Human NK Cells Research Articles

Deep phenotypic and functional characterization of NK cells during NK-mediated cytotoxicity

Abstract Natural Killer (NK) cell-mediated cytotoxicity is a critical mechanism of the immune system to respond against harmful stimuli. The development of emerging cell therapies, such as CAR-NK cells, requires a deeper characterization of cultured NK cells. Here we show a phenotyping of ex vivo and in vitro NK cells, and demonstrate how high quality and reliable reagents from BioLegend ensure a more in depth cytotoxic analysis. NK cells were isolated from fresh human PBMCs, using our MojoSort™ Human NK Cell Isolation Kit, and incubated at different ratios with Tag-it Violet™ stained K562 target cells in IMDM + BioLegend’s Cell-Vive™ T-NK Xeno-Free Serum Substitute, in the presence of CD107a-PE. Cells were then harvested for surface phenotyping and the supernatants were collected for cytokine analysis using LEGENDplex™ Human CD8/NK Panel. Tag-it Violet™ and Apotracker™ Green staining of K562 cells demonstrate significant decrease in proliferation and significant increase in apoptosis, respectively. Surface staining of CD107a reveals increased degranulation of both CD56 brightCD16 −and CD56 +CD16 +NK cells that correlates with increased apoptosis of K562 cells. Supernatant cytokine analysis revealed significant increases in Granzyme A and B, Perforin, and Granulysin, among others. Taken together, these results show a more complete picture of the phenotypical and functional state of NK cells by measuring target cell killing as well as characterizing NK surface markers and secreted molecules involved in NK-mediated cytotoxicity. BioLegend reagents are designed to address ever increasing needs for deeper understanding of immunological processes and support the demand to develop sophisticated and consistent cell-based therapies.

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.

Phenotypic and functional analysis in HER2+ targeted therapy of human NK cell subpopulation according to the expression of FcεRIγ and NKG2C in breast cancer patients.

Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ- and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ- or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C- NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ- and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C- NK cells retained greater functionality after treatment than FcεRIγ + NKG2C- NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.

Abstract LB337: Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells

Abstract The E3 ubiquitin ligase CBL-B is a master regulator of effector T cells through its downstream regulation of T-cell receptor and co-stimulatory stimulation, but it has also been shown to play a negative feedback role in NK cells. CBL-B depletion enhances the production of IFNγ and the cytotoxicity of activated NK cells. Similar to the effect of depleting CD8 T cells in mice, depletion of NK cells abolishes the tumor growth delay observed in Cbl-b-/- mice. With emerging data supporting the potential role of exogenous NK cells in anti-tumor immunity, inhibition of CBL-B represents a promising approach to enhance both adaptive and innate immunity for cancer immunotherapy. Using one of our small molecules, allosteric CBL-B inhibitors, HOT-A, we demonstrated that CBL-B inhibition enhanced NK activation, IFNγ production and granzyme B secretion in response to the various stimulation conditions, including cytokines (such as IL-15 and IL12 + IL-18) and stimulatory receptors (such as NKp30 and NKG2D + CD244). NK cell proliferation was also enhanced in the presence of HOT-A. Single cell analysis further suggested that HOT-A increased NK polyfunctional cytokine expression during IL-12 + IL-18 activation. In a coculture system of NK cells and K562 target cells, HOT-A dose-dependently promoted primary human NK cell activation, cytokine production and cytotoxicity in the K562 cells. In the in vivo CT-26 tumor model, granzyme B+ NK cells in the TIL were significantly increased in the HOT-A treated tumor bearing mice and NK function gene signature was also increased by Nanostring analysis, suggesting an enhanced NK function in the tumor microenvironment. Taken together, the pre-clinical data presented here demonstrate that our CBL-B inhibitor enhances NK cell function in vitro and in vivo, suggesting that its enhancement of anti-tumor immunity may be driven through both effector T cell and NK cell-driven biology. Citation Format: Yilin Qi, Jun Kuai, Yingzhi Bi, David Greco, Samira Jaeger, Ken Carson, Hadi Danaee, Timothy Reilly, Geraldine Harriman, Fang Wang. Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB337.

