scholarly journals Optimizing NK Cell-Based Immunotherapy in Myeloid Leukemia: Abrogating an Immunosuppressive Microenvironment

2021 ◽  
Vol 12 ◽  
Author(s):  
Natasha Mupeta Kaweme ◽  
Fuling Zhou
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Cell Function ◽  
Nk Cell ◽  
Ex Vivo ◽  
Substantial Improvement ◽  
Effector Cells ◽  
Cell Sources

Natural killer (NK) cells are prominent cytotoxic and cytokine-producing components of the innate immune system representing crucial effector cells in cancer immunotherapy. Presently, various NK cell-based immunotherapies have contributed to the substantial improvement in the reconstitution of NK cells against advanced-staged and high-risk AML. Various NK cell sources, including haploidentical NK cells, adaptive NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, chimeric antigen receptor NK cells, cytokine-induced memory-like NK cells, and NK cell lines have been identified. Devising innovative approaches to improve the generation of therapeutic NK cells from the aforementioned sources is likely to enhance NK cell expansion and activation, stimulate ex vivo and in vivo persistence of NK cells and improve conventional treatment response of myeloid leukemia. The tumor-promoting properties of the tumor microenvironment and downmodulation of NK cellular metabolic activity in solid tumors and hematological malignancies constitute a significant impediment in enhancing the anti-tumor effects of NK cells. In this review, we discuss the current NK cell sources, highlight ongoing interventions in enhancing NK cell function, and outline novel strategies to circumvent immunosuppressive factors in the tumor microenvironment to improve the efficacy of NK cell-based immunotherapy and expand their future success in treating myeloid leukemia.

scholarly journals The Advantages and Challenges of Anticancer Dendritic Cell Vaccines and NK Cells in Adoptive Cell Immunotherapy

Vaccines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1363
Author(s):  
Elena V. Abakushina ◽  
Liubov I. Popova ◽  
Andrey A. Zamyatnin ◽  
Jens Werner ◽  
Nikolay V. Mikhailovsky ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Adoptive Cell Therapy ◽  
Immune Effector ◽  
Effector Cells ◽  
Cell Immunotherapy

In the last decade, an impressive advance was achieved in adoptive cell therapy (ACT), which has improved therapeutic potential and significant value in promising cancer treatment for patients. The ACT is based on the cell transfer of dendritic cells (DCs) and/or immune effector cells. DCs are often used as vaccine carriers or antigen-presenting cells (APCs) to prime naive T cells ex vivo or in vivo. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are used as major tool effector cells for ACT. Despite the fact that NK cell immunotherapy is highly effective and promising against many cancer types, there are still some limitations, including insignificant infiltration, adverse conditions of the microenvironment, the immunosuppressive cellular populations, and the low cytotoxic activity in solid tumors. To overcome these difficulties, novel methods of NK cell isolation, expansion, and stimulation of cytotoxic activity should be designed. In this review, we discuss the basic characteristics of DC vaccines and NK cells as potential adoptive cell preparations in cancer therapy.

scholarly journals 852 Trifunctional NKp46/CD16a-NK cell engager targeting CD123 overcomes acute myeloid leukemia resistance to ADCC

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A893-A893
Author(s):  
Laurent Gauthier ◽  
Angela Virone-Oddos ◽  
Angela Virone-Oddos ◽  
Jochen Beninga ◽  
Benjamin Rossi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Antitumor Activity ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Activating Receptors ◽  
Acute Myeloid

