Natural Killer Cell-mediated Immunotherapy Research Articles

Targeting Caveolin-1 in Multiple Myeloma Cells Enhances Chemotherapy and Natural Killer Cell-Mediated Immunotherapy.

The cell membrane transport capacity and surface targets of multiple myeloma (MM) cells heavily influence chemotherapy and immunotherapy. Here, it is found that caveolin-1 (CAV1), a primary component of membrane lipid rafts and caveolae, is highly expressed in MM cells and is associated with MM progression and drug resistance. CAV1 knockdown decreases MM cell adhesion to stromal cells and attenuates cell adhesion-mediated drug resistance to bortezomib. CAV1 inhibition in MM cells enhances natural killer cell-mediated cytotoxicity through increasing CXCL10, SLAMF7, and CD112. CAV1 suppression reduces mitochondrial membrane potential, increases reactive oxygen species, and inhibits autophagosome-lysosome fusion, resulting in the disruption of redox homeostasis. Additionally, CAV1 knockdown enhances glutamine addiction by increasing ASCT2 and LAT1 and dysregulates glutathione metabolism. As a result of CAV1 inhibition, MM cells are more sensitive to starvation, glutamine depletion, and glutamine transporter inhibition, and grow more slowly in vivo in a mouse model treated with bortezomib. The observation that CAV1 inhibition modulated by 6-mercaptopurine, daidzin, and statins enhances the efficacy of bortezomib in vitro and in vivo highlights the translational significance of these FDA-approved drugs in improving MM outcomes. These data demonstrate that CAV1 serves as a potent therapeutic target for enhancing chemotherapy and immunotherapy for MM.

Death receptors 4/5 mediate tumour sensitivity to natural killer cell-mediated cytotoxicity in mismatch repair deficient colorectal cancer.

Identifying the target of natural killer (NK) cells in colorectal cancer (CRC) is critical for optimising the clinical use of NK cell-mediated immunotherapy. Mismatch repair deficiency (dMMR) is associated with high immune cell infiltration and MHC Class I defects. Whether dMMR CRC responses to NK cell therapy remains unclear. MLH1, DR4, and DR5 knockout cell lines were established using CRISPR-Cas9 system. NK92-MI or NK cell isolated from BABL/C mice were used as effector cells against tumour cells. Inflammatory cytokines secretion by CRC cells was assessed via cytokine analysis. NK-cell-deficient/proficient animal models were used to validate the NK cell sensitivity. We observed that dMMR CRC cells were more sensitive to NK cell-mediated cytotoxicity than were mismatch-repair-proficient (pMMR) CRC cells. In dMMR CRC, Death receptor (DR)4/5 was upregulated and mediated sensitivity to NK cell-mediated cytotoxicity. DR4/5-mediated secretion of interleukin -12 sustained NK cell viability in dMMR CRC. NK cell depletion induced dMMR CRC tumour growth, and NK cell transfer inhibited lung metastasis of dMMR CRC with DR4/5 expression in vivo. TP53 upregulated DR4/DR5 expression in dMMR CRC. dMMR associated with increased sensitivity to NK cell-mediated cytotoxicity in CRC. DR4/DR5 sensitise dMMR CRC to NK cell-mediated cytotoxicity.

Surface Engineering of Natural Killer Cells with CD44-targeting Ligands for Augmented Cancer Immunotherapy.

Adoptive immunotherapy utilizing natural killer (NK) cells has demonstrated remarkable efficacy in treating hematologic malignancies. However, its clinical intervention for solid tumors is hindered by the limited expression of tumor-specific antigens. Herein, lipid-PEG conjugated hyaluronic acid (HA) materials (HA-PEG-Lipid) for the simple ex-vivo surface coating of NK cells is developed for 1) lipid-mediated cellular membrane anchoring via hydrophobic interaction and thereby 2) sufficient presentation of the CD44 ligand (i.e., HA) onto NK cells for cancer targeting, without the need for genetic manipulation. Membrane-engineered NK cells can selectively recognize CD44-overexpressing cancer cells through HA-CD44 affinity and subsequently induce in situ activation of NK cells for cancer elimination. Therefore, the surface-engineered NK cells using HA-PEG-Lipid (HANK cells) establish an immune synapse with CD44-overexpressing MIA PaCa-2 pancreatic cancer cells, triggering the "recognition-activation" mechanism, and ultimately eliminating cancer cells. Moreover, in mouse xenograft tumor models, administrated HANK cells demonstrate significant infiltration into solid tumors, resulting in tumor apoptosis/necrosis and effective suppression of tumor progression and metastasis, as compared to NK cells and gemcitabine. Taken together, the HA-PEG-Lipid biomaterials expedite the treatment of solid tumors by facilitating a sequential recognition-activation mechanism of surface-engineered HANK cells, suggesting a promising approach for NK cell-mediated immunotherapy.

