NK Cells In Control Research Articles

NKG2C and NKG2A coexpression defines a highly functional antiviral NK population in spontaneous HIV control.

Elite controllers (ECs), a unique group of people with HIV (PWH), exhibit remarkable control of viral replication in the absence of antiretroviral therapy. In this study, we comprehensively characterized the NK cell repertoire in ECs after long-term viral control. Phenotypic profiling of NK cells revealed profound differences compared with other PWH, but marked similarities to uninfected individuals, with a distinctive prevalence of NKG2C+CD57+ memory-like NK cells. Functional analyses indicated that ECs had limited production of functional molecules upon NK stimulation and consequently reduced natural cytotoxicity against non-HIV target cells. Importantly, ECs showed an exceptional ability to kill primary HIV-infected cells by the antibody-dependent cell cytotoxicity adaptive mechanism, which was achieved by a specific memory-like NK population expressing CD16, NKG2A, NKG2C, CD57, and CXCR3. In-depth single-cell RNA-seq unveiled a unique transcriptional signature in these NK cells linked to increased cell metabolism, migration, chemotaxis, effector functions, cytokine secretion, and antiviral response. Our findings underscore a pivotal role of NK cells in the immune control of HIV and identify specific NK cells as emerging targets for immunotherapies.

THEMIS2 Impairs Antitumor Activity of NK Cells by Suppressing Activating NK Receptor Signaling.

NK cells are cytotoxic innate lymphocytes that play a critical role in antitumor immunity. NK cells recognize target cells by using a repertoire of activating NK receptors and exert the effector functions. Although the magnitude of activation signals through activating NK receptors controls NK cell function, it has not been fully understood how these activating signals are modulated in NK cells. In this study, we found that a scaffold protein, THEMIS2, inhibits activating NK receptor signaling. Overexpression of THEMIS2 attenuated the effector function of human NK cells, whereas knockdown of THEMIS2 enhanced it. Mechanistically, THEMIS2 binds to GRB2 and phosphorylated SHP-1 and SHP-2 at the proximity of activating NK receptors DNAM-1 and NKG2D. Knockdown of THEMIS2 in primary human NK cells promoted the effector functions. Furthermore, Themis2-deficient mice showed low metastatic burden in an NK cell-dependent manner. These findings demonstrate that THEMIS2 has an inhibitory role in the antitumor activity of NK cells, suggesting that THEMIS2 might be a potential therapeutic target for NK cell-mediated cancer immunotherapy.

CD19-Specific CAR NK Cells Coexpressing IL-21 Exhibit Superior Expansion and Antitumor Activity Against CD19-Postive Lymphoma

Background Chimeric antigen receptor (CAR) expressing NK cells induce robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. IL-15 and IL-21 promote NK cell expansion, survival, and possess both overlapping and distinctive functions. CAR NK cells engineered to express IL-15 have shown long-term persistence and potent antitumor activity, however, the pleiotropic effects of IL-21 on CAR NK cell-mediated tumor rejection are lacking. Therefore, we compared the therapeutic potency of CD19-specific human NK cells that constitutively express IL-15 or IL-21. Methods The codon-optimized minigene encoding cytokine IL-21 or IL-15, linked with CD19-CAR containing 4-1BB costimulatory domain by a P2A sequence were synthesized and sub-cloned into the pSFG retroviral vector yielding the BBz.IL21 or BBz.IL15 retroviral construct. To produce CD19-CAR retrovirus, 293T/17 cells were transfected with a combination of plasmids containing CAR constructs, RDF plasmid and PegPam3 plasmid. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. The expression of CAR, phenotype, degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 3 post transduction. To measure IL-15 and IL-21 production, supernatants were collected from CAR NK cells cultured in the absence of IL-2 for 24h and evaluated by ELISA. Results After transduction, all constructs were stably expressed by NK cells, with construct containing IL-21 (71.23 ± 4.05 %, n = 3) demonstrating higher transduction efficiency compared with the BBz.IL15 (61.13 ± 3.43 %), but slightly lower than the BBz construct (79.63 ± 3.83 %). High levels of IL-21 were found in the supernatant of BBz.IL21 NK cells after resting 24h (3464 ± 199 pg/ml), while no detectable IL-21 was observed on BBz and BBz.IL15 NK cells. NK cells expressing BBz.IL15 produced significantly more IL-15 than BBz.IL21 and BBz NK cells as baseline (BBz.IL15: 213 ± 14 pg/ml, BBz.IL21: 61.86± 4 pg/ml, BBz: 60.56 ± 11 pg/ml). Given that IL-15 or IL-21 could potentially induce antigen-independent NK cell proliferation, we compared the ability of each NK-cell group to maintain growth in the presence of IL-2. The expansion of CAR NK cells expressing IL-21 was significantly higher than that of CAR NK cells expressing IL-15, while the number of BBz.IL15 NK cells and BBz NK cells were comparable (fold expansion in 5 days: BBz.IL21: 26.6 fold, BBz.IL15: 15.9 fold, BBz: 17.2 fold), suggesting a synergistic effect of IL-21 and IL-2 on CAR NK cell expansion. BBz.IL21 NK cells shared a similar immunophenotypic profile to BBz.IL15 NK cells, as the expression of inhibitory receptors PD1, LAG3, TIM3 and NKG2A, as well as activation receptors NKG2D and CD69 were comparable. CAR engagement by CD19-postive Raji cells specifically induced cytokines production by CD19-CAR NK cells but not by control NK cells. IL-21 coexpression significantly enhanced IFN-γ and TNF-α production compared with CAR NK cells as well as CAR NK cells expressing IL-15 (BBz.IL21 vs. BBz.IL15, P < 0.05; BBz.IL21 vs. BBz, P < 0.05; BBz.IL15 vs. BBz, P > 0.05). Moreover, a higher percentage of degranulated NK cells was observed on BBz.IL21 NK cells compared to BBz.IL15 NK cells and BBz NK cells. To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. CAR NK cells expressing IL-21 showed superior cytotoxicity, compared to CAR NK cells expressing IL-15. Conclusion CD19-CAR NK cells engineered to express IL-21 gene produced high levels of IL-21 during cultivation and enabled greater ex vivo expansion of CAR NK cells compared with CAR NK cells expressing IL-15. IL-21 coexpression induced a significant increase in IFN-γ, TNF-α production and degranulation compared with IL-15 coexpression, resulting in enhanced antitumor activity of CD19-CAR NK cells against CD19-postive lymphoma.

