Memory-like NK Cells 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.

Single-cell transcriptome sequencing reveals altered peripheral blood immune cells in patients with severe tuberculosis

Tuberculosis is a serious global health burden, resulting in millions of deaths each year. Several circulating cell subsets in the peripheral blood are known to modulate the host immune response to Mycobacterium tuberculosis (Mtb) infection in different ways. However, the characteristics and functions of these subsets to varying stages of tuberculosis infection have not been well elucidated. Peripheral blood immune cells (PBICs) were isolated from healthy donors (HD group), individuals with mild tuberculosis (MI group), and individuals with severe tuberculosis (SE group). CD4+ naive T cells and CD8+ T cells were decreased in the SE and MI groups, while CD14+ monocytes were increased in the SE group. Further analysis revealed increased activated CD4+ T cells, transitional CD8+ T cells, memory-like NK cells, and IGHG3highTTNhighFCRL5high B cells were increased in all patients with tuberculosis (SE and MI group). In contrast, Th17 cells, cytotoxic NK cells, and cytotoxic CD4+ T cells were decreased. Moreover, the increase of CD14+CD16+ monocytes correlated with severe tuberculosis, and the GBP5highRSAD2high neutrophils were unique to patients with severe tuberculosis. Cellular communication analysis revealed that CD8+ T cells exhibited the highest incoming interaction strength in the SE group. The increased CD8+ T cell incoming interactions are associated with the MHC-I and LCK pathways, with HLA-(A-E)-CD8A, HLA-(A-E)-CD8B, and LCK-(CD8A+CD8B) being ligand–receptor pairs. Patients with tuberculosis, especially severe tuberculosis, have profound changes in peripheral blood immune cell profiles. CD8+ T cells showed the highest incoming interaction strength in patients with severe tuberculosis, with the main signals being MHC-I and LCK pathways.

Inhibition of MICA and MICB shedding enhances memory-like NK-cell–mediated cytotoxicity against multiple myeloma

Inhibition of MICA and MICB shedding enhances memory-like NK-cell–mediated cytotoxicity against multiple myeloma

Blockade of the TIGIT-CD155/CD112 axis enhances functionality of NK-92 but not cytokine-induced memory-like NK cells toward CD155-expressing acute myeloid leukemia

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients, we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence, we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition, we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays, CD69, CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155, one important TIGIT-ligand, is reliably expressed on AMLs, we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally, our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype, whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively, our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.

Cytokines drive the formation of memory-like NK cell subsets via epigenetic rewiring and transcriptional regulation.

Activation of natural killer (NK) cells with the cytokines interleukin-12 (IL-12), IL-15, and IL-18 induces their differentiation into memory-like (ML) NK cells; however, the underlying epigenetic and transcriptional mechanisms are unclear. By combining ATAC-seq, CITE-seq, and functional analyses, we discovered that IL-12/15/18 activation results in two main human NK fates: reprogramming into enriched memory-like (eML) NK cells or priming into effector conventional NK (effcNK) cells. eML NK cells had distinct transcriptional and epigenetic profiles and enhanced function, whereas effcNK cells resembled cytokine-primed cNK cells. Two transcriptionally discrete subsets of eML NK cells were also identified, eML-1 and eML-2, primarily arising from CD56bright or CD56dim mature NK cell subsets, respectively. Furthermore, these eML subsets were evident weeks after transfer of IL-12/15/18-activated NK cells into patients with cancer. Our findings demonstrate that NK cell activation with IL-12/15/18 results in previously unappreciated diverse cellular fates and identifies new strategies to enhance NK therapies.

NK-cell receptor modulation in viral infections.

Natural killer (NK) cells play a crucial role in controlling viral infections. The ability to kill infected cells without prior immunization, yet being tolerant to self, healthy cells, depends on the balance of germ-line encoded surface receptors. NK-cell receptors are divided into either activating, leading to activation of NK cell and its cytotoxic and pro-inflammatory activity, or inhibitory, providing tolerance for a target cell. The signals from inhibitory receptors dominate and NK-cell activation requires stimulation of activating receptors. In viral infections, NK-cell interaction with infected cells can result in activation, memory-like NK-cell differentiation, or NK-cell exhaustion, which constitutes one of the viral immune evasion mechanisms. All of these states are associated with the modulation of NK-cell receptor expression. In this review, we summarize the current knowledge of NK-cell receptors and their role in viral infection control, as well as the alterations of their expression observed in acute or chronic infections. We present recently discovered SARS-CoV-2-mediated modulation of NK-cell receptor expression and compare them with other human viral infections. Finally, since modulation of NK-cell receptor activation gives a promising addition to currently used antiviral therapies, we briefly discuss the clinical significance and future perspective of the application of agonists or antagonists of activating and inhibitory receptors, respectively. In sum, our review shows that although much is known about NK-cell receptor biology, a deeper understanding of NK-cell receptors role in viral infections is still needed.

