Dysfunctional natural killer cells can be reprogrammed to regain anti-tumor activity.

Natural killer (NK) cells are critical to the innate immune system, as they recognize antigens without prior sensitization, and contribute to the control and clearance of viral infections and cancer. However, a significant proportion of NK cells in mice and humans do not express classical inhibitory receptors during their education process and are rendered naturally "anergic", i.e., exhibiting reduced effector functions. The molecular events leading to NK cell anergy as well as their relation to those underlying NK cell exhaustion that arises from overstimulation in chronic conditions, remain unknown. Here, we characterize the "anergic" phenotype and demonstrate functional, transcriptional, and phenotypic similarities to the "exhausted" state in tumor-infiltrating NK cells. Furthermore, we identify zinc finger transcription factor Egr2 and diacylglycerol kinase DGKα as common negative regulators controlling NK cell dysfunction. Finally, experiments in a 3D organotypic spheroid culture model and an in vivo tumor model suggest that a nanoparticle-based delivery platform can reprogram these dysfunctional natural killer cell populations in their native microenvironment. This approach may become clinically relevant for the development of novel anti-tumor immunotherapeutic strategies.

Research Progress of Nanomaterials Acting on NK Cells in Tumor Immunotherapy and Imaging.

The prognosis for cancer patients has declined dramatically in recent years due to the challenges in treating malignant tumors. Tumor immunotherapy, which includes immune target inhibition and chimeric antigen receptor cell treatment, is currently evolving quickly. Among them, natural killer (NK) cells are gradually becoming another preferred cell immunotherapy after T cell immunotherapy due to their unique killing effects in innate and adaptive immunity. NK cell therapy has shown encouraging outcomes in clinical studies; however, there are still some problems, including limited efficacy in solid tumors, inadequate NK cell penetration, and expensive treatment expenses. Noteworthy benefits of nanomaterials include their chemical specificity, biocompatibility, and ease of manufacturing; these make them promising instruments for enhancing NK cell anti-tumor immune responses. Nanomaterials can promote NK cell homing and infiltration, participate in NK cell modification and non-invasive cell tracking and imaging modes, and greatly increase the effectiveness of NK cell immunotherapy. The introduction of NK cell-based immunotherapy research and a more detailed discussion of nanomaterial research in NK cell-based immunotherapy and molecular imaging will be the main topics of this review.

Ex Vivo Expanded Cord Blood Natural Killer Cells Combined with Rituximab and High-Dose Chemotherapy and Autologous Stem Cell Transplantation for B Cell Non-Hodgkin Lymphoma.

Relapse is the major cause of failure of high-dose chemotherapy (HDC) with autologous stem cell transplantation (ASCT) for B cell non-Hodgkin lymphomas (B-NHL). Improvement strategies include use in combination with effective immunotherapies. We hypothesized that the combination of rituximab/HDC/ASCT with expanded cord blood (CB)-derived natural killer (NK) cells is safe and active in B-NHL. Patients with B-NHL age 15 to 70 years and appropriate ASCT candidates were eligible for the study. The CB units were selected without considering HLA match with the recipient. The CB NK cells were expanded from day -19 to day -5. Treatment included rituximab on days -13 and -7, BEAM (carmustine/etoposide/cytarabine/melphalan) on days -13 to -7, lenalidomide on days -7 to -2, CB NK infusion (108/kg) on day -5, and ASCT (day 0). The primary endpoint was 30-day treatment-related mortality (TRM); secondary endpoints included relapse-free survival (RFS), overall survival (OS), and persistence of CB NK cells. We enrolled 20 patients. CB NK cells were expanded a median of 1552-fold with >98% purity and >96% viability. We saw no adverse events attributable to the CB NK cells and 0% 30-day TRM. At median follow-up of 47 months, the RFS and OS rates were 53% and 74%, respectively. CB NK cells were detectable in blood for 2 weeks, independent of HLA-mismatch status. CD16 expression in donor NK cells was correlated favorably with outcome, and homozygosity for the high-affinity CD16 variant (158 V/V) in CB, but not recipient, NK cells was correlated with better outcomes. Our data indicate that the combination of expanded and highly purified CB-derived NK cells with HDC/ASCT for B-NHL is safe. CD16 expression in donor NK cells, particularly if homozygous for the high-affinity CD16 variant, was correlated with better outcomes.

Breaking down barriers: The potential of smarter CAR-engineered NK cells against solid tumors.

