Primary Human Natural Killer Cells Research Articles

A Novel Method for Highly Efficient Ex Vivo Expansion and Genetic Engineering of Human Natural Killer Cells for Cancer Therapy

Cancer immunotherapy with adoptive transfer of human leukocyte antigen-mismatched, CD19-targetd chimeric antigen receptor (CAR)-transduced natural killer (NK) cells has attracted attention because of its efficacy and safety when infused in patients with refractory and relapsed B-cell lymphomas. However, generating clinical doses of CAR-NK cells is still a challenge. The methods for ex vivo expansion and genetic modification of primary human NK cells usually rely on the use of irradiated feeder cell lines, which has been restrictive due to high costs, scale-up difficulties, and licensing restrictions. Hence, novel strategies that do not require feeder cells will be beneficial in standardizing these types of cell therapies. In this study, we show the effectiveness of a novel feeder-free culture system in expanding NK cells ex vivo and generating CAR-NK cells.Unsorted peripheral blood mononuclear cells (PBMCs) collected from healthy donors were cultured with a reagent containing dissolvable microspheres that are conjugated with anti-CD2/NKp46 antibodies (Cloudz TM Human NK Cell Expansion Kit) and a combination of multiple cytokines, including interleukin (IL)-2, IL-12, IL-18, and IL-21 in medium supplemented with 10% fetal bovine serum. The activated NK cells were transduced using the RD114-pseudotyped retrovirus vector.To test whether the reagent promoted ex vivo NK cell expansion, we cultured PBMCs from 13 donors. The percentage of NK (CD56 + CD3 -) cells of initially isolated PBMCs was 15.3±7.5%. In the presence of multiple cytokine combinations, NK cell purity gradually increased and reached 91.6±7.6% by day 21. The NK cells expanded to 75.6±59.2-fold at day 10, 334±217-fold at day 14 and 1,542±913-fold at day 21. The expanded NK cells degranulated and produced intracellular cytokines upon exposure to K562 myeloid leukemia cells. The NK cells efficiently killed myeloid leukemia cells, such as K562, THP1, and KG1. The expression pattern of killer cell immunoglobulin-like receptors on NK cells remained unchanged. The expression of activating NK cell receptors, including NKp30 and NKp44, increased after 21 days of culture.Thereafter, a gene transfer to the primary human NK cells was conducted. We tested transduction efficiency and yields of modified cells on 7 days after the procedure by empty-vector transduction into NK cells expanded ex vivo for 3, 6, and 10 days (n=3, each). The results were presented as means ± standard deviation; 55.6±11.6%, 61.6±14.1%, and 73.6%±6.2% for GFP positivity in NK cells and 5.7±1.5 folds, 56.4±42.2 folds, and 12.7±5.8 folds for yields of modified cells. We selected the condition in which the transduction was carried out using NK cells expanded ex vivo for 6 days, although the differences were not statistically significant. Next, anti-CD19 CAR with a 4-1BB costimulatory and CD3z domain was transduced into NK cells. We confirmed high transduction efficiency (59.8% ±20.5%, n=3) and high CAR protein expression on the cell surface, while NK cells maintained their purity and minimal T cell outgrowth was observed. CAR-NK cells maintained their proliferative status and further expanded 15.2±6.4-fold after 7 days of the procedure.To determine whether the generated anti-CD19 CAR-NK cells had a specific effect on B cell malignancies, a CD107a mobilization assay, intracellular cytokine assay, and a flow cytometry-based cytotoxicity assay was employed. We found that CAR transduction could render NK cells to generate specific and powerful responses against CD19-positive, NK-resistant leukemia and lymphoma cell lines, such as BCR-ABL-positive acute lymphoblastic leukemia (ALL) OP-1, Burkitt lymphoma Raji, and KMT2A-rearranged ALL RS4;11, at various effector: target (ET) ratios. For example, in a 4 hour -assay, the cytotoxic effects of anti-CD19 CAR-NK cells showed 86.9±0.2% cytotoxicity against OP-1, while mock NK cells showed 17.4±2.9% cytotoxicity (ET ratio 1:1).In conclusion, this study revealed highly efficient functions of the novel feeder-free culture system, including highly efficient ex vivo expansion of primary human NK cells and generation of genetically modified NK cells for cancer immunotherapy. In future studies, we will investigate large-scale cultures using specialized flasks and GMP-grade reagents for clinical translation and the in vivo activities of the cell products in mouse xenograft models. DisclosuresImai: Juno Therapeutics: Patents & Royalties: chimeric receptor with 4-1BB signaling domain.