Memory-like differentiation enhances NK cell responses against colorectal cancer

ABSTRACT Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.

Impaired NK cell-mediated antiviral responses to HIV following BCG vaccination: implications for neonatal vaccination

Abstract Bacillus Calmette-Guérin (BCG) vaccine is the only approved vaccine against tuberculosis (TB). BCG has been shown to elicit nonspecific protection as well as detrimental effects during diseases unrelated to TB. We investigate the impact of BCG on modulating NK cell responses and its effect on altering susceptibility to HIV acquisition. The C57BL/6 mouse model was used to compare splenic NK cell responses of control (n=4) and BCG vaccinated mice (n=4) following subsequent exposure to Mycobacterium bovis (Mtb) and HIV Gag peptide antigens. Statistical comparisons were made using Wilcoxon ranked sum test. The data acquired though flowcytometry suggested that mature, BCG trained memory-like (KLRG1+ Ly49H+) NK cells showed increased proinflammatory cytokine responses and polyfunctionality upon re-exposure to Mtb antigens. However, when these trained cells were exposed to HIV Gag antigen, we observed a significant reduction in cytokine production and polyfunctionality. Specifically, these BCG trained NK cells showed marked decrease in IFNγ, TNFα, IL1β and Granzyme B production alone or in combination (p<0.05). Furthermore, negative correlations between NK polyfunctionality and mature BCG trained NK cells were observed, following HIV Gag peptide exposure. This suggests that NK cells are impaired in their antiviral effects during HIV exposure, soon after BCG vaccination. This may suggest higher risk of HIV acquisition in BCG vaccinated neonates that are frequently exposed to HIV.

Multifactorial determinants of NK cell repertoire organization: insights into age, sex, KIR genotype, HLA typing, and CMV influence.

Polymorphisms in the KIR and HLA genes contribute to the diversity of the NK cell repertoire. Extrinsic factors also play a role in modifying this repertoire. The best example is cytomegalovirus, which promotes the expansion of memory-like NK cells. However, the mechanisms governing this phenotypic structure are poorly understood. Furthermore, the influence of age and sex has been understudied. In this study, we examined these parameters in a cohort of 200 healthy volunteer blood donors, focusing on the major inhibitory KIR receptors and CD94/NKG2A, as well as the differentiation marker CD57 and the memory-like population marker NKG2C. Flow cytometry and two joint analyses, unsupervised and semi-supervised, helped define the impact of various intrinsic and extrinsic markers on the phenotypic structure of the NK cell repertoire. In the KIR NK cell compartment, the KIR3DL1 gene is crucial, as unexpressed alleles lead to a repertoire dominated by KIR2D interacting only with HLA-C ligands, whereas an expressed KIR3DL1 gene allows for a greater diversity of NK cell subpopulations interacting with all HLA class I ligands. KIR2DL2 subsequently favors the KIR2D NK cell repertoire specific to C1/C2 ligands, whereas its absence promotes the expression of KIR2DL1 specific to the C2 ligand. The C2C2Bw4+ environment, marked by strong -21T motifs, favors the expansion of the NK cell population expressing only CD57, whereas the absence of HLA-A3/A11 ligands favors the population expressing only NKG2A, a population highly represented within the repertoire. The AA KIR genotype favors NK cell populations without KIR and NKG2A receptors, whereas the KIR B+ genotypes favor populations expressing KIR and NKG2A. Interestingly, we showed that women have a repertoire enriched in CD57- NK cell populations, while men have more CD57+ NK cell subpopulations. Overall, our data demonstrate that the phenotypic structure of the NK cell repertoire follows well-defined genetic rules and that immunological history, sex, and age contribute to shaping this NK cell diversity. These elements can contribute to the better selection of hematopoietic stem cell donors and the definition of allogeneic NK cells for cell engineering in NK cell-based immunotherapy approaches.cters are displayed correctly.