Disruption of SOCS1 Promotes the Antitumor Activity of CD19-Specific CAR NK Cells Coexpressing IL-15

Background Chimeric antigen receptor (CAR) expressing NK cells exhibit robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. Interleukin-15 (IL-15) promotes NK cells expansion and survival, while IL-15 signaling is often inhibited by the suppressor of cytokine signaling (SOCS) proteins (CIS, SOCS1-7), which is rapidly induced in response to IL-15 and negatively regulates cytokine signaling through the JAK/STAT pathway in NK cells. We hypothesized that SOCS1 would be a logical checkpoint to target in human NK cells to increase their persistence and thereby enhance their antitumor potency. Here, we devised a strategy that couples targeting the SOCS1 protein, with CD19-specific human NK cells that constitutively express IL-15. Method Firstly, we evaluated the SOCS1 mRNA expression levels on NK cells after cultured with or without IL-15 for 6 or 24 hours by reverse transcription polymerase chain reaction (RT-PCR). The retroviral vector encoding CAR19.4-1BB-zeta-2A-IL-15 (named BBz.IL-15) and the production of transient retroviral supernatants have been previously described. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. CAR expression on NK cells was determined by flow cytometry, and IL-15 secretion was measured by ELISA. Knockout (KO) of SOCS1 by CRISPR/Cas9 using the Neon transfection system was performed on day 3 post transduction in both non-transduced NK cells (NT-NK) and CAR-transduced NK cells (CAR-NK). To assess KO efficiency, we used polymerase chain reaction (PCR) gel electrophoresis, western blot, and Sanger sequencing. The degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 7 post electroporation. Results The expression of SOCS1 on NK cells was significantly induced within 6 hours of IL-15 treatment. Our approach for combined retroviral transduction with the anti-CD19 CAR and RNP-mediated gene editing of SOCS1 showed that surface expression of the CAR was maintained on NK cells in culture, with construct containing BBz.IL15 (37.18 ± 11.71%) demonstrating comparable transduction efficiency compared with the BBz construct (45.48 ± 10.54%) (P＞0.05), and the efficiency of SOCS1 KO was high in both the nontransduced (NT) control and CAR-expressing NK cells by TIDE and remained stable over time. When tested against CD19-postive Raji cells, SOCS1 KO BBz.IL-15 NK cells produced more IFN-γ and TFN-α, and displayed greater degranulation (CD107a) against their targets than did their BBz.IL-15 NK controls (BBz.IL-15 SOCS1 KO vs BBz.IL-15 control: 15.75±2.05% vs 7.93±0.47% for IFN-γ, P=0.032; 6.27±1.27% vs 1.88±0.26% for TFN-α, P=0.04; 23.25±2.33% vs 12.35±2.33% for CD107a, P=0.042). However, there was no significant difference between the SOCS1 KO BBz. or NT NK cells and their respective BBz. and NT controls regarding IFN-γ and TFN-α production and degranulation (BBz.SOCS1 KO vs BBz.control: 16.95±5.30% vs 16.85±8.70% for IFN-γ, P＞0.05; 6.83±2.98% vs 4.06±2.47% for TFN-α, P＞0.05; 26.65±7.14% vs 25.8±2.12% for CD107a, P＞0.05) (NT SOCS1 KO vs NT control: 21.75±6.58% vs 7.5±2.18% for IFN-γ, P＞0.05; 7.23±3.46% vs 1.58±0.03% for TFN-α, P＞0.05; 15.05±3.46% vs 6.84±2.15% for CD107a, P＞0.05). To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. SOCS1 KO NK cells could kill the tumors more efficiently in comparison to the control. Proliferation data showed the SOCS1 KO NK cells can grow faster than the control group. Conclusion In conclusion, the results demonstrate that SOCS1 might be a potential target for genetic modification to improve activity of CAR- NK cells.

