Activated Natural Killer Cells Research Articles

MTOR signaling promotes rapid m6A mRNA methylation to regulate NK-cell activation and effector functions.

Natural killer (NK) cells can be rapidly activated in response to cytokines during host defense against malignant cells or viral infection. However, it remains unclear what mechanisms precisely and rapidly regulate the expression of the numerous genes involved in activating NK cells. In this study, we discovered that NK-cell N6-methyladenosine (m6A) methylation levels were rapidly upregulated upon short-term NK-cell activation and were repressed in the tumor microenvironment. Deficiency of methyltransferase-like 3 (METTL3) or METTL14 moderately influenced NK-cell homeostasis, while double knockout of METTL3/14 significantly impacted NK-cell homeostasis, maturation, and antitumor immunity. This suggests a cooperative role of METTL3 and METTL14 in regulating NK-cell development and effector functions. Using methylated RNA immunoprecipitation sequencing (MeRIP-seq), we demonstrated that genes involved in NK-cell effector functions, such as Prf1 and Gzmb, were directly modified by m6A methylation. Furthermore, inhibiting mTOR complex 1 (mTORC1) activation prevented m6A methylation levels from increasing when NK cells were activated, and this could be restored by S-adenosylmethionine (SAM) supplementation. Collectively, we have unraveled crucial roles for rapid m6A mRNA methylation downstream of the mTORC1-SAM signal axis in regulating NK-cell activation and effector functions.

Lingering Effects of Early Institutional Rearing and Cytomegalovirus Infection on the Natural Killer Cell Repertoire of Adopted Adolescents.

Adversity during infancy can affect neurobehavioral development and perturb the maturation of physiological systems. Dysregulated immune and inflammatory responses contribute to many of the later effects on health. Whether normalization can occur following a transition to more nurturing, benevolent conditions is unclear. To assess the potential for recovery, blood samples were obtained from 45 adolescents adopted by supportive families after impoverished infancies in institutional settings (post-institutionalized, PI). Their immune profiles were compared to 39 age-matched controls raised by their biological parents (non-adopted, NA). Leukocytes were immunophenotyped, and this analysis focuses on natural killer (NK) cell populations in circulation. Cytomegalovirus (CMV) seropositivity was evaluated to determine if early infection contributed to the impact of an atypical rearing. Associations with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), two cytokines released by activated NK cells, were examined. Compared to the NA controls, PI adolescents had a lower percent of CD56bright NK cells in circulation, higher TNF-α levels, and were more likely to be infected with CMV. PI adolescents who were latent carriers of CMV expressed NKG2C and CD57 surface markers on more NK cells, including CD56dim lineages. The NK cell repertoire revealed lingering immune effects of early rearing while still maintaining an overall integrity and resilience.

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation.

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

Abstract 6651: Compromised TGFβ signaling and DNA repair primes immune poor tumors response to immune checkpoint blockade by activating NK cells

Abstract We investigated the interplay between transforming growth factor beta (TGFβ) regulation of immunosuppressive signaling and immunogenic DNA repair. Loss of TGFβ signaling by cancer cells is accompanied by increased use of error-prone repair. We described a transcriptomic score, βAlt, that reports the TGFβ/DNA repair functional relationship. Pan-cancer analysis showed that high βAlt cancers (i.e., low TGFβ and high error-prone DNA repair) are sensitive to chemoradiation and have a greater fraction of genome altered compared to low βAlt cancers. Defective mismatch repair is associated with T cell infiltration and response to immune checkpoint blockade (ICB), hence we investigated the immune context of bAlt. Unsupervised clustering of RNAseq profiles 12 independent immunocompetent mouse tumor derived transplant (mTDT) tumors based on βAlt score indicated that high βAlt was associated with immune poor phenotype and lack of type I interferon signatures. A similar relationship was evident in the bladder, breast, and lung TCGA data. A high βAlt, immune poor mTDT in which T cells were only 20% of 10% CD45 were treated with monotherapy consisting of single fraction 10 Gy radiation, anti-PD-L1 or TGFβ inhibition, dual or triple treatment. Only monotherapy or combinations with radiation significantly increased survival. However, these ICB resistant tumors responded to triple treatment. Response was accompanied by increased tumor infiltrating CD45+ cells (P=0.02), circulating CD8 T cells (P<0.001) and natural killer (NK) cells (P<0.001). As NK cells tripled and TGFβ broadly impairs NK cell effector function, we depleted NK cells to test whether they were required for the response to triple treatment. NK depletion completely abrogated response (P<0.00001) indicating NK cells can provoke an ICB response in high βAlt immune poor tumors. Both mouse tumors and human cancers show that loss of TGFβ signaling resulting in error-prone DNA damage response is strongly associated with immune poor tumors and suggests that TGFβ inhibition can override NK cell dysfunction to create vulnerability to ICB. Citation Format: Jade T. Moore, Jim Gkantalis, Ines Guix, Mary Helen Barcellos-Hoff. Compromised TGFβ signaling and DNA repair primes immune poor tumors response to immune checkpoint blockade by activating NK cells [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 6651.