Vaccination with a combination of STING agonist-loaded lipid nanoparticles and CpG-ODNs protects against lung metastasis via the induction of CD11bhighCD27low memory-like NK cells

BackgroundNatural killer (NK) cells are effective in attacking tumor cells that escape T cell attack. Memory NK cells are believed to function as potent effector cells in cancer immunotherapy. However, knowledge of their induction, identification, and potential in vivo is limited. Herein, we report on the induction and identification of memory-like NK cells via the action of a combination of a stimulator of interferon genes (STING) agonist loaded into lipid nanoparticles (STING-LNPs) and cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs), and the potential of the inducted memory-like NK cells to prevent melanoma lung metastasis.MethodsThe antitumor effects of either the STING-LNPs, CpG-ODNs, or the combination therapy were evaluated using a B16-F10 lung metastasis model. The effect of the combined treatment was evaluated by measuring cytokine production. The induction of memory-like NK cells was demonstrated via flow cytometry and confirmed through their preventative effect.ResultsThe combination of STING-LNPs and CpG-ODNs tended to enhance the production of interleukin 12 (IL-12) and IL-18, and exerted a therapeutic effect against B16-F10 lung metastasis. The combination therapy increased the population of CD11bhighCD27low NK cells. Although monotherapies failed to show preventative effects, the combination therapy induced a surprisingly strong preventative effect, which indicates that CD11bhighCD27low cells could be a phenotype of memory-like NK cells.ConclusionAs far as could be ascertained, this is the first report of the in vivo induction, identification, and confirmation of a phenotype of the memory-like NK cells through a prophylactic effect via the use of an immunotherapeutic drug. Our findings provide novel insights into the in vivo induction of CD11bhighCD27low memory-like NK cells thus paving the way for the development of efficient immunotherapies.Graphical

Persistence of a Skewed Repertoire of NK Cells in People with HIV-1 on Long-Term Antiretroviral Therapy.

HIV-1 infection greatly alters the NK cell phenotypic and functional repertoire. This is highlighted by the expansion of a rare population of FcRγ- NK cells exhibiting characteristics of traditional immunologic memory in people with HIV (PWH). Although current antiretroviral therapy (ART) effectively controls HIV-1 viremia and disease progression, its impact on HIV-1-associated NK cell abnormalities remains unclear. To address this, we performed a longitudinal analysis detailing conventional and memory-like NK cell characteristics in n = 60 PWH during the first 4 y of ART. Throughout this regimen, a skewed repertoire of cytokine unresponsive FcRγ- memory-like NK cells persisted and accompanied an overall increase in NK surface expression of CD57 and KLRG1, suggestive of progression toward immune senescence. These traits were linked to elevated serum inflammatory biomarkers and increasing Ab titers to human CMV, with human CMV viremia detected in approximately one-third of PWH at years 1-4 of ART. Interestingly, 40% of PWH displayed atypical NK cell subsets, representing intermediate stages of NK-poiesis based on single-cell multiomic trajectory analysis. Our findings indicate that NK cell irregularities persist in PWH despite long-term ART, underscoring the need to better understand the causative mechanisms that prevent full restoration of immune health in PWH.

Abstract 5231: NK and T cells armed with EVE16, a novel synthetic immune receptor, mediate robust anti-tumor responses through dual direct cytotoxicity and ADCC with minimal trogocytosis and exhaustion

Abstract Despite the major advances, cancer patients treated with targeted chimeric antigen receptor (CAR)- based T and NK cell therapies often face the challenge of frequent disease relapse, and their efficacy in solid tumors is low. This is attributed to the inherent potency of the starting cell material, unique characteristics of CAR engineering resulting in trogocytosis and tonic signaling, and the notable absence of homogenously expressed surface markers, particularly in solid tumors. To address these limitations, we engineered a novel synthetic immune receptor termed Enhanced-Valency Engineered CD16A (EVE16) and evaluated it in memory-like NK cells and T cells against liquid and solid tumors. We hypothesized that post-activation cleavage of CD16A by ADAM17 plays a crucial role in reducing trogocytosis and tonic signaling. EVE16 utilizes the inherent potential of CD16A to trigger an activation signaling cascade through the endogenous CD3ζ adaptor signaling protein. Unexpectedly, we discovered that, aside from the CD16A transmembrane domain, the Fc-binding domain is essential for proper surface expression. After multiple rounds of optimization, we developed a receptor that surpassed the activation potency of endogenous CD16A and allowed both direct binding to the tumor targets and retained antibody-dependent cellular cytotoxicity (ADCC), and this dual 'CAR-like' and ADCC function opens the potential for combination with clinically approved antibodies to prevent immune escape. Furthermore, in contrast to the conventional CAR(BBζ), the optimized EVE16 receptor exhibited minimal tonic signaling in both T and NK cells. This translated to enhanced sensitivity towards low antigen-density targets, as demonstrated by a cytotoxicity assay performed on polyclonal cancer cells engineered with target antigen knock-down. Additionally, EVE16, but not CAR transduced, protected T and NK cells from trogocytosis and allowed for the efficient disengagement of cytotoxic lymphocytes from cancer targets due to CD16A cleavage by ADAM17 upon immune cell activation. In in vivo mouse xenograft models, CD19-targeted EVE16 memory-like NK cells, rapidly manufactured using our approach, demonstrate outstanding performance in terms of robust expansion, prolonged persistence, and effective leukemic clearance, underscoring the effectiveness of our method. In summary, we here describe a novel synthetic immune receptor called EVE16 which enables both direct (CAR-like) and ADCC functionality while preventing trogocytosis and exhaustion. Clinical trials are being planned for the near future. Citation Format: Alaa Ali, James D. Ham, Faten Zairi, Vaishvi Patel, Mubin Tarannum, Khanhlinh Dinh, Yasmin Abdulhamid, Juliana Vergara, Robert Soiffer, Jerome Ritz, John Beadle, Nikola A. Ivica, Jianzhu Chen, Rizwan Romee. NK and T cells armed with EVE16, a novel synthetic immune receptor, mediate robust anti-tumor responses through dual direct cytotoxicity and ADCC with minimal trogocytosis and exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5231.

