Cytokine-Induced Memory-like NK Cells Have a Distinct Single Cell Transcriptional Profile and Persist for Months in Adult and Pediatric Leukemia Patients after Adoptive Transfer

Abstract Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses and exhibit innate memory following stimulation with IL-12, IL-15, and IL-18, thereby differentiating into cytokine-induced memory-like (ML) NK cells. ML NK cells have well-described enhanced anti-tumor properties; however, the molecular mechanisms underlying their enhanced functionality are not well-understood. Initial reports of allogeneic donor ML NK cellular therapy for relapsed/refractory (rel/ref) acute myeloid leukemia (AML) demonstrated safety and a 47% CR/CRi rate (PMID32826231). In this setting, allogeneic ML NK cells are rejected after 3 weeks by recipient T cells, which precludes long-term evaluation of their biology. To address this limitation, we conducted a clinical trial for rel/ref AML patients that added adoptive transfer of same-donor ML NK cells on day +7 of a reduced-intensity conditioning (RIC) MHC-haploidentical HCT, followed by 4 doses of IL-15 (N-803) over 2 weeks (NCT02782546). Since the ML NK cells are from the HCT donor, they are not rejected, but remain MHC-haploidentical to the patient leukemia. Using samples from these patients, we profiled the single cell transcriptomes of NK cells using multidimensional CITE-seq, combining scRNAseq with a custom NK panel of antibodies. To identify donor ML NK cells in an unbiased fashion, we developed a CITE-seq ML NK classifier from in vitro differentiated paired conventional NK (cNK) and ML NK cells. This classifier was applied via transfer learning to CITE-seq analyzed samples from the donor (cNK cells) and patients at days +28 and +60. This approach identified 28-40% of NK cells as ML at Day +28 post-HCT. Only 1-6% of donor peripheral blood NK cells and 4-7% of NK cells in comparator leukemia patients at day +28 after conventional haplo-HCT alone were identified as ML NK cells (Fig 1A). These ML NK cells had a cell surface receptor profile analogous to a previously reported mass cytometry phenotype. Within the CITE-seq data, ML NK cells expressed a transcriptional profile consistent with enhanced functionality (GZMK, GZMA, GNLY), secreted proteins (LTB, CKLF), a distinct adhesome, and evidence of prior activation (MHC Class II and interferon-inducible genes). ML NK cells had a unique NK receptor repertoire including increased KIR2DL4, KLRC1(NKG2A), CD300A, NCAM1(CD56) , and CD2 with decreased expression of the inhibitory receptor KLRB1(CD161). Furthermore, ML NK cells upregulated HOPX, a transcription factor implicated in memory T cells and murine CMV adaptive NK cells. Additionally, ML NK cells downregulated transcription factors related to terminal maturation (ZEB2) and exhaustion (NR4A2). We next sought to identify changes during ML differentiation in patients post-HCT from day +28 to +60 post-HCT. Trajectory analysis identified a ML NK cell state distinct from cNK cells that was present at least 60 days post-HCT (Fig 1B). The ML transcriptional phenotype continued to modulate during late differentiation, including downregulation of GZMK and NCAM1, and upregulation of maturation related transcription factors, while maintaining high expression of HOPX. ML NK cells retained their enhanced functionality during in vivo differentiation, as patient ML NK cells had significantly increased IFNγ production compared to cNK cells after restimulation with leukemia targets or cytokines using mass cytometry (Fig. 2). Subsequently, we confirmed the ML CITE-seq profile in an independent clinical trial treating pediatric AML relapsed after allogenic HCT with same-donor ML NK cells (NCT03068819). In this setting, ML NK cells expressed a similar transcriptional signature and persisted for at least 2 months in the absence of exogenous cytokine support. Thus, ML NK cells possess a distinct transcriptional and surface proteomic profile and undergo in vivo differentiation while persisting within patients for at least 2 months. These findings reveal novel and unique aspects of the ML NK cell molecular program, as well as their prolonged functional persistence in vivo in patients, assisting in future clinical trial design. Figure 1 Figure 1. Disclosures Foltz: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Bednarski: Horizon Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; ImmunityBio: Research Funding; Kiadis: Other; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; OrcaBio: Other; Indapta: Other.

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Cytokine-Induced Memory-like (ML) NK Cells Persist for > 2 Months Following Adoptive Transfer into Leukemia Patients with a MHC-Compatible Hematopoietic Cell Transplant (HCT)

Blood ◽

10.1182/blood-2019-126004 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1954-1954 ◽

Cited By ~ 4

Author(s):

Jennifer A Foltz ◽

Melissa M Berrien-Elliott ◽

Carly Neal ◽

Mark Foster ◽

Ethan McClain ◽

...

Keyword(s):

