Adoptive Natural Killer Cell Therapy Research Articles

Phenotypic and transcriptomic profiling of induced pluripotent stem cell (iPSC)-derived NK cells and their cytotoxicity against cancers.

Adoptive immunotherapy using natural killer (NK) cells has attracted considerable interest in numerous clinical trials targeting both hematological and solid tumors. Traditionally, NK cells are primarily derived from either peripheral blood (PB) or umbilical cord blood (UCB). However, these methods can lead to variability and heterogeneity within the NK cell population. In contrast, induced pluripotent stem cell (iPSC)-derived NK (iNK) cells provide a more controlled and uniform cellular population, suitable for large-scale clinical applications. This makes iNK cells a promising option for developing "off-the-shelf" immunotherapeutic products. Nevertheless, current NK cell differentiation protocols, which rely on embryoid body (EB) cultures, are labor-intensive and susceptible to unwanted heterogeneity during differentiation. Here, we developed a more efficient approach for generating iNK cells by employing a monolayer and feeder-free differentiation protocol, alongside optimized culture media. The iNK cells were generated using a two-step in vitro monolayer feeder-free system following NK cell development. To evaluate their maturity, phenotypic analysis was performed using flow cytometry, comparing with PB-NK cells and the NK-92 cell line. Additionally, single-cell RNA sequencing was performed to examine their transcriptomic profiles. The cytotoxic activity of the iNK cells was evaluated by co-culturing with cholangiocarcinoma (CCA) and breast cancer (BCA) cell lines in both monolayer (2D) and tumor spheroid (3D) co-culture systems. We successfully differentiated iPSCs into mesoderm (ME), hematopoietic stem/progenitor cells (HSPCs), and NK cells. The resulting iNK cells exhibited typical NK cell markers such as CD45, CD56, and CD16, and expressed key functional proteins, including both activating and inhibitory receptors. Single-cell RNA sequencing confirmed that the transcriptomic profile of our iNK cells closely resembles that of PB-NK cells. Importantly, our iNK cells demonstrated strong cytotoxic abilities against various CCA and BCA cell lines, surpassing the NK-92 cell line in both monolayer cultures and tumor spheroid cultures. This study highlights the potential of iPSCs as an effective alternative cell source for generating NK cells. Using a two-step in vitro monolayer feeder-free system, we successfully generated iNK cells that not only expressed key NK cell markers and their receptors but also displayed a transcriptomic profile closely resembling PB-NK cells. Furthermore, iNK cells exhibited cytotoxicity against CCA and BCA cell lines comparable to that of PB-NK cells. This approach could pave the way for off-the-shelf NK cell products, potentially enhancing the effectiveness of adoptive NK cell therapy.

SKAP1 Expression in Cancer Cells Enhances Colon Tumor Growth and Impairs Cytotoxic Immunity by Promoting Neutrophil Extracellular Trap Formation via the NFATc1/CXCL8 Axis.

The mechanisms underlying the development and progression of colon cancer are not fully understood. Herein, Src kinase associated phosphoprotein 1 (SKAP1), an immune cell adaptor, is identified as a novel colon cancer-related gene. SKAP1 expression is significantly increased in colon cancer cells. High SKAP1 levels are independently predictive of poor survival in patients with colon cancer. Notably, SKAP1 expression in colon cancer cells exerted a significant tumor-promoting effect in vivo rather than in vitro. Screening of tumor-infiltrating immune cells revealed the involvement of neutrophils in SKAP1-induced colon tumor promotion. Enhanced formation of neutrophil extracellular traps (NETs) is found to be a key downstream event that contributed to the pro-tumor role of SKAP1. In colon cancer cells, SKAP1 increased the expression of C-X-C motif chemokine ligand 8 (CXCL8) via nuclear factor of activated T cells c1 (NFATc1). The blockade of CXCL8 or NFATc1 largely attenuated neutrophil infiltration, NET formation, and tumor promotion induced by SKAP1. Furthermore, inhibiting SKAP1-induced NET significantly enhanced the antitumor efficiency of adoptive natural killer cell therapy in colon tumor models. In conclusion, SKAP1 significantly promotes colon cancer growth via the cancer cell/neutrophil NFATc1/CXCL8/NET axis, suggesting that SKAP1 is a potential target for colon cancer therapy.

