KIR Expression on inVitro-Derived Natural Killer Cells Does Not Regulate Killing of Allogeneic Targets

Abstract Natural killer (NK) cells play an essential role in early innate killing of virally-infected and tumor targets. NK cell-mediated activity is regulated by a repertoire of activating and inhibitory receptors that recognize ligands on diseased, stressed, or tumor targets. Killer cell immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors that can be inhibitory or activating based on their intracellular signaling motifs. Expression of certain KIR haplotypes plays a key role in survival and relapse prevention for patients with acute myelogenous leukemia (AML) who receive allogeneic hematopoietic cell transplantation. Therefore, KIR haplotypes are an important consideration in selecting allogeneic donors for patients with AML. However, it is unclear if KIRs play a role in adoptive transfer of NK cells that are becoming more routinely utilized to treat refractory AML and other malignancies. To better address this question we used umbilical cord blood to isolate both CD34+ hematopoietic stem cells and CD45+CD56+ NK cells (UCB-56-NK) from the same umbilical cord blood unit. The CD34+ hematopoietic stem cells were then differentiated in vitro into CD56+ NK cells (UCB-34-NK). Despite originating from the same donor and sharing the same genetic background, as well as comparable expression of Fas ligand, TRAIL, NKp46, NKp44, NKG2A, and NKG2D, the UCB-34-NK cells have characteristically low KIR expression, whereas the UCB-56-NK cells have high KIR expression. This phenotype was further confirmed by mass cytometric (CyTOF) analysis of UCB-56-NK cells and UCB-34-NKcells with a panel of 36 phenotypic and functional NK cell markers. This unique system allows us to study the role of KIR expression independent of any other variations in donor or cell characteristics. The cytotoxicity and NK cell activation of UCB-34-NK cells and UCB-56-NK cells are compared to control NK cells isolated from peripheral blood (PB-NK cells) with standard in vitro cytotoxicity assays against neuroblastoma lines with varying HLA genotypes and a control K562 leukemic targets. Our data demonstrates that there is no statistical difference in NK cytotoxicity and activation of UCB-34-NK cells and UCB-56-NK cells across a spectrum of target cell HLA types, despite the differences in KIR expression. For example, at effector to target (E:T) ratios of 1:5 and 1:20 against neuroblastoma line IMR32, UCB-34-NK cells (KIR-low) demonstrated 68.5% and 84.1% maximal Caspase 3/7 activation, compared to 81.3% and 89.6% by UCB-56-NK cells (KIR-high). Additionally, we have used human induced pluripotent stem cells to derive NK cells (iPSC-NK cells) that vary in KIR expression levels. These CD45+CD56+ iPSC-NKs are differentiated from the same well-established iPSC line in the laboratory and therefore again share the same genetic background, and they have similar NK cell surface receptor expression of Fas ligand, TRAIL, NKp46, NKp44, and NKG2D, but differ in levels of KIR expression. Again in vitro cytotoxicity against hematopoietic tumor targets such as K562 and MOLM13 do not demonstrate a significant difference in killing, despite these KIR differences. For example, in targeting erythroleukemia K562 cells, iPSC-NK cells with high levels of KIR expression at E:T ratios of 1:2.5, 1:5, and 1: 10 have Caspase 3/7 activation of 21.1%, 28.2%, and 41.0%, compared to 20.1%, 22.0%, and 31.2% by iPSC-NK cells with low KIR expression. Together, these studies demonstrate that in vitro-derived NK cells do not require KIR expression to become licensed for anti-tumor activity and these cells are able to kill tumor targets whether or not they express KIRs. These studies better enable use of these allogeneic NK cell populations for off-the-shelf NK cell-based therapies without the need to optimize for KIR profiles for patients of differing HLA haplotypes. Disclosures Malmberg: Fate Therapeutics Inc.: Consultancy, Research Funding. Kaufman:Fate Therapeutics: Consultancy, Research Funding.

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CD16-IL15-CLEC12A Trispecific Killer Engager (TriKE) Drives NK Cell Expansion, Activation, and Antigen Specific Killing of Cancer Stem Cells in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-117150 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1454-1454 ◽

Cited By ~ 2

Author(s):

Upasana Sunil Arvindam ◽

Paulien van Hauten ◽

Caroline Hallstrom ◽

Daniel A. Vallera ◽

Harry Dolstra ◽

...

