A Novel Method for Highly Efficient Ex Vivo Expansion and Genetic Engineering of Human Natural Killer Cells for Cancer Therapy

Abstract Cancer immunotherapy with adoptive transfer of human leukocyte antigen-mismatched, CD19-targetd chimeric antigen receptor (CAR)-transduced natural killer (NK) cells has attracted attention because of its efficacy and safety when infused in patients with refractory and relapsed B-cell lymphomas. However, generating clinical doses of CAR-NK cells is still a challenge. The methods for ex vivo expansion and genetic modification of primary human NK cells usually rely on the use of irradiated feeder cell lines, which has been restrictive due to high costs, scale-up difficulties, and licensing restrictions. Hence, novel strategies that do not require feeder cells will be beneficial in standardizing these types of cell therapies. In this study, we show the effectiveness of a novel feeder-free culture system in expanding NK cells ex vivo and generating CAR-NK cells. Unsorted peripheral blood mononuclear cells (PBMCs) collected from healthy donors were cultured with a reagent containing dissolvable microspheres that are conjugated with anti-CD2/NKp46 antibodies (Cloudz TM Human NK Cell Expansion Kit) and a combination of multiple cytokines, including interleukin (IL)-2, IL-12, IL-18, and IL-21 in medium supplemented with 10% fetal bovine serum. The activated NK cells were transduced using the RD114-pseudotyped retrovirus vector. To test whether the reagent promoted ex vivo NK cell expansion, we cultured PBMCs from 13 donors. The percentage of NK (CD56 + CD3 -) cells of initially isolated PBMCs was 15.3±7.5%. In the presence of multiple cytokine combinations, NK cell purity gradually increased and reached 91.6±7.6% by day 21. The NK cells expanded to 75.6±59.2-fold at day 10, 334±217-fold at day 14 and 1,542±913-fold at day 21. The expanded NK cells degranulated and produced intracellular cytokines upon exposure to K562 myeloid leukemia cells. The NK cells efficiently killed myeloid leukemia cells, such as K562, THP1, and KG1. The expression pattern of killer cell immunoglobulin-like receptors on NK cells remained unchanged. The expression of activating NK cell receptors, including NKp30 and NKp44, increased after 21 days of culture. Thereafter, a gene transfer to the primary human NK cells was conducted. We tested transduction efficiency and yields of modified cells on 7 days after the procedure by empty-vector transduction into NK cells expanded ex vivo for 3, 6, and 10 days (n=3, each). The results were presented as means ± standard deviation; 55.6±11.6%, 61.6±14.1%, and 73.6%±6.2% for GFP positivity in NK cells and 5.7±1.5 folds, 56.4±42.2 folds, and 12.7±5.8 folds for yields of modified cells. We selected the condition in which the transduction was carried out using NK cells expanded ex vivo for 6 days, although the differences were not statistically significant. Next, anti-CD19 CAR with a 4-1BB costimulatory and CD3z domain was transduced into NK cells. We confirmed high transduction efficiency (59.8% ±20.5%, n=3) and high CAR protein expression on the cell surface, while NK cells maintained their purity and minimal T cell outgrowth was observed. CAR-NK cells maintained their proliferative status and further expanded 15.2±6.4-fold after 7 days of the procedure. To determine whether the generated anti-CD19 CAR-NK cells had a specific effect on B cell malignancies, a CD107a mobilization assay, intracellular cytokine assay, and a flow cytometry-based cytotoxicity assay was employed. We found that CAR transduction could render NK cells to generate specific and powerful responses against CD19-positive, NK-resistant leukemia and lymphoma cell lines, such as BCR-ABL-positive acute lymphoblastic leukemia (ALL) OP-1, Burkitt lymphoma Raji, and KMT2A-rearranged ALL RS4;11, at various effector: target (ET) ratios. For example, in a 4 hour -assay, the cytotoxic effects of anti-CD19 CAR-NK cells showed 86.9±0.2% cytotoxicity against OP-1, while mock NK cells showed 17.4±2.9% cytotoxicity (ET ratio 1:1). In conclusion, this study revealed highly efficient functions of the novel feeder-free culture system, including highly efficient ex vivo expansion of primary human NK cells and generation of genetically modified NK cells for cancer immunotherapy. In future studies, we will investigate large-scale cultures using specialized flasks and GMP-grade reagents for clinical translation and the in vivo activities of the cell products in mouse xenograft models. Disclosures Imai: Juno Therapeutics: Patents & Royalties: chimeric receptor with 4-1BB signaling domain.