PiggyBac system to co-express NKG2D CAR and IL-15 to augment the in vivo persistence and anti-AML activity of human peripheral blood NK cells

Promising progress has been made in adoptive transfer of allogeneic natural killer (NK) cells to treat relapsed or refractory acute myeloid leukemia (AML). In this regard, chimeric antigen receptor (CAR)-modification of NK cells is considered as a compelling approach to augment the specificity and cytotoxicity of NK cells against AML. Using a non-viral piggyBac transposon technology and human peripheral blood-derived primary NK cells, we generated CAR-NK cells to target NKG2D ligands and demonstrated their in vitro activity in lysing cancer cells expressing the ligands and in vivo efficacy in inhibiting tumor growth in a xenograft KG-1 AML model. We further generated CAR-NK cells co-expressing transgenes for the NKG2D CAR and interleukin-15 (IL-15). The ectopic expression of IL-15 improved the in vitro and in vivo persistence of NKG2D CAR-NK cells, leading to enhanced in vivo tumor control and significant prolongation of mouse survival in the KG-1 AML model. Collectively, our findings demonstrate the ectopic expression of IL-15 as an important means to improve the antileukemic activity of NKG2D CAR-NK cells. Our study further illustrates the feasibility of using the piggyBac non-viral platform as an efficient and cost-effective way for CAR-NK cell manufacturing.

Ex Vivo Expansion and CRISPR-Cas9 Genome Editing of Primary Human Natural Killer Cells.

Natural killer (NK) cells are potent innate immune cells that provide the surveillance and elimination of infected, stressed, and malignant cells. The unique immune recognition mechanisms and functions of NK cells make them an attractive cell type for immunology research and adoptive immunotherapy. However, primary NK cells are challenging to culture ex vivo and lack efficient genetic tools, hindering the research of NK cells and the development of NK cell therapeutics. Here we describe methods for the freeze-thaw process, feeder-free ex vivo expansion, CRISPR-Cas9 genome editing, and functional characterizations of primary human NK cells. Our protocol enables ∼30-fold and ∼2000-fold average expansion rates from 1 × 107 cryopreserved NK cells in 14 and 28 days, respectively. We also detail methods for CRISPR gene knockout and knockin by nucleofection of Cas9 ribonucleoproteins (RNP) and DNA repair templates. Gene knockout by Cas9 RNP nucleofection can be multiplexed to simultaneously target three genes. The CRISPR-edited cells can be cryopreserved and rethawed with high viability for future studies. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Thawing of natural killer cells Basic Protocol 2: Ex vivo expansion of natural killer cells Basic Protocol 3: Cryopreservation of expanded natural killer cells Basic Protocol 4: Characterization of natural killer cells: Flow cytometry and surface marker analysis Basic Protocol 5: Cytotoxicity and degranulation assays Basic Protocol 6: Preparation of homology-directed repair templates Basic Protocol 7: Nucleofection of CRISPR-Cas9 ribonucleoproteins Basic Protocol 8: Genotyping of gene-edited natural killer cells Basic Protocol 9: Phenotyping of gene-edited natural killer cells.

Non-clinical similarity of biosimilar ABP 798 with rituximab reference product

ABP 798 is a biosimilar to Rituxan® (rituximab reference product [RP]). Non-clinical assessments relevant to the primary and secondary mechanisms of action (MOA) contribute to the totality of the evidence (TOE) in supporting biosimilarity and are critical in providing scientific evidence for extrapolation of indications. Similarity of ABP 798 with rituximab RP was investigated across a range of biological activities which have potential impact on pharmacokinetics and clinical efficacy with non-clinical assessments relevant to MOA such as CD20 internalization, trogocytosis, binding to primary human natural killer (NK) cells as well as the ability to induce antibody-dependent cellular phagocytosis (ADCP) in peripheral blood mononuclear cells. Additionally, in vitro synergy of ABP 798 or RP with chemotherapeutic agents, in vivo xenograft studies in mice, and toxicological assessments in cynomolgus monkeys (including B cell depletion and toxicokinetics) were also conducted. Results from these non-clinical assessments contribute to the TOE supporting the biosimilarity between ABP 798 and rituximab RP across a range of primary and secondary MOAs and support justification for extrapolation to all indications of use for ABP 798 for which the RP is approved.

