD8α-NK cells upregulated CD8α (“new” CD8+) after IL-15 stimulation. Notably, the “new” CD8+ NK cells were the most proliferative in vitro and in vivo, and had significantly greater signaling following IL-15 stimulation. Strikingly, we also found that “new” CD8+ NK cells had significantly higher glucose uptake, nutrient receptor expression, and an enhanced capacity for glycolysis and oxidative phosphorylation. These results indicate that recent CD8α acquisition marks the ability of NK cells to respond to IL-15 signals to engage in the robust metabolic activity required for proliferation. We also investigated the role of CD8α in NK cell activation, given that CD8α can bind HLA and associate with Lck. In the absence of CD8α, NK responses (CD107a, IFNg, TNF) following activating receptor ligation were enhanced for several receptors, suggesting an intrinsic role for CD8α in inhibiting NK cell activation. Together, these results identify a novel association of CD8α with treatment outcome for NK cellular therapy and identify a previously unappreciated role of CD8α on human NK cell proliferation, metabolism, and response through activating receptors. Supported by grants from NIAID F30AI16131

Abstract 2897: Overcoming barriers to the natural killer cell response against multiple myeloma: Manipulating the NKG2A/HLA-E checkpoint and CAR engineering of memory-like natural killer cells

Abstract Recent advances in treatment options for multiple myeloma (MM) have improved patient outcomes, however, MM remains incurable and options are limited for relapsed/refractory disease. Memory-like (ML) differentiation of NK cells using brief IL-12, IL-15, and IL-18 activation generates a safe allogenic NK cellular therapy for acute myeloid leukemia that induce complete remissions. Here, we investigate pre-clinical combinations with ML NK cells for MM, including blockade of the inhibitory NKG2A/HLA-E checkpoint as well as MM-specific targeting via myeloma-targeting monoclonal antibody (mAb) and chimeric antigen receptor (CAR) engineering. Human ML NK cells generated from healthy donors display IFN-γ responses similar to conventional (cNK) cells, and only modestly increased cytotoxicity against myeloma cell lines (MM1.s and OPM2). MM cell lines and primary MM cells express high levels of HLA-E, a major negative checkpoint for ML NK cells that binds to the inhibitory receptor NKG2A expressed on NK cells. Indeed, mass cytometry analysis of MM patients compared to healthy controls demonstrated enrichment of a NK cell population expressing high levels of NKG2A, but also high levels of activation and cytotoxic markers. Disruption of HLA-E binding to NKG2A with the blocking mAb monalizumab (AstraZeneca) significantly increased healthy donor ML NK cell IFN-γ response (P=0.016), degranulation (surface CD107a, P=0.017), and killing (P=0.009) of MM1.s when compared to isotype mAb. NK cells isolated from patients with MM showed similarly enhanced functionality after ML differentiation and NKG2A blockade (IFN-γ P=0.008). In an NSG mouse model of MM using MM1.s cells, healthy donor ML NK cells combined with monalizumab showed improved control of MM tumor growth compared to ML NK cells without monalizumab (P<0.001), conventional NK cells, or control tumors alone (P<0.001). To further improve recognition of MM, ML NK cells responses were assessed in combinationwith a mAb against SLAMF7 (elotuzumab) that both targets MM cells and activates NK cells. Elotuzumab treatment increased the functionality of ML NK cells against MM cell lines (IFN-γ, P=0.004) with an additive effect of both elotuzumab and monalizumab (IFN-γ, P=0.003). In an alternative strategy to enhance MM targeting, we engineered an anti-BCMA CAR (anti-BCMA/4-1bb/z) into ML NK cells. BCMA-CAR+ NK cells exhibited increased IFN-γ (P=0.001) production and degranulation (P<0.001) against MM1.s compared to non-transduced control NK cells. These functional responses were additionally enhanced with ML differentiation. Collectively, these findings suggest that ML NK cell differentiation, combined with strategies to address inhibitory checkpoints (NKG2A) or MM-specific activation (elotuzumab, anti-BMCA CAR), are promising cellular therapy strategies for MM patients. Citation Format: Alice Y. Zhou, Melissa M. Berrien-Elliott, Ravi Vij, Mark Fiala, Michelle Becker-Hapak, Lynne Marsala, Miriam T. Jacobs, Nancy D. Marin, Jennifer Tran, Jennifer Foltz, Pamela Wong, Julie Fortier, Sarah Kelley, Carly Neal, David Russler-Germain, Timothy Schappe, Todd A. Fehniger. Overcoming barriers to the natural killer cell response against multiple myeloma: Manipulating the NKG2A/HLA-E checkpoint and CAR engineering of memory-like natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2897.