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

Blood ◽

10.1182/blood.v126.23.3090.3090 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3090-3090 ◽

Cited By ~ 1

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.

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Sustained Ex Vivo Expansion of Human Peripheral Blood NK Cells Using Artificial APCs Bearing Membrane-Bound IL-21.

Blood ◽

10.1182/blood.v114.22.3030.3030 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3030-3030

Author(s):

Cecele J Denman ◽

Lisa M. Kopp ◽

Vladimir Senyukov ◽

Sarah Hagemeister ◽

Jennifer Johnson ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Peripheral Blood ◽

Cell Expansion ◽

Nk Cell ◽

Ex Vivo ◽

Costimulatory Molecules ◽

Ex Vivo Expansion ◽

Cell Bank ◽

Membrane Bound

Abstract Abstract 3030 Poster Board II-1006 Introduction NK cells have therapeutic potential for a wide variety of human malignancies. The major obstacle for adoptive NK cell immunotherapy is obtaining sufficient cell numbers, as these cells represent a small fraction of peripheral white blood cells, expand poorly ex vivo, and have limited life spans in vivo. Common gamma-chain cytokines are important in NK cell activation, maturation, and proliferation. Others have described improved ex vivo expansion of NK cells using soluble cytokines, when cocultured with stimulated peripheral blood mononuclear cells (PBMC) or Epstein Barr Virus (EBV) lymphoblastioid cell lines, or with artificial antigen presenting cells (aAPC) engineered with costimulatory molecules and/or membrane-bound IL-15 (mIL-15). Expansion of NK cells by these methods has been limited by senescence from telomere shortening. To generate clinical-grade T cells for adoptive transfer, our group developed aAPC derived from K562 retrovirally transduced to express the costimulatory molecules CD86 and CD137L. These aAPC were produced as a master cell bank and further genetically modified to express membrane-bound cytokines. Since IL-21 signals via STAT3, and STAT3 is a known activator of telomerase transcription, we investigated whether NK cell expansion with mIL-21 would provide a sustained proliferative advantage over or in combination with mIL-15. Methods K562 aAPC were retrovirally transduced to express CD64, CD86, CD137L, CD19 (Clone 9), and mIL-15 (Clone 4). These clones were further modified by Sleeping Beauty integration of mIL-21 (Clone 9+IL-21 and Clone 4+IL-21). Freshly isolated PBMC from 5 donors were co-cultured with irradiated K562 aAPC (Clone 4, Clone 4+mIL-21, and Clone 9+mIL-21) at a ratio of 2:1 (aAPC:PBMC) in the presence of 50 IU/ml of rhIL-2. Half of the media was changed every two days and cells were re-stimulated with aAPC every seven days at ratio of 2:1. Cells were counted and phenotyped on day 0, 7, 14, and 21 for CD3, CD16, CD56, NKG2D, KIR (2DL1, 2DL2/3, and 3DL1), and NCR (NKp30, NKp44, NKp46). A preclinical SOP to expand PBMC from a 20 mL blood draw was established and additional donors of known HLA type were expanded with Clone 9+mIL-21 for up to 7 weeks. Cytotoxicity function against K562, 721.221, Raji, and AML targets was measured using the Calcien-AM assay (Invitrogen). Telomere length of expanded and fresh NK cells was measured with the FlouFish assay using the telomere specific FITC conjugated (C3TA2)3 PNA probe. Results By day 14, aAPCs bearing mIL-21 induced greater total cell expansion than those with mIL-15 alone (188, 2900, and 2281-fold for Clone 4, Clone 4+mIL-21, and Clone 9+mIL-21, respectively). However, PBMC cultured without mIL-15 contained far fewer co-expanding T cells. Exponential expansion continued for up to 7 weeks without evidence of senescence when mIL-21 was present, reaching a mean of 91,566-fold expansion of the CD3−CD16/56+ population at 4 weeks. NK cells expanded with mIL-21 had increased expression of KIR and NCR, and expressed very high CD16 and NKG2D levels. These NK cells showed much higher cytotoxicity against all targets than fresh NK cells, retained KIR inhibition, and demonstrated enhanced killing via ADCC. Furthermore, telomere lengths of NK cells expanded with Clone 9+mIL-21 were longer than that of fresh NK cells or those expanded without mIL-21, perhaps explaining the continued expansion without senescence. Thus, NK cell expansion is improved using aAPCs expressing mIL-21 rather than mIL-15. We are currently establishing a GMP-grade working cell bank of Clone 9+mIL-21 for use in clinical trials. Funding: Brenda and Howard Johnson Fund, UT MD Anderson Physician Scientist Program Disclosures No relevant conflicts of interest to declare.