Clinical Trial ◽

Nk Cells ◽

Adoptive Transfer ◽

Nk Cell ◽

Hematopoietic Cell ◽

Cell Transplant ◽

Mass Cytometry ◽

Hematopoietic Cell Transplant ◽

Cell Adoptive Transfer

Natural killer (NK) cells exhibit innate memory or memory-like responses following stimulation with haptens, viruses, or cytokines. Human memory-like (ML) NK cells differentiate following a short-term activation with IL-12, IL-15, and IL-18, and have increased anti-tumor activity against AML and other cancers in vitro, in xenograft models, and in the first-in human phase 1 clinical trial of ML NK cells in AML (PMID27655849). In this trial, CR/Cri was observed in >50% of patients treated, and mass cytometry revealed a unique multi-dimensional phenotype of in vivo differentiated ML NK cells that was confirmed using donor-specific HLA markers. Although adoptively transferred MHC-haploidentical ML NK cells expanded and differentiated over 2-3 weeks, these cells were eliminated by recipient allogeneic immune responses, a challenge observed with all allogeneic lymphocyte therapies. The immune rejection observed in the allogeneic setting precluded following ML NK phenotype, persistence, and function long-term in these patients. We hypothesized that ML NK cells could persist longer than 2-3 weeks in an MHC-compatible setting, and thus be able to assess ML NK cells durability. To test this idea, a phase 2 clinical trial was designed for relapsed/refractory AML patients, who receive a reduced-intensity HLA-haploidentical hematopoietic cell transplant (HCT), followed by same-donor ML NK cell adoptive transfer at day 7, with 2 weeks of IL-15/N-803 support (NCT02782546). Using mass cytometry, ML NK cells were confirmed as distinct from conventional NK cells, CD56hi/NKp30hi/CD62Lhi/KIR+/NKG2A+/CD57+/-, by viSNE analysis, and clearly inconsistent with immature NK cells arising from the HCT graft (CD56bright/KIR-/CD57-). ML NK cells persisted in patients for at least 2 months (n=5) following adoptive transfer, and constituted 20-50% of total NK cells at day 60 (n=3, 206±97 cells/μl; mean±SEM; peak ML NK cells = 751-1106 cells/µl, D21-D28, n=5). These ML NK cells appeared highly functional (56±8% IFN-γ+, 20±3% TNF+, 41±7% CD107a+) when stimulated with tumor targets immediately ex vivo on study day 28 (n=7). Unsupervised clustering of scRNA-seq from patient samples acquired 14-60 days after ML NK cell adoptive transfer identified a subset of NK cells transcriptionally distinct from conventional CD56bright and CD56dim. This NK cell population was the majority of NK cells at study Day 21 and remained identifiable 2 months post-transfer. In agreement with the mass cytometry data, these NK cells expressed high levels of KIRs with scRNA-seq analysis uncovering novel transcriptional changes in granzyme M, perforin, KLRG1, and IFNG suggesting ML NK cells represent a mature, activated NK cell subset distinct from conventional NK cells arising from the graft. In addition, scRNAseq analysis identified high expression of the transcription factor RUNX3, a potential regulator of ML NK cell phenotype in vivo. In conclusion, a single infusion of ML NK cells resulted in a durable population of highly functional NK cells, as evidenced by multi-dimensional analyses using mass cytometry and scRNA-sequencing. These studies provide evidence that ML NK cell therapy in the MHC-compatible setting overcomes persistence barriers and provide a platform for innovation in NK cell therapeutics. Disclosures Cashen: Celgene: Other: Speaker's Bureau; Seattle Genetics: Other: Speaker's Bureau; Novartis: Other: Speaker's Bureau. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).

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Single-Cell Multi-Omic Analysis Uncovers Comprised Immune Function and Primary Resistance Mechanism in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-149022 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 378-378

Author(s):

Jianbiao Zhou ◽

Jonathan Adam Scolnick ◽

Stacy Xu ◽

Melissa Ooi ◽

Priscella Shirley Chia ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Single Cell ◽

Research Funding ◽

Immune Escape ◽

Nk Cell ◽

Primary Resistance ◽

Bm Niche

Abstract Background: Approximately 20% of AML patients do not respond to induction chemotherapy (primary resistance) and 40-60% of patients develop secondary resistance, eventually leading to relapse followed by refractory disease (RR-AML). Diversified molecular mechanisms have been proposed for drug resistance and RR phenotype. However, we still cannot predict when relapse will occur, nor which patients will become resistant to therapy. Single-cell multi-omic (ScMo) profiling may provide new insights into our understanding of hematopoietic stem cell (HSC) differentiation trajectories, tumor heterogeneity and clonal evolution. Here we applied ScMo to profile bone marrow (BM) from AML patients and healthy controls. Methods: AML samples were collected at diagnosis with institutional IRB approval. Cells were stained with a panel of 62 DNA barcoded antibodies and 10x Genomics Single Cell 3' Library Kit v3 was used to generate ScMo data. After normalization, clusters were identified using Uniform Manifold Approximation and Projection (UMAP) and annotated using MapCell (Koh and Hoon, 2019). We analyzed 23,933 cells from 4 adult AML BM samples, and 39,522 cells from 2 healthy adults and 3 sorted CD34+ normal BM samples. Gene set enrichment analysis (GSEA) and Enrichr program were used to examine underlying pathways among differentially expressed genes between healthy and AML samples. Results: We identified 16 cell types between the AML and normal samples (Fig 1a) amongst 45 clusters in the UMAP projection (Fig 1b). Comparative analysis of the T cell clusters in AML samples with healthy BM cells identified an "AML T-cell signature" with over-expression of genes such as granzymes, NK/T cell markers, chemokine and cytokine, proteinase and proteinase inhibitor (Fig 2a). Among them, IL32 is known to be involved in activation-induced cell death in T cells and has immunosuppressive role, while CD8+ GZMB+ and CD8+ GZMK+ cells are considered as dysfunctional or pre-dysfunctional T cells. Indeed, Enrichr analysis showed the top rank of phenotype term - "decreased cytotoxic T cell cytolysis". We next examined whether NK cells, are similarly dysfunctional in the AML ecosystem. The "AML NK cell signature" includes Fc Fragment family, IFN-stimulated genes (ISGs), the effector protein-encoding genes and other genes when compared to normal NK cells (Fig 2b). GSEA analysis revealed "PD-1 signalling" among the top 5 ranked pathways in AML-NK cells, though no increase in PD-1 protein nor PDCD1 gene were identified in these cells. Inhibitory receptor CD160 was expressed higher in AML samples along with exhaustion (dysfunction) associated genes TIGIT, PRF1 and GZMB (Fig 2c). Enrichr analysis uncovered enrichment of "abnormal NK cell physiology and "impaired natural killer cell mediated cytotoxicity". Similarly, the "AML monocyte signature" was significantly enriched with genes in "Tumor Infiltrating Macrophages in Cancer Progression and Immune Escape" and "Myeloid Derived Suppressor Cells in Cancer Immune Escape". We also analyzed HSPC component in one pair of cytogenetically matched, untreated complete remission (CR) /RR AML pair (Fig 2d). Notably, half of the 10 genes overexpressed in RR-AML, CXCR4, LGALS1, S100A8, S100A9, SRGN (Serglycin), regulate cell-matrix interaction and play pivotal roles in leukemic cells homing bone marrow niche. The first 4 of these genes have been demonstrated as prognostic indicators of poor survival and associated with chemo-resistance and anti-apoptotic function. Furthermore, single-cell trajectory analysis of this CR/RR pair illustrated a change in differentiation pattern of HSPCs in CR-AML to monocytes in RR-AML. We are currently analyzing more AML samples to validate these findings. Conclusions: Our ScMo analysis demonstrates that the immune cells are systematically reprogrammed and functionally comprised in the AML ecosystem. Upregulation of BM niche factors could be the underlying mechanism for RR-AML. Thus, reversing the inhibited immune system is an important strategy for AML therapy and targeting leukemic cell-BM niche interaction should be considered for cases with high expression of these molecules on AML HSPCs. Note: J.Z. and J.A.S. share co-first authorship. Figure 1 Figure 1. Disclosures Scolnick: Proteona Pte Ltd: Current holder of individual stocks in a privately-held company. Xu: Proteona Pte Ltd: Current Employment. Ooi: Jansen: Honoraria; Teva Pharmaceuticals: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Lovci: Proteona Pte Ltd: Current Employment. Chng: Aslan: Research Funding; Takeda: Honoraria; Johnson & Johnson: Honoraria, Research Funding; BMS/Celgene: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria.