The stress response regulator HSF1 modulates natural killer cell anti-tumour immunity.

Diverse cellular insults converge on activation of the heat shock factor 1 (HSF1), which regulates the proteotoxic stress response to maintain protein homoeostasis. HSF1 regulates numerous gene programmes beyond the proteotoxic stress response in a cell-type- and context-specific manner to promote malignancy. However, the role(s) of HSF1 in immune populations of the tumour microenvironment remain elusive. Here, we leverage an in vivo model of HSF1 activation and single-cell transcriptomic tumour profiling to show that augmented HSF1 activity in natural killer (NK) cells impairs cytotoxicity, cytokine production and subsequent anti-tumour immunity. Mechanistically, HSF1 directly binds and regulates the expression of key mediators of NK cell effector function. This work demonstrates that HSF1 regulates the immune response under the stress conditions of the tumour microenvironment. These findings have important implications for enhancing the efficacy of adoptive NK cell therapies and for designing combinatorial strategies including modulators of NK cell-mediated tumour killing.

Natural killer cells in cancer immunotherapy.

Natural killer (NK) cells, as innate lymphocytes, possess cytotoxic capabilities and engage target cells through a repertoire of activating and inhibitory receptors. Particularly, natural killer group 2, member D (NKG2D) receptor on NK cells recognizes stress-induced ligands-the MHC class I chain-related molecules A and B (MICA/B) presented on tumor cells and is key to trigger the cytolytic response of NK cells. However, tumors have developed sophisticated strategies to evade NK cell surveillance, which lead to failure of tumor immunotherapy. In this paper, we summarized these immune escaping strategies, including the downregulation of ligands for activating receptors, upregulation of ligands for inhibitory receptors, secretion of immunosuppressive compounds, and the development of apoptosis resistance. Then, we focus on recent advancements in NK cell immune therapies, which include engaging activating NK cell receptors, upregulating NKG2D ligand MICA/B expression, blocking inhibitory NK cell receptors, adoptive NK cell therapy, chimeric antigen receptor (CAR)-engineered NK cells (CAR-NK), and NKG2D CAR-T cells, especially several vaccines targeting MICA/B. This review will inspire the research in NK cell biology in tumor and provide significant hope for improving cancer treatment outcomes by harnessing the potent cytotoxic activity of NK cells.

The basic biology of NK cells and its application in tumor immunotherapy.

Natural Killer (NK) cells play a crucial role as effector cells within the tumor immune microenvironment, capable of identifying and eliminating tumor cells through the expression of diverse activating and inhibitory receptors that recognize tumor-related ligands. Therefore, harnessing NK cells for therapeutic purposes represents a significant adjunct to T cell-based tumor immunotherapy strategies. Presently, NK cell-based tumor immunotherapy strategies encompass various approaches, including adoptive NK cell therapy, cytokine therapy, antibody-based NK cell therapy (enhancing ADCC mediated by NK cells, NK cell engagers, immune checkpoint blockade therapy) and the utilization of nanoparticles and small molecules to modulate NK cell anti-tumor functionality. This article presents a comprehensive overview of the latest advances in NK cell-based anti-tumor immunotherapy, with the aim of offering insights and methodologies for the clinical treatment of cancer patients.

IPSC-derived natural killer cells expressing the FcγR fusion CD64/16A can be armed with antibodies for multitumor antigen targeting

BackgroundAntibody therapies can direct natural killer (NK) cells to tumor cells, tumor-associated cells, and suppressive immune cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This antigen-specific effector function of human NK...