Keyword(s):

Stem Cells ◽

Nk Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

Nk Cell ◽

Target Cells ◽

Hematopoietic Stem ◽

Functional Assays ◽

Target Effects

Abstract Our group developed a 161533 trispecific killer engager (TriKE) molecule to target acute myeloid leukemia (AML) cells using Natural Killer (NK) cells. This molecule contains an anti-CD16 camelid nanobody to activate NK cells, an anti-CD33 single chain variable fragment (scFv) to engage cancer targets, and an IL-15 molecule that drives NK cell priming, expansion and survival. Using an earlier version of this molecule, we have shown that the CD33 TriKE is effective at activating NK cells against AML targets in vitro and in vivo. This preclinical data has lead to the establishment of a clinical trial in refractory AML patients at the University of Minnesota, set to open Q3 2018. While these previous studies have validated the use of TriKEs as an effective strategy of harnessing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen. The high mortality and poor five-year survival rates (26%) for AML patients can be attributed to chemotherapy resistance and disease relapse. A majority of chemotherapy resistant leukemia stem cells (LSCs), that are hypothesized to facilitate relapse, do not express CD33. In addition, all hematopoietic stem cells and normal myeloid cells express CD33, thus targeting this antigen can lead to severe defects in hematopoiesis and on-target/off-tumor toxicity. To address these limitations, we developed a TriKE that targets CLEC12A or C-type lectin-like molecule 1 (CLL-1). CLEC12A is highly expressed on AML cells and over 70% of CD33 negative cells express CLEC12A. It has been attributed as a stem cell marker in AML, being selectively overexpressed in LSCs. CLEC12A is expressed by CD34+/CD38- LSCs but not normal CD34+/CD38- hematopoietic stem cells in regenerating bone marrow, thus minimizing off-target effects. The CLEC12A TriKE was developed in a mammalian cell system to ensure that appropriate post-translational modifications are present. We confirmed that the TriKE binds specifically to HL-60 and THP-1 target cells that express CLEC12A compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with the CLEC12A TriKE drives a significant increase in NK cell specific proliferation over 7 days as measured by CellTrace dilution compared to treatment with a CLEC12A scFv or IL-15 alone (69.7 ± 6.7% vs 11.9 ± 2.5% vs 38.4 ± 7.3%) (Figure 1A). To measure NK cell killing, we conducted an IncuCyte Zoom assay. Here, HL-60 target cells were labeled with a caspase 3/7 reagent where a color change indicates target cell death. The CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15 as measured by number of live target cells at the end of the 48 hour assay (53.9 ± 1.9% vs 103.3 ± 3.4% vs 71.1 ± 1.4%). The CLEC12A TriKE induces an increase in NK cell degranulation, measured by CD107a expression against HL-60 AML tumor targets in a 4 hour functional assay compared to treatment with CLEC12A scFv or IL-15 alone (62.3 ± 1.1% vs 19.4 ± 3.8% vs 27.5 ± 4.9%). In this assay, there is also an increase in cytokine production, measured by IFNg and TNFa respectively (16.7 ± 4.2% vs 2.3 ± 1.5% vs 4.7 ± 1.9% and 18.0 ± 5.1% vs 2.5 ± 1.7% vs 4.6 ± 2.5%) (Figure 1B). We observe a similar enhanced functional response with THP-1 AML tumor targets. In these functional assays, treatment with the CLEC12A TriKE produced less background activation compared to the CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, we tested the efficacy of the CLEC12A TriKE against primary AML targets. AML blasts were identified as SSC low, CD45 intermediate and CD34 high cells. Out of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC12A (78.1 ± 5.2%). In functional assays with these samples, the CLEC12A TriKE was able to induce greater CD107a and IFNg expression, and enhanced killing of tumor targets as measured by a live/dead stain compared to CLEC12A scFv or IL-15 (Figure 1C). In these assays, the efficacy of the CLEC12A TriKE was comparable to the CD33 TriKE. Our data demonstrates that the CLEC12A TriKE drives NK cell specific proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. Apart from AML, CLEC12A is expressed on cancer cells and LSCs in patients with myelodysplastic syndromes (MDS). These findings highlight the clinical potential of the CLEC12A TriKE individually or in combination with the CD33 TriKE for the treatment of MDS and AML. Figure 1. Figure 1. Disclosures Vallera: GT Biopharma: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding.