Cytokine-induced memory-like (CIML) NK cells and association with circulating immune microenvironment.

e18042 Background: Cytokine-induced memory-like (CIML) NK cells are attractive for adoptive cell therapeutic approaches due to their key characteristics which include anti-tumor responses, prolonged proliferation and persistence in vivo. Several NK cell subsets have been reported in human and mice, but their heterogeneity and trajectories in human peripheral blood remains poorly characterized. We aimed to evaluate the developmental trajectories and prognosis outcomes of several CIML NK subsets between HPV+ and HPV- HNSC patients. Methods: A publicly available scRNA-seq dataset (only CD45+PBMC data sets were used) (Kürten et al., 2021) was obtained and stratified by two definitions of CIML NKs: (1) increased granzyme B mRNA expression and (2) preactivated with iterleukin-12 (IL-12), IL-15 and IL-18. Single-cell analysis was performed, followed by sub-clustering of NK populations. Results: 4,794 NK cells from 6 HPV+ and 11 HPV- HNSC patients were analyzed in our study. Expression profiles showed that these NK cells were CD56dimCD16+CD57-NKs. Further investigation showed that these cell populations exhibit a cytokine-induced memory-like phenotype, including increased granzyme B mRNA and preactivated with IL-12, IL-15 and IL-18. Transcriptomic profiles demonstrated that CIML NK cells can be classified into three subsets, including Bl1, Bl2, and Bl3, where Bl1 and Bl3 were found to be consistent with the previous studies, Bl2 was a newly identified NK cluster in this study. Pseudotime analyses suggested that CIML NK cells transitioned from Bl3 (is in cytolytic state) to Bl2, and then (maintains intermediate cell states) to Bl1 (supports cytotoxic activity), regardless of HPV status. There were 20, 60, and 26 Up-regulated genes in Bl, Bl2, and Bl3 in HPV+ patient compared to HPV- patients, respectively. Biocarta pathway analyses showed that NK cell pathway was highly enriched in all NK subsets from HPV+ patients, instead of HPV- patients, indicating a developmental independent characteristic. Survival analyses showed poor prognosis outcomes for high expressed CIML NK signatures in no matter HPV+ or HPV- patients. Conclusions: We investigated CIML NK cells in human peripheral blood and found that Bl1, Bl2, and Bl3 NK clusters showing the developmental patterns of increased cytotoxicity independent of HPV status. The Bl2 is a novel NK cell cluster which presents intermediate cell states. The cluster signatures were correlated with poor clinical outcomes, highlighting its’ potential clinical values for CIML NK-targeted immunotherapies.

Multidimensional Analyses of Donor Memory-Like NK Cells Reveal New Associations with Response after Adoptive Immunotherapy for Leukemia.

