Cell-derived Natural Killer Cells Research Articles

SynNotch-programmed iPSC-derived NK cells usurp TIGIT and CD73 activities for glioblastoma therapy

Severe heterogeneity within glioblastoma has spurred the notion that disrupting the interplay between multiple elements on immunosuppression is at the core of meaningful anti-tumor responses. T cell immunoreceptor with Ig and ITIM domains (TIGIT) and its glioblastoma-associated antigen, CD155, form a highly immunosuppressive axis in glioblastoma and other solid tumors, yet targeting of TIGIT, a functionally heterogeneous receptor on tumor-infiltrating immune cells, has largely been ineffective as monotherapy, suggesting that disruption of its inhibitory network might be necessary for measurable responses. It is within this context that we show that the usurpation of the TIGIT − CD155 axis via engineered synNotch-mediated activation of induced pluripotent stem cell-derived natural killer (NK) cells promotes transcription factor-mediated activation of a downstream signaling cascade that results in the controlled, localized blockade of CD73 to disrupt purinergic activity otherwise resulting in the production and accumulation of immunosuppressive extracellular adenosine. Such “decoy” receptor engages CD155 binding to TIGIT, but tilts inhibitory TIGIT/CD155 interactions toward activation via downstream synNotch signaling. Usurping activities of TIGIT and CD73 promotes the function of adoptively transferred NK cells into intracranial patient-derived models of glioblastoma and enhances their natural cytolytic functions against this tumor to result in complete tumor eradication. In addition, targeting both receptors, in turn, reprograms the glioblastoma microenvironment via the recruitment of T cells and the downregulation of M2 macrophages. This study demonstrates that TIGIT/CD155 and CD73 are targetable receptor partners in glioblastoma. Our data show that synNotch-engineered pluripotent stem cell-derived NK cells are not only effective mediators of anti-glioblastoma responses within the setting of CD73 and TIGIT/CD155 co-targeting, but represent a powerful allogeneic treatment option for this tumor.

Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor β-chain (ΔIL-2Rβ) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.

Early TRAIL-engagement elicits potent multimodal targeting of melanoma by CD34+ progenitor cell-derived NK cells

Umbilical cord blood (UCB) CD34+ progenitor cell-derived natural killer (NK) cells exert efficient cytotoxicity against various melanoma cell lines. Of interest, the relative cytotoxic performance of individual UCB donors was consistent throughout the melanoma panel and correlated with IFNγ, TNF, perforin and granzyme B levels. Importantly, intrinsic perforin and Granzyme B load predicts NK cell cytotoxic capacity. Exploring the mode of action revealed involvement of the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46 and most importantly of TRAIL. Strikingly, combinatorial receptor blocking led to more pronounced inhibition of cytotoxicity (up to 95%) than individual receptor blocking, especially in combination with TRAIL-blocking, suggesting synergistic cytotoxic NK cell activity via engagement of multiple receptors which was also confirmed in a spheroid model. Importantly, lack of NK cell-related gene signature in metastatic melanomas correlates with poor survival highlighting the clinical significance of NK cell therapies as a promising treatment for high-risk melanoma patients.

Advanced T and Natural Killer Cell Therapy for Glioblastoma.

Although immunotherapy has been broadly successful in the treatment of hematologic malignancies and a subset of solid tumors, its clinical outcomes for glioblastoma are still inadequate. The results could be due to neuroanatomical structures such as the bloodbrain- barrier, antigenic heterogeneity, and the highly immunosuppressive microenvironment of glioblastomas. The antitumor efficacy of endogenously activated effector cells induced by peptide or dendritic cell vaccines in particular has been insufficient to control tumors. Effector cells, such as T cells and natural killer (NK) cells can be expanded rapidly ex vivo and transferred to patients. The identification of neoantigens derived from tumor-specific mutations is expanding the list of tumor-specific antigens for glioblastoma. Moreover, recent advances in gene-editing technologies enable the effector cells to not only have multiple biological functionalities, such as cytokine production, multiple antigen recognition, and increased cell trafficking, but also relieve the immunosuppressive nature of the glioblastoma microenvironment by blocking immune inhibitory molecules, which together improve their cytotoxicity, persistence, and safety. Allogeneic chimeric antigen receptor (CAR) T cells edited to reduce graft-versushost disease and allorejection, or induced pluripotent stem cell-derived NK cells expressing CARs that use NK-specific signaling domain can be a good candidate for off-the-shelf products of glioblastoma immunotherapy. We here discuss current progress and future directions for T cell and NK cell therapy in glioblastoma.