Abstract 1017: Characterizing immune cell infiltration in colorectal cancer tumor microenvironment among Native Hawaiians

Abstract Background Colorectal cancer (CRC) is recognized as a heterogeneous disease, ranking third in new cancer diagnoses and second in cancer-related deaths globally and nationally. CRC incidence is higher in the state of Hawaii compared to the U.S. overall. This study focuses on profiling immune cell infiltration in Native Hawaiian(NH)-specific CRC tumor microenvironments. Identifying the race-specific tumor infiltrating microenvironment may uncover potential mechanisms for CRC development in the NH population. Methods Deidentified, archival formalin-fixed paraffin-embedded tumor samples from Native Hawaiian patients diagnosed with primary CRC between 1990 and 2006 were obtained from the Hawaii Tumor Registry. RNA was extracted from tumor and adjacent normal tissues. RNA-seq data from Caucasian American (CA) CRC samples were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Differentially expressed genes for each cohort were identified via DESeq2 with Benjamini-Hochberg multiple testing q-value<0.05 and a cutoff of 2-fold change. From the gene expression profiles, we applied the ESTIMATE algorithm to calculate the overall immune cell infiltration score for each sample. Based on a deconvolution algorithm, known as CIBERSORT to estimate the abundance of 22 immune cell types, we profiled the tumor-infiltrating immune cells present in each cohort’s paired samples, respectively. To improve the accuracy, p-value and root mean squared error were counted for each sample. Data with p<0.05 were filtered and selected for the next analysis. The fraction of immune cell subpopulations was evaluated and compared between paired tumor and adjacent normal tissues in NH and CA cohorts. Results In the NH cohort, we found the overall immune cell infiltration score was significantly lower in cancer tissues than the adjacent normal tissues (p<0.05), and there were significantly higher fractions of activated dendritic cells, resting natural killer (NK), and follicular helper T cells in tumor tissues compared to adjacent non-tumor tissues (p<0.05). Furthermore, the fractions of activated NK cells and plasma cells were significantly lower in tumor tissues than in adjacent non-tumor tissues in NH (p<0.05). Resting NK cells are upregulated in both the NH and the CA cohorts, while plasma cells are the only immune cell type downregulated in either cohort. In the NH cohort, activated dendritic cells and follicular helper T cells are exclusively upregulated, while activated NK cells are downregulated relative to the CA cohort. Conclusion This study demonstrates that the NH cohort displays distinctive immunological variances between tumor and adjacent non-tumor tissues which distinguishes them from the CA cohort. This emphasizes the significance of acknowledging population-specific genetic variations which may help understand race specific difference in CRC. Citation Format: Yuanyuan Fu, Gino Quintal, Masaki Nasu, Mayumi Jijiwa, Sudhir K. Rai, Isam Mohd-Ibrahim, Yu Chen, Hua Yang, Zhanwei Wang, Christina Wai, Owen Chan, Brenda Y. Hernandez, Youping Deng. Characterizing immune cell infiltration in colorectal cancer tumor microenvironment among Native Hawaiians [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 1017.