Natural killer (NK) cells are an emerging cancer cellular therapy and potent mediators of antitumor immunity. Cytokine-induced memory-like (ML) NK cellular therapy is safe and induces remissions in patients with acute myeloid leukemia (AML). However, the dynamic changes in phenotype that occur after NK-cell transfer that affect patient outcomes remain unclear. Here, we report comprehensive multidimensional correlates from ML NK cell-treated patients with AML using mass cytometry. These data identify a unique in vivo differentiated ML NK-cell phenotype distinct from conventional NK cells. Moreover, the inhibitory receptor NKG2A is a dominant, transcriptionally induced checkpoint important for ML, but not conventional NK-cell responses to cancer. The frequency of CD8α+ donor NK cells is negatively associated with AML patient outcomes after ML NK therapy. Thus, elucidating the multidimensional dynamics of donor ML NK cells in vivo revealed critical factors important for clinical response, and new avenues to enhance NK-cell therapeutics. SIGNIFICANCE: Mass cytometry reveals an in vivo memory-like NK-cell phenotype, where NKG2A is a dominant checkpoint, and CD8α is associated with treatment failure after ML NK-cell therapy. These findings identify multiple avenues for optimizing ML NK-cell immunotherapy for cancer and define mechanisms important for ML NK-cell function.This article is highlighted in the In This Issue feature, p. 1775.

CAR-modified memory-like NK cells exhibit potent responses to NK-resistant lymphomas

AbstractNatural killer (NK) cells are a promising cellular immunotherapy for cancer. Cytokine-induced memory-like (ML) NK cells differentiate after activation with interleukin-12 (IL-12), IL-15, and IL-18, exhibit potent antitumor responses, and safely induce complete remissions in patients with leukemia. However, many cancers are not fully recognized via NK cell receptors. Chimeric antigen receptors (CARs) have been used to enhance tumor-specific recognition by effector lymphocytes. We hypothesized that ML differentiation and CAR engineering would result in complementary improvements in NK cell responses against NK-resistant cancers. To test this idea, peripheral blood ML NK cells were modified to express an anti-CD19 CAR (19-CAR-ML), which displayed significantly increased interferon γ production, degranulation, and specific killing against NK-resistant lymphoma lines and primary targets compared with nonspecific control CAR-ML NK cells or conventional CAR NK cells. The 19-CAR and ML responses were synergistic and CAR specific and required immunoreceptor tyrosine-based activation motif signaling. Furthermore, 19-CAR-ML NK cells generated from lymphoma patients exhibited improved responses against their autologous lymphomas. 19-CAR-ML NK cells controlled lymphoma burden in vivo and improved survival in human xenograft models. Thus, CAR engineering of ML NK cells enhanced responses against resistant cancers and warrants further investigation, with the potential to broaden ML NK cell recognition against a variety of NK cell–resistant tumors.

A first-in-class ex vivo combination between cytokine-induced memory like (CIML) NK cells and a CD38 targeting antibody recruiting molecule (ARM) as a novel approach to target NK cells without cellular engineering for the treatment of multiple myeloma.

8523 Background: Cytokine-induced memory-like (CIML) NK cells differentiate after a brief preactivation with interleukin-12 (IL-12), IL-15, and IL-18 and exhibit enhanced responses to cytokine or activating receptor restimulation for several weeks to months. Antibody Recruiting Molecules (ARM) are bifunctional molecules that simultaneously bind a given tumor marker and engage endogenous IgG antibodies, therefore binding and activating NK cells via their FcR and targeting them to kill tumors cells. A first in class therapy that has the potential to improve the activity of CIML NK cells by combining them with KP1237, a CD38-ARM is presented for the treatment of newly diagnosed multiple myeloma (MM) patients who are minimal residual disease positive (MRD+) and eligible for autologous stem cell transplant (ASCT). Methods: Specific killing of CD38 expressing MOLP-8 cells by CIML NK cells alone or in combination with KP1237 was measured. Additionally, NK and CIML NK cells from healthy volunteers and MM patients at various stages of the disease were tested for activity against autologous and allogeneic tumor cell targets. In these experiments, surface phenotype and activation status of NK cells were determined. Results: A statistically significant increase in specific killing was observed when KP1237 was used in combination with CIML NK cells in an ADCC assay against CD38 expressing MOLP-8 (p = 0.0105, mean 61.8 ± SD 9.1 and mean 83.7 ± SD 13.58 for CIML NK alone and CIML NK+KP1237, respectively). This effect was accompanied by an increase in plasma membrane retention of CD107a. Surface phenotype of NK and CIML NK cells from healthy volunteers and MM patients was compared since the functional activity of NK cells from such patients is debated. We report surface expression of CD56, CD16, CD57, NKG2D, Nkp44, Nkp46, CD107a, KIR2DL2/3/DS2, and CD69 as well as frequencies of CD57hi CD56+ mature versus CD57lo CD56+ immature NK cell subsets. Further, autologous CIML NK cells + KP1237 exhibit cytotoxicity towards patient plasma cells. Conclusions: The activity of CIML NK cells towards CD38 expressing MM target cells is improved by the addition of KP1237 avoiding the need for cellular engineering with chimeric antigen receptors. This led to the development of the combination drug product consisting of patient autologous CIML NK cells, KP1237 and IVIG. Clinical development is under way for (MRD+) MM patients eligible for ASCT.