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Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

Blood ◽

10.1182/blood-2021-148654 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 407-407

Author(s):

Frank Cichocki ◽

Jode P Goodridge ◽

Ryan Bjordahl ◽

Svetlana Gaidarova ◽

Sajid Mahmood ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Car T Cells ◽

Car T

Abstract Treatments for B-cell malignancies have improved over the past several decades with clinical application of the CD20-specific antibody rituximab and chimeric antigen receptor (CAR) T cells targeting CD19. Despite the success of these therapies, loss of CD20 after rituximab treatment has been reported in leukemia and lymphoma patients. Additionally, up to 50% of all patients receiving anti-CD19 CAR T-cell therapy relapse within the first year with many of those patients exhibiting CD19 loss. Thus, new therapeutic approaches are needed to address tumor antigen escape. Accordingly, we generated triple gene-modified iPSC-derived NK (iNK) cells, termed "iDuo" NK cells, tailored to facilitate multi-antigen targeting. The iPSC line was clonally engineered to express high-affinity, non-cleavable CD16a (hnCD16), an anti-CD19 CAR optimized for NK cell signaling, and a membrane-bound IL-15/IL-15R fusion (IL-15RF) molecule to enhance NK cell persistence (Fig. 1A). To model antigen escape, we generated CD19 knockout AHR77 lymphoma cells alongside wild type AHR77 cells (both CD20 +) as targets in cytotoxicity assays. Activated peripheral blood NK (PBNK) cells, non-transduced iNK cells, and iDuo NK cells were tested as effectors. Unlike PBNK cells or non-transduced iNK cells, iDuo NK cells efficiently eliminated wild type AHR77 cells with or without the addition of rituximab at all tested E:T ratios. Similarly, iDuo NK cells in combination with rituximab were uniquely able to efficiently eliminate CD19 KO AHR77 cells due to enhanced antibody-dependent cellular cytotoxicity (ADCC) driven by hnCD16 (Fig. 1B-E). Cytotoxicity mediated by iDuo NK cells was also evaluated using primary chronic lymphocytic leukemia (CLL) cells. Compared to expanded PBNK cells and non-transduced iNK cells, only iDuo NK cells (in the absence of rituximab) were able to kill primary CLL cells (Fig. 1F). Expression of IL-15RF by iDuo NK cells uniquely supports in vitro expansion without the need for cytokine supplementation. To determine whether IL-15RF supports in vivo persistence of iDuo NK cells, CD19 CAR iNK cells (lacking IL-15RF) and iDuo NK cells were injected into NSG mice without the addition of cytokines or CD19 antigen availability. iDuo NK cell numbers peaked within a week after injection and persisted at measurable levels for ~5 weeks, in marked contrast to CD19 CAR iNK cell numbers that were undetectable throughout (Fig. 1G). To evaluate the in vivo function of iDuo NK cells, NALM6 leukemia cells were engrafted into NSG mice. Groups of mice received tumor alone or were treated with 3 doses of thawed iDuo NK cells. iDuo NK cells alone were highly effective in this model as evidenced by complete survival of mice in the treatment group (Fig. 1H). To assess iDuo NK cells in a more aggressive model, Raji lymphoma cells were engrafted, and groups of mice received rituximab alone, iDuo NK cells alone, or iDuo NK cells plus rituximab. Mice given the combination of iDuo NK cells and rituximab provided extended survival compared to all other arms in the aggressive disseminated Raji lymphoma xenograft model (Fig. 1I). One disadvantage of anti-CD19 CAR T cells is their inability to discriminate between healthy and malignant B cells. Because NK cells express inhibitory receptors that enable "self" versus "non-self" discrimination, we reasoned that iDuo NK cells could have higher cytotoxicity against tumor cells relative to healthy B cells. To address this, we labeled Raji cells, CD19 + B cells from healthy donor peripheral blood mononuclear cells (PBMCs) and CD19 - PBMCs. Labeled populations of cells were co-cultured with iDuo NK cells, and specific killing was analyzed. As expected, iDuo NK cells did not target CD19 - PBMCs. Intriguingly, iDuo NK cells had much higher cytotoxic activity against Raji cells compared to primary CD19 + B cells, suggesting a preferential targeting of malignant B cells compared to healthy B cells. Together, these results demonstrate the potent multi-antigen targeting capability and in vivo antitumor function of iDuo NK cells. Further, these data suggest that iDuo NK cells may have an additional advantage over anti-CD19 CAR T cells by discriminating between healthy and malignant B cells. The first iDuo NK cell, FT596, is currently being tested in a Phase I clinical trial (NCT04245722) for the treatment of B-cell lymphoma. Figure 1 Figure 1. Disclosures Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Bjordahl: Fate Therapeutics: Current Employment. Gaidarova: Fate Therapeutics, Inc: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Rogers: Fate Therapeutics, Inc: Current Employment. Huffman: Fate Therapeutics, Inc: Current Employment. Lee: Fate Therapeutics, Inc: Current Employment. Szabo: Fate Therapeutics, Inc: Current Employment. Wong: BMS: Current equity holder in publicly-traded company; Fate Therapeutics, Inc: Current Employment. Cooley: Fate Therapeutics, Inc: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment. Miller: Magenta: Membership on an entity's Board of Directors or advisory committees; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees.