Abstract 3615: Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells

Abstract Background: Neuroblastoma (NB) is a common malignant extracranial solid tumor in children, responsible for 11% of cancer deaths in pediatric patients under 15 (Smith et al, Cancer, 2014). High-risk neuroblastoma is a metastatic disease in children aged 18 months or older with L2, M, or MS disease with amplification of the MYCN oncogene (Chuang Pediatr Blood Cancer 2021). Recent analyses by COG show that HR Neuroblastoma still has a 5-year event free survival of 51% despite an extensive treatment regimen (Steven et al, ASCO, 2022). To address improved tumor targeting and overcome antigen escape, chimeric antigen receptor (CAR) technology to Natural Killer (NK) cell therapy has been adapted (Chu/Cairo, et al, Frontiers in Oncology, 2019). TGFβ is a potent mechanism of the NK cell anti-tumor resistance and is present at elevated levels in the NB tumor microenvironment (TME) (Foltz, Cancers, 2018). Studies demonstrated that blockade of TGF-β signaling in NK cells caused the accumulation of NK cells that produce IFN-γ and neutralization of TGF-β prevented NKG2D downregulation and also restored NK cell anti-tumor reactivity (Nayyar/Chu/Cairo, et al Frontierls in Oncology, 2018). ROR1, a transmembrane protein is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al Cancer Immunol Res, 2015). Objective: To determine in vitro cytotoxicity against neuroblastoma cells by ex vivo expanded TGFβ imprinting (TGFβi) NK cells. Methods: TGFβi NK cells are generated by culturing expanded NK cells with irradiated K562-41BBL-mbIL21 in 50 IU/mL IL-2 plus 10 ng/mL TGFβ every 2-3 days for two weeks. NK and TGFβi NK cells specific in-vitro cytotoxicity were compared via luciferase reporter assay against ROR1+ NB cell lines as we previously described (Chu/Cairo, et al, JITC, 2022). Cytokine/chemokine levels secreted by NK and TGFβi NK cells were compared using ELISA assays (Chu/Cairo, et al, JITC, 2022). Results: We have successfully expanded TGFβi NK cells. These TGFβi NK cells showed significantly enhanced in vitro cytotoxicity against NB cells compared to NK cells under TGFβ environment (20+2.4% vs 0%; p<0.001) at E:T=3:1 and consistently, TGFβ-imprinting NK secreted significantly high level of TNF-alpha (393.7pg/ml+16.77pg/ml) against NB cells (p<0.001) compared to NK cells (79.7pg/ml+14.7pg/ml) under TGFβ environment. Conclusion: Our data indicate TGFβi NK cells may be a promising novel therapy to overcome the immunosuppression of TGFβ in the tumor microenvironment for pediatric NB tumors. We will further engineer these TGFβi NK cells to express anti-ROR1 CAR to enhance the targeting specificity against NB tumors and investigate the anti-tumor effects of anti-ROR1 CAR TGFβi NK cells against NB tumors in-vitro and in-vivo (Funded by U54 CA232561). Citation Format: Jessica A. Ayala-Cuesta, Yaya Chu, Keira Foley, Meijuan Tian, Kayleigh Klose, Alyssa S. Mendelowitz, Timothy P. Cripe, Dean A. Lee, Mitchell S. Cairo. Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells [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 3615.

Abstract 2717: Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy

Abstract Many cancer cells upregulate the expression of programmed death ligand 1 (PD-L1) to surpass T-cell mediated immune response. Immune checkpoint blockers targeting PD-L1 or its receptor PD1 have been shown to suppress the inhibitory signals and enhance T cell function. As a new immunotherapy, immune checkpoint blockers have been used to treat multiple cancers. However, most clinically available blockers only engage with one type of immune cell: the T cells. Natural killer (NK) cells, on the other hand, play a significant role in anti-cancer immune responses. It will represent an advancement in anti-cancer immunotherapy if NK cells are engaged with immune checkpoint blockers. In this study, we designed a unique bifunctional fusion gene, PDL1sFv/MICAe, consisting of the variable regions of an anti-PD-L1 antibody and the extracellular domain of stress protein MICA and hypothesized that fusion protein PDL1sFv/MICAe will simultaneously bind to PD-L1 on tumor cells to block the inhibitory signal and extend T cells activity, and to NKG2D to activate NK Cells. cDNA sequence of fusion gene PDL1sFv/MICAe was designed based on the variable region of an established clinically used antibody, Atezolizumab, and the extracellular domain of MICA with linkers in between. After a computational confirmation of its structure and binding affinities, the fusion gene was synthesized and cloned into an expressing vector, and the fusion protein was produced for evaluation. Western blotting and fluorescent microscopic analyses confirmed that the protein binds to PD-L1 of human tumor cells, including MCF7 and MDAMB231, and NKG2D of KHYG1 cells. Preliminary results also show that the fusion protein enhances NK cell cytotoxicity against tumor cells. Studies on confirming the fusion protein’s activities on other tumor cells, NK cell IFN-γ production, T cell function, etc., are underway. In summary, fusion protein PDL1sFv/MICAe represents an improved version of the immune checkpoint blockers and could treat cancer patients quickly and effectively. Citation Format: Junyi Li, Yanzhang Wei. Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy [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 2717.