Adoptive immunotherapy with natural killer cells from peripheral blood CD34+ stem cells to prevent hepatocellular carcinoma recurrence after curative hepatectomy: a study protocol for an open-label, single-arm phase I study

IntroductionHepatocellular carcinoma (HCC) remains a major clinical problem as more than half of these cases recur after radical resection. Natural killer (NK) cells are at the forefront of the innate...

Human placental hematopoietic stem cell-derived natural killer cells (CYNK) recognize and eliminate influenza A virus-infected cells.

Influenza A virus (IAV) infections are a significant recurrent threat to public health and a significant burden on global economy, highlighting the need for developing more effective therapies. Natural killer (NK) cells play a pivotal role in the control of pulmonary IAV infection, however, little is known about the therapeutic potential of adoptively transferred NK cells for viral infections. Here, we investigated the antiviral activity of CYNK, human placental hematopoietic stem cell-derived NK cells, against IAV infection in vitro. Virus infection induced the expression of NK cell activating ligands on respiratory epithelial cells, resulting in enhanced recognition by CYNK cells. Upon co-culture with IAV-infected epithelial cells, CYNK exhibited elevated degranulation and increased production of IFN-γ, TNF-α and GM-CSF in a virus dose-dependent manner. Furthermore, CYNK showed virus dose-dependent cytotoxicity against IAV-infected cells. The antiviral activity of CYNK was mediated by NKp46 and NKG2D. Together, these data demonstrate that CYNK possesses potent antiviral function against IAV and warrant clinical investigations for adoptive NK cell therapies against viral infections.

Abstract 559: Targeting hematological malignancies and solid tumors with switchable chimeric antigen receptor-engineered iPSC-derived natural killer cells

Abstract Switchable chimeric antigen receptors (sCAR) provide an important new strategy to precisely regulate CAR-mediated anti-tumor activity. The sCAR system combines a CAR that recognizes peptide neoepitope (PNE) with an injectable “switch” molecule that consists of an anti-tumor Fab linked to PNE. Configurating PNE at different location or chain of the Fab enables us to identify best switches optimal for tumor targeting. Previous studies demonstrate that this sCAR system expressed in T cells provides tight control over anti-tumor activity. Here, we translated this approach to engineer natural killer (NK) cells with the sCAR to provide a universal, targeted cell-therapy approach in a patient-nonspecific manner. First, we engineered human induced pluripotent stem cells (iPSCs) with the sCAR combining the PNE-specific CAR scFv with our previously described NK cell optimized CAR4 signaling motifs consisting of the NKG2D transmembrane domain, 2B4 co-stimulatory domain and the CD3ζ chain. We selected clones that maintained highest level of pluripotency and most stable expression of the sCAR4 on the surface. Next, we generated mature sCAR4-expressing iPSC-derived NK cells that expressed common surface receptors similar to that of donor peripheral blood mononuclear cell-derived NK cells. We then used three panels of switches with specificity to CD19 (consisting of 9 different configurations), to CD33, CD123 and CLL1 (1 configuration each), and to FRIZZLED7 (FZD7; 6 configurations) to target CD19+ B cell lymphoma, acute myeloid leukemia (AML), and ovarian cancer, respectively. All three targets demonstrated switch-specific dose response in killing tumor cell lines. Different configurations conferred variable efficiencies in switch-mediating tumor cell killings and the optimal switch concentrations were found to be different from what was observed previously with sCAR-expressing T cells. In a comparison with the “conventional” (non-switchable) CARs (cCARs), iPSC-NK cells expressing either sCAR4 and treated with an anti-CD19 switch or iPSC-NK cells with an anti-CD19-cCAR4 demonstrated similar level of cytotoxicity against B cell leukemia cells. Finally, we have built a mouse model and are currently testing the iPSC-NK-sCAR system in vivo. Together, this iPSC-NK-sCAR strategy enables close control over CAR-mediated activity with flexibility to target multiple tumor types and a potential to become a novel off-the-shelf therapy. Citation Format: Xiao-Hua Li, Benjamin Goldenson, Jaya Lakshmi Thangaraj, Matthew Gynn, Diana Gumber, Myan Do, Karl Willert, Dan S. Kaufman. Targeting hematological malignancies and solid tumors with switchable chimeric antigen receptor-engineered iPSC-derived natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 559.