Chimeric Antigen Receptor Modified Memory-like (CAR-ML) NK Cells Exhibit Potent Responses to NK-Resistant Tumors

NK cells are cytotoxic innate lymphoid cells that mediate anti-tumor responses and traffic to most tissues. NK cells activated briefly with IL-12, IL-15, and IL-18 differentiate into memory-like (ML) NK cells with enhanced anti-tumor effects, which has been translated into clinical trials for patients with leukemia. Adoptive cellular therapy with donor ML NK cells were safe and induced CR/CRi in >50% of rel/ref AML patients in a first-in-human phase 1 clinical trial at Washington University (PMID27655849). In preliminary results, donor ML NK cells expand and can persist for months in an immune-compatible recipients during or after allogeneic hematopoietic cell transplantation (HCT), and maintain potent effector function. However, NK cell recognition of many cancer types is limited, since they depend on expression of stress-induced activating receptor ligands. We hypothesized that ML NK cells engineered with chimeric antigen receptors (CAR) would demonstrate improved anti-tumor responses against classically NK-resistant targets. To test this idea, ML NK cells were engineered to express an anti-CD19-CAR, and responses against NK-resistant B-cell malignancies evaluated in vitro and in vivo. CAR-modified primary human ML NK cells (CAR-ML) were transduced with an anti-CD19-CD8a-41BB-CD3z-GFP (CD19-CAR-ML) lentivirus. This differentiation and transfection approach resulted in approximately 15-25% of ML NK cells transduced with the CAR construct, as determined flow cytometry staining for soluble CD19 and GFP. As an additional control, αCD33-CD8a-41BB-CD3z-GFP CAR-transduced ML NK cells and GFP- internal control ML NK cells were used. Here, in vitro functional assays were used to determine if CD19-CAR enhances ML NK cells in an antigen (CD19)-specific manner. CD19-CAR-ML (GFP+) and control ML NK (GFP-) cells were evaluated for functional responses to CD19-positive or CD19-negative tumor targets in vitro. CD19-CAR-ML NK cells demonstrated significantly increased IFN-γ production (44±4% vs. 15±3%, p<0.001); mean ± SEM) and degranulation (31±4% vs. 5±1%, p<0.001) against NK-resistant CD19+ Raji targets, compared to control GFP- and CD33-CAR ML NK cells. To understand the contributions of ML differentiation on the enhanced functionality of CD19-CAR modified NK cells, we compared CD19-CAR-ML NK cells to control CD19-CAR NK cells that were treated with IL-15 only. CD19-CAR-ML NK cells also exhibited significantly increased effector responses compared to control CAR NK cells against CD19+ targets (p<0.01). CD19-CAR-ML NK cells responded similarly to GFP- ML NK cells against CD19-negative Kasumi leukemia targets. Finally, CD19-CAR-ML NK cells also exhibited significantly enhanced killing, degranulation, and IFN-γ production against primary CD19+ follicular lymphoma targets from patient lymph nodes (p<0.01). To establish the translational utility of this approach, autologous CD19-CAR-ML NK cells generated from lymphoma patients demonstrated significantly increased IFN-γ production (p<0.05) and degranulation (p<0.01) against their own CD19+ lymphoma targets, compared to control ML NK cells (GFP-). These data confirm contributions of both ML differentiation and CAR expression in the enhanced antigen-specific, anti-tumor responses observed in CAR-ML NK cells. To test the expansion and persistence of ML NK cells transduced with CD19 or CD33-CAR were transferred into CD19+ Raji-bearing NSG mice and supported in vivo with IL-15. After three weeks, mice were sacrificed and NK (CD56) cell persistence and tumor (CD19) burden assessed by flow cytometry. CD19-CAR ML recipient mice had reduced tumor burden in the BM, spleen, and blood compared to CD33-CAR ML treated mice. Notably, CD19-CAR-ML (GFP+) were increased to >70% of human NK cells from 20% in the CD33-CAR-ML recipient mice, suggesting antigen-specific CAR-expressing NK cells expand or survive better in vivo than non-transduced ML or non-specific CAR-ML NK cells in vivo. Thus, combining CAR with ML differentiation results in NK cells with enhanced responses to NK resistant tumors. These studies warrant continued CAR-ML development and provide the pre-clinical rationale for translating this combination NK cell therapy approach to the clinic. Figure Disclosures Fehniger: Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).