High-Efficiency Lentiviral Genetic Modification of Primary Human Natural Killer Cells

Natural killer (NK) cells have the unique capacity to lyse tumor cells without the need for antigen specific receptors. Modulation of NK cell cytokine, chemokine, and activating and inhibitory receptor expression using gene regimens represents an attractive strategy to further bolster NK cell anti-tumor activity for the purpose of NK cell immunotherapy. Current efforts in clinical-grade genetic modification of patient-derived cells, used in immunotherapy protocols, rely on stable transduction mediated by viral vectors. However, primary NK cells are notoriously difficult to transduce using viral vectors. Here we describe a lentiviral vector-based approach that results in an efficient, robust, and highly reproducible method of stable gene transfer into primary human peripheral blood-derived NK cells, which can subsequently be expanded ex vivo for adoptive infusion into humans. In vitro experiments established highly efficient transduction of ex vivo expanded NK cells could be achieved by activation of primary NK cells in vitro for 2-4 days in cytokine containing media followed by transduction of activated NK cells using LV vectors 2-3 days before integration into our existing ex vivo expansion protocol using irradiated EBV-LCL feeder cells. Unless NK cells were first activated in cytokine-containing media, LV transduction was unsuccessful. Cytokine activation in media containing IL-2 for 2-4 days was found to be sufficient to achieve high transduction efficiency, while additional supplementation of the media with IL-15 or IL-21 had negligible effects. NK cell transduction efficiency and viability with the use of RetroNectin® was uniformly superior to polycation-based transduction protocols. In experiments where NK cells were transduced with identical lentiviral constructs with 8 different promoter sequences driving expression of EGFP, PGK, EFS, and SV40 promoters consistently demonstrated the highest transduction efficiencies reproducibly in the range of 25-60%. Remarkably, when transduced NK cells were stimulated ex vivo using a clinical grade irradiated EBV-LCL feeder cell line, 100-1000 fold expansions of transduced NK cells could be achieved with sustained transgene expression over two weeks. Transduced and expanded primary NK cells remained highly cytotoxic against K562 tumor targets without developing functional deficiencies in degranulation, IFNγ, or TNFα production. These data establish a simple method to produce large numbers (>1011) of genetically modified clinical grade NK cells suitable for infusion. The protocol described herein overcomes previous difficulties described with common transduction techniques opening new avenues to investigate the impact of adoptive infusion of primary NK cells stably transduced to express a variety of different transgenes of interest. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clearance of Plasmodium falciparum-infected Red Blood Cells by Natural Killer cells and Monocytes

Abstract Antibodies naturally acquired during repeated exposure to the parasite Plasmodium falciparum are essential for control of blood-stage malaria. Natural killer (NK) cells are innate immune cells with an important role in containing viral infections and tumor growth, mainly by direct cytotoxicity and inflammatory cytokine production. Recently, the lab reported that primary human NK cells lyse Plasmodium falciparum-infected red blood cells (iRBCs) via antibody dependent cellular cytotoxicity (ADCC) in presence of IgG isolated from adults living in a malaria-endemic region of Mali. Using live imaging, I observed release of parasitophorous vacuoles (PV) from iRBCs after incubation with NK cells in the presence of rabbit anti-RBC Abs. I confirmed the imaging results by flow cytometric analysis. Direct interactions of NK cells with iRBCs was imaged by scanning electron microscopy, which revealed tight immunological synapses and extensive damage to iRBC membranes. This membrane damage results in formation of ghost RBCs and eventually release of PVs. We hypothesized that monocytes clear PVs, which could reduce the inflammatory response. I isolated PVs by mild saponin treatment and mechanical disruption of iRBC membranes. Binding of IgG isolated from plasma of Malian adults to PVs was orders of magnitude higher than binding to iRBCs. Phagocytosis of PVs by primary human monocytes was detected and was enhanced in the presence of Mali IgG. Similar results were obtained using PVs released by NK-dependent lysis of iRBCs in the presence of Mali IgG. These results suggest that NK-mediated ADCC inflicts severe damage to the plasma membrane of iRBCs, leading to release of PVs which bind immune serum IgG and are cleared through phagocytosis by monocytes.

Natural Killer Cell Line NK-92-Mediated Damage of Medically Important Fungi.