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Single-Cell Transcriptomic Analysis Reveals Loss of Activated Bone Marrow NK Cells in Multiple Myeloma Patients Which Associates with Disease Progression in Mice

Blood ◽

10.1182/blood-2021-150422 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1578-1578

Author(s):

Sabrin Tahri ◽

Zoltan Kellermayer ◽

Madelon M.E. de Jong ◽

Natalie Papazian ◽

Cathelijne Fokkema ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Single Cell ◽

Disease Progression ◽

Research Funding ◽

Nk Cell ◽

Newly Diagnosed ◽

Cell Compartment ◽

Tumor Load

Abstract Introduction Multiple Myeloma (MM) disease progression and therapy response are the net result of tumor cell-intrinsic features and tumor cell-extrinsic cues from the bone marrow (BM) microenvironment. Natural killer (NK) cells are mediators of the cytotoxic immune response against MM and are important effector cells in antibody-based immune therapies, especially anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided into a cytotoxic CD56 dim subset, important for antibody-dependent cellular cytotoxicity, and a cytokine-producing CD56 bright subset releasing inflammatory mediators such as IFNγ, TNFα and GM-CSF. However, accumulating evidence suggests greater heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact disease progression and response to NK cell-driven immunotherapies. Here, we combined the 5TGM1 murine model of MM with single-cell RNA sequencing of bone marrow (BM) NK cells of newly diagnosed MM patients to map NK cell heterogeneity and to investigate their role in MM progression. Results To gain insight in NK cell heterogeneity in MM disease we performed single-cell RNA sequencing on immune cells of viably frozen BM aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. Conventional CD56 bright and CD56 dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK +CD56 bright NK cells contained clusters of naïve and activated NK cells. The GZMB+CD56 dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets, we compared GZMK +CD56 bright vs GZMB+CD56 dim cluster composition and distribution between controls and MM patients. Control BM was dominated by GZMB-transcribing cytotoxic CD56 dim NK cells, resulting in a low ratio of cytokine-producing GZMK +CD56 bright vs cytotoxic GZMB+CD56 dim NK cells. In contrast, MM bone marrow was characterized by heterogeneity of this ratio with a subset of patients presenting with complete reversal of this ratio . In this subset of patients, the altered composition was due to a loss of cytotoxic GZMB +CD56 dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation. To better examine the significance of cytotoxic NK cells in MM disease course we utilized the well-established 5TGM1 mouse model. C57Bl/6 and KaLwRij mice both received 10 6 5TGM1-GFP cells intravenously. Three weeks after tumor injection all KaLwRij mice (18/18) developed MM, defined by >5% tumor cells in BM ("unrestrained tumor") and serum M-protein >2mg/ml. Interestingly, while 39% (7/18) of C57Bl/6 mice had no tumor, 44% (8/18) had low but detectable levels of MM cells (0.1-5% of BM cells, "restrained tumor") and 17% (3/18) presented with an unrestrained MM with BM tumor load similar to that seen in KaLwRij mice. With time the percentage of mice with unrestrained tumor increased (5/12, 42%) at the expense of restrained tumor (2/12, 16%). We hypothesized that C57Bl/6 mice with low tumor load could represent a model of immune-mediated tumor control. Detailed analysis of the NK cell compartment revealed an expansion of activated mature (CD69 + CD11b +CD27 +) NK cells in C57Bl/6 mice with restrained BM MM (p=0.0031). In contrast, high BM tumor burden in both genotypes was associated with a sharp decline in absolute numbers of activated NK cells. Conclusion: Through a combination of single-cell transcriptomic analyses of the BM immune microenvironment in MM patients and experimental mouse models we found a loss of activated NK cells in a subset of patients and mice. Our data suggests that loss of these activated NK cells is associated with MM progression in vivo. A subset of MM patients presented with a loss of activated cytotoxic GZMB +CD56 dim NK cells in the BM, suggestive of reduced cytotoxic anti-tumor responses. Meanwhile, in vivo, high disease burden only occurred in mice with an absence of activated NK cells. Current analyses are focused on differences in human disease progression and efficacy of Daratumumab-based therapies in patients with various NK cell phenotypes. Disclosures Broijl: Janssen, Amgen, Sanofi, Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld: Janssen: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

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Recombinant Human IL-15 Promotes in Vivo Expansion of Adoptively Transferred NK Cells in a First-in-Human Phase I Dose Escalation Study in Patients with AML

Blood ◽

10.1182/blood.v120.21.894.894 ◽

2012 ◽

Vol 120 (21) ◽

pp. 894-894 ◽

Cited By ~ 7

Author(s):

Sarah Cooley ◽

Michael R. Verneris ◽

Julie Curtsinger ◽

David McKenna ◽

Daniel J. Weisdorf ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Adoptive Transfer ◽