Natural Killer Cell Engagers (NKCEs): a new frontier in cancer immunotherapy.

Natural Killer (NK) cells are a type of innate lymphoid cells that play a crucial role in immunity by killing virally infected or tumor cells and secreting cytokines and chemokines. NK cell-mediated immunotherapy has emerged as a promising approach for cancer treatment due to its safety and effectiveness. NK cell engagers (NKCEs), such as BiKE (bispecific killer cell engager) or TriKE (trispecific killer cell engager), are a novel class of antibody-based therapeutics that exhibit several advantages over other cancer immunotherapies harnessing NK cells. By bridging NK and tumor cells, NKCEs activate NK cells and lead to tumor cell lysis. A growing number of NKCEs are currently undergoing development, with some already in clinical trials. However, there is a need for more comprehensive studies to determine how the molecular design of NKCEs affects their functionality and manufacturability, which are crucial for their development as off-the-shelf drugs for cancer treatment. In this review, we summarize current knowledge on NKCE development and discuss critical factors required for the production of effective NKCEs.

Ciclopirox olamine sensitizes leukemia cells to natural killer cell-mediated cytolysis by upregulating NKG2DLs via the Akt signaling pathway

The activating receptor natural killer group 2D (NKG2D) expressed by Natural killer (NK) cells functions as a “master-switch” in governing the awakening status of NK cells. The NKG2D-mediated cytotoxicity has been declared to be related with the expression levels of NKG2D ligands (NKG2DLs) expressed on tumor cells. Therefore, selective induction of NKG2DLs could be a reliable approach to enhance the efficacy of NK cell-mediated immunotherapy. Our existing study demonstrated that Ciclopirox Olamine (CPX), an off-patent antifungal agent, effectively elevated the expression of NKG2DLs on leukemia cells and sensitized leukemia cells to NK-cell mediated cytolysis. Induction of ROS production and AKT phosphorylation by CPX is essential for the up-regulation of NKG2DLs expressions. Inhibition of AKT by using AKT inhibitor MK2206 decreased both NKG2DLs expressions and NK cell cytotoxicity. These data indicated that increased sensitivity of CPX-treated leukemia cells to NK cell cytolysis was attributed to higher NKG2DLs expressions, resulting from activated AKT signaling pathway. Our findings support the ongoing development of CPX as an anti-tumor agent and suggest its promising immunotherapeutic value in the medication of leukemia.

Current state of NK cell-mediated immunotherapy in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has become one of the most common hematological diseases in western countries, with an annual incidence of 42/100,000. Conventional chemotherapy and targeted therapeutic drugs showed limitations in prognosis or in efficiency in high-risk patients. Immunotherapy represented is one of the most effective therapeutic approaches with the potential of better effect and prognosis. Natural killer (NK) cells are good options for immunotherapy as they can effectively mediate anti-tumor activity of immune system by expressing activating and inhibiting receptors and recognizing specific ligands on various tumor cells. NK cells are critical in the immunotherapy of CLL by enhancing self-mediated antibody-dependent cytotoxicity (ADCC), allogeneic NK cell therapy and chimeric antigen receptor-natural killer (CAR-NK) cell therapy. In this article, we reviewed the features, working mechanisms, and receptors of NK cells, and the available evidence of the advantages and disadvantages of NK cell-based immunotherapies, and put forward future study directions in this field.

IRF2 is required for development and functional maturation of human NK cells.

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.

Dysregulated Activating and Inhibitory Receptors on Natural Killer Cells Predicts Immune Escape and Poor Outcomes in Acute Myeloid Leukemia