BackgroundThere is a clear need for targeted therapies to treat acute myeloid leukemia (AML), the most common acute leukemia in adults. CD123 (IL-3 receptor alpha chain) is an attractive target for AML treatment.1 However, cytotoxic antibody targeting CD123 proved insufficiently effective in a combination setting in phase II/III clinical trials.2 T-cell engagers targeting CD123 displayed some clinical efficacy but were often associated with cytokine release syndrome and neurotoxicity.3 Interest in the use of NK cells for therapeutic interventions has increased in recent years, as a potential safer alternative to T cells. Several NK-cell activating receptors, such as CD16a, NKG2D, and the natural cytotoxicity receptors NKp30 and NKp46, can be targeted to induce antitumor immunity. We previously reported the development of trifunctional NK-cell engagers (NKCEs) targeting a tumor antigen on cancer cells and co-engaging NKp46 and CD16a on NK cells.4MethodsWe report here the design, characterization and preclinical development of a novel trifunctional NK cell engager (NKCE) targeting CD123 on AML cells and engaging the activating receptors NKp46 and CD16a on NK cells. The CD123 NKCE therapeutic molecule was engineered with humanized antibodies targeting NKp464 and CD123.5 We compared CD123-NKCE and a cytotoxic ADCC-enhanced antibody (Ab) targeting CD123, in terms of antitumor activity in vitro, ex vivo and in vivo. Pharmacokinetic, pharmacodynamic and safety profile of CD123-NKCE were evaluated in non-human primate (NHP) studies.ResultsThe expression of the high affinity Fc gamma receptor CD64 on patient-derived AML cells inhibited the ADCC of the Ab targeting CD123 in vitro and ex vivo, but not the antitumor activity of CD123-NKCE. CD123-NKCE had potent antitumor activity against primary AML blasts and AML cell lines, promoted strong NK-cell activation and induced cytokine secretion only in the presence of AML target cells. Its antitumor activity in mouse model was greater than that of the comparator antibody. Moreover, CD123-NKCE had strong and prolonged pharmacodynamic effects in NHP when used at very low doses, was well-tolerated up to high 3 mg/kg dose and triggered only minor cytokine release.ConclusionsThe data for activity, safety, pharmacokinetics, and pharmacodynamics provided here demonstrate the superiority of CD123-NKCE over comparator cytotoxic antibody, in terms of antitumor activity in vitro, ex vivo, in vivo, and its favorable safety profile, as compared to T-cell therapies. These results constitute proof-of-principle for the efficacy of CD123-NKCE for controlling AML tumors in vivo, and provide consistent support for their clinical development.ReferencesEhninger A, Kramer M, Rollig C, et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. Blood Cancer J 2014;4:e218.Montesinos P, Gail J Roboz GJ, et al. Safety and efficacy of talacotuzumab plus decitabine or decitabine alone in patients with acute myeloid leukemia not eligible for chemotherapy: results from a multicenter, randomized, phase 2/3 study. Leukemia 2021;35(1):62–74.Uy GL, Aldoss I, Foster MC, et al. Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia. Blood 2021;137(6):751–762.Gauthier L, Morel A, Anceriz N, et al. Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity. Cell 2019;177(7):1701–13.Jin L, Lee EM, Ramshaw HS, et al. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 2009;5:31–42.

scholarly journals The Tumor Microenvironment—A Metabolic Obstacle to NK Cells’ Activity

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3542
Author(s):  
Joanna Domagala ◽  
Mieszko Lachota ◽  
Marta Klopotowska ◽  
Agnieszka Graczyk-Jarzynka ◽  
Antoni Domagala ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cells ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Nk Cell ◽  
Metabolic Changes ◽  
Immune Effector ◽  
Effector Cells ◽  
Immune Effector Cells ◽  
Almost All

NK cells have unique capabilities of recognition and destruction of tumor cells, without the requirement for prior immunization of the host. Maintaining tolerance to healthy cells makes them an attractive therapeutic tool for almost all types of cancer. Unfortunately, metabolic changes associated with malignant transformation and tumor progression lead to immunosuppression within the tumor microenvironment, which in turn limits the efficacy of various immunotherapies. In this review, we provide a brief description of the metabolic changes characteristic for the tumor microenvironment. Both tumor and tumor-associated cells produce and secrete factors that directly or indirectly prevent NK cell cytotoxicity. Here, we depict the molecular mechanisms responsible for the inhibition of immune effector cells by metabolic factors. Finally, we summarize the strategies to enhance NK cell function for the treatment of tumors.