The NKcell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells.

Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cellreceptor is triggered1. Most NKcells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NKcell receptor2,3. Blockage of NKp46 inhibits NK killing of many cancer targets4. Although a few infectious NKp46 ligands have been identified, the endogenous NKp46 cell surface ligand is unknown. Here we show that NKp46 recognizes externalized calreticulin (ecto-CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane during ER stress. ER stress and ecto-CRT are hallmarks of chemotherapy-induced immunogenic cell death5,6, flavivirus infection and senescence. NKp46 recognition of the P domain of ecto-CRT triggers NKcell signalling and NKp46 caps with ecto-CRT in NK immune synapses. NKp46-mediated killing is inhibited by knockout or knockdownofCALR, the gene encoding CRT, or CRT antibodies, and is enhanced by ectopic expression of glycosylphosphatidylinositol-anchored CRT. NCR1)-deficient human (andNrc1-deficient mouse) NKcells are impaired in the killing of ZIKV-infected, ER-stressed and senescent cells and ecto-CRT-expressing cancer cells. Importantly, NKp46 recognition of ecto-CRT controls mouse B16 melanoma and RAS-driven lung cancers and enhances tumour-infiltrating NKcell degranulation and cytokine secretion. Thus, NKp46 recognition of ecto-CRT as a danger-associated molecular pattern eliminates ER-stressed cells.

Abstract 2903: Epigenetic modulation of NK cells to improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma

Abstract Osteosarcoma is the most common primary malignant bone tumor. It metastasizes almost exclusively to the lung, and this constitutes the main cause of death in these patients. Immune therapy has shown limited benefit. We investigated whether epigenetic modulation of allogeneic NK cells by the HDAC inhibitor MS-275 will improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma (OS) lung metastases. Human NK cells were purified and expanded from donor buffy coats using recombinant human IL-2 and genetically engineered K562 feeder cells and treated with MS-275 before injection into mice. RNA seq was performed in untreated and MS-275 pre-treated NK cells to determine differentially expressed genes. We also used ChiPseq to determine the histone post-translational modifications associated with the differentially expressed genes induced by the treatment. Culture media from untreated and treated NK cells was collected for cytokine analysis using the Isolight/Isoplexis system. We used our OS-17 human OS experimental lung metastases mouse model and injected untreated or pre-treated NK cells (0.5uM MS-275) IV labeled with near-infrared DiR dye twice a week. Tumor burden was assessed, and NK cells were tracked by Bioluminescence imaging (BLI). Mice were either euthanized 24 hours after the final NK cell treatment for histologic analysis of the lungs or, in a second experiment, analyzed for survival across treatment groups. Some mice from each group were selected for 3D imaging using the Xerra cryofluorescence tomography (CFT) imaging system, allowing for greater resolution of NK cell distribution as compared to the IVIS imaging system. We identified 547 differentially expressed genes between the untreated and MS-275 treated NK cells. Four immune related genes (L1CAM, IFNG, C3 and CD28) were upregulated in the MS-275 treated NK cells and histone H3 and H4 acetylation was found at the identified immunomodulatory genes. Culture media from MS-275 treated NK cells showed significantly increased MIP-1b, IL-6 and IFN-g. Furthermore, there was a significant decrease in lung micro-metastases in the MS-275 pre-treated NK cell group as compared to the untreated control group (199 vs 97 p=0.05). Lastly, Xerra CFT imaging demonstrated greater DiR signal in the lungs of the mice that received MS-275 treated NK cells compared to those that received untreated NK cells, suggesting that MS-275 might lead to greater persistence of NK cells at tumor sites. We conclude that MS-275 has an immunomodulatory effect on NK cells (increase L1CAM, IFNG, C3 and CD28) and increases H3 and H4 acetylation which leads to transcriptional activation of the immune-related genes that contribute to NK cell increased cytolytic function. Pre-treatment of NK cells with MS-275 enhances NK cell therapeutic efficacy and localization and persistence in the lungs. Citation Format: Ariana Rupp Anjier, Nancy Gordon, Shinji Maegawa, Vidya Gopalakrishnan, Juan Bournat, Donghang Cheng, Yanwen Yang, Charles Kingsley, Adam Kulp. Epigenetic modulation of NK cells to improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma [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 2903.