Cell Expansion ◽

Nk Cell ◽

Advisory Committees ◽

Dose Cohort ◽

Refractory Aml

Abstract Abstract 894 Adoptive transfer of haploidentical NK cells can induce remissions in patients with refractory AML. However, many do not expand NK cells and fail to respond. While IL-2 can promote NK cell proliferation, it also promotes the expansion of regulatory T cells, which impede NK cell expansion. Because IL-15 has different effects on regulatory T cells than IL-2, we initiated a phase I dose escalation trial of recombinant human IL-15 to enhance adoptive transfer of NK cells. Patients with refractory AML receive a lymphodepleting preparative regimen of fludarabine 25 mg/m2 × 5 days and cyclophosphamide 60 mg/kg × 2 days. Haploidentical NK cells (CD3- and CD19-depleted and overnight activated with IL-15 10 ng/ml) were infused Day 0, followed by 12 daily doses of intravenous IL-15 (Biopharmaceutical Development Program, NCI Frederick) in planned dosing cohorts of 0.25, 0.5, 1, 2 and 3 mcg/kg. To date 9 patients have been treated (Table). The first 6 patients (0.25 and 0.5 mcg/ml cohorts) received all 12 planned doses of IL-15, with no dose-limiting toxicities (DLTs). Apart from transient fevers, the IL-15 was well tolerated. While donor-derived NK cells were detected at Day 7 in all patients, none achieved the primary endpoint of >100 donor-derived NK cells/μl circulating in blood at Day 14. At the completion of IL-15 dosing, all patients in cohorts 1–2 had a lymphocytosis comprised of host T cells, which were mainly CD8+ (mean 81±6%). Five of 6 recovered neutrophils by Day 28 (range 15–26 days), and one with residual leukemia remained neutropenic leading us to conclude that IL-15 does not impede neutrophil recovery. Three of the 6 cleared their leukemic blasts, fully recovered and proceeded to allogeneic hematopoietic cell transplant (HCT). In contrast to the first 2 cohorts, the first patient at the 1 mcg/kg dose experienced a DLT (grade 4 dyspnea due to diffuse alveolar hemorrhage requiring high dose steroids) and received only 8 does of IL-15 making him not evaluable for in vivo expansion. Due to the DLT, the 1 mcg/kg dose cohort will expand to 6 evaluable patients, 2 of whom have completed IL-15 dosing. Both are evaluable, each having received the required minimum of 9 doses, but additional planned doses 10–12 were held in both patients due to high fevers and transient hypoxia possibly related to infection that did not constitute DLT. Both cleared refractory leukemia at Day 14 and successfully expanded donor NK cells (2094 and 448 cells/ml) at the end of IL-15 dosing. The in vivo expanded NK cells exhibited potent function, with 81.5% and 82.3% cytotoxicity against K562 targets at a 20:1 E:T ratio. Thus 1 mcg/ml dosing of IL-15 is significantly more likely to induce successful donor NK cell expansion at day 14 than the 0.25 or 0.5 mcg/ml doses (p = 0.04). Since endogenous IL-15 may heterodimerize with its receptor, IL-15Ra, to provide more stability and potent signalling to NK cells, batched serum samples are in process to measure free and IL-15Ra complexed IL-15. In summary, this platform of adoptive transfer of haploidentical NK cells with IL-15 has, in this early experience shown to be an effective treatment for refractory AML allowing patients to achieve remission and subsequent allogenenic HCT. The 1 mcg/kg dose is associated with more toxicity and limited ability to deliver all 12 doses, but toxicity was transient. 9 daily doses were sufficient to promote robust in vivo expansion of highly functional donor-derived NK cells. Further dose modifications (perhaps with continuous infusion or using fewer doses given subcutaneously) may be required to enhance safety. Based on this preliminary experience, IL-15 should emerge as the optimal cytokine to promote expansion and activation of adoptively transferred NK cells without Treg stimulation, which should be effective therapy for AML. In vivo expansion of donor-derived NK cells is dependent on IL-15 dosing. Subject IL-15 Dose Cohort (mcg/kg) # IL-15 Doses DLT Day 7 Day 11–14 WBC (cells/ml blood) %NK % Donor DNA Max ALC (cells/ml blood) %NK % Donor DNA %T cells %CD8+ T cells 1 0.25 12 No 100 37 QNS 1800 0.3 0% 95 86 2 0.25 12 No 100 38 25% 1200 16 0% 82 72 3 0.25 12 No 100 12 17% 1500 5.5 0% - - 4 0.5 12 No 100 44 43% 3200 0.3 2% 96 91 5 0.5 12 No <100 54 41% 100 0.2 0% 98 61 6 0.5 12 No <100 67 36% 1400 12 2% 57 93 7* 1.0 8 Yes <100 35 37% 600 0.1 0% 96 85 8 1.0 9 No 200 80 97% 2300 98 93% 1 - 9 1.0 9 No <100 92 86% 700 94 97% 1 - * Not evaluable for in vivo NK expansion due to DLT requiring IL-15 discontinuation and steroids Disclosures: Miller: Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

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Engineered Memory-like NK Cars Targeting a Neoepitope Derived from Intracellular NPM1c Exhibit Potent Activity and Specificity Against Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-134148 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 3-4

Author(s):