Trials in Progress: A Phase I/II Clinical Trial of Off the Shelf Universal Donor Natural Killer Cells Combined with Chemotherapy for Treatment of Relapsed/Refractory Acute Myeloid Leukemia (KARMA)

Background and Significance: Natural killer (NK) cells are cytotoxic lymphocytes that play a key role in recognizing malignant cells. In AML, qualitative and quantitative dysfunction of innate NK cells portends a poor prognosis and adoptive NK cell therapy has been shown to be safe and effective with minimal risk for GVHD (Ciurea et al. 2022; Silla et al. 2021). To circumvent cost and logistics restraints of per patient manufacture of NK cells, we developed a universal donor (UD) NK cell bank for “off-the-shelf” NK cells (UD FC21-NK cells). Donors with optimal NK cell characteristics are identified through the National Marrow Donor Program, and peripheral blood NK cells are expanded for two weeks by co-culture with irradiated K562 feeder cells expressing 4-1BB ligand and membrane bound IL-21 as previously described (Denman et al. 2012). We seek to study the safety and efficacy of multiple doses of UD FC21-NK cells combined with fludarabine and cytarabine (FLA) chemotherapy in patients with relapsed or refractory AML. Study Design and Methods: This open label, single-center phase I/II study will include a dose escalation phase to establish safety and determine the recommended phase II dose followed by an expansion phase to estimate efficacy (complete response rate and MRD negative response rate). Up to 20 patients ages 18-25 with relapsed or refractory AML, including isolated extramedullary disease, will be enrolled. Exclusion criteria include active GVHD, uncontrolled infections, hematopoietic stem cell transplant within 3 months of enrollment, and donor lymphocyte infusion within 30 days of enrollment. Patients will undergo FLA chemotherapy followed by six doses of UD FC21-NK cells given thrice weekly for two weeks (Figure 1). Dose levels include 1 x 10 7 UD FC21-NK cells/kg/dose, 3 x 10 7 UD FC21-NK cells/kg/dose, and 1 x 10 8 UD FC21-NK cells/kg/dose. Patients may receive up to 2 cycles if they do not achieve a CR after cycle 1 or if necessary to bridge to transplant. Peripheral blood samples will be drawn weekly for immune correlatives including in vivo NK cell expansion and persistence. The trial is currently open to enrollment to adult patients with the intent to expand to pediatrics after demonstrating preliminary safety. Clinical trial information: NCT05503134

Azacitidine, Venetoclax and Allogeneic NK Cells in Newly Diagnosed Acute Myeloid Leukemia (ADVENT-AML): An Investigator-Initiated Multicenter Phase Ib Trial