Abstract 2910: NKG2A suppression enhances the function of primary human Natural Killer cells

Abstract In this study we propose inhibition of NKG2A signaling enhances the anti-tumor response of primary human NK cells. One mechanism employed by tumor cells to evade immunosurveillance is through induction of the surface expression of unconventional HLA ligands that agonize inhibitory receptors on immune cells. Specifically on some tumors, HLA-E is either expressed or its expression is known to be induced by IFNγ and is indicative of resistance to immunotherapy. HLA-E agonizes the CD94/NKG2A inhibitory complex on NK cells and some T cells to lessen their cytotoxicity, potentially decreasing the efficacy of cellular therapy with these immune cells. In this study ex vivo-expanded primary human NK cells were shown to have significantly elevated expression of NKG2A. These expanded NK cells also secreted high levels of IFNγ and could induce expression of HLA-E on tumor cells. Suppression of NKG2A by either antibody blockade or gene deletion increased the cytotoxicity of NK cells against the lung cancer cell line A549 stably expressing HLA-E. NKG2A knockout cells had an almost 10-fold decrease in EC50 compared to wild-type NK cells (EC50 0.15 vs 0.94 at 48 h), meaning nearly log-fold fewer NKG2A knockout cells are needed to kill the same number of tumor cells. The effect of NKG2A knockout on cytotoxicity of NK cells against other cancer cell lines stably expressing HLA-E will be presented along with effects of NKG2A suppression on NK cell phenotype, cytokine secretion and degranulation. These initial data suggest that blockade of the CD94/NKG2A inhibitory complex can improve the function of ex vivo expanded human NK cells and could provide an effector population with the potential for enhanced therapeutic efficacy. Citation Format: Tayler J. Croom-Perez, Liza D. Robles-Carrillo, Md Faqrul Hasan, Alicja J. Copik. NKG2A suppression enhances the function of primary human 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 2910.

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 4: A novel humanized mouse model for study of T cells and NK cells in the tumor microenvironment

Abstract Humanized mouse models can be useful for in vivo preclinical evaluation of mechanisms underlying cancer immunotherapy. Most such models involve systemic engraftment of immunocompromised mice with human lymphoid and hematopoietic cells, often using normal donor peripheral blood mononuclear cells (PBMCs), followed by inoculation with human tumor. The ability to control and modify immune cells within the tumor microenvironment (TME), especially human NK cells, is limited in such models. Here, we report the development of a mouse model designed to control the number of human T cells and NK cells in the TME. 18 mice and pre-validated PBMCs were obtained from Charles River. Six groups of 3 mice each were inoculated with Raji lymphoma cells. Group 1 was inoculated with Raji cells only and received no PBMCs. Raji cells were mixed PBMCs for groups 2-4. Group 5 was systemically engrafted with PBMCs 12 days prior to Raji inoculation and Group 6 was systemically engrafted with PBMCs 10 days after Raji inoculation. All mice developed tumors with similar growth characteristics. Serial fine needle aspirates (FNAs) of the resulting tumors were evaluated by flow cytometry. Results of FNAs are summarized below. Tumors were harvested at the end of experiment, typically 27 days after tumor inoculation, and evaluated by immunohistochemistry. Both T cells and NK cells were evenly distributed within tumors at the time of harvest. We conclude co-injection of tumor and PBMCs results in engraftment of both T cells and NK cells within the TME for up to 27 days, with T cells and NK cells distributed evenly within the tumor. Mixing higher numbers of PBMCs with tumor cells resulted in increased numbers of T cells and NK cells in the TME throughout the experiment. Matrigel did not improve engraftment or persistence of T or NK cells in the TME. We conclude mixing PBMCs with Raji tumor cells prior to tumor inoculation into immunocompromised mice is an effective approach when a humanized model with human T cells and NK cells within the TME is desired. Group Description % T cells in tumor at 17 days % NK cells in tumor at 17 days 1 No PBMCs 0 0 2 PBMCs mixed with Raji at 10:1 ratio 8.6 1.5 3 PBMCs mixed with Raji at 2:1 ratio 20.3 2.7 4 PBMCs and Raji in matrigel 9.2 0.6 5 PBMCs systemically engrafted 12 days before Raji inoculation 23.2 0.1 6 PBMCs systemically engrafted 10 days after Raji inoculation 19.6 0.2 Citation Format: Jyoti Arora, Caitlin D. Lemke-Miltner, George J. Weiner. A novel humanized mouse model for study of T cells and NK cells in the tumor microenvironment [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 4.