Abstract 5122: Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma

Abstract Neuroblastoma (NB) is an often-lethal childhood solid tumor. Anti-GD2 immunotherapy improves survival but not all responses are durable. Transforming growth factor-beta (TGFβ) has been implicated in solid tumor immunoresistance. High-risk NBs harbor activated MYC or MYCN to coordinately expand intratumoral polyamines (PA), oncometabolites with immunomodulatory roles, to support tumor progression. Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the rate-limiting enzyme in PA synthesis, and has shown activity in preclinical NB models, including TH-MYCN+/+ transgenic mice, and is in clinical trials in children. We sought to augment anti-GD2 therapy using DFMO and the small molecule TGFβ-receptor inhibitor, galunisertib. We randomized TH-MYCN+/+ mice in two experiments (10 therapy arms; n=6-10 mice/arm) to receive combinations of DFMO, galunisertib, and anti-GD2 antibody m14G2a and to assess impact on survival. We measured 770 genes using nCounter Immuno-Oncology 360 (Nanostring), 36 cytokines using xMap bead-arrays (Luminex), and tumor-infiltrating leukocytes using flow cytometry in harvested tumors. Cell line TGFβ signaling was evaluated in tissue culture. In vitro, galunisertib consistently suppressed the upregulated TGFβ-Smad2/3 signaling cascade in MYCN-amplified neuroblastoma cell lines. In vivo, DFMO alone extends mouse survival and increases intra-tumoral neutrophils and natural killer (NK) cells. These NK cells increased surface expression of activation receptors like NKG2D (p<0.05), while tumor cells increased surface expression of NK-ligands. Tumor cells also expressed increased latency-associated peptide (LAP; p<0.05), a TGFβ precursor, suggesting NK cells were poised to kill but restrained by TGFβ. Cytotoxicity assays showed reversal of this inhibition with TGFβR1 inhibitor galunisertib in vitro. In a proof-of-concept trial, DFMO and galunisertib (DFMO/gal) demonstrated a clinical anti-tumor synergistic effect. DFMO/gal treated tumors had upregulated NK cell activity genes and downregulated TGFβ signaling genes. Cytokine analysis confirmed DFMO induced upregulation of TGFβ1 and TGFβ2 (p<0.01) that was modulated by galunisertib. IL6, GCSF, and GMCSF were markedly downregulated in tumors treated with both DFMO and galunisertib (p<0.05). An expanded trial showed DFMO/gal added to m14G2a showed no significant survival benefit over the DFMO and m14G2a combination. Ultimately, DFMO creates a pro-inflammatory cellular and cytokine milieu in TH-MYCN+/+ NBs. DFMO and TGFβR1 inhibition via galunisertib activate NK cells further and decrease the anti-inflammatory cytokine IL6. Ongoing studies are further characterizing the gene expression and cytokine levels of the NB TME under these treatment conditions. Citation Format: Om H. Gandhi, Christina S. Turn, Kangning Liu, Annette T. Vu, Hamid Bassiri, Michael D. Hogarty. Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma [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 5122.

Abstract 5786: Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC

Abstract Introduction: The prevailing histologic subtype of nasopharyngeal carcinoma (NPC) in the endemic regions is non-keratinizing carcinoma which shows almost consistent association with latent infection by Epstein-Barr virus (EBV). Previously our group developed a series of Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes, which can disrupt the EBV latency maintained by EBNA1, and such lytic induction could alert the immune cells. Methods: Natural killer (NK) cells of the innate immunity play a significant role in controlling EBV infection. In this study, we investigated whether our EBNA1 probes (ZRL5P4 & L2P4) could activate NK cells to eliminate NPC. Results: Studies of clinical specimens indicated that tumor infiltration of the cytotoxic NK cell subset is a favorable prognostic marker for NPC patients. Furthermore, the cytotoxicity of circulating NK cells derived from NPC patients is equally well the cells from healthy individuals. If those patients’ NK cells can be attracted to infiltrate into the tumors, the tumor burden should be relieved. Indeed, the NPC xenograft with the ZRL5P4 treatment showed heavy mouse NK cell infiltration and tumor size reduction. The essential role of those mouse NK cells was demonstrated. Next, we showed that ZRL5P4 could induce the production of a soluble factor by NPC cells to stimulate the NK cell cytotoxicity. Such NK cell activity was verified in a metastasis NPC model. Mechanistically, the EBNA1 probe treatment could induce the expression of EBV early lytic gene Rta and EBNA1 itself. Both Rta and EBNA1 are known transcription factors to drive the expression of LMP1, the NF-κB signaling was then activated, which turned on the production of the well-known NK cell activation cytokine IFN-β. Conclusion: Taken together, our results demonstrated that our EBNA1 probes can mobilize NK cells to the tumor sites and activate their anti-tumor activity via the EBV reactivation and stimulation of IFN-β production. Citation Format: Luo Chen, Xiao Han Liu, Hei Tung Yuen, William C. Cho, Ka-Leung Wong, Hong Lok Lung. Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC [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 5786.