Donor-Derived Memory-like NK Cells for the Treatment of Children and Young Adults with Relapsed AML Following Allo-HCT

Donor-Derived Memory-like NK Cells for the Treatment of Children and Young Adults with Relapsed AML Following Allo-HCT

Immunoregulatory functions and therapeutic potential of natural killer cell-derived extracellular vesicles in chronic diseases.

Extracellular vesicles (EVs) have been proven to play a significant immunoregulatory role in many chronic diseases, such as cancer and immune disorders. Among them, EVs derived from NK cells are an essential component of the immune cell functions. These EVs have been demonstrated to carry a variety of toxic proteins and nucleic acids derived from NK cells and play a therapeutic role in diseases like malignancies, liver fibrosis, and lung injury. However, natural NK-derived EVs (NKEVs) have certain limitations in disease treatment, such as low yield and poor targeting. Concurrently, NK cells exhibit characteristics of memory-like NK cells, which have stronger proliferative capacity, increased IFN-γ production, and enhanced cytotoxicity, making them more advantageous for disease treatment. Recent research has shifted its focus towards engineered extracellular vesicles and their potential to improve the efficiency, specificity, and safety of disease treatments. In this review, we will discuss the characteristics of NK-derived EVs and the latest advancements in disease therapy. Specifically, we will compare different cellular sources of NKEVs and explore the current status and prospects of memory-like NK cell-derived EVs and engineered NKEVs.

WU-NK-101 (W-NK), a Memory-like (ML) NK Cell, Intrinsically Overcomes Factors Restricting Adoptive Cell Therapy (ACT) in Acute Myeloid Leukemia (AML)