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RTID-07. HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS (CYNK-001) FOR TREATMENT OF RECURRENT GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.812 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii194-ii195

Author(s):

Nazanin Majd ◽

Maha Rizk ◽

Solveig Ericson ◽

Kris Grzegorzewski ◽

Sharmila Koppisetti ◽

...

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

In Vitro Cytotoxicity ◽

Orthotopic Mouse Model ◽

Hematopoietic Stem ◽

Dismal Prognosis

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with dismal prognosis. Recent advances of immunotherapy in cancer have sparked interest in the use of cell therapy for treatment of GBM. Active transfer of Natural Killer (NK) cells is of particular interest in GBM because NK cells are capable of exerting anti-tumor cytotoxicity without the need for antigen presentation and sensitization, processes that are impaired in GBM. CYNK-001 is an allogeneic, off-the-shelf product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells manufactured by Celularity. Here, we demonstrate in vitro cytotoxicity of CYNK-001 against several GBM lines and its in vivo anti-tumor activity in a U87MG orthotopic mouse model via intracranial administration resulting in 94.5% maximum reduction in tumor volume. We have developed a phase I window-of-opportunity trial of CYNK-001 in recurrent GBM via intravenous (IV) and intratumoral (IT) routes. In the IV cohort, subjects receive cyclophosphamide for lymphodepletion followed by 3-doses of IV CYNK-001 weekly. In the IT cohort, subjects undergo placement of an IT catheter with an ommaya reservoir followed by 3-doses of IT CYNK-001 weekly. Patients are monitored for 28-days after last infusion for toxicity. Once maximum safe dose (MSD) is determined, patients undergo IV or IT treatments at MSD followed by surgical resection and the tumor tissue will be analyzed for NK cell engraftment and persistence. We will utilize a 3 + 3 dose de-escalation design (maximum n=36). Primary endpoint is safety and feasibility. Secondary endpoints are overall response rate, duration of response, time to progression, progression free survival and overall survival. Main eligibility criteria include age ≥18, KPS ≥60, GBM at first or second relapse with a measurable lesion on ≤2mg dexamethasone. This is the first clinical trial to investigate CYNK-001 in GBM and will lay the foundation for future NK cell therapy in solid tumors.

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Apoptotic Effects of Corticosteroids on Pure CD34+ Hematopoietic Stem Cells and Modulation of This Effect with Cytokines

Blood ◽

10.1182/blood.v116.21.4770.4770 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4770-4770

Author(s):

Beyza Cetin ◽

Serap Erdem Kuruca ◽

Kadriye Akgun Dar ◽

Sabriye Karadenizli ◽

Ebru Gurel ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Immunohistochemical Staining ◽

Research Funding ◽

Caspase 3 ◽

Control Groups ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Apoptotic Effects

Abstract Abstract 4770 Hematopoietic stem cells which can self-renew, are very important for regeneration of hematopoietic system. Because, in chemotherapy applications, malign cells are killed but on the other hand normal cells are also damaged. It is known that corticosteroids, which is essential drug for all chemotherapy protocols, efficiently induce apoptosis not only in malign cells also in normal hematopoietic cells. But, there are no comprehensive studies about the apoptotic effects of glucocorticoids on pure CD34+ hematopoietic cell population. In several studies, it has been showed that hematopoietic cytokines can prevent apoptosis caused by chemotherapy and decrease toxic effects of this drugs. In this study, for the first time, we investigated apoptotic effects of corticosteroids on CD34+ hematopoietic stem cell population collected from human peripheral blood buffy coats by using immunomagnetic positive selection method and protective effects of hematopoietic cytokines. Buffy coats were obtained from standard blood collection bags of voluntary blood donors. Two control groups are composed that is not including cytokines in serum free medium (SFC) and induced with cytokine coctail that including interleukin 3, thrombopoietin, stem cell factor and flt3/flk2 ligand (CC). Test groups are dexamethasone group (D), prednisolone group (P) and groups induced with cytokines before drug application (CD, CP). High concentrations of dexamethasone (10-3 M) and prednisolone (10-3 M) were used in vitro in concordance with clinical treatment doses. To determine apoptotic mechanisms, immunohistochemical staining was carried out by using monoclonal antibodies against fas, caspase 3, sitocrom c, bax and bcl2. Then cells were counting and determining degree of peroxidase reactions were quantified using H-score. For statistical analysis paired t-test and one sample t-test were used. As a result, both dexamethasone and prednisolon induce apoptosis in pure CD34+ cells. When compared with control groups, corticosteroids caused significant increase for apoptotic fas, caspase 3, sitocrom c and bax (p<0,05), and a significant decrease for antiapoptotic bcl2 (p<0,05). In control that is not including cytokines (SFC) apoptosis induced and same results were determined. When cytokines were added before, it was observed a significant decrease for all apoptotic markers (p<0,05) and a significant increase for antiapoptotic bcl2 (p<0,05) (Figure 1). So that, corticosteroids induce apoptosis in human pure CD34+ hematopoietic stem cells and cytokines decrease apoptotic effects of this drugs and prevent apoptosis in vitro. As a results, our findings demonstrated that may be useful cytokine treatment in order to minimize hematopoietic toxicity and to protect CD34+ cells from high-dose corticosteroid damage.Figure 1:Pure CD34+ cells of peroxidase levels calculated using H-score by immunohistochemical stainingFigure 1:. Pure CD34+ cells of peroxidase levels calculated using H-score by immunohistochemical staining Disclosures: Cetin: Istanbul University Research Fund: Employment, Research Funding. Kuruca:Istanbul University Research Fund: Research Funding.