Role of NK cells in circadian regulation of lung inflammation

Abstract Circadian rhythms form an anticipatory system wherein various aspects of cellular physiology oscillate across a 24hrs period; these rhythms have been known to modulate various facets of immunity. We have previously shown that mice infected with Influenza A virus (IAV) at dusk had 3-fold higher mortality than littermates infected at dawn--a hallmark of circadian regulation. This time-of-day specific protection was lost when NK cells were depleted just before the infection, suggesting a role of NK cells in mediating the circadian regulation of IAV infection. We investigated the mechanism underlying the role of NK cells, both cell-intrinsic or extrinsic in circadian regulation of lung injury. We noted that viral clearance was unaffected, but immunopathology was markedly worse in the NK depleted mice than the controls. Next, we investigated the effect of the loss of the circadian clock on NK cell behavior and found that splenic and pulmonary NK cells in naïve mice with a disrupted circadian clock have a less mature phenotype than control NK cells with intact clock. Our transcriptomic profiling of NK cells sorted from clock-intact versus clock-disrupted animals revealed that several genes that regulate cell cycle progression and functional maturity of NK cells were differentially expressed across the two groups. Pulmonary NK cells from clock mutant mice were less mature than clock-intact mice. Finally, the Ncr1cre+Bmal1fl/flmice (core clock gene Bmal1deleted in NK cells) infected with IAV had higher mortality through heightened immunopathology but not delayed viral clearance. In summary, these data show that NK cell-autonomous clock boost host tolerance to mediate circadian protection from IAV infection. Supported by grants from NIH (R01HL155934-01A1, R01HL147472, R01AI137062-01)

Myc controls NK cell development, IL-15-driven expansion, and translational machinery.

MYC is a pleiotropic transcription factor involved in cancer, cell proliferation, and metabolism. Its regulation and function in NK cells, which are innate cytotoxic lymphocytes important to control viral infections and cancer, remain poorly defined. Here, we show that mice deficient for Myc in NK cells presented a severe reduction in these lymphocytes. Myc was required for NK cell development and expansion in response to the key cytokine IL-15, which induced Myc through transcriptional and posttranslational mechanisms. Mechanistically, Myc ablation in vivo largely impacted NK cells' ribosomagenesis, reducing their translation and expansion capacities. Similar results were obtained by inhibiting MYC in human NK cells. Impairing translation by pharmacological intervention phenocopied the consequences of deleting or blocking MYC in vitro. Notably, mice lacking Myc in NK cells exhibited defective anticancer immunity, which reflected their decreased numbers of mature NK cells exerting suboptimal cytotoxic functions. These results indicate that MYC is a central node in NK cells, connecting IL-15 to translational fitness, expansion, and anticancer immunity.