BACKGROUND AND SIGNIFICANCE : Older/unfit patients with acute myeloid leukemia (AML) have limited treatment options. Outcomes in adverse-risk subgroups of AML are modest with CR/CRi rates of 45% to 60% and median overall survival (OS) of 5 to 12 months with current standard frontline therapy of hypomethylating agent with venetoclax (HMA-VEN).While allogeneic stem cell transplantation (allo-SCT) can significantly improve OS in AML, only 10 to 20% of older pts are able to undergo allo-SCT (Pratz et al. Blood 2019, Maiti et al. Blood 2022) Natural killer (NK) cells bridge the innate and adaptive immune system and play a major role in graft-vs-leukemia (GVL) effect responsible for the clinical benefit noted with allo-SCT. NK cell-based adoptive cellular therapies have shown good safety profiles and promising activity in AML with responses noted in 25% to 88% of patients. Furthermore, NK cells are less likely to induce graft-versus-host disease (GVHD), severe cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). We hypothesize that the combination of azacitidine, venetoclax and allogeneic NK cells will be safe and improve cure rates in AML compared to either Aza/Ven or NK cells alone. Improved clinical activity may be mediated through multiple synergistic mechanisms including reduction of disease burden by Aza/Ven, upregulation of epigenetically silenced NKG2D ligands, dual activation of extrinsic and intrinsic apoptotic cascades, mitochondrial priming of leukemia cells by venetoclax, and bypassing significant morbidity and mortality associated allo-SCT. Significance: This will be the first trial to evaluate 1) cellular therapy in the frontline setting in AML; 2) the synergy of venetoclax with adoptive NK cell therapy; and 3) Aza/Ven as a lymphodepletion strategy. The results will have significant impact on the development of cellular therapies for hematological and solid tumors. STUDY DESIGN AND METHODS: This investigator-initiated, multicenter, open-label, phase Ib study will evaluate the combination of azacitidine, venetoclax and ex vivo-expanded, off-the-shelf cryopreserved allogeneic NK cells in patients with AML (NCT05834244). Eligibility criteria: The dose escalation phase will enroll adults with relapsed or refractory AML or MDS/AML, and the dose expansion phase will enroll older/unfit pts with newly diagnosed adverse-risk AML. Eligible Pts will be ≥ 75 years, or ≥ 18 years with at least one protocol-defined comorbidity. Patients with favorable risk AML, poor end-organ function, needing immunosuppression, uncontrolled infection or CNS leukemia will be excluded. Trial design: The dose escalation phase will enroll a maximum of 12 patients at 2 dose levels to establish a recommended phase 2 dose (RP2D) using a BOIN design. The dose expansion will enroll up to 20 patients at the RP2D ( Fig. 1). Study treatment: Patients will receive Aza/Ven as standard. Two dose levels of NK cells including 0.5 x10 9 and 1 x10 9 flat dose on day 8 and 15 of the first 4 cycles will be evaluated. Subsequently, pts will continue Aza/Ven indefinitely. Objectives: The primary objective is to determine the safety of this novel triplet combination in terms of dose-limiting toxicities including acute GVHD, CRS or ICANS. Secondary objectives include overall response rate, CR/CRi by 4 cycles, rate of negative measurable residual disease, OS and relapse-free survival. Exploratory objectives include additional response and survival endpoints, the persistence of NK cells using SNP-NGS chimerism, kinetics of allorejection, remodeling of host immune microenvironment and impact on AML stem/progenitor cell hierarchies using single cell CyTOF to understand response/resistance mechanisms. Status: Trial approved by the FDA and institutional IRB. Activation planned for 2H 2023. Funding: Gateway for Cancer Research, MDACC PRIME Award, Chimeric Therapeutics, NCI Cancer Center Support Grant.

Epigenetic Scarring Leads to Irreversible NK Cell Dysfunction in Myeloid Malignancies

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) belong to the same spectrum of myeloid malignancies and have extremely poor outcomes in the relapsed/refractory setting. Despite advances in the understanding of the pathogenesis and molecular mechanisms of these disorders, and incremental improvements in treatment regimens, patients with MDS and AML often relapse and fail to achieve cure. These and other factors underscore the urgent need for new therapeutic alternatives that will improve the clinical outcomes of these patients. Immunotherapy using checkpoint molecule inhibitors and adoptive cell therapy using autologous immune effector cells have been mostly unsuccessful in patients with MDS and AML. This could be due to the immunosuppressive tumor microenvironment in the bone marrow niche or to intrinsic dysfunction in the immune effector cells of these patients. Natural killer (NK) cells are innate lymphocytes that play an important role in cancer immune surveillance and have distinct advantages over T cells as candidates for immunotherapy. Myeloid blasts are inherently susceptible to NK cell-mediated killing as they express many of the ligands recognized by NK cell activating receptors. However, malignant myeloid blasts are capable of adapting and developing defense mechanisms that allow them to evade NK cell-mediated cytotoxicity. Little is known about the mechanisms of NK cell immune evasion developed by myeloid blasts. Here, we show that NK cells from patients with myeloid malignancies display a global dysfunction with severely impaired secretory function and killing capacity. Single cell RNAseq and mass cytometry experiments revealed that these NK cells from MDS and AML patients display an exhausted phenotype at the transcriptomic and proteomic levels. We also show that these NK cells have an altered metabolism compared with age matched healthy control NK cells. We show that this dysfunction is mediated by a crosstalk between myeloid blasts and NK cells and cell-cell contact dependent release of transforming growth factor beta (TGF-β). This crosstalk leads to a profound epigenetic reprogramming of NK cells driven by transcription factors known to mediate exhaustion and immune suppression. Myeloid blast-induced NK dysfunction and epigenetic state of exhaustion can be prevented by pharmacologically inhibiting the TGF-β pathway or knockout of TGFBR2 in NK cells. However, our data reveal that once this dysfunction occurs it is irreversible owing to epigenetic scarring driven by the transcription factor BATF. In fact, we show that TGF-β induces the expression of BATF in NK cells, which in turn mediates a gene regulatory program leading to NK cell dysfunction by driving the expression of exhaustion and inhibitory genes (e.g. HAVCR2, ENTPD1, CTLA4, TGFBR2). Collectively, our findings reveal a novel mechanism of NK cell immune evasion manifested by stable epigenetic rewiring and inactivation of NK cells by myeloid blasts. Our data support the use of allogeneic sources for adoptive NK cell therapy in combination with strategies aiming at preventing immune suppression to treat myeloid malignancies rather than therapies aiming at reversing or rescuing the function of autologous NK cells.