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Combinatorial Natural Killer Cell–based Immunotherapy Approaches Selectively Target Chordoma Cancer Stem Cells

Cancer Research Communications ◽

10.1158/2767-9764.crc-21-0020 ◽

2021 ◽

Vol 1 (3) ◽

pp. 127-139

Author(s):

Austin T.K. Hoke ◽

Michelle R. Padget ◽

Kellsye P. Fabian ◽

Anjali Nandal ◽

Gary L. Gallia ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Nk Cells ◽

Nk Cell ◽

Killer Cell ◽

Flow Cytometric Analysis ◽

In Vitro Cytotoxicity ◽

Programmed Death ◽

Tumor Types

Chordoma is a rare tumor derived from notochord remnants that has a propensity to recur and metastasize despite conventional multimodal treatment. Cancer stem cells (CSC) are implicated in chordoma's resistant and recurrent behavior; thus, strategies that target CSCs are of particular interest. Using in vitro cytotoxicity models, we demonstrated that anti-programmed death ligand 1 (anti–PD-L1; N-601) and anti-EGFR (cetuximab) antibodies enhanced lysis of chordoma cells by healthy donor and chordoma patient NK cells through antibody-dependent cellular cytotoxicity (ADCC). Treatment of NK cells with an IL15 superagonist complex (N-803) increased their cytotoxicity against chordoma cells, which was further enhanced by treatment with N-601 and/or cetuximab. PD-L1–targeted chimeric antigen receptor NK cells (PD-L1 t-haNKs) were also effective against chordoma cells. CSCs were preferentially vulnerable to NK-cell killing in the presence of N-601 and N-803. Flow cytometric analysis of a chordoma CSC population showed that CSCs expressed significantly more NK-activating ligand B7-H6 and PD-L1 than non-CSCs, thus explaining a potential mechanism of selective targeting. These data suggest that chordoma may be effectively targeted by combinatorial NK cell–mediated immunotherapeutic approaches and that the efficacy of these approaches in chordoma and other CSC-driven tumor types should be investigated further in clinical studies. Significance: Combinatory immunotherapy using NK-mediated approaches demonstrates robust antitumor activity in preclinical models of chordoma and selectively targets chordoma CSCs.

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Cytokine-Induced Memory-Like NK Cells with High Reactivity against Acute Leukemia Blasts and Solid Tumor Cells Suitable for Adoptive Immunotherapy Approaches

Cancers ◽

10.3390/cancers13071577 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1577

Author(s):

Matteo Tanzi ◽

Michela Consonni ◽

Michela Falco ◽

Federica Ferulli ◽

Enrica Montini ◽

...