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells [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 1338.

Immunotherapy Using Activated Natural Killer Cells Improves Postoperative Neutrophil-to-Lymphocyte Ratio and Long-Term Prognosis of Living Donor Liver Transplant Recipients With Hepatocellular Carcinoma

ObjectivePreoperative neutrophil-to-lymphocyte ratio (NLR) is a well-known prognostic indicator in various malignancies; however, the impact of postoperative NLR on living donor liver transplant (LDLT) recipients is unknown. Immunotherapy with donor liver-derived activated natural killer (NK) cells may improve postoperative NLR by coactivating immune cells or suppressing activated neutrophils. This study aims to clarify the clinical significance of postoperative NLR in recipients after LDLT with HCC and assess whether immunotherapy improves postoperative NLR. MethodsWe conducted a retrospective study of LDLT recipients between 2001 and 2022 to evaluate the clinical significance of postoperative NLR. Furthermore, the correlation between postoperative NLR and the activation marker of infused NK cells was also evaluated. The postoperative NLR was examined 4 weeks after LDLT. ResultsThe postoperative high NLR group (N = 78) had preoperative lower NLR and higher model for end-stage liver disease and a higher rate of postoperative infection within 30 days after LDLT than the postoperative low NLR group (N = 41). Postoperative high NLR (hazard ratio [HR], 2.62; 95% confidence interval [CI], 1.01-6.79; P = .047) and nontreatment of immunotherapy (HR, 3.10; 95% CI, 1.33-7.22; P < .01) were independent risk factors for poor overall survival in multivariate analysis. Furthermore, the activation marker of infused NK cells is inversely correlated with decreased postoperative NLR. ConclusionsThe higher level of postoperative NLR was independently associated with poor prognosis in patients after LDLT with HCC. Immunotherapy using activated NK cells may improve postoperative NLR and long-term prognosis.

Efficacy and safety of natural killer cells injection combined with XELOX chemotherapy in postoperative patients with stage III colorectal cancer in China: a prospective randomised controlled clinical trial study protocol

BackgroundColorectal cancer (CRC) is the second most frequently diagnosed cancer and the fifth leading cause of cancer-related death in China. However, resistance to multiple chemotherapeutics after surgery leads to failure...

Preclinical Characterization of the Anti-Leukemia Activity of the CD33/CD16a/NKG2D Immune-Modulating TriNKET® CC-96191.

Increasing efforts are focusing on natural killer (NK) cell immunotherapies for AML. Here, we characterized CC-96191, a novel CD33/CD16a/NKG2D immune-modulating TriNKET®. CC-96191 simultaneously binds CD33, NKG2D, and CD16a, with NKG2D and CD16a co-engagement increasing the avidity for, and activation of, NK cells. CC-96191 was broadly active against human leukemia cells in a strictly CD33-dependent manner, with maximal efficacy requiring the co-engagement of CD16a and NKG2D. A frequent CD33 single nucleotide polymorphism, R69G, reduced CC-96191 potency but not maximal activity, likely because of reduced CD33 binding. Similarly, the potency, but not the maximal activity, of CC-96191 was reduced by high concentrations of soluble CD33; in contrast, the soluble form of the NKG2D ligand MICA did not impact activity. In the presence of CD33+ AML cells, CC-96191 activated NK cells but not T cells; while maximum anti-AML efficacy was similar, soluble cytokine levels were 10- to >100-fold lower than with a CD33/CD3 bispecific antibody. While CC-96191-mediated cytolysis was not affected by ABC transporter proteins, it was reduced by anti-apoptotic BCL-2 family proteins. Finally, in patient marrow specimens, CC-96191 eliminated AML cells but not normal monocytes, suggesting selectivity of TriNKET-induced cytotoxicity toward neoplastic cells. Together, these findings support the clinical exploration of CC-96191 as in NCT04789655.

Allogeneic NK cells induce the in vitro activation of monocyte-derived and conventional type-2 dendritic cells and trigger an inflammatory response under cancer-associated conditions.