Abstract 1546: The CD30/CD16A bispecific innate immune cell engager AFM13 elicits heterogeneous single-cell NK cell responses and effectively triggers memory-like (ML) NK cells

Abstract Natural killer (NK) cells are crucial effector cells of the innate immune system capable of rapidly recognizing and eliminating infected, stressed and malignant cells. NK cells discriminate tumor targets from healthy cells via integration of activating and inhibitory receptor signals. One barrier to the broad application of NK cells across many cancer types is inconsistent cancer cell recognition, which may be overcome by immune cell engagers. AFM13 is a tetravalent bispecific antibody based on the ROCK platform characterized by bivalent binding to CD30 and CD16A with clinical efficacy in CD30+ malignancies. However, our understanding of NK cell functional responses triggered via AFM13 remain incomplete. Moreover, adoptively transferred memory-like (ML) NK cells have demonstrated enhanced anti-tumor activity (Romee R et al, Sci Transl Med, 2016) that may be receptive to AFM13-based targeting to enhance target cell recognition. To address these questions, we analyzed single-cell conventional (cNK) and ML (IL-12/15/18-induced) NK cell functional responses to NK-resistant CD30+ lymphoma cells +/- AFM13. Primary cNK cells co-incubated with AFM13-treated Hut-78 cells demonstrated increased IFN-γ, TNF, and degranulation, compared to Hut-78 cells or Raji (CD30-) targets + AFM13 as a negative control (p<0.05). To define the single-cell specificity of NK cell responses to AFM13, similar assays were performed using mass cytometry analysis of 39 lineage, maturation, activating and inhibitory receptors, and function-relevant NK cell markers. tSNE-based multidimensional analyses revealed marked distinctions between Hut-78 and AFM13-Hut-78 stimulated cNK cells, due in part to IFN-γ, MIP-1α, CD107a, and CD16. To define the impact of specific NK receptors, SPADE was used to define highly activated IFN-γ+ NK cell sub-populations. In a KIR3DL1+ donor, activation was primarily within the KIR3DL1+ subset, consistent with the lack of its inhibitory ligand (HLA-Bw4). In KIR3DL1 negative donors, responding NK cells were enriched in mature KIR2DL2/L3+CD57+ NK cells that lacked NKG2A. Additional experiments revealed that both control and ML NK cells exhibited increased IFN-γ, degranulation, and cytotoxicity with AFM13 (P<0.01), and AFM13-stimulated ML NK cells exhibited the highest IFN-γ response and killing. Collectively, these data indicate that target cell recognition of NK cells can be significantly enhanced by AFM13, yet influenced by inhibitory receptor expression, maturation state, and memory-like differentiation. Thus, these data suggest that the status and repertoire of NK cells in a patient may offer diagnostic potential for therapeutic response, and the combination of ML NK cells with AFM13 appears to be a promising therapeutic approach. Citation Format: Nancy Marin, Michelle Becker-Hapak, Joachim Koch, Melissa M. Berrien-Elliott, Mark Foster, Carly Neal, Ethan McClain, Sweta Desai, Julia A. Wagner, Timothy Schappe, Lynne Marsala, Pamela Wong, Martin Treder, Todd A. Fehniger. The CD30/CD16A bispecific innate immune cell engager AFM13 elicits heterogeneous single-cell NK cell responses and effectively triggers memory-like (ML) NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1546.