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3090-3090 ◽  
Author(s):  
Folashade Otegbeye ◽  
Nathan Mackowski ◽  
Evelyn Ojo ◽  
Marcos De Lima ◽  
David N. Wald
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

scholarly journals Daratumumab induces mechanisms of immune activation through CD38+ NK cell targeting

2019 ◽  
Author(s):  
Domenico Viola ◽  
Ada Dona ◽  
Enrico Caserta ◽  
Estelle Troadec ◽  
Emine Gulsen Gunes ◽  
...  
Keyword(s):  
Nk Cells ◽  
Immune Modulation ◽  
Cell Activation ◽  
Plasma Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Targeting ◽  
Effector Cells ◽  
Cd38 Expression

AbstractDaratumumab (Dara), a multiple myeloma (MM) therapy, is an antibody against the surface receptor CD38, which is expressed not only on plasma cells but also on NK cells and monocytes. Correlative data have highlighted the immune-modulatory role of Dara, despite the paradoxical observation that Dara regimens decrease the frequency of total NK cells. Here we show that, despite this reduction, NK cells play a pivotal role in Dara anti-MM activity. CD38 on NK cells is essential for Dara-induced immune modulation, and its expression is restricted to NK cells with effector function. We also show that Dara induces rapid CD38 protein degradation associated with NK cell activation, leaving an activated CD38-negative NK cell population. CD38+ NK cell targeting by Dara also promotes monocyte activation, inducing an increase in T cell costimulatory molecules (CD86/80) and enhancing anti-MM phagocytosis activity ex-vivo and in vivo. In support of Dara’s immunomodulating role, we show that MM patients that discontinued Dara therapy because of progression maintain targetable unmutated surface CD38 expression on their MM cells, but retain effector cells with impaired cellular immune function. In summary, we report that CD38+ NK cells may be an unexplored therapeutic target for priming the immune system of MM patients.

scholarly journals 171 Targeting the Immunomodulatory roles of T-cell immunoglobulin– and mucin domain–containing (TIM)–3 on natural killer cells in glioblastoma

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A182-A182
Author(s):  
Tram Dao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Glucose Metabolism ◽  
Nk Cells ◽  
Cytokine Production ◽  
Cell Function ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Metabolism ◽  
Knock Out ◽  
Cytotoxic Function

BackgroundNatural killer (NK) cells have emerged as a viable alternative to T cells in adoptive cell transfer for cancer treatment. NK cell activity is driven by the balance between inhibitory and activating receptors, many of which remain elusive. In addition, NK cell metabolism is also a driver of NK cell fitness in tumor settings, where changes in NK metabolic states with the tumor microenvironment in vivo, or with stimulants ex vivo, further confounds the NK cells’ cytotoxic function in cancer settings. One receptor that lies at the intersection between NK cell function and metabolism is TIM-3, with its expression having consequences on NK cell cytokine production and glucose metabolism. However, the contribution of TIM-3 to NK cell anti-tumor immunity is unclear and its role in driving NK cell function so far not fully defined.MethodsNK cells were isolated from healthy adult peripheral blood and expanded in feeder-cell media. NK cell metabolism and function were evaluated by different flow cytometric assays to measure glucose uptake, cytotoxicity, degranulation, and cytokine production. TIM-3 knock-out cells were generated using the CRISPR-Cas9 system. Patient samples, including whole blood and tumor, were also processed and phenotyped to compare expression level with healthy donor samples.ResultsPreviously, we discovered that TIM-3 downregulation was associated with decreased cytokine production and target cytotoxicity, and that maintenance of expression above a certain threshold was needed for NK cell function. As cytokine production reflects immune cell metabolic state, we hypothesized that TIM-3 participates in regulation of ex vivo-activated NK cell metabolism, which in turn affect the production of the cytokine IFN- γ to sensitize cancer targets to NK cell-mediated lysis. Here, we report the consequences of glucose starvation on TIM-3 expression, and how knock-out of TIM-3 on human NK cells affects NK cell metabolism and functionalities against glioblastoma targets. We also cross-reference TIM-3 expression level with glioblastoma patient samples, which provide clinical context for microenvironmental cues and nutrient deprivation.ConclusionsOur findings suggest that TIM-3 expression is associated with both ex vivo-activated NK cell glucose metabolism and cytotoxic function against glioblastoma. As ex vivo-activated NK cells are considered to be highly glycolytic, and as such associated with higher cytotoxicity, TIM-3’s involvement with glucose uptake could prove crucial in sustaining NK cytotoxic phenotype in the tumor microenvironment. This information is shedding further light on the immunomodulatory roles of TIM-3, and aiding in leveraging this receptor usage in future NK cell-based immunotherapies.Ethics ApprovalAll procedures performed in studies involving human participants were approved by Purdue University’s Institutional Review Board (IRB) in August 2018 (#1804020540). All institutional safety and biosecurity procedures were adhered to.