Potentiation of natural killer cells to overcome cancer resistance to NK cell-based therapy and to enhance antibody-based immunotherapy.

Natural killer (NK) cells are cellular components of the innate immune system that can recognize and suppress the proliferation of cancer cells. NK cells can eliminate cancer cells through direct lysis, by secreting perforin and granzymes, or through antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC involves the binding of the Fc gamma receptor IIIa (CD16), present on NK cells, to the constant region of an antibody already bound to cancer cells. Cancer cells use several mechanisms to evade antitumor activity of NK cells, including the accumulation of inhibitory cytokines, recruitment and expansion of immune suppressor cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), modulation of ligands for NK cells receptors. Several strategies have been developed to enhance the antitumor activity of NK cells with the goal of overcoming cancer cells resistance to NK cells. The three main strategies to engineer and boost NK cells cytotoxicity include boosting NK cells with modulatory cytokines, adoptive NK cell therapy, and the employment of engineered NK cells to enhance antibody-based immunotherapy. Although the first two strategies improved the efficacy of NK cell-based therapy, there are still some limitations, including immune-related adverse events, induction of immune-suppressive cells and further cancer resistance to NK cell killing. One strategy to overcome these issues is the combination of monoclonal antibodies (mAbs) that mediate ADCC and engineered NK cells with potentiated anti-cancer activity. The advantage of using mAbs with ADCC activity is that they can activate NK cells, but also favor the accumulation of immune effector cells to the tumor microenvironment (TME). Several clinical trials reported that combining engineered NK cells with mAbs with ADCC activity can result in a superior clinical response compared to mAbs alone. Next generation of clinical trials, employing engineered NK cells with mAbs with higher affinity for CD16 expressed on NK cells, will provide more effective and higher-quality treatments to cancer patients.

Direct Tumor Irradiation Potentiates Adoptive NK Cell Targeting Against Parental and Stemlike Cancer in Human Liver Cancer Models

Radiation therapy (RT) has been shown to effectively induce the expression of intercellular adhesion molecule-1 (ICAM-1), which is recognized by lymphocyte function-associated antigen 1 (LFA-1) expressed on natural killer (NK) cells. However, the potential synergistic antitumor immune response of tumor irradiation and administered NK cells has not been explored in intractable human liver cancers. Furthermore, NK cell targeting against both parental and cancer stemness has never been investigated. Highly activated ex vivo NK cells were administered into the human liver tumor-bearing mice. Tumor direct RT was optimized according to tumor bearing site. HepG2 and Hep3B ICAM-1 knockout cells were generated using CRISPR/CAS9. Stemness tumor spheres were generated. NK cell cytolysis against parental and tumor sphere was evaluated using flow cytometry and real-time cytotoxicity assay. A combination of adoptive NK cell therapy with RT significantly improved therapeutic efficacy over monotherapies against subcutaneous, orthotopic, and metastatic human liver tumor models. Direct tumor irradiation potentiated NK cell recognition and conjugation against liver cancer through the LFA-1/ICAM-1 axis. Suppression of immune synapse formation on NK cells using high-affinity LFA-1 inhibitors or ICAM-1 knockout liver cancer induced "outside-in" signal blocking in NK cells, resulting in failure to eliminate liver tumor despite the combination therapy. NK cells effectively recognized and targeted triple-high epithelial cell adhesion molecule+CD133+CD24+ liver cancer expressing upregulated ICAM-1 in the irradiated tumor microenvironment, which led to prevention of the initiation of metastasis, improving survival in a metastatic model. In addition, the LFA-1/ICAM-1 axis interruption between NK cells and stemness liver tumor spheres significantly diminished NK cell cytolysis. Consistent with our preclinical data, the LFA-1/ICAM-1 axis correlated with survival outcomes in patients with metastatic cancer from the The Cancer Genome Atlas databases. NK cells in combination with tumor irradiation can provide synergistic therapeutic effects for NK cell recognition and elimination against both parental and stemlike liver cancer through LFA-1/ICAM-1.