Abstract 5704: Mass cytometry identifies the expansion, persistence, and immune checkpoints of adoptively transferred memory-like NK cells in patients with leukemia

Abstract 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.

Formation of FcRγ-deficient NK cell subset associated with HCMV-reactivation in hematopoietic cell transplant recipients.

131 Background: We have previously identified a novel subset of human NK cells that lacks FcRγ, a transmembrane signaling adaptor associated with CD16, the Fc receptor for IgG. These FcRγ-deficient NK cells (termed g-NK cells) display enhanced functional activity against target cells, including virus-infected cells, in concert with target-specific antibody molecules. The presence of g-NK cells in humans is associated with prior infection by human cytomegalovirus (HCMV). Additionally, g-NK cells are activated and expand robustly against HCMV-infected cells in the presence of HCMV-specific antibodies, consistent with predicted features of memory-like or adaptive NK cells. Recent studies have reported that expansion of a specific subset of NK cells with an adaptive phenotype can occur in response to HCMV reactivation after hematopoietic cell transplantation. Remarkably, patients with the expanded subset of NK cells showed reduced incidence of leukemia relapse and improved disease-free survival. Methods: Using blood samples obtained from BMT patients pre- and post-transplant, we have performed FACS-based phenotyping and ELISA-based serotyping analyses to explore whether HCMV-reactivation correlates with the generation of FcRγ-deficient NK cells in a bone marrow transplantation (BMT) setting. Results: Our data demonstrate that FcRγ-deficient NK cells appear in transplant patients following HCMV-reactivation, but not in the BMT patients without HCMV-reactivation. Phenotypic analysis indicates that these FcRγ-deficient NK cells display characteristics consistent with those observed in g-NK cells obtained from healthy HCMV-seropositive individuals, e.g. decreased expression of tyrosine kinase SYK. Conclusions: These results provide evidence in support of a causal relationship between HCMV infection and the generation of g-NK cells. Given the enhanced functional capability of g-NK cells against HCMV-infected target cells, the formation and functional activity of g-NK cells within BMT patients may contribute to the control of HCMV infection during the transplant procedure, and impact recovery from other diseases.

Human Cytokine-Induced Memory-Like (CIML) NK Cells Exhibit Potent Anti-Leukemia Cytotoxicity and Maintain Memory-Like Functionality After Adoptive Transfer Into Immunodeficient NOD-SCID-Gc-/- (NSG) Mice