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Donor-Derived Ex-Vivo Activated NK Cells in Management of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation in High-Risk Acute Leukemia

Blood ◽

10.1182/blood.v124.21.312.312 ◽

2014 ◽

Vol 124 (21) ◽

pp. 312-312

Author(s):

Xing-Yu Cao ◽

Tong Wu ◽

Bi-Ping Deng ◽

Rui-Juan Sun ◽

Yue Lu ◽

...

Keyword(s):

High Risk ◽

Cell Therapy ◽

Acute Leukemia ◽

Nk Cells ◽

Culture System ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Original Culture ◽

Nk Cell Therapy

Abstract Introduction: Relapse remains the main cause of failure of hematopoietic stem cell transplantation (HSCT) in acute leukemia. NK cells have the property of killing leukemia cells without GVHD aggravation theoretically. Moreover, in some cases, leukemia cells may lost HLA-I and/HLA-II antigens which would result in poor response to the immunotherapy except NK-based adoptive effectors. Objective: In present study, the safety and efficacy of donor-derived ex-vivo activated NK cells in management of relapse after allogeneic HSCT in high-risk acute leukemia were examined. Patients and methods: Between July 2012 and July 2014, 29 patients with acute leukemia who received NK cell infusion after HSCT were analyzed retrospectively. Some cases failed to chemotherapy combined with donor lymphocyte infusion (DLI) before NK cell therapy. The diagnosis were ALL (10 cases), AML (18 cases) and mixed acute leukemia (1 case). All patients were high-risk leukemia. The disease status before transplant was CR1 in 8 cases, CR2 in 7, CR3 in 1 and non-remission in 13. The types of donor included identical sibling (5 cases), haploientical family member (21 cases) and unrelated donor (3 cases). The conditioning and GVHD prophylactic regimens were reported previously (Lu DP et al., Blood 2006; 107:3065). Minimal residual disease (MRD) was detected by either quantitative RT-PCR for fusion genes or flow cytometry or both. The expression of HLA-I and HLA-II antigens in leukemia cells was evaluated by flow cytometry. Donor-derived either peripheral blood stem cells or lymphocytes were cultured for 6 days using original culture system (AIM-V medium with IL-2, IL-12, IL-15 and IL-21) or modified culture system (SCGM medium with IL-2, IL-12, IL-15, IL-18 and IL-21). Escalated dosage of NK cells were infused starting with 1×105 cells/kg (recipient’s body weight) with or without IL-2 injection. Nine patients were in prevention group and 20 cases were in treatment group. The patients with hematologic relapse received NK cells 3 days later after chemotherapy. Results: Compared with our original culture system, the modified culture system enhanced approximately 10% to 20% of the purity and 4 to 8 fold in number of NK cells by day 6. Furthermore, our modified culture system elevated the expression of function phenotype including TRAIL, NKG2D and CD62L on NK cells in approximately 8 to 10 folds at day 6 and simultaneously stimulated higher level of IFN-γ. One to 4 NK cell infusions were given in each case with two week interval. Two of 29 cases developed mild skin GVHD. No transfusion-related side effects were noted. In prevention group, four of 9 cases remain complete remission, and the other 5 patients became MRD positive or relapse. In treatment group, seven of 20 cases have response to NK cell therapy, and two out of 7 cases who response to NK cells had failed to chemotherapy plus DLI before. Among 11 patients who had response to NK cells, eight of them are AML, and the remaining 3 patients are ALL. Higher response rate (10/23 cases) was seen with NK cell therapy by our modified culture system compared with the one (1/6 cases) by our original culture system. Conclusions: Our preliminary results have demonstrated that donor-derived ex-vivo activated NK cells are safe and effective modality in the management of relapse after allogeneic HSCT in high-risk acute leukemia even failed to chemotherapy combined with DLI. Optimal culture system has improved not only NK cell’s purity, number and function phenotype but also clinical efficacy. Disclosures No relevant conflicts of interest to declare.