IPSC-Derived Natural Killer Cell Therapies - Expansion and Targeting.

Treatment of cancer with allogeneic natural killer (NK) cell therapies has seen rapid development, especially use against hematologic malignancies. Clinical trials of NK cell-based adoptive transfer to treat relapsed or refractory malignancies have used peripheral blood, umbilical cord blood and pluripotent stem cell-derived NK cells, with each approach undergoing continued clinical development. Improving the potency of these therapies relies on genetic modifications to improve tumor targeting and to enhance expansion and persistence of the NK cells. Induced pluripotent stem cell (iPSC)-derived NK cells allow for routine targeted introduction of genetic modifications and expansion of the resulting NK cells derived from a clonal starting cell population. In this review, we discuss and summarize recent important advances in the development of new iPSC-derived NK cell therapies, with a focus on improved targeting of cancer. We then discuss improvements in methods to expand iPSC-derived NK cells and how persistence of iPSC-NK cells can be enhanced. Finally, we describe how these advances may combine in future NK cell-based therapy products for the treatment of both hematologic malignancies and solid tumors.

Making a Killer: Selecting the Optimal Natural Killer Cells for Improved Immunotherapies.

Over the past 20 years natural killer (NK) cell-based immunotherapies have emerged as a safe and effective treatment option for patients with relapsed or refractory leukemia. Unlike T cell-based therapies, NK cells harbor an innate capacity to eliminate malignant cells without prior sensitization and can be adoptively transferred between individuals without the need for extensive HLA matching. A wide variety of therapeutic NK cell sources are currently being investigated clinically, including allogeneic donor-derived NK cells, stem cell-derived NK cells and NK cell lines. However, it is becoming increasingly clear that not all NK cells are endowed with the same antitumor potential. Despite advances in techniques to enhance NK cell cytotoxicity and persistence, the initial identification and utilization of highly functional NK cells remains essential to ensure the future success of adoptive NK cell therapies. Indeed, little consideration has been given to the identification and selection of donors who harbor NK cells with potent antitumor activity. In this regard, there is currently no standard donor selection criteria for adoptive NK cell therapy. Here, we review our current understanding of the factors which govern NK cell functional fate, and propose a paradigm shift away from traditional phenotypic characterization of NK cell subsets towards a functional profile based on molecular and metabolic characteristics. We also discuss previous selection models for NK cell-based immunotherapies and highlight important considerations for the selection of optimal NK cell donors for future adoptive cell therapies.