Han Dong ◽

Guozhu Xie ◽

Yong Liang ◽

James Dongjoo Ham ◽

Juliana Vergara ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

Nk Cell ◽

Leukemia Cells ◽

Tumor Resistance ◽

Cell Therapies ◽

Car T

Introduction: Acute myeloid leukemia (AML) continues to be a major therapeutic challenge. There is an emerging need to develop less toxic and more effective targeted therapies. Natural Killer (NK) cells possess many of the key attributes critical for effective cancer therapies- "born to kill" but without apparent risk of graft versus host disease, cytokine release syndrome, or neurotoxicity. Furthermore, their intrinsic propensity to target myeloid blasts makes them particularly attractive for AML. Despite promising clinical results in blood cancer, the development of NK cell-based therapy remains challenging mostly due to NK cells' short lifespan, inadequate proliferation and lack of specific tumor targeting. Here, we utilized a new approach to arm NK cells for adoptive immunotherapy based on innate cell memory. Chimeric antigen receptors (CARs) significantly enhance anti-tumor specificity and activity of immune effector cells. Our innovative CAR-NK cells target a tumor-specific neoepitope in AML and harness potent function pathways in their design to enhance efficacy and minimize toxicity. Methods: 1. Mutated NPM1c as a CAR Target in AML. Most CAR-T cell therapies target tumor-associated antigens (TAAs), which could lead to on-target/off-tumor toxicity as well as tumor resistance. One way to overcome these drawbacks is to target tumor-specific oncogenic driver mutations. The four-nucleotide duplication in nucleophosmin, referred to as NPM1c, is a driver oncogene mutation in about 35% of AML. The mutation creates a neoepitope that is presented by the most common HLA-A2 allele. Using yeast surface display, we have isolated a human single-chain variable fragment (scFv) that specifically binds to the NPM1c epitope-HLA-A2 complex, but not HLA-A2 alone or HLA-A2 loaded with control peptides. 2. Cytokine-Induced Memory-Like (CIML) NK Cells as a CAR Platform. CIML NK cells can provide a unique platform for development of NK cell CARs based on the favorable safety profile, increased proliferation, prolonged persistence and enhanced anti-leukemia function that we have observed in pre-clinical models (Romee et al, Blood 2012) and in patients (Romee et al, Science Trans Med 2016) treated with un-modified CIML NK cells. 3. Efficient Gene Editing in Primary NK Cells. We have overcome the transduction block in primary human and mouse NK cells by utilizing an unconventional pseudotyped lentivirus based on a unique protein with high expression on CIML NK cells. Results: 1. Engineered CAR-T cells with the isolated scFv exhibit potent cytotoxicity both in vitro and in vivo against NPM1c+HLA-A2+ leukemia cells (OCI-AML3) and primary AML blasts, but not NPM1c-HLA-A2+ leukemia cells (OCI-AML2) or HLA-A2- tumor cells (PC-3). 2. The in vivo anti-leukemia efficacy of anti-NPM1c CAR-T cells was however transient (overall survival extended from 28 to 42 days, median survival extended from 21 to 37 days, compared with the control mice adoptively transferred with untraduced T cells), with unneglectable toxicity. 3. Utilizing an unconventional pseudotyped lentivirus to transduce CIML NK cells from healthy donor blood (n = 5 donors), we have successfully generated anti-NPM1c CAR-NK cells with high transduction efficiency (using MOI = 10: transduction rate mean 48%, range 32% to 65%; compared with 2%, range 0.8% to 4.5% for the conventional approach with VSVG pseudotyped lentivirus). 4. Harnessing key cytokine pathways in the CAR design substantially promoted CAR-NK cell survival (indicated by the enhanced cell viability from 29.7% to 75.2%) and proliferation (marked by the increased levels of ki-67 from 60.2% to 94.5%). 5. Anti-NPM1c CAR significantly promoted anti-tumor function (represented by CD107a, IFN-gamma) and tumor-specific killing (measured by annexin V and 7-AAD) of CIML NK cells against AML with NPM1c oncogene (OCI-AML3). 6. Dual-armed CIML NK cells with CAR and cytokine signaling exhibited optimal specificity and sustainability against AML targets. Conclusion: These results demonstrate that the innovative CAR-CIML NK cells could be developed as an efficient cellular immunotherapy for treating NPM1c+HLA-A2+ AML with potentially reduced on-target/off-tumor toxicity and tumor resistance. Our study should drive novel conception and design of CAR-NK cell therapies against myeloid malignancies in the clinic. Figure Disclosures Ritz: Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy.

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A Phase I Clinical Trial Testing the Safety of IL-21-Expanded, Universally Alloreactive Donor-Derived Natural Killer Cells for Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

Blood ◽

10.1182/blood-2021-150750 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1732-1732

Author(s):

Sumithira Vasu ◽

Nidhi Sharma ◽

Alison R. Walker ◽

Sarah A Wall ◽

James S. Blachly ◽

...

Keyword(s):

Clinical Trial ◽

Nk Cells ◽

Board Of Directors ◽

Dose Level ◽

Research Funding ◽

Nk Cell ◽

Third Party ◽

Advisory Committees ◽

Refractory Aml

Abstract Background: Natural Killer (NK) cells are cytotoxic lymphocytes that are able to exert an anti-tumor effect in an MHC-I independent manner, but are dysfunctional and reduced in number in patients with leukemia. Several studies have shown therapeutic potential for related donor NK cells expanded and/or activated ex vivo when administered to cancer patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, personalized, donor-derived cell therapies require time for donor identification, manufacturing and product release, resulting in patient attrition. As NK alloreactivity and NKG2C expression play critical roles in mediating anti-tumor effects, we identified 'ideal' NK cell donors (in partnership with "Be the Match Biotherapies") for collection. Human leukocyte antigen (HLA) and Killer immunoglobulin receptor (KIR) genotyping were done to screen and select donors. Following this, a sample was collected from donors to expand NK cells under small-scale conditions. If donor NK cells showed robust expansion, they proceeded with apheresis. To ensure these therapeutic cells would be readily-available, we established a third-party NK cell bank through scalable, affordable mass-production with membrane-bound IL-21 feeder cells (FC21), and cryopreserved large numbers of these NK cells for immediate 'off-the-shelf' administration to recipients. Here, we report our initial experience in a Phase I trial of these off-the-shelf (OTS) alloreactive NK cells as immunotherapy for patients with relapsed/refractory AML and MDS. Methods: Patients were treated at Dose level 1, to allow infusion at a dose of 1 x 10e7 NK cells/ kg. Each dose level had two cohorts, stratified by age. Patients were enrolled into Cohort 1 (patients <60 yrs. received Fludarabine (Flu) 30 mg/m2/day & Cytarabine 2 g/m2/day on days -6 to -2) and Cohort 2 (patients ≥ 60 yrs. received Flu 30 mg/m2/day (days -5 to -2) & Decitabine 20 mg/m2/day (days -6 to -2). Patients were evaluated to see if they had any donor-directed antibodies. After chemotherapy, NK cells were infused thrice weekly for 6 doses, to be given over a period of 2 weeks (Clinicaltrials.gov: NCT04220684). Persistence of donor NK cells was determined by flow cytometry using haplotype-discriminating anti-HLA antibodies (Figure 1, 2). NK cells were completely HLA-mismatched between donor and recipients. Results: 6 patients (3/cohort) were treated at the first dose level of 1x10 7 NK cells/kg/dose. All 6 patients had relapsed/refractory AML and had received at least two prior lines of treatment. One patient (005) withdrew from the study prior to infusion of NK cells. 6 patients received the first NK cell dose as an inpatient and were discharged to receive the remaining 5 doses as an outpatient. 5 patients tolerated infusion of all 6 doses of NK cells administered over 2 weeks. One patient (006) developed a Cytokine response (CRS) - like syndrome in the context of streptococcus bacteremia and received only one dose of NK cells. Symptoms suggestive of CRS responded fully with steroids and patient was able to successfully wean off steroids and discharged to the outpatient setting. One patient proceeded to allogeneic hematopoietic cell transplant. No infusion-related reactions, neurotoxicity or graft versus host disease was observed. In vivo persistence/expansion NK cells were superior with FLU/CY lymphodepletion (cohort 1). Conclusions: Cryopreserved, third-party donor-derived NK cells are safe and feasible in relapsed/refractory AML patients, and even at this first dose level, completely HLA-mismatched NK cells can persist and expand to high levels in vivo. Figure 1 Figure 1. Disclosures Vasu: Kiadis, Inc.: Research Funding; Seattle Genetics: Other: travel support; Boehringer Ingelheim: Other: Travel support; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Walker: Newave: Other: clinical trial support; Geron: Other: clinical trial support; Novartis: Other: clinical trial support. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. de Lima: Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding. Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.