Natural killer (NK) cells are innate lymphocytes capable to recognize and kill virus-infected and cancer cells. In the past years, the use of allogeneic NK cells as anti-cancer therapy gained interest due to their ability to induce graft-versus-cancer responses without causing graft-versus-host disease and multiple protocols have been developed to produce high numbers of activated NK cells. While the ability of these cells to mediate tumor kill has been extensively studied, less is known about their capacity to influence the activity of other immune cells that may contribute to a concerted anti-tumor response in the tumor microenvironment (TME). In this study, we analyzed how an allogeneic off-the-shelf cord blood stem cell-derived NK-cell product influenced the activation of dendritic cells (DC). Crosstalk between NK cells and healthy donor monocyte-derived DC (MoDC) resulted in the release of IFNγ and TNF, MoDC activation, and the release of the T-cell-recruiting chemokines CXCL9 and CXCL10. Moreover, in the presence of prostaglandin-E2, NK cell/MoDC crosstalk antagonized the detrimental effect of IL-10 on MoDC maturation leading to higher expression of multiple (co-)stimulatory markers. The NK cells also induced activation of conventional DC2 (cDC2) and CD8+ T cells, and the release of TNF, GM-CSF, and CXCL9/10 in peripheral blood mononuclear cells of patients with metastatic colorectal cancer. The activated phenotype of MoDC/cDC2 and the increased release of pro-inflammatory cytokines and T-cell-recruiting chemokines resulting from NK cell/DC crosstalk should contribute to a more inflamed TME and may thus enhance the efficacy of T-cell-based therapies.

Abstract B032: Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers

Abstract We investigated the interplay between immunosuppressive TGFβ signaling and immunogenic DNA repair in response to immune checkpoint inhibition (ICI) in a new model of breast cancer. Transforming growth factor beta (TGFβ) is a canonical suppressor. Cancer cells evade TGFβ either by selectively overcoming TGFβ proliferative barrier or completely shutting down signaling. Loss of TGFβ signaling is accompanied by high immunosuppressive TGFβ activity. We have shown that loss of TGFβ is critical to genomic integrity. It endorses classical DNA repair and inhibits error-prone repair. We described a score, βAlt, that reports the TGFβ/DNA repair functional relationship. Pan-cancer analysis showed that high βAlt cancers, in which the TGFβ target signature is low and DNA repair signature is high, use error-prone DNA repair, are sensitive to chemoradiation and have a greater fraction of the genome altered compared to low βAlt cancers, which maintain TGFβ signaling. We established a novel murine tumor derived transplant model (mTDT) of triple-negative breast cancer, similar to a human PDX, in immunocompetent BALB/cJ mice. Transcriptomics analysis showed that mTDT range from high to low βAlt and from immune poor to immune rich, which was confirmed by multiplex immunstaining. Unsupervised clustering of mTDT based on βAlt score demonstrated that low βAlt was associated with immune rich tumors while high βAlt was associated with immune poor tumors. A similar relationship is evident in the TCGA breast cancers. We then asked how mTDT respond to ICI. Mice bearing mTDT were randomized to monotherapy consisting of single fraction 10 Gy radiation, anti-PD-L1 or TGFβ inhibition, dual or triple treatment. Neither anti-PD-L1 or TGFβ inhibition affected survival, alone or in combination, whereas radiation significantly increased survival. Unexpectedly immune poor, high βAlt mTDT were the most responsive. To evaluate biological correlates, mice were stratified as a function of treatment as responders, in which tumor growth was controlled for 7 days post-treatment, or non-responders, in which tumor growth was uncontrolled. Tumor infiltrating CD45+ cells increased (P=0.02) as did circulating CD8 T cells (P&amp;lt;0.001) in responders. Natural killer (NK) cells were also significantly expanded in responders (P&amp;lt;0.001). TGFβ broadly downregulates chemokine receptor expression necessary to recruit NK cells and impairs NK cell cytotoxic effector function. We depleted NK cells to test whether they were necessary for the pronounced response to triple treatment. NK depletion was completely abrogated response (P&amp;lt;0.00001) indicating NK cells can provoke an ICI response in high βAlt mammary tumors devoid of lymphocytes. This preclinical model and human breast cancer shows that loss of TGFβ signaling resulting in error-prone DNA damage response is strongly associated with immune poor tumors, and suggests that TGFβ inhibition can override NK cell dysfunction to create vulnerability to ICI and anti-tumor immunity. Citation Format: Jade Moore, Jim Gkantalis, Ines Guix, Colin Foster, Mary Helen Barcellos-Hoff. Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B032.