Invasive fungal disease (IFD) in hematopoietic stem cell transplantation is associated with high morbidity and mortality. As the antifungal host response determines risk and outcome of IFD, there is growing interest in adoptive immunotherapy using T cells or natural killer (NK) cells. Although the NK-92 cell line has been tested as anticancer therapy in clinical trials, data on the antifungal activity of NK-92 cells are lacking. Here, we show that the NK-92 cell line exhibits considerable fungal damage on all medically important fungi tested, such as different species of Aspergillus, Candida, mucormycetes, and Fusarium. The extent of fungal damage differs across various species of mucormycetes and Fusarium, whereas it is comparable across different species of Aspergillus and Candida. Interferon (IFN)-γ levels in the supernatant were lower when NK-92 cells are co-incubated with Aspergillus fumigatus, Candida albicans, or Rhizopus arrhizus compared to the levels when NK-92 cells are incubated alone. Different to primary human NK cells, no increase of perforin levels in the supernatant was observed when the fungi were added to NK-92 cells. Our in vitro data demonstrated that the NK-92 cell line could be a feasible tool for antifungal immunotherapy, but data of animal models are warranted prior to clinical trials.

Systematic improvements in lentiviral transduction of primary human natural killer cells undergoing ex vivo expansion

Transduction of primary human natural killer (NK) cells with lentiviral vectors has historically been challenging. We sought to evaluate multiple parameters to optimize lentiviral transduction of human peripheral blood NK cells being expanded to large numbers using a good manufacturing practice (GMP)-compliant protocol that utilizes irradiated lymphoblastoid (LCL) feeder cells. Although prestimulation of NK cells with interleukin (IL)-2 for 2 or more days facilitated transduction with vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentivirus, there was a subsequent impairment in the capacity of transduced NK cells to proliferate when stimulated with LCL feeder cells. In contrast, incubation of human NK cells with LCL feeder cells plus IL-2 before transduction in the presence of the TBK1 inhibitor BX795 resulted in efficient lentiviral integration (mean of 23% transgene+ NK cells) and successful subsequent proliferation of the transduced cells. Investigation of multiple internal promoter sequences within the same lentiviral vector revealed differences in percentage and level of transgene expression per NK cell. Bicistronic lentiviral vectors encoding both GFP and proteins suitable for the isolation of transduced cells with magnetic beads led to efficient transgene expression in NK cells. The optimized approaches described herein provide a template for protocols that generate large numbers of fully functional and highly purified lentivirus-transduced NK cells for clinical trials.

A robust platform for expansion and genome editing of primary human natural killer cells.

Genome editing is a powerful technique for delineating complex signaling circuitry and enhancing the functionality of immune cells for immunotherapy. Natural killer (NK) cells are potent immune effectors against cell malignancy, but they are challenging to modify genetically by conventional methods due to the toxicity of DNA when introduced into cells coupled with limited transfection and transduction efficiency. Here, we describe an integrated platform that streamlines feeder-free ex vivo expansion of cryopreserved primary human NK cells and nonviral genome editing by the nucleofection of CRISPR-Cas9 ribonucleoproteins (Cas9 RNPs). The optimized Cas9 nucleofection protocol allows efficient and multiplex gene knockout in NK cells while preserving high cell viability and negligible off-target effects. Cointroduction of a DNA template also enables in-frame gene knock-in of an HA affinity tag and a gfp reporter across multiple loci. This work demonstrates the advantages and flexibility of working with cryopreserved NK cells as potential off-the-shelf engineered therapeutic agents.

Optimization and Validation of CAR Transduction into Human Primary NK Cells Using CRISPR and AAV

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity, but genetic modification of primary human NK cells has been problematic compared to the ease in which primary human T cells are transduced. Despite some progress made in using lentiviral and retroviral vectors, the efficiency of viral transduction in NK cells remains relatively low. Additionally, using these viral vectors results in random integration events which can potentially affect the safety or efficacy of transduced immune cells. Recently, we described an efficient method for gene targeting in primary human NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined that approach with Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus, and optimized transduction efficiency using various AAV pseudotypes, single-stranded (ss) or self-complementary (sc) with a wide variety of homology arms, and homology repair (HR) andor non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For initial optimization we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms for HR resulted in optimal transduction efficiency. To validate the process for generating large numbers of CAR-NK cells needed for clinical application, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered by the transgene, and the NK cells maintained high CAR expression (mean 68% CAR+) during expansion. The expanded CD33-CAR-NK cells showed enhanced activation and antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML). Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome and low off-target insertion. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

TXNIP Regulates Natural Killer Cell-Mediated Innate Immunity by Inhibiting IFN-γ Production during Bacterial Infection.