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.852 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A893-A893

Author(s):

Laurent Gauthier ◽

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.

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Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽

10.3390/cells9040811 ◽

2020 ◽

Vol 9 (4) ◽

pp. 811

Author(s):

Pranav Oberoi ◽

Kathrina Kamenjarin ◽

Jose Francisco Villena Ossa ◽

Barbara Uherek ◽

Halvard Bönig ◽

...

Keyword(s):

Nk Cells ◽

Progenitor Cells ◽

Peripheral Blood ◽

Cell Expansion ◽

Nk Cell ◽

Ex Vivo ◽

Feeder Cells ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

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Immunostimulatory Properties of the Human Ortholog of the GMCSF/IL2 Fusion Protein for Cell Based Therapy.

Blood ◽

10.1182/blood.v110.11.4894.4894 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4894-4894

Author(s):

Claudia Penafuerte Graduate ◽

Jacques Galipeau

Keyword(s):

Fusion Protein ◽

Nk Cells ◽

Cell Expansion ◽

Cell Activation ◽

Nk Cell ◽

Ex Vivo ◽

Potential Candidate ◽

Activation Markers ◽

Cell Based Therapy

Abstract NK cells constitute a potential candidate for cancer cell therapy because they express a diverse array of inhibitory and activating receptors, which recognize and kill infected or tumor cells without prior immune sensitization. However, autologous NK cell mediated adoptive immunotherapy is restricted due to insufficient cytolytic activity of NK cells from patient with aggressive malignancies. In contrast, the infusion of alloreactive NK cells has shown more successful outcomes in the treatment of cancer, but this approach also presents difficulties such as the high doses of cytokines required to induce NK cell expansion ex vivo, which may also sensitize NK cells to apoptosis. Therefore, a critical issue for NK cell based therapy is the use of appropriate growth factors or cytokines that promote NK cell expansion and activation. We have previously shown that a murine GM-CSF/IL-2 fusion protein (aka GIFT2) displays novel antitumor properties in vivo compared to both cytokines in combination regarding tumor site recruitment of macrophages and significant functional NK cell infiltration [Stagg et al., Cancer Research (December 2004)]. In the present work, we found that human GIFT2 will lead to a substantial two fold proliferation of human blood-derived NK cells which is significantly (p<0.05) superior to either IL2 or GMCSF single cytokine treatment or both cytokines combined at equimolar concentration. In addition, we observed that GIFT2 leads to robust expression of NK-cell activation markers CD69 and CD107a. In conclusion, the human GIFT2 fusokine is a novel and potent tool for ex vivo expansion of activated NK cells which may be of use in cell-based immunotherapy of cancer.

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Ex Vivo Expansion of Highly Purified Human NK Cells..