Natural killer (NK) cells are considered to be the foremost fighters of our innate immune system against foreign invaders and thus tend to promptly latch onto the virus-infected and tumor/cancerous cells, killing them through phagocytosis. At present, the application of genetically engineered Chimeric antigen receptor (CAR) receptors ensures a guaranteed optimistic response with NK cells and would not allow the affected cells to dodge or escape unchecked. Hence the specificity and uniqueness of CAR-NK cells over CAR-T therapy make them a better immunotherapeutic choice to reduce the load of trafficking of numerous tumor cells near the healthy cell populations in a more intact way than offered by CAR-T immunotherapy. Our review mainly focuses on the preclinical, clinical, and recent advances in clinical research trials and further strategies to achieve an augmented and efficient cure against solid tumors.

Optimizing the cryopreservation and post-thaw recovery of natural killer cells is critical for the success of off-the-shelf platforms.

Natural killer (NK) cells are a promising allogeneic, off-the-shelf, cellular immunotherapy product. These cells can be manipulated ex vivo, genetically edited to enhance tumor targeting and expanded to produce large cell banks for multiple patient infusions. Therapeutic efficacy of these products depends on the recovery of viable and functional cells post-thaw. Post-thaw loss of viability and cytolytic activity results in large, and often variable, discrepancies between the intended cell dose (based on counts at cryopreservation) and the actual dose administered. Compared to their highly activated state in fresh culture, post-thaw NK cells demonstrate critical changes in cytokine production, cytotoxic activity, in vivo proliferation and migration. When these NK cells are introduced into the highly immunosuppressive tumor microenvironment, the functional changes induced by cryopreservation further limits the clinical potential of these products. This report will review the impact of cryopreservation on ex vivo expanded NK cells and outlines strategies described in published studies to recover post-thaw function.

Visualization of natural killer cell-mediated killing of cancer cells at single-cell resolution in live zebrafish.

Tumor immunotherapy has been an important advancement in cancer treatment in recent years. Compared with T cell-based therapy, natural killer (NK) cell-based therapy does not require human leukocyte antigen matching and has fewer side effects; thus, NK cell therapy has gradually attracted the attention of researchers and clinicians. Reliable and effective animal models are essential for evaluating the effects of NK cell therapy. NK cells kill cancer cells mainly through apoptosis. In this study, we first established a 3D coculture model using fluorescence resonance energy transfer (FRET)-based lung or breast cancer cells and tdTomato-labeled NK cells. We observed that cancer cells changed from green to blue when undergoing apoptosis induced by red NK cells. We then coinjected these green cancer cells with red NK cells into zebrafish to visualize the interaction between them and the killing process of NK cells against cancer cells in real-time and at single-cell resolution in circulation. Using this model, we found that NK cells can quickly kill cancer cells in zebrafish circulation in 40 min and the caspase-3 can be activated in 5-10 min. This FRET-based zebrafish tumor model can serve as a powerful in vivo tool that can facilitate the development of NK cell-based therapy. More importantly, cancer cells from cancer patients can be labeled with our apoptotic biosensor and then transplanted into zebrafish to evaluate the sensitivity of the cancer cells to NK cells to help clinicians make treatment plans that can benefit patients.

Adoptive NK Cell Transfer as a Treatment in Colorectal Cancer Patients: Analyses of Tumour Cell Determinants Correlating With Efficacy In Vitro and In Vivo.

Background: Colorectal cancer (CRC) is a heterogeneous disease with variable mutational profile and tumour microenvironment composition that influence tumour progression and response to treatment. While chemoresistant and poorly immunogenic CRC remains a challenge, the development of new strategies guided by biomarkers could help stratify and treat patients. Allogeneic NK cell transfer emerges as an alternative against chemoresistant and poorly immunogenic CRC. Methods: NK cell-related immunological markers were analysed by transcriptomics and immunohistochemistry in human CRC samples and correlated with tumour progression and overall survival. The anti-tumour ability of expanded allogeneic NK cells using a protocol combining cytokines and feeder cells was analysed in vitro and in vivo and correlated with CRC mutational status and the expression of ligands for immune checkpoint (IC) receptors regulating NK cell activity. Results: HLA-I downmodulation and NK cell infiltration correlated with better overall survival in patients with a low-stage (II) microsatellite instability-high (MSI-H) CRC, suggesting a role of HLA-I as a prognosis biomarker and a potential benefit of NK cell immunotherapy. Activated allogeneic NK cells were able to eliminate CRC cultures without PD-1 and TIM-3 restriction but were affected by HLA-I expression. In vivo experiments confirmed the efficacy of the therapy against both HLA+ and HLA- CRC cell lines. Concomitant administration of pembrolizumab failed to improve tumour control. Conclusions: Our results reveal an immunological profile of CRC tumours in which immunogenicity (MSI-H) and immune evasion mechanisms (HLA downmodulation) favour NK cell immunosurveillance at early disease stages. Accordingly, we have shown that allogeneic NK cell therapy can target tumours expressing mutations conferring poor prognosis regardless of the expression of T cell-related inhibitory IC ligands. Overall, this study provides a rationale for a new potential basis for CRC stratification and NK cell-based therapy.