Background: Although improvements have been made in understanding the pathophysiology of Acute Myeloid Leukemia (AML), heading to development of emerging therapies targeting leukemic stem cells, relapse is still a major problem leading to poor long-term survival and low cure rates. Of particular interest, Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms underlying the control of hematopoietic neoplasms by NK cells remains unclear and not yet explored as therapeutical options. Aims: We hypothesized that functionally impaired NK cells due to imbalanced expression of inhibitory and activating receptors can predict poor outcomes in AML. Methods: NK cell phenotypes based on the expression of DNAM-1 and NKG2D (activating) and NKG2A and KIR2DL1 (inhibitory) receptors on CD56+, CD56bright CD16- and CD56dim CD16+ NK subsets were profiled by multiparametric flow cytometry in 100 de novo AML bone marrow (BM) samples (Table 1), followed up from February 2016 to June 2022 and 17 healthy bone marrows (HBM). Correlations between NK phenotype with ELN2017 risk stratification, complete remission (CR), relapse (R), measurable residual disease (MRD), overall survival (OS) and relapse-free survival (RFS) were verified. Six BM from AML patients in remission after induction chemotherapy (AML-CR) were used to evaluate NK cell frequency and receptors recovery. Co-culture of AML-exposed CD56+ NK cells and K562 cell line was used to assay for killing (1:1, 5:1, 10:1 E:T ratios) and degranulation (CD107a). Mann Whitney, Kruskal-Wallis and mixed effects tests were performed to determine associations, and Log Rank test and Kaplan Meir curves were used for survival analysis. Results: The frequency of all NK cell subsets were decreased in AML (P < .01). AML CD56+ NK cells showed decreased expression of NKG2D (P < .01), a trending to decreased DNAM-1 and increased NKG2A (P = .04) and KIR2DL1 (P < .01), while recovering to near healthy values in AML-CR. Expression of both NKG2D and DNAM-1 activating receptors was decreased (P < .01), while inhibitory NKG2A (P = .02) and KIR2DL1 (P < .01) were considerably increased in CD56dim NK, which is known to carry out the most cytotoxic activity. The expression of both activating receptors by CD56dim subset was found to be decreased in ELN2017 adverse versus low/intermediate risk (NKG2D, P = .05; DNAM-1, P < .01), in relapse (P < .01), and with similar trends in MRD+ patients after remission induction. The opposite was observed for inhibitory receptors, found to be increased in adverse risk (P < .01), relapse (NKG2A, P < .01; KIRDL1, P < .05) and MRD+ (NKG2A, P = .03) patients. Degranulation (P < .01) and specific target cell death (5:1, P = .01; 10:1, P = .002) were significantly decreased in AML. Patients with lower frequency of CD56dim NK expressing low NKG2D and DNAM-1 showed less degranulation and specific target cell death (P = .03), whereas patients with higher inhibitory receptors demonstrated lower degranulation (NKG2A, P=0.06) and cytotoxicity (KIR2DL1, P=.005). Of note, RFS was decreased in patients with lower frequency of CD56+ NK cells (P = .02) and both OS and RFS were decreased in patients with lower frequency of CD56dim NK cells (OS, P = .04; RFS, P = .03) and in patients with lower expression of NKG2D (OS, P = .03; RFS, P = .008) and DNAM-1 (OS, P = .02; RFS, P = .11) activating receptors, strongly suggesting an imbalance between activation and inhibition of NK cell activity. Conclusion: Our data indicate that CD56dim NK show an exhaustion immunophenotype with high expression of KIR2DL1 and NKG2A inhibitory receptors and low expression of NKG2D and DNAM-1 activating receptors and that this imbalance leads to impaired cytotoxicity, compromised anti-leukemic activity and can be predictive of poor outcomes in AML, such as response to treatment, relapse and MRD positivity. Additionally, this NK phenotype at diagnosis could be considered an adverse risk prognostic factor associated with lower survival rates. Closing these gaps in the knowledge of NK-mediated immune evasion in leukemia is of significant interest for targeting the leukemic microenvironment by NK cell-mediated immunotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IMBALANCED ACTIVATING AND INHIBITORY RECEPTORS ON CD56DIM NATURAL KILLER CELLS PREDICTS POOR OUTCOMES IN ACUTE MYELOID LEUKEMIA