Abstract 1823: Overcoming siglec-9-mediated immunosuppression by using glycomimetics that block the binding of this receptor with its natural ligands

Abstract MUC16, a 3-5 million Da mucin overexpressed by ovarian cancer cells binds to Siglec-9, an inhibitory receptor on immune cells. Upon binding to Siglec-9, MUC16 triggers inhibitory signaling in the immune cells that allows ovarian cancer cells to evade cytolytic immune responses. Ovarian and other tumors express a plethora of cell surface as well as secreted glycoproteins with glycans that are terminated with the Siglec-9 ligand, α-2,3-linked sialic acid. Therefore, targeting the sialic acid-Siglec-9 axis has been of major interest to augment antitumor immunity. Here, we demonstrate that synthetic glycomimetic Siglec-9 ligands have the potential to enhance the immune recognition of ovarian cancer cells. We first demonstrate, using siglec-9 chimeras, that the glycomimetics developed by our group inhibit the binding of Siglec-9 to its natural ligands. Our data showed that 50-fold excess of the glycomimetics over Siglec-9 chimera prevented the Siglec-9 chimera from binding to the OVCAR-3 cell line (P.value, 0.039). Additionally, we demonstrate that glycomimetics (#153 and 157) developed by our group, enhance the recognition of ovarian cancer cell targets by human NK cells. In healthy donor 1, CD107a expression was increased (21, 17, 19 %) when 0.01, 0.025, 0.05 mM concentration of glycomimetic 153 was used, respectively. A statistically significant increase (5%) in CD107a expression was observed when the NK cells were treated with 0.05 mM concentration of glycomimetic 157. At higher concentrations of glycomimetic 153 and 157, inhibition of CD107a was repeatedly observed. These experiments, therefore, suggest that there is likely a certain concentration range of the glycomimetic that can be used to overcome NK cell suppression occurring via Siglec-9. To our knowledge, these data are among the first to demonstrate that specially designed glycomimetics can be used to overcome the suppression of NK cells against ovarian cancer cells. Future studies will employ murine and humanized models to demonstrate the therapeutic potential of the Siglec-9-targeting glycopeptides in enhancing immune responses against epithelial ovarian cancer. Citation Format: Lojain I. AlJohani, Manish Patankar, Weiping Tang, Christopher Stevens, Peng Teng. Overcoming siglec-9-mediated immunosuppression by using glycomimetics that block the binding of this receptor with its natural ligands [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 1823.