Comparable transforming growth factor beta-mediated immune suppression in ex vivo-expanded natural killer cells from cord blood and peripheral blood: implications for adoptive immunotherapy.

T cell-based therapies like genetically modified immune cells expressing chimeric antigen receptors have shown robust anti-cancer activity in vivo, especially in patients with blood cancers. However, extending this approach to an "off-the-shelf" setting can be challenging, as allogeneic T cells carry a significant risk of graft-versus-host disease (GVHD). By contrast, allogeneic natural killer (NK) cells recognize malignant cells without the need for prior antigen exposure and have been used safely in multiple cancer settings without the risk of GVHD. However, similar to T cells, NK cell function is negatively impacted by tumor-induced transforming growth factor beta (TGF-ß) secretion, which is a ubiquitous and potent immunosuppressive mechanism employed by most malignancies. Allogeneic NK cells for adoptive immunotherapy can be sourced from peripheral blood (PB) or cord blood (CB), and the authors' group and others have previously shown that ex vivo expansion and gene engineering can overcome CB-derived NK cells' functional immaturity and poor cytolytic activity, including in the presence of exogenous TGF-ß.  However, a direct comparison of the effects of TGF-ß-mediated immune suppression on ex vivo-expanded CB- versus PB-derived NK cell therapy products has not previously been performed. Here the authors show that PB- and CB-derived NK cells have distinctive gene signatures that can be overcome by ex vivo expansion. Additionally, exposure to exogenous TGF-ß results in an upregulation of inhibitory receptors on NK cells, a novel immunosuppressive mechanism not previously described. Finally, the authors provide functional and genetic evidence that both PB- and CB-derived NK cells are equivalently susceptible to TGF-ß-mediated immune suppression. The authors believe these results provide important mechanistic insights to consider when using ex vivo-expanded, TGF-ß-resistant PB- or CB-derived NK cells as novel immunotherapy agents for cancer.

Venetoclax enhances NK cell killing sensitivity of AML cells through the NKG2D/NKG2DL activation pathway.

BACKGROUND: Venetoclax, a selective B-cell lymphoma-2 (BCL2) inhibitor, has a potential therapeutic effect when combined with demethylating agents in the first-line setting of unfit elderly patients with acute myeloid leukaemia (AML); however, efficacy is still limited in refractory/recurrent AML. Therefore, exploration of a suitable novel treatment scheme is urgently needed.However, combining venetoclax with NK cell-based immunotherapy has not been studied. METHODS: The cytotoxicity of NK cell combined with venetoclax was assessed in vitro using flow cytometry. Venetoclax-induced natural killer group 2 member D (NKG2D) ligand (NKG2DL) expression was detected by flow cytometry and western blotting. Mechanisms underlying venetoclax-induced NKG2DL expression were found by GSE127200 analysis and investigated using real-time PCR (Q-PCR) and western blotting. RESULTS: Flow cytometric analysis showed that combining venetoclax with NK cells produced synergistic anti-leukaemia effects similar to those of venetoclax + azacitidine. Venetoclax could render AML cell lines and primary AML cells sensitive to NK cell killing by promoting NK cell degranulation, NK-AML cell recognition and NK cell secretion of interferon (IFN)-γ and granzyme B. The synergistic effect resulted from venetoclax-induced NKG2DL upregulation in AML cells and could be undermined by blocking NKG2D on NK cells. This finding suggests that venetoclax enhances NK cell killing activity by activating the NKG2D/NKG2DL ligand-receptor pathway. Furthermore, the nuclear factor-kappa-B (NFKB) signalling pathway was involved in venetoclax-induced NKG2DL upregulation. CONCLUSIONS: Collectively, our data confirm that venetoclax combined with NK cells induces synergistic AML cell cytolysis and preliminarily revealed that venetoclax could selectively induce NKG2DLs on AML cells via NFKB signalling pathway.