scholarly journals Adoptively Transferred Donor-Derived Cytokine Induced Memory-like NK Cells Persist and Induce Remission in Pediatric Patient with Relapsed Acute Myeloid Leukemia after Hematopoietic Cell Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3307-3307
Author(s):  
Jeffrey J. Bednarski ◽  
Clare Zimmerman ◽  
Amanda F Cashen ◽  
Sweta Desai ◽  
Mark Foster ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Hematopoietic Cell Transplantation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Salvage Chemotherapy ◽  
Acute Myeloid ◽  
Refractory Aml

Acute myeloid leukemia (AML) accounts for 18% of pediatric leukemias. For high-risk AML, standard treatment includes multi-agent chemotherapy followed allogeneic hematopoietic cell transplantation (HCT). Despite a high remission rate, 50% of patients relapse and have a poor prognosis with < 20% of patients surviving more than 3 years. Salvage chemotherapy alone or combined with donor lymphocyte infusions (DLI) have little curative potential, and new treatment strategies are needed for relapsed-refractory AML. Previous studies have shown that natural killer (NK) cells can be stimulated ex vivo with IL-12/15/18 to generate a memory-like phenotype with enhanced anti-leukemia effect. In adults with relapsed-refractory AML, adoptive transfer of MHC-haploidentical cytokine-induced memory-like (CIML or ML) NK cells induced remission in 54% of patients (PMID27655849). The infused donor ML NK cells expand in vivo but are rapidly eliminated following recovery of recipient T cells, providing a window of therapeutic activity of 2-3 weeks. We sought to test the safety and efficacy of ML NK cells for treatment of pediatric/young adult patients with post-HCT relapsed AML. We hypothesized that ML NK cells derived from the HCT donor would be well-tolerated, exhibit anti-leukemia activity, and expand with prolonged persistence following transfer into pediatric AML patients. Here, we report the results of the first pediatric patient treated on a phase I clinical trial using ML NK cell therapy for relapsed AML after allogeneic HCT (NCT03068819). Briefly, patients are treated with FLAG (fludarabine, cytarabine and granulocyte colony stimulating factor) salvage chemotherapy to reduce the bulk of AML and provide lymphodepletion for ML NK cell expansion. Two weeks after chemotherapy, a non-mobilized leukapheresis product is collected from the original HCT donor and processed into a T cell-based DLI and ML NK cells. The T cell DLI (1 x 106 T cells/kg) is immediately infused, and the ML NK cells are generated by stimulation with IL-12/15/18 ex vivo for 12-16 hours and then infused (10x106/kg). An 18-month-old male with infant AML had relapse of his leukemia 3 months after MHC-haploidentical HCT. Treatment with chemotherapy, including mitoxantrone and daunorubicin-cytarabine liposome, failed to induce remission. At the time of enrollment on the phase I trial, he had AML blasts in his bone marrow (Table 1). He was treated with FLAG chemotherapy followed by infusion of DLI and ML NK cells from the original haploidentical HCT donor. Assessment at 30 days, 3 months and 6 months post NK cell infusion showed complete remission with no evidence of leukemia and full donor engraftment. Remarkably, donor-derived ML NK cells expanded to 77% of donor lymphocytes on day 28 and still comprised 24% percent of lymphocytes at 6 months post infusion (Figure 1A-C). The expanded donor NK cell phenotype was consistent with ML NK cells (e.g., NKG2A+KIR+) utilizing CyTOF multidimensional analysis previously confirmed to identify ML NK cells (Figure 1D). The ML NK cells were functional as demonstrated by leukemia-triggered IFN-γ production immediately ex vivo from day 7-28 samples (Figure 1E-F). The patient's clinical course was complicated by mild gastrointestinal graft-versus-host disease that resolved with low-dose steroids and tociluzimab. These early results demonstrate proof-of-principle that adoptive transfer of donor-derived ML NK cells in combination with DLI is feasible and offers a novel immunotherapy option for patients with relapsed AML after HCT. Moreover, in this T and NK cell compatible immune environment post-HCT, donor ML NK cells expand and persist robustly in vivo for > 6 months without exogenous cytokine support and have potent anti-leukemic activity. Thus, ML NK cells are a cellular therapy platform to treat AML that has relapsed after allogeneic HCT. Disclosures Cashen: Celgene: Other: Speaker's Bureau; Seattle Genetics: Other: Speaker's Bureau; Novartis: Other: Speaker's Bureau. Fehniger:Horizon Pharma PLC: Other: Consultancy (Spouse); Cyto-Sen Therapeutics: Consultancy.