Abstract 890: ADAM17 knockout NK or CAR NK cells augment antibody dependent cellular cytotoxicity (ADCC) and anti-tumor activity

Abstract Natural killer (NK) cells are a critical component of the innate immune system and do not elicit graft-versus-host disease, making them an attractive source of ‘off-the-shelf’ allogeneic cell therapy. NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Another important anti-tumor role of NK cells is via antibody-dependent cell mediated cytotoxicity (ADCC). The process is mediated by CD16a of NK cells, which binds the Fc portion of antibodies. After NK cell activation, the CD16a ectodomain is rapidly cleaved from the NK cell surface by a disintegrin, Metalloprotease-17 (ADAM17), that is produced by both tumor cells and activated NK cells. This dampens NK cell activity by preventing NK cell attachment to antibody-coated target cells and diminishes CD16a signaling. Inhibiting ADAM17-mediated CD16a cleavage leads to augmented ADCC activity and increased cytokine production by human NK cells. Other NK cell activating receptors are known to be regulated by a similar mechanism1. In this study, we have utilized CRISPR-Cas9 ribonucleoproteins (RNPs) to disrupt the ADAM17 gene in isolated peripheral blood NK cells from healthy donors. FACS and Amplicon NGS Sequencing data confirmed successful disruption of the gene. Edited NK cells were expanded using IL-2 and stimulation with NKSTIM, a modified K562 stimulatory cell line expressing a membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation. Furthermore, we transduced ADAM17 edited NK cells to express a CD70 CAR2 or CD19 CAR3 construct and mbIL-15. CAR expression was assessed by FACS. In vitro cytotoxicity was measured by IncuCyte. Using an artificial activation system (phorbol 12-myristate 13-acetate [PMA]), we showed that ADAM17 KO NK cells maintain dramatically higher surface expression of CD16a and CD62L than control NK cells. In IncuCyte cytotoxicity assays against Raji tumor cells in the presence of rituximab (anti-CD20), ADAM17 KO NK cells and ADAM17 KO CD19 CAR NK cells demonstrated higher cytotoxicity compared to control NK cells. Similarly, ADAM17 KO NK cells and ADAM17 KO CD70 CAR NK cells also showed enhanced cytotoxicity against 786-O tumor cells in the presence of anti-EGFR antibody, cetuximab. Furthermore, ADAM17/CISH/CBLB triple KO CD70 CAR NK cells exhibited enhanced antitumor efficacy in vivo in a 786-O RCC tumor and a HL60 AML xenograft model. Taken together, we show ADAM17 KO NK or ADAM17 KO CAR NK cells demonstrate improved ADCC and ADAM17 KO can enhance in vivo anti-tumor activity of CISH/CBLB KO CD70 CAR NK cells against relevant tumor models. These data support the further exploration of ADAM17 gene knockout NK cells for clinical application and to improve efficacy of therapeutic antibodies in combination with adoptive transfer of engineered NK cells. Citation Format: Chao Guo, Yanying Fan, Alexander Aronov, Nikki Kimura, Damie Juat, Jessica Sood, Ivan H. Chan, James B. Trager. ADAM17 knockout NK or CAR NK cells augment antibody dependent cellular cytotoxicity (ADCC) and anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 890.

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

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

The co-inhibitory receptor TIGIT regulates NK cell function and is upregulated in human intrahepatic CD56bright NK cells.

The crosstalk between NK cells and their surrounding environment is enabled through activating and inhibitory receptors, which tightly control NK cell activity. The co-inhibitory receptor TIGIT decreases NK cell cytotoxicity and is involved in NK cell exhaustion, but has also been associated with liver regeneration, highlighting that the contribution of human intrahepatic CD56bright NK cells in regulating tissue homeostasis remains incompletely understood. A targeted single-cell mRNA analysis revealed distinct transcriptional differences between matched human peripheral blood and intrahepatic CD56bright NK cells. Multiparameter flow cytometry identified a cluster of intrahepatic NK cells with overlapping high expression of CD56, CD69, CXCR6, TIGIT and CD96. Intrahepatic CD56bright NK cells also expressed significantly higher protein surface levels of TIGIT, and significantly lower levels of DNAM-1 compared to matched peripheral blood CD56bright NK cells. TIGIT+ CD56bright NK cells showed diminished degranulation and TNF-α production following stimulation. Co-incubation of peripheral blood CD56bright NK cells with human hepatoma cells or primary human hepatocyte organoids resulted in migration of NK cells into hepatocyte organoids and upregulation of TIGIT and downregulation of DNAM-1 expression, in line with the phenotype of intrahepatic CD56bright NK cells. Intrahepatic CD56bright NK cells represent a transcriptionally, phenotypically, and functionally distinct population of NK cells that expresses higher levels of TIGIT and lower levels of DNAM-1 than matched peripheral blood CD56bright NK cells. Increased expression of inhibitory receptors by NK cells within the liver environment can contribute to tissue homeostasis and reduction of liver inflammation.