ML-NK cells are generated from conventional (c) NK cells activated with cytokines IL-12/15/18 which results in enhanced fitness, in vivo persistence, and function against tumor targets. While effective in early phase clinical trials, a 1 donor to 1 recipient cell product hinders commercial development as a cellular therapeutic. Here, W-NK is a cytokine-reprogrammed, expanded, cryopreserved ML-NK product derived from healthy human peripheral blood (PBMC) cNK cells, with scalable manufacturability. W-NKs harness the benefits of ML-NKs, and further overcome challenges previously reported for adoptive cell therapy (ACT) in AML: antigen escape, limited tumor trafficking/infiltration, and restricted function in tumor microenvironment (TME). W-NK characterization was performed by scRNA-seq (10X Genomics) and multidimensional flow and mass cytometry.Functionality was evaluated by in vitro cytotoxicity assays against AML cell lines (THP-1, and HL-60) and bioenergetics assays (Seahorse XF), under different conditions: conventional (N) media (pH 7, glucose 11 mM), or two models for TME (TME-aligned media - pH 5.9, glucose 6 mM, immune suppressive agents, e.g., PGE2, TGFb1, and malignant ascites), and in hypoxia (0.1% O 2; AVATAR). In vivo efficacy was evaluated in NSG mice bearing THP-1 AML tumors. Proteomic data was acquired by tandem-mass spectrometry (Sciex Zeno TOF 7600). Cell trafficking was measured by fluorescent-labeled W-NK in NSG mice. W-NK cells are transcriptionally distinct from cNKs. Sc-RNA-seq analysis revealed important transcriptional and compositional differences compared to cNK cells, including novel NK clusters expressing metabolic ( GLUL), proliferation-associated ( Ki67) and IL-15-inducible genes ( TXNIP) known to enhance NK responses. Additionally, W-NKs express higher levels of activating receptors ( i.e. 2B4, DNAM-1, NKG2D, NKp30, etc.), cytotoxic effector proteins ( i.e. granzyme B), and lower levels of inhibitory receptors leading to increased potency in vitro against HL-60 cells (mEC50 1.7 and 5.2; p= 0.032; W-NK vs. cNK respectively) and demonstrated a dose dependent reduction of tumor burden in THP-1 xenografts ( p=0.015). W-NK also express high levels of CXCR4 (81.6% positive, MFI 5552), contributing to improved bone marrow (BM) homing (16.5 ± 2.4%) compared to data previously reported for IL-2-expanded NK cells (3%; Sato et al.CCR. 2020). Once in the TME, unlike cNK and T-cells, W-NK cytotoxic function is preserved (TME-media vs. N-media-; W-NK: 96.5% vs. 88.2%, p=0.45; cNK: 44.1% vs.13.5%, p=0.009; T-cell: 53.2 % vs. 25.2%, p=0.041, respectively). This is potentially due to W-NK resistance to hypoxia, and improved metabolic fitness/flexibility. W-NK cytotoxic activity was also preserved in hypoxic conditions (90% vs. 89.5%, p=0.2) compared to cNK (24% vs. 6.9%, p=0.02; normoxia vs. hypoxia). Metabolically, W-NK express higher levels of cell surface nutrient transporters, implying better adaptation to adverse TMEs ( i.e. upregulation of amino acids, lactate, and pyruvate). This might enable utilization of diverse macronutrients, activation of metabolic pathways, and optimal ATP manufacturing, and is supported by upregulation of REACTOME pathways capturing amino-acid and lipid metabolism, as well as mitochondrial function. Lastly, W-NK did not attack cells from healthy human tissues including PBMCs, indicating that W-NK are not indiscriminate killers. This observation would predict low myelosuppression and a favorable safety profile. In summary, W-NK are effective against AML cells, robustly home to the BM, overcome hypoxia, immunosuppression, and nutritional scarcity to survive/function within the leukemia TME. Moreover, they are not restricted to a single antigen target thereby mitigating antigen escape. These features, which overcome challenges for ACT, herald the promise of NK cell therapy and augur positively for patients being treated with W-NK in a Phase 1 study in R/R AML (NCT# 05470140).

Mechanistic Studies of Cytokine Release Syndrome (CRS) with Roles of Interferon-Gamma (IFN-g) and Tumor Necrosis Factor Alpha (TNF-α) While Maintaining CAR-T Function in Vitro