Abstract 2907: Development of a CAR-NK cellular platform for cancer immunotherapy

Abstract Although Chimeric Antigen Receptor (CAR)-T cell therapy has proven to be successful in treating multiple hematological malignancies, their use is limited due to the potential life-threatening adverse events, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). CAR natural killer (CAR-NK) cells are rapidly emerging as a promising alternative cellular therapeutic with improved efficacy and reduced adverse effects. Moreover, due to the intrinsic characteristics of NK cells, whereby they lack antigen-specific cell surface receptors, CAR-NK cells offer the additional advantage of being an “off-the-shelf” product, thus satisfying the requirement for large-scale production for cancer immunotherapy, giving it the potential to reach more patients at a reduced cost. Here we outline the method to generate, expand and enrich human primary CD19 CAR-NK cells, and demonstrate their efficacy in-vitro against CD19-expressing target cancer cells. Human primary NK cells were isolated from healthy donors and transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV, as an empty vector transduced control. Transduction efficiency greater than 10% or 20% was achieved following transduction and expansion over a 7-day period in response to CD19 CAR LV or GFP LV respectively. Following sufficient expansion, cytotoxicity of the CD19 CAR-NK cells was tested in-vitro in a Tumor Killing Assay (TKA) against CD19+ (Raji) or CD19- (SKOV-3) target cells. As expected, CD19 CAR-NK cells displayed increased cytotoxicity towards CD19+ target cells when compared to non-transduced or GFP transduced controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting specific CD19 CAR mediated killing. Moreover, CD19 CAR-NK cells showed increased degranulation and IFNꝩ secretion when compared to both non-transduced and GFP-transduced controls, suggesting increased NK cell activation and cytotoxicity. Additionally, when degranulation of CD19 CAR positive and CD19 CAR negative NK populations was directly compared, the level of NK cell degranulation was analogous between the two populations, suggesting that CD19 CAR NK cells can enhance the cytotoxic activity of neighboring non-transduced cells, resulting in enhanced killing of CD19+ target cells by the total NK population present. Taken together these data demonstrate the generation, expansion and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This culture system provides the scientific community a platform to screen novel CAR-constructs and to test novel immunomodulators which could enhance NK cell expansion or functional potential to further develop new, more widely available cellular therapeutics to treat cancer. Citation Format: Karina Di Gregoli, Ravindra Mode, Kaitlin Mitchell, David Cobeta-Lopez, Sabrina de Munnik, Rene McLaughlin, Gemma Moiset, Louise Brackenbury, Lauren Schewitz-Bowers, Robert Nunan. Development of a CAR-NK cellular platform for cancer immunotherapy [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 2907.

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.

Abstract 2900: To develop a next generation NK therapy with deep and durable response by engineered CD16 enhancing NK cytotoxicity

Abstract Introduction: Allogeneic NK cell-based therapies have been demonstrated to elicit clinical efficacy against hematopoietic malignancies. Unlike T-cell therapies, allogeneic NK cells do not cause toxicities such as serious cytokine release syndrome, neurotoxicity, or graft-vs-host-disease, and are less expensive. We believe NK cell-based therapies provide an important platform for “off-shelf” allogeneic cell therapies, so we designed a panel of arming strategies to enhance NK cytotoxicity and elicit deep and durable response. CD16a is a transmembrane protein that co-localizes with CD3ζ and FcεRIγ on NK cells and binds to the Fc portion of IgG antibodies. Upon ligation, it induces a potent series of signals resulting in cytokine production and cytotoxic effector activity via ADCC. Here we use the term CD16 to refer to CD16a on NK cells. CD16 is a potent activating receptor and is necessary for NK cell cytotoxic effector, so engineered CD16 is the cornerstone of NK enhancing strategy. CD16 displays a functional allelic dimorphism generating allotypes with either a phenylalanine (F) or a valine (V) residue at amino acid position 158. CD16VV is associated with higher ADCC activity and clinical response than CD16VF and CD16FF. However, only 15% of the population is CD16VV that maximizes ADCC. Several studies reported that tumor microenvironment may cause the shedding of CD16 which suppresses ADCC though ADAM17-mediated proteolytic cleavage upon NK cell activation. Therefore, selective inhibition of ADAM17 can reduce the shedding of CD16 in NK cells. Method: We designed a library of CD16 point mutations in the cleavage region of ADAM17 to screen for ADAM17-resistant CD16 mutations to prevent CD16 shedding and improve the efficacy of NK therapy. NK92 cells lacking expression of endogenous CD16 were transduced to express wild type CD16 or CD16 mutations, then activated with PMA. The surface expression of CD16 on these cells was then examined using flow cytometry. Result: We found 3 mutations effectively blocked CD16 cleavage in cell-based assays, while wild-type CD16 and other CD16 mutations underwent a marked down-regulation of expression. We also demonstrated these 3 mutations were resistant to cleavage when expressed in the human primary NK cells and did not disrupt IgG binding. Conclusion: We developed a non-cleavable CD16 with high affinity to Fc receptor, which can be expressed in engineered NK cells to develop a next generation NK therapy to elicit more potent and durable cytotoxic response. Citation Format: Cuiqing Yang, Fuwei Jiang, Yifang Wang, Tingting Liu, Qingyang Wang, Qin Wang, Gang Ye, Renhong Tang, Zhuoxiao Cao. To develop a next generation NK therapy with deep and durable response by engineered CD16 enhancing NK cytotoxicity [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 2900.