The function of natural killer (NK) cell-derived interferon-γ (IFN-γ) expands to remove pathogens by increasing the ability of innate immune cells. Here, we identified the critical role of thioredoxin-interacting protein (TXNIP) in the production of IFN-γ in NK cells during bacterial infection. TXNIP inhibited the production of IFN-γ and the activation of transforming growth factor β-activated kinase 1 (TAK1) activity in primary mouse and human NK cells. TXNIP directly interacted with TAK1 and inhibited TAK1 activity by interfering with the complex formation between TAK1 and TAK1 binding protein 1 (TAB1). Txnip−/− (KO) NK cells enhanced the activation of macrophages by inducing IFN-γ production during Pam3CSK4 stimulation or Staphylococcus aureus (S. aureus) infection and contributed to expedite the bacterial clearance. Our findings suggest that NK cell-derived IFN-γ is critical for host defense and that TXNIP plays an important role as an inhibitor of NK cell-mediated macrophage activation by inhibiting the production of IFN-γ during bacterial infection.

Black Raspberries Suppress Colorectal Cancer by Enhancing Smad4 Expression in Colonic Epithelium and Natural Killer Cells.

Innate immune cells in the tumor microenvironment have been proposed to control the transition from benign to malignant stages. In many cancers, increased infiltration of natural killer (NK) cells associates with good prognosis. Although the mechanisms that enable NK cells to restrain colorectal cancer (CRC) are unclear, the current study suggests the involvement of Smad4. We found suppressed Smad4 expression in circulating NK cells of untreated metastatic CRC patients. Moreover, NK cell-specific Smad4 deletion promoted colon adenomas in DSS-treated ApcMin/+ mice and adenocarcinomas in AOM/DSS-treated mice. Other studies have shown that Smad4 loss or weak expression in colonic epithelium associates with poor survival in CRC patients. Therefore, targeting Smad4 in both colonic epithelium and NK cells could provide an excellent opportunity to manage CRC. Toward this end, we showed that dietary intervention with black raspberries (BRBs) increased Smad4 expression in colonic epithelium in patients with FAP or CRC and in the two CRC mouse models. Also, benzoate metabolites of BRBs, such as hippurate, upregulated Smad4 and Gzmb expression that might enhance the cytotoxicity of primary human NK cells. Of note, increased levels of hippurate is a metabolomic marker of a healthy gut microbiota in humans, and hippurate also has antitumor effects. In conclusion, our study suggests a new mechanism for the action of benzoate metabolites derived from plant-based foods. This mechanism could be exploited clinically to upregulate Smad4 in colonic epithelium and NK cells, thereby delaying CRC progression.

Arsenite suppresses IL-2-dependent tumoricidal activities of natural killer cells

Chronic exposure to arsenic causes cancers in various organs including the skin, liver, lung, and bladder in humans, but the mechanisms of the multi-organ carcinogenicity of arsenic remain unknown. Natural killer (NK) cells play important roles in the immune surveillance and elimination of tumor cells. Although accumulating evidence has indicated that arsenic has immunosuppressive properties, little is known about the effects of arsenic on the tumoricidal functions of NK cells. We examined the effects of arsenite on the cytotoxic activities of human and mouse NK cells toward target tumor cells. Exposure of human NK-92 cells and primary mouse NK cells to sublethal doses of arsenite reduced the IL-2-activated cytotoxic activities toward human K562 cells and murine YAC-1 cells, respectively. NK cells recognize target cells via integrated signals from both activating and inhibitory receptors and induce apoptosis of target cells via a granzyme/perforin system. We found that exposure of NK-92 cells to arsenite diminished the IL-2-activated down-regulation of the inhibitory receptors, KIR2DL2 and KIR2DL3, and the up-regulation of granzyme B and lymphotoxin-α. The IL-2-activated increases in secretion of interferon-γ and IL-10 were also slightly reduced by arsenite. Thus, arsenite suppressed the IL-2-activated cytotoxic activity of NK cells by disrupting multiple pathways required for the recognition and killing of target tumor cells. Our findings provide new insights into the roles of NK cell-mediated tumor immunity in cancer development by arsenic.

CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity

AbstractMultiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

Histone deacetylase 8 inhibition suppresses mantle cell lymphoma viability while preserving natural killer cell function

Mantle Cell Lymphoma (MCL) is a non-Hodgkin lymphoma with a median survival rate of five years. Standard treatment with high-dose chemotherapy plus rituximab (anti-CD20 antibody) has extended overall survival although, the disease remains incurable. Histone deacetylases (HDAC) are a family of enzymes that regulate multiple proteins and cellular pathways through post-translational modification. Broad spectrum HDAC inhibitors have shown some therapeutic promise, inducing cell cycle inhibition and apoptosis in leukemia and non-Hodgkin’s lymphoma. However, the therapeutic effects of these broad-spectrum HDAC inhibitors can detrimentally dampen Natural Killer (NK) cell cytotoxicity, reduce NK viability, and downregulate activation receptors important for NK mediated anti-tumor responses. Impairment of NK function in MCL patients during therapy potentially limits therapeutic activity of rituximab. Thus, there is an unmet need to decipher specific roles of individual HDACs in order to preserve and/or enhance NK function, while, directly impairing MCL viability. We investigated the impact of HDAC8 in MCL cell lines. Inhibition or genetic loss of HDAC8 caused MCL cells to undergo apoptosis. In contrast, exposure of primary human NK cells to an HDAC8 inhibitor does not alter viability, receptor expression, or antibody dependent cellular cytotoxicity (ADCC). However, an increase in effector cytokine interferon-gamma (IFNγ) producing NK cells was observed in response to HDAC8 inhibition. Taken together these data suggest that selective HDAC8 inhibitors may simultaneously preserve NK functional activity, while impairing MCL tumor growth, establishing a rationale for future clinical evaluation.

Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

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

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.

Genetic and epigenetic modification of human primary NK cells for enhanced antitumor activity

Cancer immunotherapy using genetically modified immune cells such as those expressing chimeric antigen receptors has shown dramatic outcomes in patients with refractory and relapsed malignancies. Natural killer (NK) cells as a member of the innate immune system, possessing both anticancer (cytotoxic) and proinflammatory (cytokine) responses to cancers and rare off-target toxicities have great potential for a wide range of cancer therapeutic settings. Therefore, improving NK cell antitumor activity through genetic modification is of high interest in the field of cancer immunotherapy. However, gene manipulation in primary NK cells has been challenging because of broad resistance to many genetic modification methods that work well in T cells. Here we review recent successful approaches for genetic and epigenetic modification of NK cells including epigenetic remodeling, transposons, mRNA-mediated gene delivery, lentiviruses, and CRISPR gene targeting.

Augmentation of NK Cell Proliferation and Anti-tumor Immunity by Transgenic Expression of Receptors for EPO or TPO

Many investigational adoptive immunotherapy regimens utilizing natural killer (NK) cells require the administration of interleukin-2 (IL-2) or IL-15, but these cytokines cause serious dose-dependent toxicities. To reduce or preclude the necessity for IL-2 use, we investigated whether genetic engineering of NK cells to express the erythropoietin (EPO) receptor (EPOR) or thrombopoietin (TPO) receptor (c-MPL) could be used as a method to improve NK cell survival and function. Viral transduction of NK-92 cells to express EPOR or c-MPL receptors conveyed signaling via appropriate pathways, protected cells from apoptosis, augmented cellular proliferation, and increased cell cytotoxic function in response to EPO or TPO ligands invitro. In the presence of TPO, viral transduction of primary human NK cells to express c-MPL enhanced cellular proliferation and increased degranulation and cytokine production toward target cells invitro. In contrast, transgenic expression of EPOR did not augment the proliferation of primary NK cells. In immunodeficient mice receiving TPO, invivo persistence of primary human NK cells genetically modified to express c-MPL was higher compared with control NK cells. These data support the concept that genetic manipulation of NK cells to express hematopoietic growth factor receptors could be used as a strategy to augment NK cell proliferation and antitumor immunity.

Charge-altering releasable transporters enable phenotypic manipulation of natural killer cells for cancer immunotherapy

AbstractChimeric antigen receptor (CAR) natural killer (NK) cells are an emerging cell therapy with promising results in oncology trials. However, primary human NK cells are difficult to transfect, hampering both mechanistic studies and clinical applications of NK cells. Currently, NK cell CAR modification relies on viral vectors or cell activation. The former raises cost and tolerability issues, while the latter alters NK cell biology. Here, we report that readily synthesized and inexpensive nonviral charge-altering releasable transporters (CARTs) efficiently transfect primary human NK cells with messenger RNA without relying on NK cell activation. Compared with electroporation, CARTs transfect NK cells more efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype and function. We use CARTs to generate cytotoxic primary anti-CD19 CAR NK cells, demonstrating this technology can drive clinical applications of NK cells. To our knowledge, CARTs represent the first efficacious transfection technique for resting primary human NK cells that preserves NK cell phenotype and can enable new biological discoveries and therapeutic applications of this understudied lymphocyte subset.