Progress and prospects for use of cellular immunotherapy in pancreatic cancer.

Pancreatic cancer (PC) is a highly malignant tumor with an increasing incidence rate in recent years. Because pancreatic cancer has an insidious onset, unknown pathophysiology, and poor prognosis, the overall survival rate of pancreatic cancer patients has not improved considerably even with extensive treatment methods such as surgery, radiation, biotherapy, and targeted therapy. Therefore, finding and developing more effective and safe treatments for pancreatic cancer is critical. Cellular immunotherapy has achieved considerable advances in the field of oncology in recent years. Technology is continuously advancing, with new breakthroughs virtually every month, and pancreatic cancer eradication is expected to improve considerably. This article examines the advance of chimeric antigen receptor NK cell immunotherapy (CAR-NK) cell immunotherapy for pancreatic cancer research, as well as research ideas for pancreatic cancer treatment.

ERAP1 Controls the Interaction of the Inhibitory Receptor KIR3DL1 With HLA-B51:01 by Affecting Natural Killer Cell Function.

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by major histocompatibility complex (MHC) class I molecules. Previously, we have shown that genetic or pharmacological inhibition of ERAP1 on murine and human tumor cell lines perturbs the engagement of NK cell inhibitory receptors Ly49C/I and Killer-cell Immunoglobulin-like receptors (KIRs), respectively, by their specific ligands (MHC class I molecules), thus leading to NK cell killing. However, the effect of ERAP1 inhibition in tumor cells was highly variable, suggesting that its efficacy may depend on several factors, including MHC class I typing. To identify MHC class I alleles and KIRs that are more sensitive to ERAP1 depletion, we stably silenced ERAP1 expression in human HLA class I-negative B lymphoblastoid cell line 721.221 (referred to as 221) transfected with a panel of KIR ligands (i.e. HLA-B*51:01, -Cw3, -Cw4 and -Cw7), or HLA-A2 which does not bind any KIR, and tested their ability to induce NK cell degranulation and cytotoxicity. No change in HLA class I surface expression was detected in all 221 transfectant cells after ERAP1 depletion. In contrast, CD107a expression levels were significantly increased on NK cells stimulated with 221-B*51:01 cells lacking ERAP1, particularly in the KIR3DL1-positive NK cell subset. Consistently, genetic or pharmacological inhibition of ERAP1 impaired the recognition of HLA-B*51:01 by the YTS NK cell overexpressing KIR3DL1*001, suggesting that ERAP1 inhibition renders HLA-B*51:01 molecules less eligible for binding to KIR3DL1. Overall, these results identify HLA-B*51:01/KIR3DL1 as one of the most susceptible combinations for ERAP1 inhibition, suggesting that individuals carrying HLA-B*51:01-like antigens may be candidates for immunotherapy based on pharmacological inhibition of ERAP1.

The Biological Role and Therapeutic Potential of NK Cells in Hematological and Solid Tumors.

NK cells are an attractive target for cancer immunotherapy due to their potent antitumor activity. The main advantage of using NK cells as cytotoxic effectors over T cells is a reduced risk of graft versus host disease. At present, several variants of NK-cell-based therapies are undergoing clinical trials and show considerable effectiveness for hematological tumors. In these types of cancers, the immune cells themselves often undergo malignant transformation, which determines the features of the disease. In contrast, the current use of NK cells as therapeutic agents for the treatment of solid tumors is much less promising. Most studies are at the stage of preclinical investigation, but few progress to clinical trials. Low efficiency of NK cell migration and functional activity in the tumor environment are currently considered the major barriers to NK cell anti-tumor therapies. Various therapeutic combinations, genetic engineering methods, alternative sources for obtaining NK cells, and other techniques are aiming at the development of promising NK cell anticancer therapies, regardless of tumorigenesis. In this review, we compare the role of NK cells in the pathogenesis of hematological and solid tumors and discuss current prospects of NK-cell-based therapy for hematological and solid tumors.

Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy.

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation-expansion process and its validation on clinical-scale. METHODS: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. RESULTS: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. CONCLUSIONS: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

Enhanced Natural Killer Cell Immunotherapy by Rationally Assembling Fc Fragments of Antibodies onto Tumor Membranes.