Genetically Engineered Pluripotent Cell-Derived Natural Killer Cell Therapy Provides Enhanced Antibody Dependent Cellular Cytotoxicity Against Hematologic Malignancies and Solid Tumors in Combination with Monoclonal Antibody Therapy

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism mediated by natural killer (NK) cells that has been shown to significantly contribute to the anti-tumor effect of therapeutic monoclonal antibodies (mAbs) including rituximab (anti-CD20), cetuximab (anti-EGFR) and trastuzumab (anti-ErbB2). ADCC is induced by the crosslinking of the FcγRIIIA (CD16a) receptor expressed on NK cells with cell surface-bound IgG antibodies. Upon NK cell activation, CD16a is proteolytically cleaved from the surface of NK cells, a process that decreases ADCC. There exist two allelic variants of CD16a, with the high-affinity CD16a 158V allele found in about 10% of the population. Additionally, we previously identified a single substitution mutation that renders CD16a resistant to activation-induced cleavage. Engineering of human induced pluripotent stem cells (hiPSCs) with a non-cleavable version of the high-affinity CD16a 158V variant, followed by differentiation of hiPSCs to NK cells, enables us to develop an off-the-shelf NK cell therapy with improved ADCC properties when combined with monoclonal antibody therapy for treatment of both hematologic malignancies and solid tumors. We evaluated cell surface phenotype, as well as the in vitro and in vivo tumor-killing function, of these high affinity, non-cleavable CD16a iNK (hnCD16-iNK) cells alone and in combination with anti-CD20 mAb against multiple B-cell leukemia and lymphoma targets. In contrast to the endogenous expression of CD16a on peripheral blood NK (PB-NK) cells, which is variable depending on the donor, the derivation of NK cells from a clonal hiPSC line engineered to express hnCD16a produced a uniform population of CD16+ NK cells (>95% CD16+ for hnCD16-iNK vs. 40-60% CD16+ PB-NK cells). These hnCD16-iNK cells are phenotypically mature, highly functional in vitro, and exhibit enhanced ADCC when combined with anti-CD20 mAb against multiple lymphoma targets, including Raji, Nalm6 and Jeko-1. Both direct and CD16-mediated activation of hnCD16-iNK cells result in production of the inflammatory cytokines tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). Furthermore, in contrast to PB-NK cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16 in response to Raji cells coated with anti-CD20 (68±26% cleavage for PB-NK vs. 8.0±4.2% cleavage for hnCD16-iNK cells). To extend these findings to an in vivo model, we treated Raji-lymphoma bearing mice with either anti-CD20 mAb (rituximab) alone or in combination with either PB-NK cells or hnCD16-iNK cells. Combination treatment with hnCD16-iNK cells enhanced the efficacy of anti-CD20 compared to mAb treatment only, and provided improved tumor regression compared to mAb plus PB-NK (p<0.001; tumor burden measured six days after NK cell injection). Additionally, mice that received combination treatment with hnCD16-iNK cells had significantly better survival than mice treated with mAb alone (median survival rate of 39 days for rituximab treatment only group vs. >50 days for combination treatment with hnCD16-iNK). Similar results were seen with the combination of anti-HER2 antibody and hnCD16-iNK cells in an SKOV3 ovarian cancer mouse model. In summary, these data demonstrate that combination therapy hnCD16-iNK cells with monoclonal antibody therapy is highly functional against various tumor cell lines in vitro and in vivo, and justify further investigation into their clinical application to enhance ADCC and the anti-tumor effects of therapeutic mAbs. DisclosuresBjordahl:Fate Therapeutics: Employment, Equity Ownership. Gaidarova:Fate Therapeutics Inc.: Employment, Equity Ownership. Rogers:Fate Therapeutics Inc.: Employment, Equity Ownership. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics: Employment, Equity Ownership.

Optimizing NK Cell-Based Immunotherapy in Myeloid Leukemia: Abrogating an Immunosuppressive Microenvironment.