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Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer Immunotherapy

Cancers ◽

10.3390/cancers11060769 ◽

2019 ◽

Vol 11 (6) ◽

pp. 769 ◽

Cited By ~ 30

Author(s):

Kyle B. Lupo ◽

Sandro Matosevic

Keyword(s):

T Cells ◽

Nk Cells ◽

Cancer Immunotherapy ◽

Natural Killer ◽

Adoptive Transfer ◽

Nk Cell ◽

Human Leukocyte ◽

Hematologic Malignancies ◽

Published Evidence

Natural killer (NK) cells are attractive within adoptive transfer settings in cancer immunotherapy due to their potential for allogeneic use; their alloreactivity is enhanced under conditions of killer immunoglobulin-like receptor (KIR) mismatch with human leukocyte antigen (HLA) ligands on cancer cells. In addition to this, NK cells are platforms for genetic modification, and proliferate in vivo for a shorter time relative to T cells, limiting off-target activation. Current clinical studies have demonstrated the safety and efficacy of allogeneic NK cell adoptive transfer therapies as a means for treatment of hematologic malignancies and, to a lesser extent, solid tumors. However, challenges associated with sourcing allogeneic NK cells have given rise to controversy over the contribution of NK cells to graft-versus-host disease (GvHD). Specifically, blood-derived NK cell infusions contain contaminating T cells, whose activation with NK-stimulating cytokines has been known to lead to heightened release of proinflammatory cytokines and trigger the onset of GvHD in vivo. NK cells sourced from cell lines and stem cells lack contaminating T cells, but can also lack many phenotypic characteristics of mature NK cells. Here, we discuss the available published evidence for the varying roles of NK cells in GvHD and, more broadly, their use in allogeneic adoptive transfer settings to treat various cancers.

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Natural Killer Cell Adoptive Immunotherapy Is Hampered by A Rapid Development of NK Cell Dysfunction Characterized by Loss of Effector Mechanisms and Downregulation of the Transcription Factors T-Bet and Eomesodermin; Definition and Possible Mechanism of NK Cell Exhaustion

Blood ◽

10.1182/blood.v118.21.1000.1000 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1000-1000

Author(s):

Saar I. Gill ◽

Adrianne E Vasey ◽

Jeanette B Baker ◽

Aaron Smith ◽

Holbrook E Kohrt ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Transcription Factors ◽

Nk Cells ◽

Nk Cell ◽

Homeostatic Proliferation ◽

Chromium Release ◽

Tumor Bearing

Abstract Abstract 1000 Natural killer (NK) cells exhibit in vitro cytotoxicity against many tumor cell types and have an important role in controlling tumor growth, as depletion of NK cells from tumor-bearing mice hastens tumor growth and impairs survival. These data, in combination with results from clinical trials of haploidentical killer immunoglobulin-like receptor (KIR)-ligand mismatched bone marrow transplantation, led to interest in the use of adoptive NK immunotherapy for the treatment of malignancy. Recent clinical results have shown that allogeneic NK cells can be safely administered after chemotherapy and/or irradiation but have also demonstrated limited persistence of the infused NK cells without clear evidence of efficacy. We traced the fate of adoptively infused NK cells in order to delineate the barriers to successful NK immunotherapy using several NK-sensitive murine tumor models. Mice bearing established lymphoma or leukemia received an intravenous infusion of 0.5–1.0×106 (approximately 2.5–5×107/kg body weight) NK cells from allogeneic or syngeneic donors after or concurrently with total body irradiation and bone marrow rescue. Using luciferase +ve NK cells, we first showed that in animals bearing subcutaneous tumors, NK cells homed to lymphoid organs in the first week, followed by progressive localization to and accumulation within the tumor site (Figure 1). In contrast, in non-tumor bearing animals NK cells homed to lymph nodes, spleen and liver with maximal proliferation at the end of the second week. These observations indicated that NK cells fail to eradicate the tumor despite prior demonstration of in vitro sensitivity, successful homing and local accumulation. As expected from these findings, survival was not prolonged by the NK cell infusion. Reisolation of donor NK cells within 18 hours of transfer showed enhanced cytotoxicity (14% vs 2%, p = 0.004) and IFNγ production (48% vs 22%, p = 0.04) compared with naive resting NK cells. In contrast, NK cells isolated from tumor-bearing mice at later time-points beginning d+5 showed loss of IFNγ production (48% early vs 3% late, p = 0.01), decreased expression of the activating receptor NKG2D, and impaired cytotoxicity in chromium release assays. These observations did not relate to over-stimulation through NKG2D, as NK cells from NKG2D−/− animals were also susceptible to acquired dysfunction (although their baseline cytotoxicity was lower than WT NK toward A20 lymphoma).Fig. 1Adoptively transferred luciferase-transgenic NK cells accumulate within the tumor over timeFig. 1. Adoptively transferred luciferase-transgenic NK cells accumulate within the tumor over time Eomesodermin and T-bet are transcription factors with important roles in effector functions of CD8+ T cells and NK cells. In T cells T-bet downregulation has been shown to correlate with exhaustion (Kao et al, Nat Imm 2011). Flow cytometry of reisolated NK cells revealed downregulation of Eomesodermin (naive splenic control, d+1 reisolated and d+10 reisolated cells showing 85%, 96%, and 29% expression, respectively) and T-bet (naive splenic control, d+1 reisolated and d+10 reisolated cells showing 82%, 99%, and 59% expression, respectively), correlating with loss of IFNγ production. The phenotype described herein was most dramatic within the tumor and within mice carrying high tumor burdens, but was also present in NK cells reisolated from non-tumor bearing animals that received NK cells, suggesting that homeostatic proliferation after transfer of mature NK cells could also contribute to exhaustion. CFSElo proliferated NK cells showed the most dramatic loss of effector function (chromium release = 42% in unproliferated vs 18% in proliferated cells, p = 0.03) and transcription factor expression (Eomesodermin positive 83% in unproliferated cells vs 18% in proliferated cells, p = 0.002). Collectively, our results suggest that the success of NK cell immunotherapy is limited by an acquired dysfunction that occurs within days after homeostatic proliferation and target encounter and that may be related to the downregulation of transcription factors required for NK effector function. These findings illuminate a previously unappreciated phenomenon and explain why short-term in vitro killing assays have limited utility in predicting the in vivo behaviour of transferred NK cells. Hence, these findings suggest that transferred NK cells become dysfunctional in vivo and that novel approaches may be required in order to circumvent the described dysfunction phenotype. Disclosures: No relevant conflicts of interest to declare.

