4-1BBL-Expressing aAPCs Attenuate IL-15-Induced NK Cell Expansion and Cytokine Production In Vitro but Induce NK Cell-Mediated Gvhd In Vivo

Abstract Abstract 2536 Methods to expand natural killer (NK) cells ex vivo for adoptive cell therapy are being explored to improve outcomes after allogeneic blood and marrow transplant (alloBMT). Artificial antigen presenting cells (aAPCs) can present cytokines and/or co-stimulatory molecules that can potentially improve expansion and activity. 4-1BBL (CD137L) has demonstrated mixed results on murine and human NK cells, but the impact on murine NK cell biology after alloBMT has not been explored. NK cells were harvested from either C57BL/6 (B6) or CB6F1 spleens and cultured ex vivo with a recombinant interleukin (IL)-15/IL-15 receptor alpha (Ra) complex in the presence or absence of a CD137L+ aAPC. Because IL-15 is typically presented in trans by IL-15Ra, the complex was utilized to potently increase agonist bioactivity. NK cells cultured with IL-15/IL-15Ra alone showed a peak of 20-fold expansion, but this expansion was decreased with the addition of CD137L+ aAPCs if the ratio of aAPC to NK cells was greater than 1:1. In the presence of IL-15/IL-15Ra, the impact of CD137L+ aAPCs on expression of the inhibitory receptors, Ly49C+I and activating receptor Ly49H was variable and strain dependent, with increased expression in B6 NK cells, but decreased expression in CB6F1 NK cells. The expression of major histocompatibility complex (MHC) class I was not affected in NK cells from either strain by the presence of CD137L+ aAPCs. The production of gamma interferon and tumor necrosis factor-a was robust in NK cells expanded by IL-15/IL-15Ra alone, but attenuated with the addition of CD137L+ aAPCs. Animal experiments showed that administration of NK cells expanded ex vivo with IL-15/IL-15Ra alone was well tolerated after T cell depleted MHC-mismatched alloBMT (CB6F1–>B6), but surprisingly the addition of CD137L+ aAPCs to cultures caused NK cells to induce GVHD-associated weight loss. In summary, IL-15/IL-15Ra expanded murine NK cells demonstrate increased cytokine production and do not cause toxicity when infused after alloBMT. The presence of CD137L+ aAPCs attenuated cytokine production and increased Ly49 receptor expression in NK cells from B6 mice. Remarkably, NK cells expanded by IL-15/IL-15Ra in the presence of CD137L+ aAPCs demonstrate increased propensity to cause GVHD. Ongoing studies are exploring the anti-tumor efficacy of IL-15/IL-15Ra expanded murine NK cells cultured in the presence and absence of CD137L. Disclosures: No relevant conflicts of interest to declare.

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Binding of Excreted and/or Secreted Products of Adult Hookworms to Human NK Cells in Necator americanus-Infected Individuals from Brazil

Infection and Immunity ◽

10.1128/iai.00419-08 ◽

2008 ◽

Vol 76 (12) ◽

pp. 5810-5816 ◽

Cited By ~ 15

Author(s):

Andréa Teixeira-Carvalho ◽

Ricardo T. Fujiwara ◽

Erik J. Stemmy ◽

Denise Olive ◽

Jesse M. Damsker ◽

...

Keyword(s):

Nk Cells ◽

Transforming Growth Factor ◽

Nk Cell ◽

Ex Vivo ◽

Interleukin 10 ◽

Receptor Expression ◽

Necator Americanus ◽

Ifn Γ ◽

The Impact

ABSTRACT The impact of the interaction between excreted and/or secreted (ES) Necator americanus products and NK cells from Necator-infected individuals was analyzed. We investigated the binding of ES products to NK cells, the expression of NK cell receptors (CD56, CD159a/NKG2A, CD314/NKG2D, CD335/NKp46, and KLRF1/NKp80), the frequency of gamma interferon (IFN-γ)-producing NK cells after whole-blood in vitro stimulation, and the capacity of N. americanus ES products to induce NK cell chemotaxis. In contrast to those from noninfected individuals, NK cells from Necator-infected individuals demonstrated no binding with N. americanus ES products. This phenomenon was not due to alterations in NK cell receptor expression in infected subjects and could not be reproduced with NK cells from uninfected individuals by incubation with immunoregulatory cytokines (interleukin-10/transforming growth factor β). Further, we found that a significantly greater percentage of NK cells from infected subjects than NK cells from uninfected individuals spontaneously produced IFN-γ upon ex vivo culture. Our findings support a model whereby NK cells from Necator-infected individuals may interact with ES products, making these cells refractory to binding with exogenous ES products. During N. americanus infection, human NK cells are attracted to the site of infection by chemotactic ES products produced by adult Necator worms in the gut mucosa. Binding of ES products causes the NK cells to become activated and secrete IFN-γ locally, thereby contributing to the adult hookworm's ability to evade host immune responses.