Natural killer (NK) cells are prominent cytotoxic and cytokine-producing components of the innate immune system representing crucial effector cells in cancer immunotherapy. Presently, various NK cell-based immunotherapies have contributed to the substantial improvement in the reconstitution of NK cells against advanced-staged and high-risk AML. Various NK cell sources, including haploidentical NK cells, adaptive NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, chimeric antigen receptor NK cells, cytokine-induced memory-like NK cells, and NK cell lines have been identified. Devising innovative approaches to improve the generation of therapeutic NK cells from the aforementioned sources is likely to enhance NK cell expansion and activation, stimulate ex vivo and in vivo persistence of NK cells and improve conventional treatment response of myeloid leukemia. The tumor-promoting properties of the tumor microenvironment and downmodulation of NK cellular metabolic activity in solid tumors and hematological malignancies constitute a significant impediment in enhancing the anti-tumor effects of NK cells. In this review, we discuss the current NK cell sources, highlight ongoing interventions in enhancing NK cell function, and outline novel strategies to circumvent immunosuppressive factors in the tumor microenvironment to improve the efficacy of NK cell-based immunotherapy and expand their future success in treating myeloid leukemia.

CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy

BackgroundTumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and...

Exploring the NK cell platform for cancer immunotherapy.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that are capable of killing virally infected and/or cancerous cells. Nearly 20 years ago, NK cell-mediated immunotherapy emerged as a safe and effective treatment approach for patients with advanced-stage leukaemia. Subsequently, the field of NK cell-based cancer therapy has grown exponentially and currently constitutes a major area of immunotherapy innovation. In general, the development of NK cell-directed therapies has two main focal points: optimizing the source of therapeutic NK cells for adoptive transfer and enhancing NK cell cytotoxicity and persistence in vivo. A wide variety of sources of therapeutic NK cells are currently being tested clinically, including haploidentical NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, cytokine-induced memory-like NK cells and chimeric antigen receptor NK cells. A plethora of methods to augment the cytotoxicity and longevity of NK cells are also under clinical investigation, including cytokine-based agents, NK cell-engager molecules and immune-checkpoint inhibitors. In this Review, we highlight the variety of ways in which diverse NK cell products and their auxiliary therapeutics are being leveraged to target human cancers. We also identify future avenues for NK cell therapy research.

Induced pluripotent stem cell-derived natural killer cells gene-modified to express chimeric antigen receptor-targeting solid tumors.

The use of allogeneic, pluripotent stem cell-derived immune cells for cancer immunotherapy has been the subject of recent research, including clinical trials. The use of pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, regarding efficacy, safety, and producibility. In this review, we have described the characteristics of natural killer (NK) cells from multiple cell sources, including pluripotent stem cells, the chimeric antigen receptor (CAR)-modification method and strategy for these NK cells, and the current and planned clinical trials of CAR-modified induced pluripotent stem cell-derived NK cells.

Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes InVivo Persistence and Enhances Anti-tumor Activity.

Cytokine-inducible SH2-containing protein (CIS; encoded by the gene CISH) is a key negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. Here, we develop human CISH-knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-NK cells demonstrate increased IL-15-mediated JAK-STAT signaling activity. Consequently, CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations. CISH-/- iPSC-NK cells display significantly increased invivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by increased basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration, and spare respiration capacity mediated by mammalian target of rapamycin (mTOR) signaling that directly contributes to enhanced NK cell function. Together, these studies demonstrate that CIS plays a key role to regulate human NK cell metabolic activity and thereby modulate anti-tumor activity.

Metabolic Reprograming Due to Deletion of &lt;i&gt;CISH&lt;/i&gt; in Human Natural Killer Cells Promotes &lt;i&gt;in vivo&lt;/i&gt; Persistence and Enhances Anti-Tumor Activity

Cytokine-inducible SH2-containing protein (CIS, encoded by the gene CISH) is a key negative regulator of IL-15 signaling in natural killer (NK) cells. Here, we developed human CISH knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-derived NK cells demonstrate increased IL-15 mediated JAK-STAT pathway signaling activity. Consequently CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentration. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by a significant increase in basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration and spare capacity respiration which are mediated by mTOR signaling and directly contribute to enhanced NK cell function. In addition, we demonstrate that deletion of CISH in human NK cells increases single-cell polyfunctionality by secreting multiple effector cytokines, a profile has been proven to be positively associated with clinical outcome of CAR-T cells. Together, these studies demonstrate CIS plays a key role to regulate human NK cell metabolic activity and deletion of CIS leads to enhanced anti-tumor activity.