Background: Chimeric Antigen Receptor (CAR) T cell therapy in patients with hematologic malignancies is limited by immune-related toxicities such as Cytokine Release Syndrome (CRS) in over 30% of patients, whereas no CRS was observed in patients receiving NK cell therapy. CRS is characterized by an early phase with fever, hypotension, and elevations of cytokines, including IFN-γ, GM-CSF, TNF-α , IL-10, and IL-6. Methods: To study the mechanisms of CRS, we developed a model of in vitro CRS assay in which different effector cells, tumor target cells, and immature dendritic cells (iDC) were co-cultured, and the induction of inflammatory cytokines was measured by ELISA. To generate immature dendritic cells (iDC) for the assay, monocytes were cultured in the presence of GM-CSF and IL-4 for 4 to 6 days with the addition of fresh cytokines every 2 or 3 days, and the assay was set up on day 5 of iDC culture. This “priming” step was essential for the production of IL-6 by iDC (confirmed by scRNA-seq) after exposure of CART to target cells. Effector cells including CAR-T cells (targeting CD19 or BCMA), CAR- Memory-Like NK cells (CAR-ML-NK), and CAR invariant NKT (CAR-iNKT) cells were evaluated in our model. Results: In our initial in vitro CRS assay, we conducted an experiment by using CART19 (19-28BBζ, 25,000 cells), which was co-cultured with CD19+ tumor (Ramos CBR/GFP, 50,000 cells) and iDC (5000 cells) for 24 to 48hrs. Secreted IL-6, a surrogate marker of CRS, was determined using a human IL-6 ELISA. In vitro killing efficacy of CART19 was determined by bioluminescent imaging (BLI) 24 hrs post co-culture of CART19 with Ramos CBR/GFP targets. A marked induction of CRS-related cytokines (IL-6, IFNγ, TNFα) was observed 24 and 48 hr post-coculture. Similar results were also found when we used CAR-T cell targeting BCMA with BCMA+ MM.1S target and iDC. In contrast, neglibile levels of IL-6, IFNγ, and TNFα secretion were detected when BCMA CAR Memory-Like Natural Killer cells (MLNK) were used compared to BCMA CAR T cells in spite of comparable levels of in vitro killing of BCMA+ MM.1S target cells by both BCMA-CART and BCMA-CAR-MLNK. With the BCMA CAR-iNKT, levels of IL-6, IFN-γ, and TNF-α were intermediate to those of BCMA CAR T cells and BCMA-CAR-MLNK again in spite of identical killing of BCMA+ targets in vitro. To test the effect of kinase inhibitors (Amatya et al, Blood,140:12686) and neutralizing antibodies to block CRS (IL-6 secretion by iDCs) and their impact on CAR-T cell killing, we performed the CRS assay in the presence or absence of small JAK1/2 inhibitors and PI3Kg/d inhibitors (ruxolitinib and duvelisib) and neutralizing antibodies (against IFN-γ, TNF-α, GM-CSF). Both ruxoliltinib and duvelisib and neutralizing antibodies (anti-IFN-γ and anti-TNF-α but not GM-CSF) significantly reduced IL-6 levels (Fig 1). In contrast to dasatinib which completely blocked both CRS and CART killilng (data not shown), no significant attenuation of CAR-T killing efficacy was found with any of these kinase inhibitors or the neutralizing antibodies. The CART CRS assay was adapted to measure both CRS (IL-6 secretion) and target cell killing in response to BiTEs such as blinatumomab. Similar to CART19 blinatumomab induced a dose-dependent increase in IL-6 secretion in the presence of T cells, target Ramos cells, and iDC. CRS (in vitro production of IL-6) was dependent on the presence of GM-CSF/IL-4 “primed” iDC and abolished by duvelisib while maintaining blinatumomab-induced Ramos killing (Fig 2). Finally, we also developed and in vitro mouse CRS assay by coculturing mouse CART19, target (A20 CBR/GFP) cells and mouse bone marrow-derived macrophages. Marked increase in secreted mouse IL-6 was observed which was completely inhibited by duvelisib, ruxolitinib, anti-IFNγ and anti-TNFα. Conclusion: We have developed in vitro CRS co-culture models using cells from both man and mouse and tested the role of multiple effector cell types including CART cells, CARMLNK cells, and CAR-iNK cells and multiple inhibitors on CRS and CART function in vitro. We show that JAK1/2 and PI3Kg/d kinase inhibitors as well as neutralizing antibodies to IFNγ and TNFα all block in vitro CRS without attenuating the anti-tumor efficacy of CAR-T cells. Our result suggests IFNγ, TNFα, JAK1/2 and PI3Kg/d kinases inhibitors are rational targets for CRS mitigation approaches without compromising CART-mediated anti-tumor efficacy in the clinic.

Expansion, Persistence, and Characteristics of Autologous, Bhv-1100 Armored Memory-like NK Cells Infused Prior to Autologous Stem Cell Transplant in MRD+ Multiple Myeloma Patients: A First-in-Human Trial

Introduction/Background Autologous stem cell transplant (ASCT) improves MRD negativity and prolongs progression-free survival in patients with multiple myeloma (MM) in their first or second remission following induction chemotherapy. MM NK cells are dysfunctional, negatively impacting outcomes. BHV-1100, a novel Antibody Recruiting Molecule ( ARM), binds to CD38 and recruits NK cells for antibody-dependent cell cytotoxicity (ADCC) without inducing fratricide. Allogeneic, cytokine induced memory-like (CIML) NK cells effectively treat myeloid disorders, however, it is not known if autologous CIML NK cells, when coated with BHV-1100, would further improve ASCT outcomes in MM. Methods We designed a first-in-human study of autologous CIML NK cells coated ex-vivo with BHV-1100 for MRD+, MM patients undergoing ASCT following first or second remission. NK cells were isolated from non-mobilized leukapheresis on day -1 (prior to melphalan for HCT) using CD3 depletion followed by CD56 positive selection with Miltenyi's CliniMACS. The NK cells were incubated overnight (12-16 hours) with IL-12 (10ng/ml), IL-15 (100ng/ml), and IL-18 (50ng/ml) to induce CIML differentiation, washed and subsequently coated with BHV-1100 for one hour prior to infusion. The product was infused fresh on D0 after standard melphalan 200 mg/m2 myeloablative conditioning and followed by stem cell infusion. Low dose IL2 (1 mIU/m 2) was administered SQ starting on D+1, QOD for a total of 7 doses. Results This is an ongoing trial (NCT04634435) with a median follow-up of 191 days. We are herein reporting data on the in vivo expansion and functional characterization of ARMored CIML NK for the first 5 patients. CIML NK cells were manufactured with a 100% success rate and infused at a target dose of 5-10x10 6 cells/kg-body weight, 24 hours after 200 mg/m 2 melphalan administration. Patients received between 3.9-6.0x10 6/Kg stem cells. Engraftment based on recovery of neutrophil count occurred on D+12-D+14. There was a 3-fold expansion of NK cells in the peripheral blood from D+7 (from 12% to 42%) to D+28 that persisted until D+60 (25% total PBMC, Fig. 1A). Most expanded NK cells were CD56 dim, CD16 high, KIR high and CD57 high. CD57 and KIR expression increased over time from D+7 to D+60, whereas NKG2A expression decreased, indicating the expansion of mature, activated, and cytotoxic NK cells. Regulatory T cells increased by D+7 (3% vs 15% total PBMC) and returned to baseline after D+14 most likely reflecting the effect of IL-2 treatment. The functional capacity of the infused product was tested in vitro against MOLP8 MM cell line. The BHV-1100 ARMored CIML NK cells showed increased IFN (53% vs 48% at 0H and 53% vs 44% at 24H) and CD107a (Fig.1B) (26% vs 13% at 0H and 34% vs 15% at 24H) production compared to untreated CIML NK cells and the product was stable for 24 hours. Conclusion Autologous, BHV-1100 ARMored CIML NK cells have enhanced anti-MM activity as well as expand and persist in vivo peaking at D+28 after infusion. This represents an innovative approach to boost autologous cancer immunosurveillance in the context of ASCT. Aside from anticipated infusion reactions, no severe/unexpected adverse events were noted; longer follow-up is required to assess safety and efficacy.