Abstract B040: Trispecific killer engagers against IGF1R and fetal form of insulin receptor induce human natural killer cell mediated killing of hormone receptor positive breast cancer in vitro and in vivo

Abstract Patients with hormone receptor-positive (HR+) breast cancers frequently recur with metastatic disease 5-20 years after the initial diagnosis and completion of chemotherapy and prolonged treatment with hormonal therapy. Thus, patients with metastatic HR+ breast cancer need new therapeutic options. A potential mechanism of therapeutic resistance in HR+ patients is the presence of slow-growing, well-differentiated cancer cells that result in clinically detectable metastases after a prolonged latency. One way to eliminate these cancer cells is with immunotherapy (IT). Unfortunately HR+ breast cancers have not responded to current T-cell based IT. The objective of this study is to target HR+ breast cancers using a natural killer (NK) cell based approach that targets cell surface receptors in HR+ breast cancer. The type I IGF receptor (IGF1R) is expressed on HR+ breast cancers and its expression is tightly related to ER function. Endocrine therapy resistance is associated with increased expression of the A-isoform or fetal form of IR (IR-A) and depend on IR signaling. We hypothesized that a drug that would target NK cells to IGF1R or IR-A expressing HR+ cells will be effective in killing them. We generated IGF1R or IR-A specific Tri specific Killer Engagers (TriKE) that leverage expression of IGF1R and IR-A on HR+ cells to cause NK directed death. The IGF1R TriKE consists of humanized single domain antibody (VHH) sequence of a CD16 nanobody to engage and activate NK cells, a humanized VHH of a nanobody against IGF1R to target HR+ breast cancer cells, and an IL-15 molecule between them to drive NK cell expansion and survival. The IGF1R TriKE did not bind embryonal fibroblasts from IGF1R knock out mice (R- cells) but bound R-/IGF1R cells (R- cells engineered to express human IGF1R) and to IGF1R+ MCF-7 and T47D breast cancer cells indicating specific binding to IGF1R on cells. It did not cause downregulation of IGF1R levels in multiple HR+ breast cancer cells and did not inhibit signaling via IGF1R. It specifically induced NK cell degranulation, measured by surface CD107a expression and intracellular IFNγ, against IGF1R+ target breast cancer cells (MCF-7, T-47D, ZR-75-1) in a functional assay compared to the anti-IGF1R nanobody. It did not induce degranulation of NK cells against IGF1R negative cells suggesting that the functionality was specific for IGF1R expressing targets. The IGF1R TriKE enhanced cell killing of HR+ breast cancer cells as measured by cytotoxicity assays. IGF1R TriKE combined with human NK cells inhibited tumor growth of MCF-7 tumors in NSG mice compared to control groups. Human NK cells persisted in peripheral blood three weeks after final NK infusion based on flow cytometry assessment. The IR-A TriKE incorporated a novel engineered non-immunoglobulin IR-A specific peptide binder. Our preliminary data show that the IR-A TriKE degranulated NK cells against breast cancer cells overexpressing IR-A. These data indicate that this strategy could be an effective treatment for HR+ metastatic breast cancer and make them responsive to IT. Citation Format: Courtney Baar, Emily Chiu, Yvette Soignier, Behiye Kodal, Jeffrey S Miller, Martin Felices, Deepali Sachdev. Trispecific killer engagers against IGF1R and fetal form of insulin receptor induce human natural killer cell mediated killing of hormone receptor positive breast cancer in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B040.

Identification and Analysis of Gene Biomarkers for Ovarian Cancer.