Abstract 2 Development of CAR-NK Cell Therapy for Hematologic Malignancies

IntroductionNatural killer (NK) cells can kill tumor cells without priming or prior activation through their complement of activating and inhibitory surface molecules. Chimeric antigen receptor (CAR) expression by engineered NK cells can improve both specificity and potency of the NK cells’ anti-tumor efficacy. CAR-NK cell therapy may be a safer, more clinically accessible, and cost effective allogeneic cellular therapy in comparison to autologous CAR-T cell therapy, as NK cells do not cause graft versus host disease (GvHD) or cytokine release syndrome (CRS).ObjectiveWe aimed to develop an allogeneic, cryopreserved, off-the-shelf CAR-NK cell product for the treatment of hematologic malignancies.MethodsOur CD56+ NK cells are generated from cord blood-derived CD34+ cells, which undergo expansion and priming on a proprietary Notch ligand cell culture platform followed by a second culture phase of NK cell differentiation. These cells were transduced using a viral vector to express a CAR specific for an antigen expressed on the cell surface of acute lymphoblastic leukemia (ALL) cells. The CAR-NK cells were assessed for CAR expression, NK cell phenotype, and in vitro cytotoxicity both pre- and post-cryopreservation. An ALL xenograft mouse model was treated using repeat doses of cryopreserved CAR-NK cell product, with readouts of body weight, tumor growth, and survival.ResultsThe transduced CAR-NK cell product displayed viability and phenotyping comparable to the untransduced control NK cell product but possessed significantly enhanced cytotoxicity against ALL target cells in vitro. Following cryopreservation and thaw, the CAR-NK cell product retained CAR expression and maintained enhanced anti-tumor function in vitro. Cryopreserved CAR-NK cell product was safely given in up to 8 repeat doses to ALL xenograft mice and significantly inhibited tumor growth as well as increased survival compared with control NK cell product.DiscussionWe have demonstrated in this proof-of-concept study that our cord blood CD34+ cell-derived CAR-NK cell product can maintain CAR expression, with specific and enhanced potency both in vitro and in vivo, following cryopreservation and thaw. Additional preclinical studies are planned to develop CAR-NK off-the-shelf cell therapy for AML and other hematologic malignancies.

Abstract 1536: Functional characterization of EDIT-201, a multiplexed CRISPR-Cas12a gene edited healthy donor derived NK cell therapy, reveals increased granzyme B and degranulation supporting improved serial killing capacity

Abstract We have previously described EDIT-201, a healthy donor derived NK cell therapy with a CISH and TGFBR2 knockout mediated by CRISPR-Cas12a that results in resistance to TGF-β inhibition and increased effector function. EDIT-201 kills tumor cells more efficiently and for a longer duration than control NK cells in the presence of TGF-β. Based on these data, we hypothesized that EDIT-201 has increased serial killing capacity. First, we explored the transcriptional changes contributed by each gene edit with a focus on transcripts critical for NK cell effector function and metabolism using Nanostring analysis. Unedited, mock electroporated, and control edited (targeting a biologically irrelevant site) NK cells were included as controls in addition to CISH and TGFBR2 single and double gene knockout (KO) NK cells to interrogate the potential impact of electroporation and double-stranded DNA breaks on NK cell function. All samples included in the analysis were cultured for 3 days in IL-15 post-electroporation. No significant transcriptional changes were detected in all control conditions, while samples that contained CISH editing clearly upregulated transcripts relevant for NK cell effector function, including contents of cytolytic granules (GZMB, GZMA and GZMH). EDIT-201 expressed an average of 22 fold more GZMB transcript than control NK cells as measured by RT-qPCR in four unique NK cell donors. Furthermore, EDIT-201 showed significantly higher levels of CD107a, a marker of degranulation, after 14hrs of co-culture with SKOV-3 tumor cells, suggesting that EDIT-201 had an increased capacity to degranulate relative to control NK cells. To determine the presence of granzyme proteins within tumor cells following engagement with NK cells, we developed a novel GzmB reporter gene and used lentiviral vectors to introduce this reporter into tumor cell lines. Using SK-OV-3 tumor cells transduced with our reporter, we demonstrated that upon coculture of EDIT-201 with tumor cells, intratumoral GzmB activity was identified 4 hours sooner than when the target cells were cultured with control NK cells. In addition, EDIT-201 affected 80% more SK-OV-3 tumor cells with granzyme B compared to control NK cells over a 20 hour period. Most significantly, our data demonstrated that EDIT-201 not only discharged GzmB more rapidly than control NK cells, but also the amount of GzmB degranulated is greater as well, confirming that enhanced degranulation is a key mechanism by which EDIT-201 has superior functional capacity relative to control NK cells. Together, these data demonstrate one mechanism by which EDIT-201 demonstrates superior cytotoxicity during in vitro killing of SK-OV-3 tumor targets, and continues to support the advancement of EDIT-201 as a potential novel gene edited NK cell-based treatment for cancer. Citation Format: Christopher M. Borges, Shuqi Zhang, Kevin Wasko, Kelly Donahue, Lincy Prem Antony, Jared Nasser, Steven Sexton, Amanda Pfautx, Richard A. Morgan, Karrie K. Wong. Functional characterization of EDIT-201, a multiplexed CRISPR-Cas12a gene edited healthy donor derived NK cell therapy, reveals increased granzyme B and degranulation supporting improved serial killing capacity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1536.