WU-NK-101 (W-NK), a Memory-like (ML) NK Cell, Naturally Overcomes Tumor Microenvironment (TME) Metabolic Challenges, Retaining Anti-Tumor Potency

Cellular engineering revolutionized adoptive cell therapy (ACT) through improved antigen recognition and cellular activation. Cancer cells are metabolically active and extensively consume nutrients from the TME, rendering it nutrient-deprived, acidic, and hypoxic, and resulting in intratumoral metabolic demands which negatively impact ACT antitumor activity. This is central to T cells, however, some of these challenges can be overcome through engineering, an ideal solution would be to have a cellular product that is inherently adept at overcoming these challenges. W-NK is a cytokine-reprogrammed, expanded, cryopreserved, ML-NK cell product derived from healthy human peripheral blood mononuclear cells; it comes with naturally effective potency towards tumor cell killing, and importantly can overcome metabolic challenges present in the TME. We aim at deciphering what metabolic features make W-NK particularly potent in adverse TME. W-NK characterization of metabolic fitness/adaptability was with transcriptomics (sc-RNA-seq; 10X Genomics) flow cytometry, bioenergetics (Seahorse Real-Time Cell Metabolic Analysis; Agilent), and proteomic (Sciex Zeno TOF 7600 tandem-mass spectrometry). The aforementioned assays evaluated the effect of differing media/TME on W-NK: 1) conventional (N) media (pH 7, glucose 11 mM); 2) TME-aligned media (pH 5.9, glucose 6 mM, immune suppressive agents, e.g., PGE2, TGFb1); and 3) ascites derived from patients with malignancy. Media composition was analyzed biochemically using clinical assays (Core Lab Clinical Studies at Washington University, St. Louis, USA), and by immune secretome profiling (Nomic Bio, Quebec, CA). At baseline, W-NK cells have a unique metabolic phenotype, with higher expression of cell surface nutrient transporters, e.g., GLUT1, CD98, ASCT2, MCT1 and PiT1, compared to conventional NK cells (cNK); additionally, W-NK metabolism was consistent with aerobic (“Warburg”) glycolysis, with ~80% of ATP coming from glycolysis vs. ~30% for cNK. In TME/ascites media, W-NK surface nutrient receptor expression adapted to meet metabolic demand. For example, transporters for amino acids, lactate and pyruvate are upregulated in these hypoglycemic medias. This coincided with a 50% shift in ATP manufacturing from glycolysis to mitochondria, suggesting that nutrients bypassed glycolysis to enter tricarboxylic acid cycle and oxidative phosphorylation. Proteomic analyses of W-NK cells cultured with either TME-aligned media or ascites supported this hypothesis, with up-regulation of metabolic pathways (ShinyGO) capturing amino-acid and lipid metabolism, and mitochondrial function. Overall, W-NK retained cytotoxic function in TME and ascites, as well as in native TME-aligned 3D assays from primary surgical tumor samples. Conversely, cNKs did not adapt to TME media, whether in nutrient transporter expression or ATP manufacturing, and demonstrated reduced survival and function, including cytotoxicity. W-NK has enhanced metabolic fitness, flexibility, and plasticity. This enables W-NK to utilize diverse macronutrients, engage different metabolic pathways, and maintain optimal ATP manufacturing. Overall, W-NK inherently survives and maintains function in the TME, a limiting factor for immune cell-based ACT. These data herald the promise of NK cell therapy; a Phase 1 clinical study of W-NK in acute myeloid leukemia is currently open and enrolling patients (NCT# 05470140).