Boosting cytotoxicity of adoptive allogeneic NK cell therapy with an oncolytic adenovirus encoding a human vIL-2 cytokine for the treatment of human ovarian cancer

Despite good results in the treatment of hematological malignancies, Natural killer (NK) cells have shown limited effectiveness in solid tumors, such as ovarian cancer (OvCa). Here, we assessed the potential of an oncolytic adenovirus expressing a variant interleukin-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, to enhance NK cell therapy efficacy in human OvCa ex vivo. Human OvCa surgical specimens were processed into single-cell suspensions and NK cells were expanded from healthy blood donors. OvCa sample digests were co-cultured ex vivo with NK cells and vIL-2 virus and cancer cell killing potential assessed in real time through cell impedance measurement. Proposed therapeutic combination was evaluated in vivo with an OvCa patient-derived xenograft (PDX) in mice. Addition of vIL-2 virus significantly enhanced NK cell therapy killing potential in treated OvCa co-cultures. Similarly, vIL-2 virus in combination with NK cell therapy promoted the best in vivo OvCa tumor control. Mechanistically, vIL-2 virus induced higher percentages of granzyme B in NK cells, and CD8+ T cells, while T regulatory cell proportions remained comparable to NK cell monotherapy in vivo. Ad5/3-E2F-d24-vIL2 virus treatment represents a promising strategy to boost adoptive NK cell therapeutic effect in human OvCa.

Adoptive NK Cell Therapy - a Beacon of Hope in Multiple Myeloma Treatment.

Major advances in the treatment of multiple myeloma (MM) have been achieved by effective new agents such as proteasome inhibitors, immunomodulatory drugs, or monoclonal antibodies. Despite significant progress, MM remains still incurable and, recently, cellular immunotherapy has emerged as a promising treatment for relapsed/refractory MM. The emergence of chimeric antigen receptor (CAR) technology has transformed immunotherapy by enhancing the antitumor functions of T cells and natural killer (NK) cells, leading to effective control of hematologic malignancies. Recent advancements in gene delivery to NK cells have paved the way for the clinical application of CAR-NK cell therapy. CAR-NK cell therapy strategies have demonstrated safety, tolerability, and substantial efficacy in treating B cell malignancies in various clinical settings. However, their effectiveness in eliminating MM remains to be established. This review explores multiple approaches to enhance NK cell cytotoxicity, persistence, expansion, and manufacturing processes, and highlights the challenges and opportunities associated with CAR-NK cell therapy against MM. By shedding light on these aspects, this review aims to provide valuable insights into the potential of CAR-NK cell therapy as a promising approach for improving the treatment outcomes of MM patients.

NK cell subsets and dysfunction during viral infection: a new avenue for therapeutics?

In the setting of viral challenge, natural killer (NK) cells play an important role as an early immune responder against infection. During this response, significant changes in the NK cell population occur, particularly in terms of their frequency, location, and subtype prevalence. In this review, changes in the NK cell repertoire associated with several pathogenic viral infections are summarized, with a particular focus placed on changes that contribute to NK cell dysregulation in these settings. This dysregulation, in turn, can contribute to host pathology either by causing NK cells to be hyperresponsive or hyporesponsive. Hyperresponsive NK cells mediate significant host cell death and contribute to generating a hyperinflammatory environment. Hyporesponsive NK cell populations shift toward exhaustion and often fail to limit viral pathogenesis, possibly enabling viral persistence. Several emerging therapeutic approaches aimed at addressing NK cell dysregulation have arisen in the last three decades in the setting of cancer and may prove to hold promise in treating viral diseases. However, the application of such therapeutics to treat viral infections remains critically underexplored. This review briefly explores several therapeutic approaches, including the administration of TGF-β inhibitors, immune checkpoint inhibitors, adoptive NK cell therapies, CAR NK cells, and NK cell engagers among other therapeutics.