Keyword(s):

Acute Leukemia ◽

Nk Cells ◽

Tumor Cells ◽

Cell Function ◽

Nk Cell ◽

Autologous Tumor ◽

Lymphoid Leukemia ◽

Hematopoietic Stem ◽

Solid Malignancies

The limited efficacy of Natural Killer (NK) cell-based immunotherapy results in part from the suboptimal expansion and persistence of the infused cells. Recent reports suggest that the generation of NK cells with memory-like properties upon in vitro activation with defined cytokines might be an effective way of ensuring long-lasting NK cell function in vivo. Here, we demonstrate that activation with IL-12, IL-15 and IL-18 followed by a one-week culture with optimal doses of Interleukin (IL-2) and IL-15 generates substantial numbers of memory-like NK cells able to persist for at least three weeks when injected into NOD scid gamma (NSG) mice. This approach induces haploidentical donor-derived memory-like NK cells that are highly lytic against patients’ myeloid or lymphoid leukemia blasts, independent of the presence of alloreactive cell populations in the donor and with negligible reactivity against patients’ non-malignant cells. Memory-like NK cells able to lyse autologous tumor cells can also be generated from patients with solid malignancies. The anti-tumor activity of allogenic and autologous memory-like NK cells is significantly greater than that displayed by NK cells stimulated overnight with IL-2, supporting their potential therapeutic value both in patients affected by high-risk acute leukemia after haploidentical hematopoietic stem cell transplantation and in patients with advanced solid malignancies.

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Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma

Frontiers in Immunology ◽

10.3389/fimmu.2021.668307 ◽

2021 ◽

Vol 12 ◽

Author(s):

Paul D. Bates ◽

Alexander L. Rakhmilevich ◽

Monica M. Cho ◽

Myriam N. Bouchlaka ◽

Seema L. Rao ◽

...

Keyword(s):

Tumor Growth ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Tnf Α

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

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Centromeric KIR AA Individuals Harbor Particular KIR Alleles Conferring Beneficial NK Cell Features with Implications in Haplo-Identical Hematopoietic Stem Cell Transplantation

Cancers ◽

10.3390/cancers12123595 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3595

Author(s):

Léa Dubreuil ◽

Bercelin Maniangou ◽

Patrice Chevallier ◽

Agnès Quéméner ◽

Nolwenn Legrand ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Killer Cell ◽

Allelic Polymorphism ◽

Functional Effect ◽

Cell Frequency ◽

Hematopoietic Stem ◽

Cell Responses ◽

Allele Expression

We have recently shown a broad disparity of Natural Killer (NK) cell responses against leukemia highlighting good and bad responders resting on the Killer cell Immunoglobulin-like Receptors (KIR) and HLA genetics. In this study, we deeply studied KIR2D allele expression, HLA-C recognition and functional effect on NK cells in 108 blood donors in combining high-resolution KIR allele typing and multicolor flow cytometry. The KIR2DL1*003 allotype is associated with centromeric (cen) AA motif and confers the highest NK cell frequency, expression level and strength of KIR/HLA-C interactions compared to the KIR2DL1*002 and KIR2DL1*004 allotypes respectively associated with cenAB and BB motifs. KIR2DL2*001 and *003 allotypes negatively affect the frequency of KIR2DL1+ and KIR2DL3+ NK cells. Altogether, our data suggest that cenAA individuals display more efficient KIR2DL alleles (L1*003 and L3*001) to mount a consistent frequency of KIR2DL+ NK cells and to confer an effective NK cell responsiveness. The transposition of our in vitro observations in the T-replete haplo-identical HSCT context led us to observe that cenAA HSC grafts limit significantly the incidence of relapse in patients with myeloid diseases after T-replete haplo-identical HSCT. As NK cells are crucial in HSCT reconstitution, one could expect that the consideration of KIR2DL1/2/3 allelic polymorphism could help to refine scores used for HSC donor selection.

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Cyclosporine A (CSA) Significantly Reduces the Cytotoxic Effects of In Vitro Expanded NK Cells: Implications for Adoptive NK Cell Therapy in the Setting of Allogeneic Hematopoietic Stem Cell Transplantation (HCT).

Blood ◽

10.1182/blood.v110.11.4899.4899 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4899-4899

Author(s):

Hisayuki Yokoyama ◽

Maria Berg ◽

Andreas Lundqvist ◽

J. Philip McCoy ◽

Shivani Srivastava ◽

...