Even with advances in understanding the physiopathology of Acute Myeloid Leukemia (AML), most patients still relapse leading to poor long-term prognosis and cure rates. Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms by which they control hematopoietic neoplasms remain unclear. We hypothesize that functionally impaired NK cells can predict poor outcomes in AML. We analyzed the expression of DNAM-1 and NKG2D (activating) and NKG2A and KIR2DL1 (inhibitory) receptors on CD56 + , CD56 bright CD16 - and CD56 dim CD16 + NK subsets, by flow cytometry, in 100 de novo AML bone marrow (BM), followed up from 02/2016 to 06/2022, 17 healthy BM (HBM) and 6 BM from remission after induction chemotherapy (AML-CR). Correlations with ELN2017 risk, complete remission (CR), relapse (R), measurable residual disease (MRD), overall survival (OS), and relapse-free survival (RFS) were verified. Co-culture of AML-exposed NK cells and K562 cell line was used to assay for killing and degranulation. Proteomics and genomics were accessed by CITE-seq single-cell sequencing. All NK cell subsets were decreased in AML (p < .01). AML CD56+ NK cells showed decreased NKG2D (p <.01) and increased NKG2A (p = .04) and KIR2DL1 (p < .01), recovering to near healthy values in AML-CR. NKG2D and DNAM-1 activating receptors were decreased (p < .01), while inhibitory NKG2A (p = .02) and KIR2DL1 (p < .01) were increased in CD56 dim NK (Most cytotoxic subset). Both activating receptors within CD56 dim NK were decreased in ELN2017 adverse risk (NKG2D, p =.05; DNAM-1, p < .01), in relapse (p < .01), and in MRD+ patients after remission induction. Inhibitory receptors were increased in adverse risk (p < .01), relapse (NKG2A, p < .01; KIRDL1, p < .05), and MRD+ (NKG2A, p = .03) patients. Degranulation (p < .01) and target cell death (5:1, p = .01; 10:1, p = .002) were decreased in AML. Patients with lower frequency of CD56 dim NK expressing low NKG2D and DNAM-1 showed less degranulation and target cell death (p = .03), whereas in those with higher inhibitory receptors lower degranulation (NKG2A, p = .06) and cytotoxicity (KIR2DL1, p = .005) was found. Both OS and RFS were decreased in patients with lower CD56 dim NK (OS, p = .04; RFS, p = .03) and patients with lower expression of NKG2D (OS, p = .03; RFS, p = .008) and DNAM-1 (OS, p = .02; RFS, p = .11). Our single cell preliminary analysis showed that NK cells from AML display a distinct pro-inflammatory gene signature compared to HBM, which is strongly correlated to its impaired function. Our data points that CD56 dim NK shows an exhaustion phenotype with high expression of inhibitory and low expression of activating receptors. This imbalance leads to impaired cytotoxicity, compromised anti-leukemic activity, and can predict worst response to treatment, relapse, and MRD positivity, and so, can be considered an adverse risk prognostic factor associated with lower survival rates. On top of that, identifying a pro-inflammatory gene signature on AML NK cells can lead to the identification of pathways that prevent their activation and functionality, making it possible to intervene and prevent the onset of the disease. Closing these gaps in the knowledge of NK-mediated immune evasion in leukemia is of significant interest for targeting the leukemic microenvironment by NK cell-mediated immunotherapy.

MMP-2 Inhibitor-Mediated Tumor Microenvironment Regulation Using a Sequentially Released Bio-Nanosystem for Enhanced Cancer Photo-Immunotherapy.

Combining photodynamic therapy (PDT) with natural killer (NK) cell-based immunotherapy has shown great potential against cancers, but the shedding of NK group 2, member D ligands (NKG2DLs) on tumor cells inhibited NK cell activation in the tumor microenvironment. Herein, we assembled microenvironment-/light-responsive bio-nanosystems (MLRNs) consisting of SB-3CT-containing β-cyclodextrins (β-CDs) and photosensitizer-loaded liposomes, in which SB-3CT was considered to remodel the tumor microenvironment. β-CDs and liposomes were linked by metalloproteinase 2 (MMP-2) responsive peptides, enabling sequential release of SB-3CT and chlorin e6 triggered by the MMP-2-abundant tumor microenvironment and 660 nm laser irradiation, respectively. Released SB-3CT blocked tumor immune escape by antagonizing MMP-2 and promoting the NKG2D/NKG2DL pathway, while liposomes were taken up by tumor cells for PDT. MLRN-mediated photo-immunotherapy significantly induced melanoma cell cytotoxicity (83.31%), inhibited tumor growth (relative tumor proliferation rate: 1.13% of that of normal saline) in the xenografted tumor model, and enhanced tumor-infiltrating NK cell (148 times) and NKG2DL expression (9.55 and 16.52 times for MICA and ULBP-1, respectively), achieving a synergistic effect. This study not only provided a simple insight into the development of new nanomedicine for programed release of antitumor drugs and better integration of PDT and immunotherapy but also a novel modality for clinical NK cell-mediated immunotherapy against melanoma.

Bioactivity of Exosomes Derived from Trained Natural Killer Cells versus Non-Trained One: More Functional and Antitumor Activity.