Abstract 2915: Intratumoral co-injections of NK cells and neutralizing antibodies against the NKG2A inhibitory pathway achieve efficacious anti-tumor responses in a CD8 T cell and cDC1-dependent manner

Abstract Local intratumoral NK cell activity is known to foster adaptive T cell-mediated immunity against cancer, that conceivably is inhibited by the interactions of the inhibitory receptor NKG2A with the MHC class I molecule HLA-E in humans or Qa-1b in mice. In this study, we found that intratumoral delivery of NK cells significantly attains therapeutic effects only if co-injected with anti-NKG2A and anti-Qa1b blocking monoclonal antibodies (mAb) in two solid mouse tumors models. Such therapeutic activity was contingent on endogenous CD8 T cells and on type-1 conventional dendritic cells (cDC1), which mediate cross-priming of tumor antigens. Moreover, the anti-tumor effects can be enhanced upon combination with anti-PD-1 mAb. This combination achieves at least partial abscopal efficacy against distant untreated tumors. In xenografted mice with HLA-E-expressing human cancer cells, intratumoral co-injection of allogeneic human NK cells and the clinical-grade anti-NKG2A mAb monalizumab synergistically achieved therapeutic effects and gave rise to stronger antibody-dependent cellular cytotoxicity (ADCC) in conjunction with cetuximab. In conclusion, these studies provide evidences for the therapeutic potential of on-site NK cell-based immunotherapies that may recruit endogenous T-cell responses. Citation Format: Maite Alvarez, Maria C. Ochoa, Carmen Molina, Alvaro Teijeira, Saray Garasa, Sandra Sanchez-Gregorio, Irene Olivera, Javier Glez-Vaz, Asunta Cirella, Gabriel Gomis, Jose Gonzalez-Gomariz, Pedro Berraondo, Ignacio Melero. Intratumoral co-injections of NK cells and neutralizing antibodies against the NKG2A inhibitory pathway achieve efficacious anti-tumor responses in a CD8 T cell and cDC1-dependent manner [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 2915.