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A Phase I Study of Intravenous NCI IL-15 to Enhance Adoptively Transferred NK Cells Uncovers Defects in CD16 Triggered IFNγ Production and Competition Between Donor NK and Recipient T Cells

Blood ◽

10.1182/blood.v126.23.566.566 ◽

2015 ◽

Vol 126 (23) ◽

pp. 566-566 ◽

Cited By ~ 1

Author(s):

Jeffrey S. Miller ◽

Michael R. Verneris ◽

Julie Curtsinger ◽

Todd E. DeFor ◽

David McKenna ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Target Cell ◽

Adoptive Transfer ◽

Cell Expansion ◽

Nk Cell ◽

Donor Chimerism ◽

The Mean ◽

Stimulation Of

Abstract Adoptive transfer of IL-2 driven NK cells can induce remissions in patients with refractory AML but this may be limited by IL-2 induced expansion of regulatory T cells (Tregs). Because IL-15 does not stimulate Tregs, we performed a phase I dose escalation trial of recombinant human IL-15 (NCI Frederick) to enhance adoptive NK cell transfer. Patients with refractory AML received a lymphodepleting regimen of cyclophosphamide and fludarabine, followed by infusion of haploidentical NK cells activated overnight with IL-15 and intravenous IL-15 for 12 daily doses in cohorts of 0.25, 0.5, 1 and 0.75 mcg/kg. We treated 24 patients and deterimined the maximum tolerated dose (MTD) to be 0.75 mcg/kg. IL-15 serum levels peaked 1 hour after dosing with a significant drop at 4 hours and no evidence of drug accumulation between doses 1 and 6 (Figure, A). Fevers were common, possibly attributable to the rise in multiple inflammatory cytokines 4 hours after IL-15 dosing (Figure, B). Higher fevers occurred after 6 doses, corresponding to higher cytokine levels. No patients experienced hypotension or vascular leak. Dose limiting toxicity was observed in 2 subjects at 1.0 mcg/kg who did not recover neutrophils 42 days after NK cell infusion, one of whom died of infection 60 days after adoptive NK cell transfer at which time donor NK cells persisted in blood and pleural fluid. Clearance of refractory leukemia was seen in 38% of patients 14 days after adoptive transfer. Three patients with CRp went on to best donor allogeneic transplant with overall survival of those receiving IL-15 (n=24) of 32% at 12 months. We observed two patterns of donor cell expansion and autologous count recovery. In the 5 of 24 (21%) patients with detectable NK cell donor chimerism, the mean ALC measured at day 7 after NK infusion was 160/µL with an mean of 56% donor chimerism. By day 14 the mean ALC increased to 1200/µL with 87% donor chimerism, where virtually all donor lymphocytes were NK cells. The remaining 19 patients who did not expand NK cells had a mean ALC of 68/µL with 27% donor chimerism at day 7, which increased to 900/µL and only 2.5% donor chimerism by day 14. In the non-NK expanders, nearly all of the day 14 lymphocytes were recipient CD8+ T cells, suggesting host T cell mediated rejection of donor NK cells. We extensively evaluated the function of the successfully in vivo expanded donor NK cells collected from the recipient day 14 post-NK infusion and IL-15 administration, and directly compared it to that of NK cells at steady state from the same donor. Although we expected degranulation to be increased in the IL-15 expanded NK cells, CD107a expression was similar in steady state donor and in vivo IL-15 expanded NK cells (Figure, C). In contrast, IFNγ production triggered by CD16 signalling with Rituxan against antibody-dependent cellular cytotoxicity sensitive Raji targets was significantly decreased in the in vivo IL-15 expanded NK cells (Figure, D). This did not reflect an inherent defect in IFNγ production as IL-12/IL-18 stimulation of day 14 in vivo IL-15 expanded donor NK cells induced significantly more IFNγ compared to the study state donor cells (Figure, E). These characteristics (decreased target cell-induced INFg and increased IL-12/IL-18 induced IFNγ) suggest that donor NK cells expanded in vivo with IL-15 are functionally similar to the CD56bright population. This effect is not unique to IL-15 as a similar functional pattern was observed in patients with in vivo IL-2 expanded NK cells. This suggests that robust, cytokine driven in vivo NK proliferation may limit the target cell-induced cytokine production of the expanded population. In summary, NCI IL-15 led to robust donor NK cell expansion in some patients, but competitive stimulation of recipient CD8+ T cells was common. Even when in vivo expansion of donor NK cell was successful, the cells exhibited defects in target cell and CD16-induced IFNγ production. Long-lived CMV-driven adaptive NK cells with memory like properties are associated with enhanced anti-leukemia activity and CD16 signalling. Novel methods to expand adaptive NK cells may be of value for the next generation of NK cell therapy. Additionally, the NCI CITN trial of subcutaneous IL-15 administration suggests that compared to IV dosing, it may favor donor NK vs. host T cell expansion based on different affinities of trans-presented IL-15 to these cell populations, and these strategies should be tested to optimize NK cell adoptive transfer. Figure 1. Figure 1. Disclosures Miller: Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau.

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