Prospects and Advances in Adoptive Natural Killer Cell Therapy for Unmet Therapeutic Needs in Pediatric Bone Sarcomas.

Malignant bone tumors are aggressive tumors, with a high tendency to metastasize, that are observed most frequently in adolescents during rapid growth spurts. Pediatric patients with malignant bone sarcomas, Ewing sarcoma and osteosarcoma, who present with progressive disease have dire survival rates despite aggressive therapy. These therapies can have long-term effects on bone growth, such as decreased bone mineral density and reduced longitudinal growth. New therapeutic approaches are therefore urgently needed for targeting pediatric malignant bone tumors. Harnessing the power of the immune system against cancer has improved the survival rates dramatically in certain cancer types. Natural killer (NK) cells are a heterogeneous group of innate effector cells that possess numerous antitumor effects, such as cytolysis and cytokine production. Pediatric sarcoma cells have been shown to be especially susceptible to NK-cell-mediated killing. NK-cell adoptive therapy confers numerous advantages over T-cell adoptive therapy, including a good safety profile and a lack of major histocompatibility complex restriction. NK-cell immunotherapy has the potential to be a new therapy for pediatric malignant bone tumors. In this manuscript, we review the general characteristics of osteosarcoma and Ewing sarcoma, discuss the long-term effects of sarcoma treatment on bones, and the barriers to effective immunotherapy in bone sarcomas. We then present the laboratory and clinical studies on NK-cell immunotherapy for pediatric malignant bone tumors. We discuss the various donor sources and NK-cell types, the engineering of NK cells and combinatorial treatment approaches that are being studied to overcome the current challenges in adoptive NK-cell therapy, while suggesting approaches for future studies on NK-cell immunotherapy in pediatric bone tumors.

Prospects for NK-based immunotherapy of chronic HBV infection.

Effective and long-term treatment is required for controlling chronic Hepatitis B Virus (HBV) infection. Natural killer (NK) cells are antiviral innate lymphocytes and represent an essential arm of current immunotherapy. In chronic HBV (CHB), NK cells display altered changes in phenotypes and functions, but preserve antiviral activity, especially for cytolytic activity. On the other hand, NK cells might also cause liver injury in the disease. NK -based immunotherapy, including adoptive NK cell therapy and NK -based checkpoint inhibition, could potentially exploit the antiviral aspect of NK cells for controlling CHB infection while preventing liver tissue damage. Here, we review recent progress in NK cell biology under the context of CHB infection, and discuss potential NK -based immunotherapy strategies for the disease.

Natural Killer Cells: A Promising Kit in the Adoptive Cell Therapy Toolbox.

As an important component of the innate immune system, natural killer (NK) cells have gained increasing attention in adoptive cell therapy for their safety and efficacious tumor-killing effect. Unlike T cells which rely on the interaction between TCRs and specific peptide-MHC complexes, NK cells are more prone to be served as "off-the-shelf" cell therapy products due to their rapid recognition and killing of tumor cells without MHC restriction. In recent years, constantly emerging sources of therapeutic NK cells have provided flexible options for cancer immunotherapy. Advanced genetic engineering techniques, especially chimeric antigen receptor (CAR) modification, have yielded exciting effectiveness in enhancing NK cell specificity and cytotoxicity, improving in vivo persistence, and overcoming immunosuppressive factors derived from tumors. In this review, we highlight current advances in NK-based adoptive cell therapy, including alternative sources of NK cells for adoptive infusion, various CAR modifications that confer different targeting specificity to NK cells, multiple genetic engineering strategies to enhance NK cell function, as well as the latest clinical research on adoptive NK cell therapy.