Abstract 1532: EDIT-201: CRISPR-Cas12a gene editing enhances functional metabolism of natural killer cells and enables tumor cell cytolysis in metabolically stressful conditions that inhibit effector cell function

Abstract We have previously described EDIT-201, a healthy donor derived NK cell therapy with CRISPR-Cas12a mediated CISH and TGFBR2 double gene knockout that demonstrated resistance to TGF-β inhibition and increased tumor control in vitro and in vivo. The tumor microenvironment (TME) is known to be nutrient-deprived due to active tumor cell metabolism leading to competition for essential nutrients with infiltrating effector cells, while at the same time being enriched in immunosuppressive metabolites such as lactic acid due to Warburg Metabolism. To explore whether EDIT-201 is functional in such hostile metabolic conditions, we aimed to more accurately model the metabolic microenvironment in a long-term tumor spheroid model. To model this, we generated SK-OV-3 ovarian tumor spheroids in decreasing concentrations of glucose (10-0.5mM) or glutamine (2-0.1mM), two important fuels for NK cell metabolism, as well as increasing concentrations of inhibitory metabolite lactate (0-50mM), or decreasing pH (7.2-6.5). Each of these metabolic conditions are known to suppress effector cell function; we additionally performed spheroid cells co-cultures in the presence of 10ng/mL TGF-β to increase the potential for NK inhibition. We demonstrated that, relative to control NK cells, EDIT-201 demonstrated rapid and sustained tumor killing in all conditions tested including, the absence of critical nutrients. We next created a multifactorial matrix of metabolic conditions where we combined deprivation of multiple nutrients, in the presence of lactate and/or acidic cell culture media. Remarkably, in all combinations tested, we surprisingly found that EDIT-201 continued to demonstrate increased cytotoxicity against SK-OV-3 spheroids relative to control NK cells, suggesting a clear and robust metabolic advantage of EDIT-201 over control NK cells. We additionally observed a corresponding increase in the concentrations of IFN-γ and TNF-α by EDIT-201 in all of these conditions relative to control NK cells. Given that mitochondrial respiration is key to NK cell persistence and function, we next interrogated the mitochondrial function of EDIT-201. We find that EDIT-201 consistently demonstrated greater spare respiratory capacity (SRC) relative to control NK cells, suggesting enhanced mitochondrial reserve as a result of CISHand TGFBR2 knockout. The increase in SRC likely enables EDIT-201 to meet enhanced energy demands necessary to mediate effector function in metabolically challenging conditions, thus sustaining superior cytotoxic capacity and cytokine production. In summary, our data demonstrate enhanced metabolic function of EDIT-201, resulting in superior cytotoxicity in metabolically unfavorable conditions. These data further support the development of EDIT-201 as a novel cell therapy for cancer. Citation Format: Karrie K. Wong, Steven Sexton, Lincy Prem Antony, Kevin Wasko, Jared Nasser, Kelly Donahue, Amanda Pfautz, Shuqi Zhang, Richard A. Morgan, Christopher M. Borges. EDIT-201: CRISPR-Cas12a gene editing enhances functional metabolism of natural killer cells and enables tumor cell cytolysis in metabolically stressful conditions that inhibit effector cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1532.

Cutaneous Squamous Cell Carcinoma Development Is Associated with a Temporal Infiltration of ILC1 and NK Cells with Immune Dysfunctions

NK cells and tissue-resident innate lymphoid cells (ILCs) are innate effectors found in the skin. To investigate their temporal dynamics and specific functions throughout the development of cutaneous squamous cell carcinoma (cSCC), we combined transcriptomic and immunophenotyping analyses in mouse and human cSCCs. We identified an infiltration of NK cells and ILC1s as well as the presence of a few ILC3s. Adoptive transfer of NK cells in NK cell‒ and ILC-deficient Nfil3-/- mice revealed a role for NK cells in early control of cSCC. During tumor progression, we identified a population skewing with the infiltration of atypical ILC1 secreting inflammatory cytokines but reduced levels of IFN-γ at the papilloma stage. NK cells and ILC1s were functionally impaired, with reduced cytotoxicity and IFN-γ secretion associated with the downregulation of activating receptors. They also showed a high degree of heterogeneity in mouse and human cSCCs with the expression of several markers of exhaustion, including TIGIT on NK cells and PD-1 and TIM-3 on ILC1s. Our data show an enrichment in inflammatory ILC1 at the precancerous stage together with impaired antitumor functions in NK cells and ILC1 that could contribute to the development of cSCC and thus suggest that future immunotherapies should take both ILC populations into account.