Abstract 5175: DX-002, a dual EP2 and EP4 antagonist, alters the tumor microenvironment to enhance tumor immunogenicity and responses to immune checkpoint inhibitors

Abstract Prostaglandin E2 (PGE2) generated by the cyclooxygenase (COX) pathways regulates both inflammation and tumor microenvironment (TME). Among the four E-type prostanoid receptors EP1-4, it has been demonstrated that PGE2 exhibits its immune suppression and tumorigenic function through engagement with EP2 and EP4. Mechanistically, tumor-derived PGE2 acts on EP2 and EP4 in immune cells (e.g., NK cells and dendritic cells) and elicits immunosuppression in TME1,2. Importantly, the deletion of both EP2 and EP4, but not deletion of either receptor, is required to reverse PGE2-mediated immune evasion. Furthermore, high levels of PGE2-EP2/EP4 associated inflammatory signature have been shown in multiple human cancer types to correlate with poor prognosis and worse response to immune checkpoint blockade in the clinic1. Therefore, PGE2-EP2/EP4 signaling likely acts as a key regulatory node linking inflammation and immunosuppression in TME, and its inhibition may lead to a less immunosuppressive TME and enhance the response to immune checkpoint inhibitors. COX inhibitors have been used to block PGE2 synthesis but cause cardiovascular and gastrointestinal risks due to the inhibition of all prostanoids synthesis3. Thus, therapeutically targeting both EP2 and EP4 is an effective and more selective approach to limit the immunosuppressive functions of PGE2 while avoiding potential side effects associated with COX inhibition. Here we describe the discovery of a potent and selective EP2/EP4 dual antagonist DX-002. At 10 nM concentration, it displayed >90% inhibition of PGE2 binding to EP2 or EP4, whereas no significant inhibition was seen towards PGE2 binding to EP1 or EP3. In human NK cells DX-002 reversed PGE2-mediated inhibition of IFN-γ production more potently than selective EP2 or EP4 antagonist. DX-002 has excellent pharmacokinetics profiles across all animal species. In the mouse tumor model, treatment of DX-002 produced superior tumor regression as a monotherapy over selective EP2 or EP4 antagonists. In addition, DX-002 also provided significant synergistical effect for tumor regression in combination with anti-PD1 in both immune checkpoint inhibitor insensitive and sensitive models, in association with increased tumor infiltration of CD8+ T cells. Based on its in vitro and in vivo profiles along with other properties, DX-002 is being profiled as a development candidate as a potential cancer immunotherapeutic agent. Citation Format: Lintong Li, Guorui Yao, Wei Zheng, Yongshuai Chai, Zhongying Huang, Guojian Ma, Linlin Nie, Dengfeng Xu, Jiuquan Yang, Nanxin Li. DX-002, a dual EP2 and EP4 antagonist, alters the tumor microenvironment to enhance tumor immunogenicity and responses to immune checkpoint inhibitors. [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 5175.

Recombinant Human Interleukin-2 Corrects NK Cell Phenotype and Functional Activity in Patients with Post-COVID Syndrome.

Post-COVID syndrome develops in 10-20% of people who have recovered from COVID-19 and it is characterized by impaired function of the nervous, cardiovascular, and immune systems. Previously, it was found that patients who recovered from infection with the SARS-CoV-2 virus had a decrease in the number and functional activity of NK cells. The aim of the study was to assess the effectiveness of recombinant human IL-2 (rhIL-2) administered to correct NK cell phenotype and functional activity in patients with post-COVID syndrome. Patients were examined after 3 months for acute COVID-19 of varying severity. The phenotype of the peripheral blood NK cells was studied by flow cytometry. It was found that disturbances in the cell subset composition in patients with post-COVID syndrome were characterized by low levels of mature (p = 0.001) and cytotoxic NK cells (p = 0.013), with increased release of immature NK cells (p = 0.023). Functional deficiency of NK cells in post-COVID syndrome was characterized by lowered cytotoxic activity due to the decreased count of CD57+ (p = 0.001) and CD8+ (p < 0.001) NK cells. In the treatment of patients with post-COVID syndrome with recombinant IL-2, peripheral blood NK cell count and functional potential were restored. In general, the effectiveness of using rhIL-2 in treatment of post-COVID syndrome has been proven in patients with low levels of NK cells.

Natural killer cells suppress human cutaneous squamous cell carcinoma cancer cell survival and tumor growth.

