Interleukin-15 Complex Treatment Protects Mice from Cerebral Malaria by Inducing Interleukin-10-Producing Natural Killer Cells.

Cerebral malaria is a deadly complication of Plasmodium infection and involves blood brain barrier (BBB) disruption following infiltration of white blood cells. During experimental cerebral malaria (ECM), mice inoculated with Plasmodium berghei ANKA-infected red blood cells develop a fatal CM-like disease caused by CD8+ T cell-mediated pathology. We found that treatment with interleukin-15 complex (IL-15C) prevented ECM, whereas IL-2C treatment had no effect. IL-15C-expanded natural killer (NK) cells were necessary and sufficient for protection against ECM. IL-15C treatment also decreased CD8+ T cell activation in the brain and prevented BBB breakdown without influencing parasite load. IL-15C induced NK cells to express IL-10, which was required for IL-15C-mediated protection against ECM. Finally, we show that ALT-803, a modified human IL-15C, mediates similar induction of IL-10 in NK cells and protection against ECM. These data identify a regulatory role for cytokine-stimulated NK cells in the prevention of a pathogenic immune response.

Ablation of SYK Kinase from Expanded Primary Human NK Cells via CRISPR/Cas9 Enhances Cytotoxicity and Cytokine Production.

CMV infection alters NK cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the FcRγ-chain (gene: FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced Ab-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives enhanced ADCC and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. We ablated genes that encode molecules in the ADCC pathway, such as FcRγ, CD3ζ, SYK, SHP-1, ZAP70, and the transcription factor PLZF, and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ-chain caused a modest increase in TNF-α production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced cytotoxicity, cytokine production, and target cell conjugation, whereas ZAP70 kinase ablation diminished function. Ablating the phosphatase SHP-1 enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. We found the lack of SYK expression could improve target cell conjugation through enhanced CD2 expression or limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production.

Dual antigen-targeted off-the-shelf NK cells show durable response and prevent antigen escape in lymphoma and leukemia.

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition, we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies.

Anti-NKG2C/IL-15/anti-CD33 killer engager directs primary and iPSC-derived NKG2C+ NK cells to target myeloid leukemia.

Natural killer (NK) cells mediate the cytolysis of transformed cells and are currently used as an adoptive cellular therapy to treat cancer. Infection with human cytomegalovirus has been shown to expand a subset of "adaptive" NK cells expressing the activation receptor NKG2C that have preferred functional attributes distinct from conventional NK cells. Because NKG2C delivers a strong activating signal to NK cells, we hypothesized that NKG2C could specifically trigger NK-cell-mediated antitumor responses. To elicit a tumor-directed response from NKG2C+ NK cells, we created an anti-NKG2C/IL-15/anti-CD33 killer engager called NKG2C-KE that directs NKG2C+ cells to target CD33+ cells and tumor-associated antigen expressed by acute myelogenous leukemia cells. The NKG2C-KE induced specific degranulation, interferon-γ production, and proliferation of NKG2C-expressing NK cells from patients who reactivated cytomegalovirus after allogeneic transplantation. The NKG2C-KE was also tested in a more homogeneous system using induced pluripotent stem cell (iPSC)-derived NK (iNK) cells that have been engineered to express NKG2C at high levels. The NKG2C-KE triggered iNK-cell-mediated cytotoxicity against CD33+ cells and primary AML blasts. The NKG2C-KE-specific interaction with adaptive NK and NKG2C+ iNK cells represents a new immunotherapeutic paradigm that uniquely engages highly active NK cells to induce cytotoxicity against AML through redirected targeting.

CD16xCD33 Bispecific Killer Cell Engager (BiKE) as potential immunotherapeutic in pediatric patients with AML and biphenotypic ALL.