Background Allogeneic NK cells are anti-leukemia immune effector lymphocytes, with evidence of activity in patients in adoptive transfer clinical studies. What is the optimal approach to prepare allogeneic NK cells for maximal effector function remains an open question for adoptive NK cell therapy. Recently described cytokine-induced memory-like (CIML) NK cells are generated following a brief (16 hour) pre-activation with a combination of IL-12+IL-15+IL-18 in both mice and humans. Following weeks or months of rest, CIML NK cells exhibit an enhanced recall IFN-g response when restimulated with K562 cells or cytokines. However, their anti-leukemic cytotoxic activity and identification of key supporting cytokines for survival and sustained functionality have not been reported. We hypothesized that CIML NK cells may have enhanced effector function against AML, providing a potential rationale for future clinical studies of CIML NK cells in AML patients. To test this hypothesis we investigated the CIML NK cell response to myeloid leukemia, including primary AML blasts, and evaluated their function following transfer into NSG mice. Methods Normal human donor NK cells (>95% purity) were cultured with low dose (1 ng/mL) IL-15 alone (control) or pre-activated with IL-12 (10 ng/ml) + IL-15 (1 ng/ml) + IL-18 (50 ng/ml) for 16 hours. After washing, the cells were cultured for 7 days in low dose IL-15 (to support survival). Following this prolonged rest period in vitro, NK cell responses were assessed after 6-hour re-stimulation with K562 leukemia cells or primary AML blasts. NK cell functional responses assessed include IFN-g production and cytotoxicity (using flow based killing assays). For adoptive transfer experiments, 5-8 x 106 human CIML NK cells or control cells were injected into sub-lethally irradiated NSG mice, and assessed for persistence, expansion and function of the adoptively transferred CIML NK or control NK cell. Additional experiments included evaluating CIML NK cells for cytokine receptor expression and effector proteins after the 7 day rest period. Results As described previously, CIML NK cells had a significantly increased IFN-g response to K562 leukemia cells (15.5 ± 3% vs. 7 ± 1%, P=0.03). CIML NK cells also exhibited a more potent IFN-g response to primary blasts from untreated, newly diagnosed AML patients (N=4 AML samples, P< 0.0001). Further, CIML NK cells demonstrated a significantly greater cytotoxic response, compared to control NK cells, upon co-incubation with K562 leukemia cells (Figure 1). Consistent with this enhanced cytotoxicity, CIML NK cells had significantly increased expression of granzyme A (P=0.005) and granzyme B (P=0.006) proteins. Further, we noted a marked induction of CD25 (IL-2Ra) after IL-12+IL-15+IL-18 pre-activation, which via the IL-2Rabg resulted in enhanced functional responses to picomolar concentrations of IL-2. This included enhanced cytotoxicity against leukemia cells, IFN-g production in response to co-stimulation with IL-12, and proliferation. To assess persistence and expansion upon adoptive transfer, NSG mice were injected with control or CIML NK cells. After 7 days (during which 75,000IU of IL-2 was injected qOD) there was a preferential expansion of the CIML NK cells in blood (11±2.6 vs. 5±1.3, P=0.01) and bone marrow (0.6±0.14 vs. 0.21±0.06, P= 0.03) in these mice as assessed by the ratio of human to mouse CD45 positive cells. Further, CIML NK cells supported in vivo in NSG mice exhibited enhanced IFN-g responses upon re-stimulation with K562 leukemia cells (10±1.5% vs. 2.5±1% IFN-g positive, P= 0.03) or IL-12+IL-15 (15±2% vs. 2±0.5%, P= 0.001). Conclusions Brief (16 hour) pre-activation with a combination of IL-12+IL-15+IL-18 results in the generation of CIML NK cells that have an enhanced IFN-g and cytotoxic response to K562 leukemia cells and primary allogeneic AML blasts. Further, CD25 is induced on CIML NK cells, which in the context of the high affinity IL-2Rabg confers selective responsiveness to low concentrations of IL-2 for proliferation, enhanced cytotoxicity, and enhanced IFN-g production. CIML NK cells may develop in vivo in NSG xenografts, and CIML NK cells appear to be selectively supported by exogenous low dose IL-2 in this context. These pre-clinical data support CIML NK cells as a novel optimization approach for NK cell adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.