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

Vaccines ◽

10.3390/vaccines9111363 ◽

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.

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CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity

Blood ◽

10.1182/blood.2020006200 ◽

2020 ◽

Vol 136 (21) ◽

pp. 2416-2427 ◽

Cited By ~ 2

Author(s):

Meisam Naeimi Kararoudi ◽

Yuya Nagai ◽

Ezgi Elmas ◽

Marcelo de Souza Fernandes Pereira ◽

Syed Abbas Ali ◽

...

Keyword(s):

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Ex Vivo ◽

Human Monoclonal Antibody ◽

Metabolic Reprogramming ◽

Target Cells ◽

Plasma Cell Neoplasm ◽

Cd38 Expression ◽

The Impact

Abstract Multiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

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Dysregulation of Natural Killer Cells in Obesity

Cancers ◽

10.3390/cancers11040573 ◽

2019 ◽

Vol 11 (4) ◽

pp. 573 ◽

Cited By ~ 15

Author(s):

Donal O’Shea ◽

Andrew E. Hogan

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Biology ◽

Viral Infections ◽

Nk Cell ◽

Viral Immunity ◽

Host Protection ◽

Cell Functions ◽

Innate Lymphocytes ◽

The Impact

Natural killer (NK) cells are a population of lymphocytes which classically form part of the innate immune system. They are defined as innate lymphocytes, due to their ability to kill infected or transformed cells without prior activation. In addition to their cytotoxic abilities, NK cells are also rapid producers of inflammatory cytokines such as interferon gamma (IFN-γ) and are therefore a critical component of early immune responses. Due to these unique abilities, NK cells are a very important component of host protection, especially anti-tumour and anti-viral immunity. Obesity is a worldwide epidemic, with over 600 million adults and 124 million children now classified as obese. It is well established that individuals who are obese are at a higher risk of many acute and chronic conditions, including cancer and viral infections. Over the past 10 years, many studies have investigated the impact of obesity on NK cell biology, detailing systemic dysregulation of NK cell functions. More recently, several studies have investigated the role of NK cells in the homeostasis of adipose tissue and the pathophysiology of obesity. In this review, we will discuss in detail these studies and focus on emerging data detailing the metabolic mechanisms altering NK cells in obesity.

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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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KIR Ligation During Ex Vivo Expansion Allows Molding Of The KIR Repertoire

Blood ◽

10.1182/blood.v122.21.2288.2288 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2288-2288

Author(s):

Dean A. Lee ◽

Vladimir V Senyukov ◽

Jerome R Trembley

Keyword(s):