Similar to pediatric acute myeloid leukemia (AML) the subgroup of biphenotypic acute lymphoblastic leukemia (ALL) is a rare complex entity with adverse outcome, characterized by the surface expression of CD33. Despite novel and promising anti-CD19 targeted immunotherapies such as chimeric antigen receptor T cells and bispecific anti-CD19/CD3 antibodies, relapse and resistance remain a major challenge in about 30% to 60% of patients. To investigate the potential role of the fully humanized bispecific antibody CD16 × CD33 (BiKE) in children with CD33+ acute leukemia, we tested whether the reagent was able to boost NK cell effector functions against CD33+ AML and biphenotypic ALL blasts. Stimulation of primary NK cells from healthy volunteers with 16 × 33 BiKE led to increased cytotoxicity, degranulation and cytokine production against CD33+ cell lines. Moreover, BiKE treatment significantly increased degranulation, IFN-γ and TNF-α production against primary ALL and AML targets. Importantly, also NK cells from leukemic patients profited from restoration of effector functions by BiKE treatment, albeit to a lesser extent than NK cells from healthy donors. In particular, those patients with low perforin and granzyme expression showed compromised cytotoxic function even in the presence of BiKE. In patients with intrinsic NK cell deficiency, combination therapy of CD16xCD33 BiKE and allogeneic NK cells might thus be a promising therapeutic approach. Taken together, CD16xCD33 BiKE successfully increased NK cell effector functions against pediatric AML and biphenotypic ALL blasts and constitutes a promising new option for supporting maintenance therapy or "bridging" consolidation chemotherapy before hematopoietic stem cell transplantation.

Cytomegalovirus and systemic inflammation at time of surgery is associated with worse outcomes in serous ovarian cancer.

OBJECTIVES: Cytomegalovirus (CMV) is a common infection that establishes latency in healthy people. CMV has been associated with alterations of the immune compartment leading to improved responses, while inflammation has been shown to adversely impact outcomes. We investigated whether CMV serostatus predicts outcomes in ovarian cancer in the presence or absence of inflammation. METHODS: A total of 106 patients with serous ovarian cancer from 2006 to 2009 were analyzed. CMV and systemic inflammation was measured using CMV immunoglobulin G (IgG) and C-reactive protein (CRP), respectively, in serum collected prior to cytoreduction. Patients were stratified by CMV IgG (non-reactive, reactive/borderline) and CRP (≤10, >10 mg/L) status. Overall survival (OS) and recurrence-free survival (RFS) were compared by group using log-rank tests and Cox proportional hazards regression models adjusting for age at surgery. RESULTS: Of 106 eligible patients, 40 (37.7%) were CMV+/CRP+, 24 (22.6%) CMV+/CRP-, 19 (17.9%) CMV-/CRP+, and 23 (21.7%) CMV-/CRP-. CRP+ had higher CA-125 levels (P = 0.05) and higher rates of suboptimal debulking (P = 0.03). There were no other significant differences in demographic, surgical, or pathologic factors between groups. CMV+/CRP+ patients median RFS and OS were 16.9 months (95% CI: 9.0-21.1) and 31.7 months (95% CI: 25.0-48.7), respectively, with a significantly worse RFS (aHR: 1.85, 95% CI: 1.05-3.24, P = 0.03) and OS (aHR: 2.12, 95% CI: 1.17-3.82, P = 0.01) compared to CMV-/CRP- (RFS = 31.2 months (95% CI: 16.0-56.4) and OS = 63.8 months (95% CI: 50.7-87.0)). CMV+/CRP- group displayed the longest OS (89.3 months). CONCLUSIONS: Previous exposure to CMV and high CRP at surgery portended worse RFS and OS compared to women who tested negative. The CMV+/CRP- group had the longest OS, indicating that CMV status alone, in the absence of inflammation, may be protective.

A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy.

Enhancing natural killer (NK) cell cytotoxicity by blocking inhibitory signaling could lead to improved NK-based cancer immunotherapy. Thus, we have developed a highly efficient method for editing the genome of human NK cells using CRISPR/Cas9 to knock out inhibitory signaling molecules. Our method efficiently edits up to 90% of primary peripheral blood NK cells. As a proof-of-principle we demonstrate highly efficient knockout of ADAM17 and PDCD1, genes that have a functional impact on NK cells, and demonstrate that these gene-edited NK cells have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Furthermore, we were able to expand cells to clinically relevant numbers, without loss of activity. We also demonstrate that our CRISPR/Cas9 method can be used for efficient knockin of genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Our platform represents a feasible method for generating engineered primary NK cells as a universal therapeutic for cancer immunotherapy.

Natural killer cells unleashed: Checkpoint receptor blockade and BiKE/TriKE utilization in NK-mediated anti-tumor immunotherapy.