scholarly journals Ruxolitinib partially reverses functional NK cell deficiency in patients withSTAT1gain-of-function mutations

2017 ◽  
Author(s):  
Alexander Vargas-Hernandez ◽  
Emily M. Mace ◽  
Ofer Zimmerman ◽  
Christa S. Zerbe ◽  
Alexandra F. Freeman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Function ◽  
Viral Infections ◽  
Nk Cell ◽  
Cell Phenotype ◽  
Effector Cells ◽  
Viral Susceptibility ◽  
And Function

AbstractBackgroundNatural Killer (NK) cells are critical innate effector cells whose development is dependent on the JAK-STAT pathway. NK deficiency can result in severe or refractory viral infections. Patients with Signal Transducer and Activator of Transcription (STAT)1 gain of function (GOF) mutations have increased viral susceptibility.ObjectiveWe sought to investigate NK cell function in STAT1 GOF patients. Methods: NK cell phenotype and function were determined in 16 STAT1 GOF patients.MethodsNK cell phenotype and function were determined in 16 STAT1 GOF patients.NK cell lines expressing patient mutations were generated with CRISPR-Cas9 mediated gene editing. STAT1 GOF NK cells were treated in vitro with ruxolitinib.ResultsPeripheral blood NK cells from of STAT1 GOF patients had impaired terminal maturation. Specifically, patients withSTAT1 GOFmutations have immature CD56dimNK cells with decreased expression of CD16, perforin, CD57 and impaired cytolytic function. STAT1 phosphorylation was elevated but STAT5 was aberrantly phosphorylated in response to IL-2 stimulation. Upstream inhibition of STAT signaling with the small molecule JAK1/2 inhibitor ruxolitinibin vitroandin vivorestored perforin expression in CD56dimNK cells and partially restored NK cell cytotoxic function.ConclusionsProperly regulated STAT1 signaling is critical for NK cell maturation and function. Modulation of elevated STAT1 phosphorylation with ruxolitinib is an important option for therapeutic intervention in patients withSTAT1 GOFmutations.

scholarly journals 799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A834-A834
Author(s):  
Xue Yao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

scholarly journals Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3607-3607
Author(s):  
Grace Lee ◽  
Sheela Karunanithi ◽  
Zachary Jackson ◽  
David Wald
Keyword(s):  
Cell Therapy ◽  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Akt Activation ◽  
Nk Cell Cytotoxicity ◽  
Rock Inhibition ◽  
Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'&gt;0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

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