Keyword(s):

Cell Proliferation ◽

Nk Cells ◽

Nk Cell ◽

Immunofluorescent Staining ◽

Hematopoietic Stem ◽

Trail Expression ◽

And Function ◽

The Impact ◽

Cells Cultured

Abstract The ability to expand NK cells in vitro has led to the recent initiation of protocols incorporating adoptive NK cell infusions after HCT. Calcineurin inhibitors such as CSA are commonly used to prevent graft versus host disease (GVHD) in HCT recipients. Recently, Hong et al found the phenotype and function of fresh NK cells cultured in vitro with CSA was altered, with CSA treated NK cell cultures having enhanced cytotoxicity against tumor targets. However, the impact of CSA on in vitro expanded NK cell function and phenotype has not been explored. We analyzed cell proliferation, IFN-gamma production, cell surface immunofluorescent staining and cytotoxicity against K562 and renal cell carcinoma cell lines by in vitro expanded vs freshly isolated NK cells cultured in physiological doses of CSA (40ng/ml, 200ng/ml, 1000ng/ml for 18hrs). Fresh NK cells were obtained from the PBMC of healthy donors using immunomagnetic beads to isolate CD56+/ CD3− cells. NK cells were expanded in vitro using irradiated EBV transformed B cells as feeder cells in media containing IL-2 [500U/ml] for 12–14 days. Comparing CSA containing cultures to controls, there was a significant reduction in IL-2 stimulated fresh NK cell proliferation (stimulation index 0.51± 0.1) and TRAIL expression (MFI 10.4 vs 3.01). Furthermore, an ELISA assay showed fresh NK cells treated with CSA had a significant reduction in IL-2 induced IFN-g production compared to controls (median 231 vs 57 pg/ml, p=0.025). In contrast, in vitro expanded NK cells cultured in CSA showed no significant reduction of proliferation or TRAIL expression. At the highest doses of CSA (1000ng/ml), minimal inhibition of K562 killing of freshly isolated NK cells was observed. In contrast, expanded NK cells cultured in CSA for 18 hours compared to controls had a significant reduction in the killing of K562 cells (E:T=10:1, median 66 vs 43% lysis, p=0.011) and RCC tumor cells (E:T=20:1, 14.8 vs 8.8%, p=0.043). Figure Figure These data confirm CSA alters the phenotype and function of CD3−/CD56 + NK cells. Importantly, CSA appears to have a deleterious effect on expanded NK cell tumor cytotoxicity that was not observed with fresh NK cells. These finding suggest the anti-tumor effects of in vitro expanded NK cells could be hindered when adoptively infused in HCT patients receiving CSA.

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In Vitro Generation of NK22 Cells From Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v116.21.108.108 ◽

2010 ◽

Vol 116 (21) ◽

pp. 108-108

Author(s):

Qin Tang ◽

Ahn Yong-Oon ◽

Peter Southern ◽

Bruce R. Blazar ◽

Jeffrey S Milller ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Nk Cells ◽