Natural killer (NK) cells have long been known to mediate anti-tumor responses without prior sensitization or recognition of specific tumor antigens. However, the tumor microenvironment can suppress NK cell function resulting in tumor escape and disease progression. Despite recent advances in cytokine therapy and NK cell adoptive transfer, tumor expression of ligands to NK - expressed checkpoint receptors can still suppress NK mediated tumor lysis. This review will explore many of the checkpoint receptors tumors utilize to manipulate the NK cell response as well as some of the current and upcoming pharmacological solutions to limit tumor suppression of NK cell function. Furthermore, we will discuss the potential to use these drugs in combinational therapies with novel antibody reagents such as bi- and tri-specific killer engagers (BiKEs and TriKEs) against tumor-specific antigens to enhance NK cell-mediated tumor rejection.

Cytokine-induced memory-like natural killer cells have enhanced function, proliferation, and in vivo expansion against ovarian cancer cells.

OBJECTIVE: Natural killer (NK) cells are lymphocytes well suited for adoptive immunotherapy. Attempts with adoptive NK cell immunotherapy against ovarian cancer have proven unsuccessful, with the main limitations including failure to expand and diminished effector function. We investigated if incubation of NK cells with interleukin (IL)-12, IL-15, and IL-18 for 16h could produce cytokine-induced memory-like (CIML) NK cells capable of enhanced function against ovarian cancer. METHODS: NK cells were preactivated briefly with IL-12, IL-15, and IL-18, rested, then placed against ovarian cancer targets to assess phenotype and function via flow cytometry. Real-time NK-cell-mediated tumor-killing was evaluated. Using ascites cells and cell-free ascites fluid, NK cell proliferation and function within the immunosuppressive microenvironment was evaluated in vitro. Finally, CIML NK cells were injected intraperitoneal (IP) into an in vivo xenogeneic mouse model of ovarian cancer. RESULTS: CIML NK cells demonstrate enhanced cytokine (IFN-γ) production and NK-cell-mediated killing of ovarian cancer. NK cells treated overnight with cytokines led to robust activation characterized by temporal shedding of CD16, induction of CD25, and enhanced proliferation. CIML NK cells proliferate more with enhanced effector function compared to controls in an immunosuppressive microenvironment. Finally, human CIML NK cells exhibited potent antitumor effects within a xenogeneic mouse model of ovarian cancer. CONCLUSIONS: CIML NK cells have enhanced functionality and persistence against ovarian cancer in vitro and in vivo, even when exposed to ascites fluid. These findings provide a strategy for NK cell-based immunotherapy to circumvent the immunosuppressive nature of ovarian cancer.

Early Reconstitution of NK and γδ T Cells and Its Implication for the Design of Post-Transplant Immunotherapy.

Relapse is the most frequent cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Natural killer (NK) cells and γδ T cells reconstitute early after allo-HSCT, contribute to tumor immunosurveillance via major histocompatibility complex-independent mechanisms and do not induce graft-versus-host disease. Here we performed a quantitative and qualitative analysis of the NK and γδ T cell repertoire in healthy individuals, recipients of HLA-matched sibling or unrelated donor allo-HSCT (MSD/MUD-HSCT) and umbilical cord blood-HSCT (UCB-HSCT). NK cells are present at high frequencies in all allo-HSCT recipients. Immune reconstitution (IR) of vδ2+ cells depended on stem cell source. In MSD/MUD-HSCT recipients, vδ2+ comprise up to 8% of the total lymphocyte pool, whereas vδ2+ T cells are barely detectable in UCB-HSCT recipients. Vδ1+ IR was driven by CMV reactivation and was comparable between MSD/MUD-HSCT and UCB-HSCT. Strategies to augment NK cell mediated tumor responses, similar to IL-15 and antibodies, also induced vδ2+ T cell responses against a variety of different tumor targets. Vδ1+ γδ T cells were induced less by these same stimuli. We also identified elevated expression of the checkpoint inhibitory molecule TIGIT (T cell Ig and ITIM domain), which is also observed on tumor-infiltrating lymphocytes and epidermal γδ T cells. Collectively, these data show multiple strategies that can result in a synergized NK and γδ T cell antitumor response. In the light of recent developments of low-toxicity allo-HSCT platforms, these interventions may contribute to the prevention of early relapse.

NK cells pave the road for alloengraftment.