Recent advances in cancer immunotherapy have exploited the efficient potential of natural killer (NK) cells to kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC). However, this therapeutic strategy is seriously limited by tumor antigen heterogeneity since antibodies can only recognize specific antigens. In this work, modified antibodies or their Fc fragments that can target solid tumors without the necessity of specific antigen presentation on tumors are developed. Briefly, Fc fragments or therapeutic monoclonal antibodies are conjugated with the N-terminus of pH low insertion peptide so that they will selectively assemble onto the membrane of solid tumor cells via the conformational transformation of the peptide by responding to the acidic tumor microenvironment. The inserted Fc fragments or antibodies can efficiently activate NK cells, initiating ADCC and killing multiple types of tumor cells, including antigen-negative cancer cells. In vivo therapeutic results also exhibit significant efficacy on both primary solid tumors and tumor metastasis. These modified Fc fragments and antibodies present strong potential to overcome the limitation of tumor antigen heterogeneity, broadening the applications of NK cell immunotherapy on solid tumor treatment.

In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer.

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) act by inhibiting EGFR downstream signaling and by eliciting a natural killer (NK) cell-mediated antitumor response. The IgG1 mAb cetuximab has been used for treatment of RASwt metastatic colorectal cancer (mCRC) patients, showing limited efficacy. In the present study, we address the potential of adoptive NK cell therapy to overcome these limitations investigating two allogeneic NK cell products, i.e., allogeneic activated peripheral blood NK cells (A-PBNK) and umbilical cord blood stem cell-derived NK cells (UCB-NK). While cetuximab monotherapy was not effective against EGFR- RASwt, EGFR+ RASmut, and EGFR+ BRAFmut cells, A-PBNK were able to initiate lysis of EGFR+ colon cancer cells irrespective of RAS or BRAF status. Cytotoxic effects of A-PBNK (but not UCB-NK) were further potentiated significantly by coating EGFR+ colon cancer cells with cetuximab. Of note, a significantly higher cytotoxicity was induced by UCB-NK in EGFR-RASwt (42 ± 8 versus 67 ± 7%), EGFR+ RASmut (20 ± 2 versus 37 ± 6%), and EGFR+ BRAFmut (23 ± 3 versus 43 ± 7%) colon cancer cells compared to A-PBNK and equaled the cytotoxic efficacy of the combination of A-PBNK and cetuximab. The antitumor efficacy of UCB-NK cells against cetuximab-resistant human EGFR+ RASmut colon cancer cells was further confirmed in an in vivo preclinical mouse model where UCB-NK showed enhanced antitumor cytotoxicity against colon cancer independent of EGFR and RAS status. As UCB-NK have been proven safe in a recently conducted phase I clinical trial in acute myeloid leukemia, a fast translation into clinical proof of concept for mCRC could be considered.

Efficient mRNA-Based Genetic Engineering of Human NK Cells with High-Affinity CD16 and CCR7 Augments Rituximab-Induced ADCC against Lymphoma and Targets NK Cell Migration toward the Lymph Node-Associated Chemokine CCL19.

For more than a decade, investigators have pursued methods to genetically engineer natural killer (NK) cells for use in clinical therapy against cancer. Despite considerable advances in viral transduction of hematopoietic stem cells and T cells, transduction efficiencies for NK cells have remained disappointingly low. Here, we show that NK cells can be genetically reprogramed efficiently using a cGMP-compliant mRNA electroporation method that induces rapid and reproducible transgene expression in nearly all transfected cells, without negatively influencing their viability, phenotype, and cytotoxic function. To study its potential therapeutic application, we used this approach to improve key aspects involved in efficient lymphoma targeting by adoptively infused ex vivo-expanded NK cells. Electroporation of NK cells with mRNA coding for the chemokine receptor CCR7 significantly promoted migration toward the lymph node-associated chemokine CCL19. Further, introduction of mRNA coding for the high-affinity antibody-binding receptor CD16 (CD16-158V) substantially augmented NK cell cytotoxicity against rituximab-coated lymphoma cells. Based on these data, we conclude that this approach can be utilized to genetically modify multiple modalities of NK cells in a highly efficient manner with the potential to improve multiple facets of their in vivo tumor targeting, thus, opening a new arena for the development of more efficacious adoptive NK cell-based cancer immunotherapies.