Lymphoid Tissue ◽

Culture System ◽

Stage Iv ◽

Stage Iii ◽

Hematopoietic Stem ◽

Secondary Lymphoid Tissue

Abstract Abstract 108 NK cells are the first lymphocytes to recover after allogeneic hematopotiec cell transplantation (allo-HCT). Rapid NK recovery after allo-HCT is associated with reduced treatment related mortality. Because NK cells elaborate inflammatory cytokines (IFN-g) and mediate cytotoxic killing of malignant cells, they are also implicated in graft vs. leukemia reactions. Curiously early after transplant, donor-derived NK cells are hypofunctional and immature. Over the past year, investigators identified a new category of NK cells, called NK22 cells. These cells are present in secondary lymphoid tissue, such as tonsils, lymph nodes and Peyer's patches. Previous investigators have not been able to identify NK22 cells in adult blood or UCB, likely due to lymphoid tissue homing receptor expression (CCR6 and CCR7). NK22 cells are CD56+/−CD117highCD94−IL-1bR+, a phenotype which overlaps with one previously used to describe NK progenitors (i.e., stage III immature NK cells). At present, it is not known whether NK22 cells are a distinct branch of the NK lineage or are NK developmental intermediates. NK22 cells are present in secondary lymphoid tissue at vanishingly small quantities (<1% of all mononuclear cells), thereby making the study of these cells challenging. Functionally, NK22 cells lack of “classical” NK functions (cytotoxicity and IFN-g production) and instead elaborate IL-22 in response to dendritic cell derived IL-1 and/or IL-23. IL-22 does not act on hematopoietic cells, but rather on mucosal tissues to induce proliferation, anti-apoptotic functions and the production of antimicrobial proteins (b defensins). NK22 cells also increase the expression of adhesion molecules on MSCs after co-culture, suggesting a role in secondary lymphoid generation and homeostasis. We have previously used a stromal cell based culture system to study NK development from hematopoietic stem cells. Briefly, CD34+ cells are cultured in the presence of IL-3 (for the first week), FLT-3L, SCF, IL-7 and IL-15 for ~4-5 weeks. At the end of this culture period, functional mature NK cells are obtained. Because this system closely recapitulates ontongeny, we hypothesized that it could be used to study NK22 development. At D28 of culture, we found that 90% (range=88-94%) of cells expressed CD56. Approximately 22% (range=16-28%) had a stage III immature NK cell phenotype (i.e., CD56+CD117highCD94−), of which ~87% (range=77-93%) also expressed IL-bR, a phenotype consistent with NK22 cells (n=5). We next purified CD56− and CD56+ cell populations in these cultures and neither showed IL-22 expression at rest. Following IL-1 and/or IL-23 stimulation, the CD56+ fraction made IL-22 transcripts (by qPCR) and protein by ELISA. We next purified the stage III (CD56+CD117highCD94−) and stage IV (CD56+CD117lowCD94+) fractions and found that only the stage III cells were capable of IL-22 production following IL-1/23 stimulation. Co-culture of NK22 cells (or their supernatant) with MSCs resulted in a >2 log increase in ICAM. Likewise, the supernatant of from activated stage III cells induced keritinocyte proliferation and production of antimicrobial compounds. In vitro derived NK22 cells were compared to freshly isolated NK22 cells from human tonsils and nearly identical staining patterns for ROR-gt, Acyl hydrocarbon receptor, NKp44, NKp46, CD127, CD161, CCR6 and CCR7 were observed. Lastly, purified CD56+CD117highCD94− cells could acquire IL-bR and then further differentiate into stage IV cells (CD56+CD117lowCD94+) in the presence of IL-15. However, this was less likely in the presence of IL-15 and IL-1b, suggesting that NK22 cells are developmental intermediates with specific functions (SLT homeostasis and maintenance of mucosal surfaces and immunity). Depending upon the environmental stimuli, these cells will either maintain their IL-22 producing capacity or develop into cytotoxic lymphocytes. These studies are the first studies to describe the generation of NK22 cells from hematopoietic stem cells. They also allow a better understanding of the developmental requirements and functions of these rare cells. Lastly, this simple culture system creates a new opportunity to use NK22 cells therapeutically to enhance SLT tissue repair and mucosal immunity after allo-HCT. Disclosures: No relevant conflicts of interest to declare.

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Irradiated NK-92 Targets AML Leukemic Stem Cells in Vivo and Gene-Modified CD16+NK-92 Mediates Antibody Dependent Cell Mediated Cytotoxicity (ADCC) Against CD123+ Cells

Blood ◽

10.1182/blood.v120.21.1909.1909 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1909-1909

Author(s):

Brent A. Williams ◽

Xing-Hua Wang ◽

Sonam Maghera ◽

Jeffrey V. Leyton ◽

Raymond Reilly ◽

...

Keyword(s):