Blood ◽

10.1182/blood.v114.22.2157.2157 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2157-2157

Author(s):

Chunji Gao ◽

Xiaohong Li ◽

Jian Ma ◽

Xiaoxiong Wu ◽

Feifei Wang ◽

...

Keyword(s):

Nk Cells ◽

High Purity ◽

Cell Expansion ◽

Ex Vivo ◽

Granzyme B ◽

Ex Vivo Expansion ◽

Biological Functions ◽

Hematopoietic Stem ◽

Significant Difference ◽

Ifn Γ

Abstract Abstract 2157 Poster Board II-134 Object To optimize the expansion of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after Ex vivo expansion. Methods NK cells were isolated from PBMNC by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II(Miltenyi Biotec, Germany), then they were cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were fed with fresh media and cytokines every 3 days, and were evaluated for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-γ secretion at the end of the culture period. Results In group IL2+IL15 and IL2+IL15+IL12, cells were expanded 50.46±4.31 and 52.35±6.72 fold, respectively, much more higher than others(P<0.01), but no significant difference between them (P>0.05). And the purity of CD3−CD56+NK cells was over 94% in all groups except the control. The expressions of perforin and granzyme B mRNA of expanded NK cells cultured with cytokines was significantly higher than the starting population(P<0.01), although IL2+IL15+IL12 group was slightly higher than that of IL2+IL15 group, without significant difference (P>0.05). There was great increase in IFN-γ levels in the supernatants of NK cells culture in the presence of cytokines; IL2+IL15+IL12 group and IL2+IL12 group was significantly higher than others(P<0.01). Conclusion High purity NK cells could be efficiently expanded in culture with IL2+IL15, and its biological functions were enhanced in this condition. Disclosures: No relevant conflicts of interest to declare.

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RANKL Expressed by Acute Myeloid Leukemia Cells Impairs NK Cell-Mediated Immune Surveillance

Blood ◽

10.1182/blood.v116.21.2164.2164 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2164-2164

Author(s):

Benjamin J Schmiedel ◽

Constantin M Wende ◽

Tina Baessler ◽

Carolin Scheible ◽

Stefan Wirths ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Conflicts Of Interest ◽

Immune Surveillance ◽

Surface Protein ◽

Leukemia Cells ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Abstract 2164 NK cells play an important role in tumor immunosurveillance, especially of leukemia. Their reactivity is governed by various activating and inhibitory molecules expressed by their targets including multiple members of the TNF family. The TNF family member Receptor Activator of NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but recently have also been shown to influence progression of hematopoetic malignancies. Here we studied the yet unkown role of the RANK/RANKL molecule system in NK cells and their reactivity against acute myeloid leukemia (AML). Primary leukemia cells from AML patients were found to substantially express RANKL mRNA and surface protein in 75% of the investigated cases (n=40). Reverse signaling via surface-expressed RANKL into AML blasts induced the release of soluble factors including the immunoregulatory cytokines TNF and IL-10, which impaired NK cell anti-tumor reactivity. Moreover, we observed upregulation of RANK on NK cells among PBMC of healthy donors upon exposure to IL-10. This was not caused by direct effects on NK cells, but was rather due to yet unidentified factors released by monocytes among the PBMC upon IL-10 exposure and could be prevented by the activating cytokine IL-2. Furthermore, functional experiments with NK cells and RANKL transfectants or RANKL-negative controls revealed that forward signaling into RANK-expressing NK cells by tumor-expressed RANKL also directly impaired NK cytotoxicity and IFN-γ production. In line, blocking RANK-RANKL interaction using anti-RANKL antibodies or RANK-Fc fusion protein increased cytotoxicity and cytokine production of allogenic NK cells in cultures with RANKL-positive primary AML cells. Our data indicate that RANKL expression enables immune evasion of leukemia cells both by directly inhibiting reactivity of RANK-expressing NK cells and by orchestrating a reciprocal interplay between AML cells, monocytes and NK cells resulting in an immunosuppressive cytokine milieu. Thus, therapeutic modulation of the RANK/RANKL system, e.g. with Denosumab/AMG162, which is presently being evaluated for treatment of both non-malignant and malignant osteolysis, holds promise to reinforce NK reactivity against hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