In this issue of Blood, Alvarez et al describe a novel inhibitory receptor-mediated role for unlicensed natural killer (U-NK) cells in allogeneic graft facilitation.

The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT).

Natural killer (NK) cells were first identified for their capacity to reject bone marrow allografts in lethally irradiated mice without prior sensitization. Subsequently, human NK cells were detected and defined by their non-major histocompatibility complex (MHC)-restricted cytotoxicity toward transformed or virally infected target cells. Karre et al. later proposed 'the missing self hypothesis' to explain the mechanism by which self-tolerant cells could kill targets that had lost self MHC class I. Subsequently, the receptors that recognize MHC class I to mediate tolerance in the host were identified on NK cells. These class I-recognizing receptors contribute to the acquisition of function by a dynamic process known as NK cell education or licensing. In the past, NK cells were assumed to be short lived, but more recently NK cells have been shown to mediate immunologic memory to secondary exposures to cytomegalovirus infection. Because of their ability to lyse tumors with aberrant MHC class I expression and to produce cytokines and chemokines upon activation, NK cells may be primed by many stimuli, including viruses and inflammation, to contribute to a graft-versus-tumor effect. In addition, interactions with other immune cells support the therapeutic potential of NK cells to eradicate tumor and to enhance outcomes after hematopoietic cell transplantation.

Enhanced ADCC and NK Cell Activation of an Anticarcinoma Bispecific Antibody by Genetic Insertion of a Modified IL-15 Cross-linker.

Previously, we constructed a bispecific NK-cell-engager (BiKE) bearing single-chain variable fragments (scFv) against CD16 on NK cells and EpCAM on tumor cells. This BiKE facilitated antigen-specific antibody-dependent cell-mediated cytotoxicity (ADCC) but did not induce NK cell expansion. We incorporated a modified interleukin-15 cross-linker to create a trispecific construct (TriKE) in order to improve activation, proliferation, and survival of NK cells. Synthesis and assembly of hybrid genes encoding the TriKE was accomplished using DNA-shuffling and DNA-ligation techniques. The TriKE was tested for specificity, efficacy, proliferative capability, and cytokine profile using functional assays. The molecular modifications improved yield without compromising binding to EpCAM(+) HT-29 colorectal carcinoma cells. (51)Chromium-release and degranulation assays showed better killing rates with TriKE compared to BiKE. TriKE was more active in a variety of different carcinoma cell lines. TriKE showed the ability to stimulate expansion of CD56(+)CD3(-) NK cells. BiKE and TriKE showed enhanced but not supraphysiologic levels of cytokine secretion. 1615EpCAM TriKE drives enhanced ADCC while significantly improving proliferation, activation, and survival of NK cell effectors. The TriKE provides a selectively delivered self-sustaining signal at the NK/tumor cell synapse. Targeted cytokine stimulation, rather than systemic cytokine administration, may impact toxicity in patients rendering the TriKE a promising new off-the-shelf carcinoma therapy.

Functional NK cell repertoires are maintained through IL-2Rα and Fas ligand.

Acquisition of a functional NK cell repertoire, known as education or licensing, is a complex process mediated through inhibitory receptors that recognize self. We found that NK cells containing self-killer Ig-like receptors for cognate HLA ligand in vivo were less susceptible to apoptosis. In vitro IL-15 withdrawal showed that uneducated NK cells upregulated Bim and Fas. Conversely, educated NK cells upregulated Fas ligand (FasL) under these conditions. Induction of cell death and Bim expression on uneducated cells correlated with increased IL-2Rα expression. Overexpression and knockdown studies showed that higher IL-2Rα limits NK cell survival in a novel manner that is independent from the role of IL-2 in activation-induced cell death. To study the role of FasL in induction of IL-2Rα(hi) NK cell death, a coculture assay with FasL-blocking Abs was used. IL-15 withdrawal led to FasL-dependent killing of IL-2Rα(hi) NK cells by more educated IL-2Rα(lo) NK cells. Finally, CMV reactivation induces a potent long-lasting population of licensed NK cells with enhanced survival. These findings show that education-dependent NK cell survival advantages and killing of uneducated NK cells result in the maintenance of a functional repertoire, which may be manipulated to exploit NK cells for cancer immunotherapy.

Notch signaling at later stages of NK cell development enhances KIR expression and functional maturation.