Human placental hematopoietic stem cell-derived natural killer cells (CYNK) recognize and eliminate influenza A virus-infected cells.

Influenza A virus (IAV) infections are a significant recurrent threat to public health and a significant burden on global economy, highlighting the need for developing more effective therapies. Natural killer (NK) cells play a pivotal role in the control of pulmonary IAV infection, however, little is known about the therapeutic potential of adoptively transferred NK cells for viral infections. Here, we investigated the antiviral activity of CYNK, human placental hematopoietic stem cell-derived NK cells, against IAV infection in vitro. Virus infection induced the expression of NK cell activating ligands on respiratory epithelial cells, resulting in enhanced recognition by CYNK cells. Upon co-culture with IAV-infected epithelial cells, CYNK exhibited elevated degranulation and increased production of IFN-γ, TNF-α and GM-CSF in a virus dose-dependent manner. Furthermore, CYNK showed virus dose-dependent cytotoxicity against IAV-infected cells. The antiviral activity of CYNK was mediated by NKp46 and NKG2D. Together, these data demonstrate that CYNK possesses potent antiviral function against IAV and warrant clinical investigations for adoptive NK cell therapies against viral infections.

A novel membrane-bound interleukin-2 promotes NK-92 cell persistence and anti-tumor activity

ABSTRACT A major challenge in natural killer (NK) cell immunotherapy is the limited persistence of NK cells in vivo. However, the proliferation of NK cells is dependent on cytokines such as interleukin-2 (IL-2). Although IL-2 is a critical cytokine for NK cell activation and survival, IL-2 administration in adoptive NK cell therapy can induce adverse toxicities. To improve the persistence of NK cells and attenuate the systemic toxicity of IL-2, we constructed a cell-restricted artificial IL-2, named membrane-bound IL-2 (mbIL-2), comprising human IL-2 and human IL-2Rα joined by a classic linker. We found that mbIL-2-activated NK-92 cells can survive and proliferate in vitro and in vivo, independent of exogenous IL-2, while mbIL-2-expressing NK-92 cells do not support bystander cell survival or proliferation. Additionally, mbIL-2 enhanced NK-92 cell-mediated antitumor activity by tuning the IL-2 receptor downstream signals and NK cell receptor repertoire expression. To conclude, our novel mbIL-2 improves NK-92 cell persistence and enhances NK-92 cell-mediated antitumor activity. NK-92 cells genetically modified to express the novel mbIL-2 with potential significance for clinical development.

Chemo-immune cell therapy by intratumoral injection of adoptive NK cells with capecitabine in gastric cancer xenograft model.

Gastric cancer is one of the most commonly known malignancies and is the fifth cancer-related death globally. Whereas natural killer (NK) cells play a critical role in tumor elimination; therefore, adoptive NK cell therapy has become a promising approach in cancer cytotherapy. Hence, this study investigated the chemo-immune cell therapy in MKN-45 derived xenograft gastric cancer model. Three groups of animals have received the following treatments separately: activated NK cells, capecitabine, the combination of capecitabine and activated NK cells, and one was considered as the control group. Morphometric properties of tumor samples were evaluated at the end of the study. NK cells infiltration was evaluated by immunohistochemistry (IHC) of hCD56. Mitotic count and treatment response was assessed by hematoxylin and eosin (H&E) staining. The proliferation ratio to apoptosis was determined by IHC assessment of Ki67 and caspase 3. The results indicated that the NK cell therapy could effectively decrease the mitotic count in pathology assessment, but the tumor was not completely eradicated. In combination with metronomic chemotherapy (MC) of capecitabine, NK cell therapy demonstrated a significant difference in tumor morphometric properties compared to the control group. The proliferation ratio to apoptosis was also in line with pathology data. Although NK cell therapy could effectively decrease the mitotic count in vivo, the obtained findings indicated lesser potency than MC despite ex vivo activation. In order to enhance NK cell therapy effectiveness, suppressive features of the tumor microenvironment and inhibitory immune checkpoints blockade should be considered.