Cutaneous squamous cell carcinoma (CSCC), which develops in response to ultraviolet irradiation exposure, is among the most common cancers. CSCC lesions can be removed by surgical excision, but 4.5% of these cancers reappear as aggressive and therapy-resistant tumors. CSCC tumors display a high mutation burden, and tumor frequency is dramatically increased in immune-suppressed patients, indicating a vital role for the immune system in controlling cancer development. Natural killer cells (NK cells) play a key role in cancer immune surveillance, and recent studies suggest that NK cells from healthy donors can be expanded from peripheral blood for use in therapy. In the present study, we test the ability of ex vivo expanded human NK cells to suppress the CSCC cell cancer phenotype and reduce tumor growth. We expanded human NK cells from multiple healthy donors, in the presence of IL-2, and tested their ability to suppress the CSCC cell cancer phenotype. NK cell treatment produced a dose-dependent reduction in SCC-13 and HaCaT cell spheroid growth and matrigel invasion and induced SCC-13 and HaCaT cell apoptosis as evidenced by increased procaspase 9, procaspase 3, and PARP cleavage. Moreover, two important CSCC cell pro-cancer signaling pathways, YAP1/TAZ/TEAD and MEK1/2-ERK1/2, were markedly reduced. Furthermore, tail-vein injection of NK cells markedly suppressed the growth of SCC-13 xenograft tumors in NSG mice, which was also associated with a reduction in YAP1 and MEK1/2-P levels and enhanced apoptosis. These findings show that NK cell treatment suppresses CSCC cell spheroid formation, invasion, viability, and tumor growth, suggesting NK cell treatment may be a candidate therapy for CSCC.

Delivering mRNA to a human NK cell line, NK-92 cells, by lipid nanoparticles

In cancer immunotherapy, therapeutic methods targeting NK are highly expected. NK cell-based therapy using NK-92, a human NK cell line, has been clinically evaluated. Delivering mRNA into NK-92 cells is a potent strategy for enhancing its functions. However, the use of lipid nanoparticles (LNP) for this purpose has not yet been evaluated. We previously developed a LNP that was composed of CL1H6 (CL1H6-LNP) for the efficient delivery of siRNA to NK-92 cells, and the use of this material for delivering mRNA to NK-92 cells is reported in this study. Compared with a DLin-MC3-DMA based LNP, used as a benchmark, the CL1H6-LNP caused a high mRNA expression intensity and a cell transfection efficiency of 100%. The efficient mRNA delivery by this CL1H6-LNP is attributed to the high affinity for NK-92 cells and the intense, rapid fusion with the endosomal membrane. It therefore appears that the CL1H6-LNP could be a useful non-viral vector for modifying the NK-92 functions by mRNA. Our findings also provide some insights into the design and development of LNPs for delivering mRNA to NK-92 and NK cells.

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

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

Deciphering the localization and trajectory of human natural killer cell development.

Innate immune cells represent the first line of cellular immunity, comprised of both circulating and tissue-resident natural killer cells and innate lymphoid cells. These innate lymphocytes arise from a common CD34+ progenitor that differentiates into mature natural killer cells and innate lymphoid cells. The successive stages in natural killer cell maturation are characterized by increased lineage restriction and changes to phenotype and function. Mechanisms of human natural killer cell development have not been fully elucidated, especially the role of signals that drive the spatial localization and maturation of natural killer cells. Cytokines, extracellular matrix components, and chemokines provide maturation signals and influence the trafficking of natural killer cell progenitors to peripheral sites of differentiation. Here we present the latest advances in our understanding of natural killer and innate lymphoid cell development in peripheral sites, including secondary lymphoid tissues (i.e. tonsil). Recent work in the field has provided a model for the spatial distribution of natural killer cell and innate lymphoid cell developmental intermediates in tissue and generated further insights into the developmental niche. In support of this model, future studies using multifaceted approaches seek to fully map the developmental trajectory of human natural killer cells and innate lymphoid cells in secondary lymphoid tissues.