The Notch signaling pathway plays a substantial role in human NK cell development. However, the role of Notch on killer Ig-like receptor (KIR) upregulation and acquisition of effector function has not been explored. To evaluate how Notch influences terminal differentiation, cord blood-derived NK cells or sorted peripheral blood NK cells were cultured with IL-15 for 7 d with inhibitory or activating Notch signals. Inhibition of Notch signaling significantly decreased KIR expression, whereas activation enhanced it. Overexpression of activated Notch on cord blood-derived NK cells resulted in a 2-fold increase in KIR expression, indicating that Notch signaling plays a direct, cell-intrinsic role in KIR regulation. Moreover, Notch-mediated KIR expression on NK cells is regulated through cis inhibition by delta-like ligand 1. Notch signaling also enhances CD16 upregulation that precedes KIR expression. Concomitant with the upregulation of KIR and CD16, Notch signaling induces increased cytolytic effector capacity and cytokine secretion, even in posttransplant samples in which NK cell function is inherently defective. Given these attributes of Notch signaling, we propose that Notch agonists may enhance NK cell maturation and tumor killing in a posttransplant setting.

Adaptive Natural Killer Cell and Killer Cell Immunoglobulin-Like Receptor-Expressing T Cell Responses are Induced by Cytomegalovirus and Are Associated with Protection against Cytomegalovirus Reactivation after Allogeneic Donor Hematopoietic Cell Transplantation.

Cytomegalovirus (CMV) reactivates in >30% of CMV-seropositive patients after allogeneic hematopoietic cell transplantation (HCT). Previously, we reported an increase of natural killer (NK) cells expressing NKG2C, CD57, and inhibitory killer cell immunoglobulin-like receptors (KIRs) in response to CMV reactivation after HCT. These NK cells persist after the resolution of infection and display "adaptive" or memory properties. Despite these findings, the differential impact of persistent/inactive versus reactivated CMV on NK versus T cell maturation after HCT from different graft sources has not been defined. We compared the phenotype of NK and T cells from 292 recipients of allogeneic sibling (n = 118) or umbilical cord blood (UCB; n = 174) grafts based on recipient pretransplantation CMV serostatus and post-HCT CMV reactivation. This cohort was utilized to evaluate CMV-dependent increases in KIR-expressing NK cells exhibiting an adaptive phenotype (NKG2C(+)CD57(+)). Compared with CMV-seronegative recipients, those who reactivated CMV had the highest adaptive cell frequencies, whereas intermediate frequencies were observed in CMV-seropositive recipients harboring persistent/nonreplicating CMV. The same effect was observed in T cells and CD56(+) T cells. These adaptive lymphocyte subsets were increased in CMV-seropositive recipients of sibling but not UCB grafts and were correlated with lower rates of CMV reactivation (sibling 33% versus UCB 51%; P < .01). These data suggest that persistent/nonreplicating recipient CMV induces rapid production of adaptive NK and T cells from mature cells from sibling but not UCB grafts. These adaptive lymphocytes are associated with protection from CMV reactivation.

Tim-3 is an inducible human natural killer cell receptor that enhances interferon gamma production in response to galectin-9.

NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-γ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3(+) population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-γ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-γ production, and Tim-3 cross-linking induced ERK activation and degradation of IκBα. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-γ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-γ production, which has important implications for control of infectious disease and cancer.

Enhanced IL-15-mediated NK cell activation and proliferation by an ADAM17 function-blocking antibody involves CD16A, CD137, and accessory cells.