Mechanisms of Cytokine-Induced NK Cell Therapy: IL-12/15/18 Induce a Unique Transcriptional, Epigenetic, and Functional Human NK Cell Memory Program

NK cells are innate lymphoid cells critical for mediating anti-tumor responses and are being advanced as a cancer immunotherapy. While conventional (c) NK cell-based therapies have been explored in clinical trials, challenges related to cancer target recognition, in vivo persistence, and functionality result in limited activity. To overcome these challenges, NK cell memory programs have been investigated. Cytokine-induced memory-like (CIML or ML) NK cells are generated after brief activation with IL-12, IL-15, and IL-18, resulting in enhanced functionality, cytotoxicity, and in vivo persistence. ML NK cells have been advanced in multiple clinical trials for leukemia with promising results. Despite this translational progress, the underlying cellular dynamics and molecular mechanisms of ML reprogramming remain unclear. Elucidating these molecular programs may optimize NK therapeutics and reveal new translational strategies. We hypothesized that transcriptional and epigenetic mechanisms contribute to ML NK cell heterogeneity arising after IL-12/15/18 activation. To test this, NK cells from healthy donors were activated with IL-12/15/18 (to become ML) or IL-15 (to maintain cNK) and differentiated in vitro for 7 days (Fig 1, n=3-40 donors), followed by the assessment of their transcriptome (CITE-seq) and epigenome (ATAC-seq). CITE-seq clustering revealed unexpected heterogeneity with ~50% clustering with cNK cells (effector, effcNK) and ~50% forming a unique enriched ML cluster (eML). eML clusters were significantly increased in frequency in the IL-12/15/18 condition, compared to cNK (Mean±SEM=53±10.5% for IL-12/15/18, 6.2±1.1% for IL-15, p<0.0001) and discovered to expressed ENTPD1 (CD39). Based on CITE-seq, a flow cytometry strategy was developed to distinguish eML NK cells from effector cNK and cNK cells. Next, we identified eML NK cells 3-4 weeks after adoptive transfer of IL-12/15/18 activated donor NK cells across three different clinical trials (mean=13.3±1.9%, p<0.001). eML NK cells produced significantly more IFNg compared to cNK cells and effector cNK cells when stimulated with leukemia targets (Fig. 2, p<0.0001), and had enhanced cytotoxicity compared to cNK cells (EC50 of eML=0.862, EC50 of cNK=2.223, p<0.0001). Bulk ATAC-seq on flow-sorted populations revealed that eML NK cells have a distinct global epigenetic profile with >4,500 significant differentially accessible regions (DARs) compared to effcNK, while effcNK and cNK cells were epigenetically similar. Further, we interrogated which transcription factors are enriched in DARs of eML NK cells revealing TBOX and IRF family motifs. Within CITE-seq, the eML cluster was comprised of two subsets (eML-1 and eML-2). eML1 had increased expression of the transcription factors TCF7 and TOX2. In contrast, eML-2 had increased expression of IRF4 and PRDM1, as well as terminal exhaustion genes, including LAG3 and TIGIT (p<0.05). To evaluate eML-1 and eML-2 function, we performed CITE-seq after stimulation of eML NK cells with leukemia targets. eML-1 NK cells were the most functional, with significantly increased IFNG compared to eML-2, while eML-2 was hypofunctional and marked by high TOX expression (p<0.05). These findings suggest that optimizing strategies that lead to eML-1 differentiation may enhance the therapeutic activity of IL-12/15/18-induced ML NK cells. In summary, we identify heterogeneity following IL-12/15/18 activation of NK cells, resulting in multiple cellular fates. Further, these data identify an eML NK cell population with a distinct single-cell transcriptional and epigenetic program that directly manifests as enhanced NK cell functional response to leukemia.

P-004 Expansion, persistence, and characteristics of autologous, BHV-1100 ARMored memory-like NK cells infused prior to autologous stem cell transplant in MRD+, newly diagnosed multiple myeloma patients

P-004 Expansion, persistence, and characteristics of autologous, BHV-1100 ARMored memory-like NK cells infused prior to autologous stem cell transplant in MRD+, newly diagnosed multiple myeloma patients