Background Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system, capable of killing viral-infected and cancerous cells. NK cell-mediated immunotherapy has remarkably changed the current paradigm of cancer treatment in recent years. It emerged as a safe and effective therapeutic approach for patients with advanced-stage leukemia. Several immune-escape mechanisms can be enacted by cancer cells to avoid NK-mediated killing. Exosomes released by NK cells that carry proteins and miRNAs can exert an antitumor effect. In the present study, we hypothesized that maybe exosomes derived from trained natural killer cells show more antitumor effect in comparison to non-trained one. Methods PBMC was separated by the Ficoll method and cultured with IL-2 for 21 days to expand NK cells. The NK cells were co-cultured with K562 for 72 hours and exosome-derived co-cultured (as trained) and natural killer cell-derived exosomes (as non-trained) were extracted by Exo kit. The exosomes were confirmed by dynamic light scattering (DLS), transmission electron microscopy (TEM), flow cytometry, and western blotting. The K562 cells were separately treated by trained and non-trained exosomes and MTT assay, apoptosis, and real-time PCR were performed. Results Based on flow cytometry, CD56 marker was 89.7% and 40.1% for NK cells and NK-derived exosomes, respectively. CD63 and CD9 were positive for exosomes by western blotting. The morphology of exosome was confirmed by TEM. Treated K562 cells by trained exosomes indicated the diminished cell viability and higher apoptosis. Furthermore, the trained exosomes showed up-regulation in both P53 and caspase3 genes as compared with non-trained sample. Discussion. Trained Exos showed a potent inhibitory effect on proliferation and induced apoptosis on K562 cell lines compared to the same dose of non-trained Exos. According to the results of qRT-PCR, trained Exos exerted an antitumor activity through up-regulation of caspase 3 and P53 in the apoptotic signaling pathway in tumor cells. Our findings indicate an effective action of trained Exos against cancer cells.

P396: DYSREGULATED DNAM-1 AND KIR2DL1 EXPRESSION IN NATURAL KILLER CELLS CORRELATES WITH POOR OUTCOME IN ACUTE MYELOID LEUKEMIA

Background: Although improvements have been made in understanding the physiopathology of Acute Myeloid Leukemia (AML), most patients still relapse leading to poor long-term prognosis and cure rates. Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms underlying the control of hematopoietic neoplasms by these cells remain unclear. Aims: We hypothesized that FLT3-ITD mutation, a molecular marker of adverse prognosis, is associated with phenotypically and functionally impaired NK cells due to imbalanced expression of inhibitory and activating receptors during AML. Methods: We analyzed the expression of DNAM-1 and KNG2D activating and NKG2A, KIR2DL1 inhibitory receptors on CD56+, CD56bright CD16- and CD56dim CD16+ NK subsets and its correlation with FLT3-ITD mutation in 70 AML patients and 12 healthy bone marrows (NBM) by flow cytometry. Seven marrows from AML patients in complete remission after induction chemotherapy (AML-CR) were used to evaluate NK cell and receptor recovery. Mann Whitney or Kruskal-Wallis tests (P < 0.05) were performed using GraphPad Prism (V8.0.2). Results: A decreased frequency of all NK cell subsets was found in AML (CD56+, P=0.03; CD56bright, P=0.04, CD56dim, P<0.01). Decreased expression of NKG2D (P<0.01) and a tendency of decreasing DNAM-1 (P=0.38) was found in CD56+ NK in AML, recovering to values closer to normal in AML-CR. Expression of NKG2D was decreased in both CD56bright and CD56dim NK subsets (P=0.03 and P<0.01), while DNAM-1 was found to be considerably decreased in CD56dim cytotoxic NK (P=0.02). FLT3 mutated patients showed even lower expression of DNAM-1 (P=0.02) and NKG2D (P=0.13) in CD56+ NK. Even though the latter was not statistically significant, we observed a decreasing pattern in these patients, that was confirmed by an important decrease of both activating receptors uniquely in the CD56dim subset (DNAM-1, P=0.03; NKG2D, P=0.05), which are known to contain the most cytotoxic activity. As for the inhibitory receptors, our cohort demonstrated a trending increase in NKG2A expression on all NK subsets, although not significant, and no difference was found in FLT3 mutated patients. Meanwhile, KIR2DL1 expression was higher in CD56+, CD56bright and CD56dim NK cells in AML (P<0.01; P=0.06 and P<0.01), while AML-CR showed frequencies comparable to healthy In FLT3-ITD mutated patients, CD56dim NK had increased expression of KIR2DL1 (P=0.03), strongly correlating with an imbalance between activation and inhibition of NK cell activity. Summary/Conclusion: Our data indicate that CD56dim NK show an exhaustion immunophenotype in FLT3-ITD mutated AML, with high expression of KIR2DL1 inhibitory receptor and low expression of both NKG2D and DNAM-1 activating receptors and that this imbalance likely leads to impaired cytotoxicity, compromised anti-leukemic activity and could be contributing to worst disease outcome, which will be confirmed through functional assays. Closing these gaps in knowledge informs the fundamental characteristics of NK dysfunction during leukemia and is of significant interest for targeting, therapeutic interventions and NK cell-mediated immunotherapy.