Background: NK cells are being extensively studied as a cell therapy for cancer. Their effector functions are induced by the recognition of ligands on tumor cells and by various cytokines. IL-15 is broadly used to stimulate endogenous and adoptively transferred NK cells in cancer patients. These stimuli activate the membrane protease ADAM17, which then cleaves assorted receptors on the surface of NK cells as a negative feedback loop to limit their activation and function. We have shown that ADAM17 inhibition can enhance IL-15-mediated NK cell proliferation in vitro and in vivo . In this study, we investigated the underlying mechanism of this process. Methods: PBMCs or enriched NK cells from human peripheral blood, either unlabeled or labeled with a cell proliferation dye, were cultured for up to 7 days in the presence of rhIL-15 +/- an ADAM17 function-blocking antibody. Different versions of the antibody were generated; Medi-1 (IgG1), Medi-4 (IgG4), Medi-PGLALA, Medi-F(ab') 2 , and TAB16 (anti-ADAM17 and anti-CD16 bispecific) to modulate CD16A engagement on NK cells. Flow cytometry was used to assess NK cell proliferation and phenotypic markers, immunoblotting to examine CD16A signaling, and IncuCyte-based live cell imaging to measure NK cell anti-tumor activity. Results: The ADAM17 function-blocking mAb Medi-1 markedly increased initial NK cell activation by IL-15. Using different engineered versions of the antibody revealed that the activating Fcγ receptor CD16A, a well-described ADAM17 substrate, was critical for enhancing IL-15 stimulation. Hence, Medi-1 bound to ADAM17 on NK cells can be engaged by CD16A and block its shedding, inducing and prolonging its signaling. This process did not promote evident NK cell fratricide, phagocytosis, or dysfunction. Synergistic activity by Medi-1 and IL-15 enhanced the upregulation of CD137 on CD16A + NK cells and augmented their proliferation in the presence of PBMC accessory cells. Conclusions: Our data reveal for the first time that CD16A and CD137 underpin Medi-1 enhancement of IL-15-driven NK cell activation and proliferation, respectively. The use of Medi-1 represents a novel strategy to enhance IL-15-driven NK cell proliferation, and it may be of therapeutic importance by increasing the anti-tumor activity of NK cells in cancer patients. What is already known on this topic: NK cell therapies are being broadly investigated to treat cancer. NK cell stimulation by IL-15 prolongs their survival in cancer patients. Various stimuli including IL-15 activate ADAM17 in NK cells, a membrane protease that regulates the cell surface density of various receptors as a negative feedback mechanism. What this study adds: Treating NK cells with the ADAM17 function-blocking mAb Medi-1 markedly enhanced their activation and proliferation. Our study reveals that the Fc and Fab regions of Medi-1 function synergistically with IL-15 in NK cell activation. Medi-1 treatment augments the upregulation of CD137 by NK cells, which enhances their proliferation in the presence of PBMC accessory cells. How this study might affect research practice or policy: Our study is of translational importance as Medi-1 treatment in combination with IL-15 could potentially augment the proliferation and function of endogenous or adoptively transferred NK cells in cancer patients.

Natural Killer Cell Infiltration in Prostate Cancers Predict Improved Patient Outcomes.

BACKGROUND: Natural killer (NK) cells are non-antigen specific innate immune cells that can be redirected to targets of interest using multiple strategies, although none are currently FDA-approved. We sought to evaluate NK cell infiltration into tumors to develop an improved understanding of which histologies may be most amenable to NK cell-based therapies currently in the developmental pipeline. METHODS: DNA (targeted/whole-exome) and RNA (whole-transcriptome) sequencing was performed from tumors from 45 cancer types (N = 90,916 for all cancers and N = 3365 for prostate cancer) submitted to Caris Life Sciences. NK cell fractions and immune deconvolution were inferred from RNA-seq data using quanTIseq. Real-world overall survival (OS) and treatment status was determined and Kaplan-Meier estimates were calculated. Statistical significance was determined using X2 and Mann-Whitney U tests, with corrections for multiple comparisons where appropriate. RESULTS: In both a pan-tumor and prostate cancer (PCa) -specific setting, we demonstrated that NK cells represent a substantial proportion of the total cellular infiltrate (median range 2-9% for all tumors). Higher NK cell infiltration was associated with improved OS in 28 of 45 cancer types, including (PCa). NK cell infiltration was negatively correlated with common driver mutations and androgen receptor variants (AR-V7) in primary prostate biopsies, while positively correlated with negative immune regulators. Higher levels of NK cell infiltration were associated with patterns consistent with a compensatory anti-inflammatory response. CONCLUSIONS: Using the largest available dataset to date, we demonstrated that NK cells infiltrate a broad range of tumors, including both primary and metastatic PCa. NK cell infiltration is associated with improved PCa patient outcomes. This study demonstrates that NK cells are capable of trafficking to both primary and metastatic PCa and are a viable option for immunotherapy approaches moving forward. Future development of strategies to enhance tumor-infiltrating NK cell-mediated cytolytic activity and activation while limiting inhibitory pathways will be key.