PLGA nanodepots co-encapsulating prostratin and anti-CD25 enhance primary natural killer cell antiviral and antitumor function.

Natural killer (NK) cells are attractive effector cells of the innate immune system against human immunodeficiency virus (HIV) and cancer. However, NK cell therapies are limited by the fact that target cells evade NK cells, for example, in latent reservoirs (in HIV) or through upregulation of inhibitory signals (in cancer). To address this limitation, we describe a biodegradable nanoparticle-based "priming" approach to enhance the cytotoxic efficacy of peripheral blood mononuclear cell-derived NK cells. We present poly(lactic-co-glycolic acid) (PLGA) nanodepots (NDs) that co-encapsulate prostratin, a latency-reversing agent, and anti-CD25 (aCD25), a cell surface binding antibody, to enhance primary NK cell function against HIV and cancer. We utilize a nanoemulsion synthesis scheme to encapsulate both prostratin and aCD25 within the PLGA NDs (termed Pro-aCD25-NDs). Physicochemical characterization studies of the NDs demonstrated that our synthesis scheme resulted in stable and monodisperse Pro-aCD25-NDs. The NDs successfully released both active prostratin and anti-CD25, and with controllable release kinetics. When Pro-aCD25-NDs were administered in an in vitro model of latent HIV and acute T cell leukemia using J-Lat 10.6 cells, the NDs were observed to prime J-Lat cells resulting in significantly increased NK cell-mediated cytotoxicity compared to free prostratin plus anti-CD25, and other controls. These findings demonstrate the feasibility of using our Pro-aCD25-NDs to prime target cells for enhancing the cytotoxicity of NK cells as antiviral or antitumor agents.

Deletion of CISH in Human Pluripotent Stem Cell-Derived Natural Killer Cells Enhances Anti-Tumor Activity Via Metabolic Reprogramming

Cellular metabolism is now recognized to play a key role in regulating function and differentiation of immune cells. Immune cells undergo dynamic metabolic shifts to support their activity. Recent studies have shown the importance of metabolism to regulate NK cell function under different conditions. IL-15 can regulate NK cell metabolism in a dose- and time- dependent manner. However, whether improving metabolic fitness of NK cells can lead to improved anti-tumor activity is largely unknown. Here, we deleted CIS (encoded by the CISH gene in human induced pluripotent stem cells (iPSC)-derived NK cells (CISH-/- iPSC-NK) using CRISPR/Cas9 technology. CIS is known to function as a critical negative regulator of IL-15 signaling and a potent inhibitory checkpoint in NK cell-mediated anti-tumor activity. Surprisingly, more than 3000 differentially expressed genes were identified in CISH-/- iPSC-NK cells through RNA sequencing analysis. Gene ontology (GO) enrichment analysis showed that the top 2 up-regulated signaling pathways were associated with leukocyte differentiation and activation. Notably, more than 50 genes that involved in positive regulation of lymphocyte activation including genes associated with NK cell effector functions were significantly up-regulated in CISH-/- iPSC-NK cells compared with unmodified iPSC-NK cells. As expected, CISH-/- iPSC-NK cells exhibited increased sensitivity to IL-15 stimulation. Specifically, IL-15 stimulated JAK1 tyrosine phosphorylation, STAT3 and STAT5 phosphorylation were all dramatically increased in CISH-/- iPSC-NK cells, compared with unmodified iPSC-NK cells. In addition, when incubated with low concentrations of IL-15 (5 ng/ml), a concentration that does not support the growth and functions of unmodified iPSC-NK cells, CISH-/- iPSC-NK cells maintained significantly better expansion with &amp;gt;10 fold expansion for CISH-/- iPSC-NK cells in 3 weeks, whereas most of WT iPSC-NK cells died under these conditions (p&amp;lt;0.001). These CISH-/- iPSC-NK cells exhibited improved cytotoxic against acute myeloid leukemia (AML) cell lines K562 and Molm-13. More importantly, in an AML (MOLM-13) xenograft model, CISH-/- iPSC-NK cells displayed significantly increased in vivo persistence thus lead to significantly better control of tumor progression. To further understand the underling mechanisms contributing to the increased functionality in CISH-/- iPSC-NK cells, we then analyzed the metabolic profiles of these cells using Agilent Seahorse assays. After treatment with low concentration IL-15 for 7 days, CISH-/- iPSC-NK cells displayed both increased glucose metabolism and increased mitochondrial respiration profiles when compared with unmodified iPSC-NK cells. Increased glucose metabolism include a significant increase in basal glycolysis (p=0.0003, 84.5 ± 1.0 vs 61.1 ± 1.8 mpH/min, n=6), and glycolytic capacity (p=0.0049, 15.2±0.4 vs 11.7±0.5 mpH/min, n=6). And increased mitochondrial respiration profile is characterized by an increase in the spare respiratory capacity (p=0.0009, 16.3±1.9 vs 6.9±0.5 pmol/min, n=6), maximal respiration (p&amp;lt;0.0001, 31.6±1.9 vs 15.7±0.8 pmol/min, n=6) and ATP-linked respiration (p&amp;lt;0.0001, 11.5 ± 0.4 vs 7.0±0.3 pmol/min, n=6). Interestingly, limiting the increase of glucose metabolism and mitochondrial respiration via utilization of an mTOR inhibitor (rapamycin) attenuated the increased functionality in CISH-/- iPSC-NK cells. The findings presented here show that deletion of CISH in iPSC-NK cells enhance anti-tumor activity, at least in part via metabolic reprogramming. These findings suggest that improving NK cell functions for cancer immunotherapy through metabolic reprogramming provides a promising therapeutic strategy. Disclosures Guan: OncoImmune: Equity Ownership. Kaufman:FATE Therapeutics: Consultancy, Research Funding.