Stem Cells ◽

Treatment Group ◽

Nk Cell ◽

Xenograft Model ◽

In Vitro Cytotoxicity ◽

Class I ◽

Leukemic Stem Cells ◽

Nsg Mice ◽

Dependent Cell

Abstract Abstract 1909 Introduction: Patients with acute myeloid leukemia (AML) in remission relapse frequently from residual leukemic stem cells (LSCs) that are CD34+CD38-CD123+. NK-92 is an infusible CD16- malignant NK cell line cytotoxic against AML and after irradiation for prevention of NK cell malignancy has minimal adverse events in Phase I trials. Here, we tested irradiated NK-92 (iNK-92) against primary AML in a NOD/SCID gamma null (NSG) xenograft model. To optimize killing of LSCs in vitro we utilized a gene modified CD16+NK-92 against the CD123+ leukemia cell line OCI/AML5 treated with and without an anti-CD123 monoclonal antibody (7G3) to facilitate antibody dependent cell mediated cytotoxicity (ADCC). Methods: NSG mice were irradiated with 225 or 325 cGy before infusion with primary AML blasts (1 or 3×10e6 cells). iNK-92 was started 1 or 10 days post AML infusion and given 1–3 times weekly (15 or 20×10e6 cells/dose) to a total dose of 60–75 ×10e6 cells/mouse. In vitro cytotoxicity was measured by the chromium release assay. OCI/AML5 or primary AML was pretreated with 7G3 or anti-Class I antibody respectively at 10 ug/ml 2 hours before ADCC assays. Results: We developed an NSG AML xenograft model using a CD34+CD38+ primary AML sample containing a small fraction of CD34+CD38- cells that were predominantly CD123+. 3×10e6 primary cells induced leukemia in NSG mice by 6 weeks and maintained comparable potency up to quaternary transplantations with a stable immunophenotype. In vitro cytotoxicity against LSCs was assessed by treating 1×10e6 first passage BM derived primary AML +/− 5×10e6 iNK-92 ×4 hours and injecting into 2 cohorts of 5 mice. At 6 weeks mice were sacrificed and bone marrow harvested. Average leukemic engraftment in the iNK-92 group (79.8, 3.48, 92.1, 81.3, 86.1, Av= 68.6%) was less than untreated AML inoculated group (95.0, 93.4, 19.4, 95, 97.3, Av=80.0%), but not statistically significant (p=0.62). Removing one poorly engrafted outlier mouse from each group yielded a higher engraftment in the control (Av=95.1%) versus the treatment group (Av=84.8%) and was significant (p=0.011). To test iNK-92 to treat engrafted leukemia, NSG mice were infused with 3×10e6 AML cells starting day 10 with 20×10e6 iNK-92 weekly ×6 doses. Survival was improved in the treatment group to near statistical significance (p=0.055), but all mice ultimately succumbed to disease. Addition of IL-2 to iNK-92 did not improve outcomes (p=0.13). 3×10e6 primary AML cells were also infused into 2 cohorts of 4 mice and treated +/− iNK-92 from day 2 and given 15×10e6 cells twice weekly to 75×10e6 total dose. BM (1×10e6 cells) from each of 4 primary recipients in control and treatment was serially transplanted 1:1 into four new NSG mice. BM engraftment occurred in all AML only cohort secondary mice (80.8, 93.3, 80.4, 96.4 Av=87.7%) while one mouse from iNK-92 group was leukemia free with engraftment at background levels of non-injected mice (96.4, 94.7, 1.8, 95.7 Av=72.2%). Proportion of LSCs in secondary transplanted mice was: AML (8.01, 9.48, 8.66, 5.25, Av=7.85%) and AML + iNK-92 (7.13, 3.46, 0.03, 4.0 Av=3.66%) which was statistically significant (p=0.05). CD16+NK-92 killed CD123+ OCI/AML5 cells at effector:target (E:T) ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis +/−SD: 35.0 +/−4.0, 9.0+/−6.6, -2.0 +/−0.1, 1.7 +/−3.3) and was significantly enhanced (2–6x) when targets were coated with anti-CD123 mAb (% lysis +/−SD: 64.3 +/−3.1, 48.5 +/−4.1, 20.9 +/−0.1, 10.1+/−3.3). Further, CD16+NK-92 also killed primary AML at E:T ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis +/−SD: 34.7 +/−4.6, 15.6 +/− 4.7, 11.5 +/− 2.0, 7.7 +/−1.6) which was significantly enhanced (2–3x) by coating with anti-Class I monoclonal antibodies (% lysis +/−SD: 64.8 +/−10.4, 31.1 +/− 8.9, 30.2 +/− 9.4, 23.9+/−2.8). Conclusion: iNK-92 reduces engraftment of AML and improves survival in a primary AML xenograft model. CD16+NK-92 cytotoxicity against primary AML is enhanced with anti-class I antibodies via ADCC, demonstrating that CD16+NK-92 can be redirected against bulk primary leukemia using a highly expressed cell surface marker. Finally, CD16+NK-92 cytotoxicity can be improved against OCI/AML5 with anti-CD123 antibodies via ADCC, providing the first proof-of-principle for the targeting of leukemic stem cells by combining humoral and cellular approaches. Disclosures: No relevant conflicts of interest to declare.

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