Down-regulation of MLLT1 super elongation complex subunit impairs the anti-tumor activity of natural killer cells in esophageal cancer

Natural killer (NK) cells actively participate in anti-tumor immunity and are thus regarded as a promising tool in immunotherapy against esophageal cancer (EC). However, the mechanisms regulating NK cell activation and exhaustion have not been completely elucidated. In this study, we characterized the expression and function of MLLT1 super elongation complex subunit (MLLT1) in esophageal NK cells in a mouse EC model. MLLT1 was down-regulated in esophageal NK cells, especially NK cells expressing both T cell immunoglobulin and mucin-domain containing-3 (TIM-3) and lymphocyte activation gene3(LAG-3). In vitro knockdown of MLLT1 in NK cells resulted in significant decreases in the expression of IFN-γ and perforin, as well as impaired NK cell cytotoxicity on tumor cells. Adoptive transfer of MLLT-deficient NK cells into EC-bearing mice showed consistent impairment of NK cell anti-tumor activity, as evidenced by decreases in IFN-γ and perforin but not granzyme B. Furthermore, EC tissue cells, which were enriched from the esophagus of EC-bearing mice, induced down-regulation of MLLT1 in splenic NK cells. This down-regulation was partially restored by a TIM-3 blocking antibody. Therefore, this study indicated that TIM-3 signaling down-regulated MLLT1 in esophageal NK cells, and MLLT1 down-regulation undermined the tumoricidal function of NK cells in EC. Our study unveils a novel mechanism underlying NK cell exhaustion/dysfunction in the EC microenvironment. MLLT1 could be a potential target in future NK cell-mediated immunotherapy against EC.

Glioblastoma multiforme expresses cell surface PCNA, a potential target for NK cell-mediated immunotherapy.

Abstract Glioblastoma multiforme (GBM) is the most common form of primary brain cancer and carries a dreadful five-year survival rate of 9%. Current treatment options include surgery, chemotherapy, and radiation. Recently, there has been a move to pursue immunotherapy options to improve patient outcomes. These therapies often depend on the identification of molecular markers that are distinctive to the tumor cells. Some markers, such as HER2/Neu and EGFR, are overexpressed on a significant percentage of GBM tumors and are used as targets for immunotherapies. However, to address GBM tumors that do not overexpress HER2/Neu and EGFR, our lab set out to identify novel markers on GBM as future candidates for Natural Killer (NK) cell-mediated immunotherapy. Previously, our lab demonstrated that Lectin-like Transcript-1 (LLT1), membrane-bound Proliferating Cell Nuclear Antigen (PCNA), NKp44 Ligand (NKp44L) and CS1 (CD319) are targets of NK cell-mediated killing of various cancers. Based on these prior studies, we examined their expression on the well-known GBM cell lines LN-229 and LN-18 by flow cytometry using PE-labeled antibodies specific for each marker. PCNA was the lone marker identified to be highly expressed on both LN-229 and LN-18 cells. Based on our results, we concluded that cell surface PCNA is an ideal candidate for NK cell-mediated immunotherapy for GBM. Currently, we are evaluating blocking inhibitory signals to NK cells from the PCNA-NKp44 interaction to target GBM for NK-mediated killing. Recent findings demonstrating the ability to transiently open the blood-brain barrier further increase the feasibility of targeting GBM by NK cells in the future. Supported by funding from the Osteopathic Scholars in Cancer Research Program grant from the Cancer Prevention and Research Institute of Texas (RP170301) and UNTHSC Intramural grants.

CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity

Natural killer (NK) cells are known to mediate killing of various cancer types, but tumor cells can develop resistance mechanisms to escape NK cell-mediated killing. Here, we use a “two cell type” whole genome CRISPR-Cas9 screening system to discover key regulators of tumor sensitivity and resistance to NK cell-mediated cytotoxicity in human glioblastoma stem cells (GSC). We identify CHMP2A as a regulator of GSC resistance to NK cell-mediated cytotoxicity and we confirm these findings in a head and neck squamous cells carcinoma (HNSCC) model. We show that deletion of CHMP2A activates NF-κB in tumor cells to mediate increased chemokine secretion that promotes NK cell migration towards tumor cells. In the HNSCC model we demonstrate that CHMP2A mediates tumor resistance to NK cells via secretion of extracellular vesicles (EVs) that express MICA/B and TRAIL. These secreted ligands induce apoptosis of NK cells to inhibit their antitumor activity. To confirm these in vitro studies, we demonstrate that deletion of CHMP2A in CAL27 HNSCC cells leads to increased NK cell-mediated killing in a xenograft immunodeficient mouse model. These findings illustrate a mechanism of tumor immune escape through EVs secretion and identify inhibition of CHMP2A and related targets as opportunities to improve NK cell-mediated immunotherapy.

Liposome-based nanocomplexes with pH-sensitive second near-infrared photothermal property for combinational immunotherapy

Natural killer (NK) cell-mediated immunotherapy has shown a great promise for treatments of tumors, while some strategies that can improve its therapeutic efficacy and reduce potential adverse events are highly desired. We herein report a pH-sensitive second near-infrared (NIR-II) photothermal liposomal nanocomplex for enhanced NK cell-based immunotherapy of breast cancer. Such nanocomplex (LNCDS) consists of charge-transfer nanoparticles (CTN) with pH-sensitive NIR-II photothermal effect, deoxyribonuclease I (DNase I), NK cell stimulant (SIS3) and a surface coated thermal-responsive liposome shell. Upon 1064 nm laser irradiation, LNCDS generates mild heat in a controlled manner, which results in destruction of thermal-responsive liposome shell to allow on-demand release of stimulants and DNase I in tumor sites. The released DNase I not only kills cancer cells, but also induces immunogenic cell death (ICD), which synergizes with the action of the released SIS3 to promote activation of NK cells and cytotoxic T lymphocytes, contributing to enhanced therapeutic efficacy of immunotherapy. As a result, a single treatment of LNCDS upon NIR-II photoactivation greatly inhibits the growths of subcutaneously implanted primary and distant tumors in a breast cancer murine model, and even completely prevents lung metastasis. This study thus offers a photo-controlled drug delivery nanosystem for efficacy and precise NK cell-mediated immunotherapy of cancer.

AFM26 - Targeting B Cell Maturation Antigen (BCMA) for NK Cell-Mediated Immunotherapy of Multiple Myeloma

Significant progress in multiple myeloma (MM) treatment has been made in recent years and achieving sustained minimal residual disease (MRD) negativity now constitutes a major goal of treatment. High dose melphalan combined with autologous stem cell transplantation (ASCT) is currently regarded the standard of care in 1st line treatment of MM in eligible patients, but fails to eradicate MRD in the majority of patients ultimately leading to disease relapse. Consequently, additional therapies are needed to target residual disease to delay or potentially prevent relapse.The cell surface antigen B cell maturation antigen (BCMA, CD269) constitutes a highly attractive target antigen for immunotherapy because of its restricted expression in non-malignant tissue but almost universal expression on MM cells.AFM26 is a BCMA-targeted tetravalent bispecific antibody that selectively engages natural killer (NK) cells to induce myeloma cell lysis. Through its high-affinity bivalent binding to CD16A (FcγRIIIa), AFM26 possesses prolonged cell retention times, and has been shown to be less prone to interference by high levels of circulating IgG than antibody Fc-domains. Thus, AFM26 can be used to “arm” NK cells for effective killing of tumor cells. AFM26 also induces markedly lower levels of pro-inflammatory cytokines in peripheral blood mononuclear cell (PBMC) cultures in the presence of target cells compared to BCMA-directed T cell activating approaches. In contrast to other monoclonal antibodies (mAbs) such as daratumumab and elotuzumab, AFM26 does not confer target-independent NK cell activation or NK cell depletion.NK cells are promising effectors to target MRD. Due to their early reconstitution following ASCT and their ability to rapidly destroy malignant cells through direct cytotoxicity. Redirecting NK cells through AFM26 may therefore offer an effective treatment of MM patients early after ASCT.Employing a wide range of functional assays, we demonstrate here that AFM26 exhibits potent cytotoxic activity against primary myeloma cells using patient (autologous) and donor-derived (allogeneic) NK cells as well as against a panel of high and low BCMA expressing cell lines. In particular, we show that AFM26 induces NK cell cytotoxicity towards cells expressing almost undetectable surface levels of BCMA, suggesting the potential for broad and deep anti-MM activity. In summary, AFM26 is well-differentiated from other mAbs and a promising therapeutic candidate to address the unmet medical need of MRD positivity in MM. DisclosuresGantke:Affimed: Employment. Reusch:Affimed: Employment. Kellner:Affimed: Research Funding. Klausz:Affimed: Research Funding. Haneke:Affimed: Employment. Knackmuss:Affimed: Employment. Müller:Affimed: Employment. Ellwanger:Affimed: Employment. Fucek:Affimed: Employment. Schniegler-Mattox:Affimed: Employment. Koch:Affimed: Employment. Kerber:Affimed: Employment. Peipp:Affimed: Research Funding. Treder:Affimed: Employment.