Objective: To identify potential diagnostic markers for ovarian cancer (OC) and explore the contribution of immune cells infiltration to the pathogenesis of OC. Methods: As the study cohort, two gene expression datasets of human OC (GSE27651 and GSE26712, taken as the metadata) taken from the Gene Expression Omnibus (GEO) database were combined, comprising 228 OC and 16 control samples. Analysis was performed to identify the differentially expressed genes between the OC and control samples, while support vector machine analysis using the recursive feature elimination algorithm and least absolute shrinkage and selection operator regression were performed to identify candidate biomarkers that could discriminate OC. In addition, immunohistochemistry staining was performed to verify the diagnostic value and protein expression levels of the candidate biomarkers. The GSE146553 dataset (OC n = 40, control n = 3) was used to further validate the diagnostic values of those biomarkers. Further, the proportions of various immune cells infiltration in the OC and control samples were evaluated using the CIBERSORT algorithm. Results: CLEC4M, PFKP, and SCRIB were identified as potential diagnostic markers for OC in both the metadata (area under the receiver operating characteristic curve [AUC] = 0.996, AUC = 1.000, AUC = 1.000) and GSE146553 dataset (AUC = 0.983, AUC = 0.975, AUC = 0.892). Regarding immune cell infiltration, there was an increase in the infiltration of follicular helper dendritic cells, and a decrease in the infiltration of M2 macrophages and neutrophils, as well as activated natural killer (NK) cells and T cells in OC. CLEC4M showed a significantly positive correlation with neutrophils (r = 0.57, p < 0.001) and resting NK cells (r = 0.42, p = 0.0047), but a negative correlation with activated dendritic cells (r = -0.33, p = 0.032). PFKP displayed a significantly positive correlation with activated NK cells (r = 0.36, p = 0.016) and follicular helper T cells (r = 0.32, p = 0.035), but a negative correlation with the naive B cells (r = -0.3, p = 0.049) and resting NK cells (r = -0.41, p = 0.007). SCRIB demonstrated a significantly positive correlation with plasma cells (r = 0.39, p = 0.01), memory B cells (r = 0.34, p = 0.025), and follicular helper T cells (r = 0.31, p = 0.04), but a negative correlation with neutrophils (r = -0.46, p = 0.002) and naive B cells (r = -0.48, p = 0.0012). Conclusion: CLEC4M, PFKP, and SCRIB were identified and verified as potential diagnostic biomarkers for OC. This work and identification of the three biomarkers may provide guidance for future studies into the mechanism and treatment of OC.

CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation

BackgroundThe antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric...

CTLA-4 Blockade of Natural Killer Cells Increases Cytotoxicity against Acute Lymphoid Leukaemia Cells Neda.

There is interest in using cytotoxic T lymphocyte antigen-4 (CTLA-4) immunotherapy to treat blood cancers. Unfortunately, patients with acute lymphoblastic leukaemia (ALL) frequently exhibit resistance to treatment and natural killer (NK) cell exhaustion. This study aims to increase the cytotoxic potency of natural killer cells by using CTLA-4 to block the Nalm-6 leukaemia cell line. In this experimental study, NK cells were purified from the peripheral blood mononuclear cells (PBMCs) of 10 healthy people and assessed by flow cytometry for purity and viability. The purified cells were activated overnight at 37°C and 5% CO2 with interleukin-15 (IL-15, 10 ng/ml) followed by evaluation of expressions of CTLA-4, activating and inhibitory receptors, and the release of interferon gamma (IFN-γ) and granzyme B (GZM B). CTLA-4 expression on NK cells from recurrent ALL patients was also evaluated. Finally, the cytotoxic activity of NK cells was assessed after the CTLA-4 blockade. The purity of the isolated cells was 96.58 ± 2.57%. Isolated NK cells activated with IL-15 resulted in significantly higher CTLA-4 expression (8.75%, P<0.05). Similarly, CTLA-4 expression on the surface of NK cells from patients with ALL was higher (7.46%) compared to healthy individuals (1.46%, P<0.05). IL-15 reduced NKG2A expression (P<0.01), and increased expressions of NKP30 (P<0.05) and NKP46 (P<0.01). The activated NK cells released more IFN-γ (P<0.5) and GZM B (P<0.01) compared to unactivated NK cells. Blockade of CTLA-4 enhanced the NK cell killing potential against Nalm-6 cells (56.3%, P<0.05); however, IFN-γ and GZM B levels were not statistically different between the blocked and non-blocked groups. Our findings suggest that CTLA-4 blockage of Nalm-6 cells causes an increase in antitumour activity of NK cells against these cells. Our study also provides evidence for the potential of cancer immunotherapy treatment using blocking anti-CTLA-4 mAbs.

Networked Cluster Formation via Trigonal Lipid Modules for Augmented Ex Vivo NK Cell Priming.

Current cytokine-based natural killer (NK) cell priming techniques have exhibited limitations such as the deactivation of biological signaling molecules and subsequent insufficient maturation of the cell population during mass cultivation processes. In this study, we developed an amphiphilic trigonal 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) lipid-polyethylene glycol (PEG) material to assemble NK cell clusters via multiple hydrophobic lipid insertions into cellular membranes. Our lipid conjugate-mediated ex vivo NK cell priming sufficiently augmented the structural modulation of clusters, facilitated diffusional signal exchanges, and finally activated NK cell population with the clusters. Without any inhibition in diffusional signal exchanges and intrinsic proliferative efficacy of NK cells, effectively prime NK cell clusters produced increased interferon-gamma, especially in the early culture periods. In conclusion, the present study demonstrates that our novel lipid conjugates could serve as a promising alternative for future NK cell mass production.