The transcription factor ETS1 is an important regulator of human NK cell development and terminal differentiation

AbstractNatural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation–sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.

De novo polyamine synthesis supports metabolic and functional responses in activated murine NK cells.

Cellular metabolism is dynamically regulated in NK cells and strongly influences their responses. Metabolic dysfunction is linked to defective NK cell responses in diseases such as obesity and cancer. The transcription factors, sterol regulatory element binding protein (SREBP) and cMyc, are crucial for controlling NK cell metabolic and functional responses, though the mechanisms involved are not fully understood. This study reveals a new role for SREBP in NK cells in supporting de novo polyamine synthesis through facilitating elevated cMyc expression. Polyamines have diverse roles and their de novo synthesis is required for NK cell glycolytic and oxidative metabolism and to support optimal NK cell effector functions. When NK cells with impaired SREBP activity were supplemented with exogenous polyamines, NK cell metabolic defects were not rescued but these NK cells displayed significant improvement in some effector functions. One role for polyamines is in the control of protein translation where spermidine supports the posttranslational hypusination of translation factor eIF5a. Pharmacological inhibition of hypusination also impacts upon NK cell metabolism and effector function. Considering recent evidence that cholesterol-rich tumor microenvironments inhibit SREBP activation and drive lymphocyte dysfunction, this study provides key mechanistic insight into this tumor-evasion strategy.

Targeting intracellular check points to unleash natural killer cell cytotoxicity

Abstract Natural killer (NK) cells are a potent weapon of the immune system against viral infections and tumor growth. The actomyosin network generates forces through the activity of actin filaments and myosin motors. This machinery is responsible for the conversion of mechanical forces into biochemical signals in a process termed mechanotransduction. However, the mechanism by which mechanotransduction controls the immune response, and specifically lymphocyte activity, is poorly understood. Here, we demonstrate that actomyosin retrograde flow (ARF) controls NK cell response through a novel interaction between beta-actin with the SH2-domain containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, thereby regulating NK cell cytotoxicity. Actin dynamics govern SHP-1 conformational structure dictating its catalytic activity. Indeed, blocking actin dynamics results in reduced SHP-1 activity, by confining SHP-1 to its inactivated “closed” conformation. This reduced enzymatic activity of SHP-1 leads to increased phosphorylation of SHP-1 substrates, an elevation of intracellular calcium flux, and NK cell cytotoxicity. Using multidisciplinary approaches including functional and advanced molecular imaging, we followed the signaling events in individual NK cells from the moment of target cell encounter until the final outcome – target cell killing or NK cell inhibition. Our data suggest that SHP-1 plays a major role as a sensor of ARF-generated forces in the process of mechanotransduction, and reveal a novel mechanism by which regulation of SHP-1 by ARF dictates NK cell killing decisions. Our data identify ARF as a master regulator of the lymphocyte response.

MicroRNA-155 governs SHIP-1 expression and localization in NK cells and regulates subsequent infiltration into murine AT3 mammary carcinoma.

NK cell migration and activation are crucial elements of tumor immune surveillance. In mammary carcinomas, the number and function of NK cells is diminished, despite being positively associated with clinical outcome. MicroRNA-155 (miR-155) has been shown to be an important regulator of NK cell activation through its interaction with SHIP-1 downstream of inhibitory NK receptor signaling, but has not been explored in regard to NK cell migration. Here, we explored the migratory potential and function of NK cells in subcutaneous AT3 in mice lacking miR-155. Without tumor, these bic/miR-155-/- mice possess similar numbers of NK cells that exhibit comparable surface levels of cytotoxic receptors as NK cells from wild-type (WT) mice. Isolated miR-155-/- NK cells also exhibit equivalent cytotoxicity towards tumor targets in vitro compared to isolated WT control NK cells, despite overexpression of known miR-155 gene targets. NK cells isolated from miR-155-/- mice exhibit impaired F-actin polymerization and migratory capacity in Boyden-chamber assays in response chemokine (C-C motif) ligand 2 (CCL2). This migratory capacity could be normalized in the presence of SHIP-1 inhibitors. Of note, miR-155-/- mice challenged with mammary carcinomas exhibited heightened tumor burden which correlated with a lower number of tumor-infiltrating NK1.1+ cells. Our results support a novel, physiological role for SHIP-1 in the control of NK cell tumor trafficking, and implicate miR-155 in the regulation of NK cell chemotaxis, in the context of mammary carcinoma. This may implicate dysfunctional NK cells in the lack of tumor clearance in mice.