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Transfer of Ex Vivo Expanded NK and γδT Cells from Untouched Posttransplant PBMCs to Clear Minimal Residual Disease in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.5294.5294 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5294-5294

Author(s):

Patrick Schlegel ◽

Chihab Klose ◽

Christina Kyzirakos ◽

Ursula J.E. Seidel ◽

Kai Witte ◽

...

Keyword(s):

Cell Culture ◽

Acute Myeloid Leukemia ◽

Nk Cells ◽

Cell Expansion ◽

Myeloid Leukemia ◽

Nk Cell ◽

Residual Disease ◽

Cell Transfer ◽

Minimal Residual ◽

Acute Myeloid

Abstract GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded >1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

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Manufacture of Clinical-Grade Universal Antigen Presenting Cells (UAPC) for the Ex Vivo Expansion and Activation of Natural Killer (NK) Cells

Blood ◽

10.1182/blood-2018-99-118804 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2061-2061

Author(s):

Enli Liu ◽

Sonny Ang ◽

Yijiu Tong ◽

Li Li ◽

Rafet Basar ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Cell Activation ◽

Nk Cell ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

High Avidity ◽

Clinical Grade ◽

Antigen Presenting ◽

Promising Source

Abstract NK cells are potent cellular immunotherapeutic agents against a wide array of human malignancies. Ex vivo expansion of NK cells to achieve clinically relevant numbers must overcome poor in vitro growth kinetics, low starting percentages within the mononuclear cell fraction (especially from autologous donors with active disease), and limited in vivo life span. We targeted three universally critical NK signaling pathways, namely IL-21, 4-1BB, and SLAM family member 4 (SLAMF4), to increase NK cell proliferation and enhance survival. We genetically-engineered HLA-A-ve and -B-ve K562 cells to enforce expression of membrane-bound IL-21 (mbIL21), 4-1BB-L, and CD48, forming a universal antigen presenting cell (UAPC) to generate highly potent clinical-grade umbilical cord blood (CB-NK) or peripheral blood NK cells (PB-NK). While the mbIL21 and 4-1BB signaling nexuses have been utilized previously, we highlight here SLAMF-mediated immunological sculpting of NK cells for clinical applications. SLAMF triggering of co-receptors modulate NK cell activation, in particular through high-avidity interactions between SLAMF4 (2B4/CD244) and its heterophilic, robust affinity, and physiological ligand CD48, a glycosyl-phosphatidyl-inositol (GPI)-anchored cell surface protein. Upon ligand binding and receptor phosphorylation, SLAMF4 recruits PTPN11/SHP-2 and SH2D1A/SAP for downstream signaling, including significant increases in NK cell-mediated cytotoxicity, granule exocytosis, and production of IFN-γ and IL-2. Other than a subset of low surface density expressers from aging subjects consistently associated with inefficient and impaired activating signal transduction, the majority of NK cells express SLAMF4. The functional prominence of SLAMF4, presently recognized as an activating co-receptor, is also evidenced by loss-of-function mutations associated with X-linked lymphoproliferative (XLP) disease. We achieved log-scale expansion of NK cells with UAPC (>1000 fold in 2 weeks), with excellent purity (>99% CD56+ve/CD3−ve and < 1% CD3+ve cells), without indications of senescence/exhaustion, even after 4 weeks of culture. Surface molecular phenotypes of UAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded without SLAMF4. Synergistic signals from IL-21/STAT3, 4-1BB/4-1BBL, and SLAMF4 drive tonic NK propagation, supporting their clinical application. Our novel and clinically accessible platform technology for generation of high purity CB-NKs, a promising source of fresh and cryopreserved allogeneic NK cells, is well-suited for adoptive cancer immunotherapy. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

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