Engineered human pluripotent stem cell-derived natural killer cells: the next frontier for cancer immunotherapy.

Adoptive immunotherapy using immune effector cells has revolutionized cancer treatments with approval of two autologous chimeric antigen receptor (CAR) T cell therapies by the US FDA. Clinical trials using natural killer (NK) cell-based adoptive immunotherapy have been shown to be safe and effective for treatment of multiple malignancies, especially acute myelogenous leukemia. However, most of these trails use primary NK cells isolated from peripheral or cord blood which can have donor-dependent variability and can be challenging to genetic engineer to improve antitumor functions, limiting the widespread use of this promising new therapy. NK cells can now be routinely produced from human pluripotent stem cells, both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). These pluripotent stem cells are homogenous, easy to genetically modify on a clonal level and can be used as unlimited source of NK cells, making them ideal population to develop standardized, off-the-shelf adoptive NK cell therapy products. In this review, we discuss recent advances of obtaining and expanding hESC and iPSC-derived NK cells and novel genetic engineering strategies that are being applied to improve their antitumor functions.

Induction of human pluripotent stem cell-derived natural killer cells for immunotherapy under chemically defined conditions

Natural killer (NK) cells are innate lymphocytes and show cytotoxicity against tumor cells without prior antigen specific stimulation. Because of their innate properties, NK cells are being considered for immunotherapies against various malignancies or leukemia. Human pluripotent stem cells (hPSCs) are capable of inducing enough NK cells for allogeneic transplantation. However, current induction protocols require feeder cells or human or bovine serum for the differentiation and expansion of NK cells, which incurs potential risk for contamination and may cause lot dependency in the cells. To address these issues, here we established a differentiation protocol for developing functional NK cells from hPSCs under a completely chemically-defined condition. The resultant PSC-derived NK cells show comparable phenotypes to those produced under serum-containing condition, exerting strong killing potential against a leukemia cell line in vitro and resistance to tumor growth in vivo. Our protocol can be a useful tool for applying PSC-derived NK cells to future cellular cancer immunotherapies.