NK cells in pancreatic cancer demonstrate impaired cytotoxicity and a regulatory IL-10 phenotype

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is one of the most common tumor subtypes and remains associated with very poor survival. T cell infiltration into tumor tissue is associated with improved clinical outcome but little is known regarding the potential role of NK cells in disease control. Here we analyze the phenotype and function of NK cells in the blood and tumor tissue from patients with PDAC. Peripheral NK cells are present in normal numbers but display a CD16hiCD57hi phenotype with marked downregulation of NKG2D. Importantly, these cells demonstrate reduced cytotoxic activity and low levels of IFN-γ expression but instead produce high levels of intracellular IL-10, an immunoregulatory cytokine found at increased levels in the blood of PDAC patients. In contrast, NK cells are largely excluded from tumor tissue where they display strong downregulation of both CD16 and CD57, a phenotype that was recapitulated in primary NK cells following co-culture with PDAC organoids. Moreover, expression of activatory proteins, including DNAM-1 and NKP30, was markedly suppressed and the DNAM-1 ligand PVR was strongly expressed on tumor cells. As such, in situ and peripheral NK cells display differential features in patients with PDAC and indicate local and systemic mechanisms by which the tumor can evade immune control. These findings offer a number of potential options for NK-based immunotherapy in the management of patients with PDAC.

Patrolling Monocytes Control NK Cell Expression of Activating and Stimulatory Receptors to Curtail Lung Metastases.

The role of nonclassical, patrolling monocytes in lung tumor metastasis and their functional relationships with other immune cells remain poorly defined. Contributing to these gaps in knowledge is a lack of cellular specificity in commonly used approaches for depleting nonclassical monocytes. To circumvent these limitations and study the role of patrolling monocytes in melanoma metastasis to lungs, we generated C57BL/6J mice in which the Nr4a1 superenhancer E2 subdomain is ablated (E2 -/- mice). E2 -/- mice lack nonclassical patrolling monocytes but preserve classical monocyte and macrophage numbers and functions. Interestingly, NK cell recruitment and activation were impaired, and metastatic burden was increased in E2 -/-mice. E2 -/- mice displayed unchanged "educated" (CD11b+CD27+) and "terminally differentiated" (CD11b+CD27-) NK cell frequencies. These perturbations were accompanied by reduced expression of stimulatory receptor Ly49D on educated NK cells and increased expression of inhibitory receptor NKG2A/CD94 on terminally differentiated NK cells. Thus, our work demonstrates that patrolling monocytes play a critical role in preventing lung tumor metastasis via NK cell recruitment and activation.

Functional recovery of natural killer cell activity by nanoparticle‐mediated delivery of transforming growth factor beta 2 small interfering RNA

AbstractNatural killer (NK) cells are at the forefront of immunotherapies, as they have potent innate cytolytic effects on cancer cells. The success of NK cell therapies requires that they overcome immunosuppression in the tumor microenvironment. Tumors produce immunosuppressive factors like transforming growth factor beta (TGF‐β) that inhibit the effector functions of NK cells. Silencing of TGF‐beta signaling in NK cells is a potential approach to enhance their functions. However, transfection of NK cells by conventional methods is challenging. Here, we report the development of a nanoparticle (NP) system that delivers small interfering RNA for the TGF‐β receptor 2 (TGFBR2) into NK cells to restore their activation against cancer cells. Manganese dioxide NPs were synthesized by the reduction of potassium permanganate by poly (allylamine), which effectively complexed siRNA and protected it from degradation. The NPs were cytocompatible with NK cells and, upon loading with TGFBR2 siRNA, resulted in a 90% knockdown of the TGFBR2 receptor. NP‐mediated TGFBR2 receptor knockdown protected NK cells against TGF‐β suppression, which was studied in both two‐dimensional and three‐dimensional lung cancer cell culture systems. Namely, NK cells treated with TGFBR2 siRNA loaded NPs demonstrated higher interferon gamma production, infiltration, and killing of lung cancer cells compared with control NK cells. This study demonstrates the feasibility of NP‐mediated RNA interference in NK cells to increase their resilience to the immunosuppressive environments in solid tumors.