Combined use of cisplatin plus natural killer cells overcomes immunoresistance of cisplatin resistant ovarian cancer

Standard chemotherapy for ovarian cancers is often abrogated by drug resistance. Specifically, resistance to cisplatin is a major clinical obstacle to successful treatment of ovarian cancers. The aim of this study was to develop a therapeutic strategy using natural killer (NK) cells to treat cisplatin-resistant ovarian cancers. First, we compared the responses of ovarian cancer cell line A2780 and its cisplatin-resistant counterpart, A2780cis, to treatment with cisplatin plus NK92MI cells. Although combined treatment induces apoptosis of ovarian cancer cells via ROS-dependent and -independent mechanisms, A2780cis were resistant to NK92MI cell-mediated cytotoxicity. We found that A2780cis cells showed markedly higher expression of immune checkpoint protein, PD-L1, than the parental cells. Although pretreatment of A2780cis cells with cisplatin stimulated further expression of PD-L1, it also increased expression of ULBP ligands, which are activating receptors on NK92MI cells, both in vitro and in vivo. These findings suggest that combined use of cisplatin plus NK cell-mediated immunotherapy could overcome immunoresistance of chemoresistant ovarian cancers.

Expression profiles of Natural Killer Group 2D Ligands (NGK2DLs) in colorectal carcinoma and changes in response to chemotherapeutic agents.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Natural Killer Group 2D Receptor (NKG2D) and their ligands (NKG2DLs) play crucial roles in natural killer (NK) cell-mediated cytotoxicity. Tumorigeneses cause increased NKG2DLs expression on tumor cell surfaces, thereby these cells individually eliminated by NK cells. However, CRC cells can reduce their NKG2DL expression to escape from NK-mediated immune surveillance which is associated with poor prognosis. Therefore, previous studies suggest that up-regulation of NKG2DLs can contribute to promising NK cell-mediated immunotherapy strategies. We aimed to analyze NKG2DLs expression profiles in response to chemotherapeutic drugs and increased MHC class I polypeptide-related sequence A (MICA) expression, which is related to favorable prognosis in CRC, using low doses of bortezomib and epirubicin combination without causing direct cytotoxicity. Results showed that MICA expression sligthlyincreased following drug treatment in the CRC cells but not for the normal cells. Also, we enriched our study with Gene Expression Omnibus (GEO) datasets including expression profiles of various NKG2DLs using in silico analyses. Accordingly, NKG2DL expression in CRC was screened in proportion to other cancers, histologic subtypes, TNM stages and metastatic samples to compare with our data. Overall, the analyzed data showed that NKG2DLs demonstrate different expression profiles in response to chemotherapeutic agents and a combination of low-dose bortezomib and epirubicin slightly increased MICA mRNA expression in CRC cell lines. However, performing further analysis of the combination therapy for MICA protein expression and studying its interaction with NK cells will make the results more meaningful.

Exploring the NK cell platform for cancer immunotherapy.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that are capable of killing virally infected and/or cancerous cells. Nearly 20 years ago, NK cell-mediated immunotherapy emerged as a safe and effective treatment approach for patients with advanced-stage leukaemia. Subsequently, the field of NK cell-based cancer therapy has grown exponentially and currently constitutes a major area of immunotherapy innovation. In general, the development of NK cell-directed therapies has two main focal points: optimizing the source of therapeutic NK cells for adoptive transfer and enhancing NK cell cytotoxicity and persistence in vivo. A wide variety of sources of therapeutic NK cells are currently being tested clinically, including haploidentical NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, cytokine-induced memory-like NK cells and chimeric antigen receptor NK cells. A plethora of methods to augment the cytotoxicity and longevity of NK cells are also under clinical investigation, including cytokine-based agents, NK cell-engager molecules and immune-checkpoint inhibitors. In this Review, we highlight the variety of ways in which diverse NK cell products and their auxiliary therapeutics are being leveraged to target human cancers. We also identify future avenues for NK cell therapy research.