Nk Cells ◽

Cell Population ◽

Nk Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Cell Populations ◽

Hematopoietic Stem ◽

Hla Ligand ◽

Cell Subpopulations ◽

Hla Haplotypes

Abstract NK Cell subpopulations express tremendous diversity through polymorphisms, haplotypes, differential expression, and licensing of the Killer Immunoglobulin-like Receptors (KIR). KIR diversity affects both the predisposition to cancer, and the response to therapies such as hematopoietic stem cell transplantation. Clinical trials that take advantage of the anti-cancer properties of NK cells have been limited to choosing donors on the basis of KIR genotypes and/or HLA haplotypes. Moreover, adoptive immunotherapy approaches have been limited by low NK cell doses. The latter hurdle has been recently mitigated by methods for expanding clinical grade NK cells ex vivo. These approaches for growing large numbers of cells now enable investigation into selecting more potent NK cell subsets for increased therapeutic efficacy. We hypothesized that the desired KIR repertoire could be molded through inhibition of undesirable KIR populations by crosslinking with relevant anti-KIR antibodies during expansion with our previously described method, which produces a mean 30,000-fold expansion of NK cells in 3 weeks. First, we determined that maximum inhibition was obtained when anti-KIR antibodies were applied to previously activated NK cells, crosslinked with secondary antibody, and then restimulated for proliferation. Robust reduction of targeted KIR-positive populations could be achieved for each inhibitory KIR (Fig. 1). When pre-activated with anti-KIR2DL1 for one stimulation cycle, NK cells expressing this KIR were decreased by a median of 70.4% ± 19.3%. Similarly, KIR2DL2/3+ NK cells could be reduced by 56% ± 17.5%, and KIR3DL1+ NK cells could be reduced by 53.5% ± 16.3%. When anti-KIR antibodies were combined, similar suppression of multiple-KIR subpopulations was observed. Other NK cell receptors were not significantly affected during targeted KIR inhibition. We then assessed the resulting NK cell populations for degranulation responses to targets with selected HLA as KIR ligands. Inhibition of KIR-expressing subpopulations during expansion resulted in NK cell populations with enhanced degranulation against tumor cells expressing the HLA ligand of the targeted KIR. Importantly, the cytotoxicity of the bulk NK cell population against HLA-negative targets remained. These results indicate that KIR crosslinking during NK cell propagation enables significant reduction in the targeted KIR subpopulations, resulting in an NK cell population with a selective decrease in KIR inhibition. By utilizing antibody-controlled expansion for molding of the KIR repertoire according to patient HLA type, a personalized NK cell product may be produced with enhanced potency, improving NK cell immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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Cyclosporin Influences NK Cell Expansion, Activating and Inhibitory Receptor Expression and Cytokine Synthesis.

Blood ◽

10.1182/blood.v104.11.3868.3868 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3868-3868

Author(s):

Hongbo Wang ◽

Alisa Lee ◽

Michael R. Verneris

Keyword(s):

Nk Cells ◽

Multiplex Pcr ◽

Hematopoietic Cell Transplantation ◽

Cell Function ◽

Nk Cell ◽

Receptor Expression ◽

Activating Receptors ◽

Kir Receptors ◽

Cytokine Synthesis ◽

Cells Cultured

Abstract Mouse models demonstrate that natural killer (NK) cells play an important role after allogeneic BMT by mediating GVL effects. Human haploidentical transplantation extends these observations since KIR receptor mismatch between donor and recipient is associated with improved DFS for AML patients. In contrast to the above observations, most patients undergoing hematopoietic cell transplantation receive GVHD prophylaxis with immune suppressive drugs, such as cyclosporin A (CsA). Little is known about the effects of CsA on NK cells and to investigate this, mature peripheral blood NK cells were cultured in IL-2 (1,000 U/ml) with either CsA (1 μg/ml or 10 μg/ml) or vehicle (EtOH) for 7 days. Under these conditions, CsA resulted in a significant inhibition in NK cell (CD3−CD56+) expansion (p<0.05). Cell cycle analysis showed that compared to EtOH, more CsA treated cells were in G1, and less cells were in G2-M phase, demonstrating that CsA reduces the number of NK cells in cycle. Since NK cells recognize malignant targets using both activating and inhibitory cell surface receptors, we used FACS to investigate the expression of KIR receptors (CD158a, CD158b and NKB1) and activating receptors (NKG2D, NKp30, NKp44 and NKp46) on cells cultured with and without CsA. CsA induced changes in the intensity of one or more of the above receptors for all donors tested (n=12). When analyzed in aggregate, we found that compared to EtOH control, NK cells cultured in CsA frequently had reduced expression of KIR receptors (66.7% for CD158a, 50% for CD158b and 33.3% for NKB1) and rarely increased KIR expression (0% for CD158a, 16.6% for CD158b and 0% for NKB1). In contrast, when cells were cultured in CsA the change in expression of NK cell activating receptors was more variable since some receptors increased (33.3% for NKG2D, 33.3% for NKp30, 75% for NKp44 and 16.7% for NKp46) while others receptors decreased (25% for NKG2D, 50% for NKp30, 0% for NKp44 and 16.7% for NKp46). Because CsA affected NK cell receptor density, we performed cytotoxicity assays using both NK cell sensitive (K562) and NK cell resistant, LAK sensitive targets (Raji). NK cells cultured with CsA (for 1 week) had a slightly reduced capacity to kill both targets (E:T 5:1, 60.9%, 36.2%, 35.2% for K562 and 72.4%, 53.3%, 40.7% for EtOH, CsA 1μg/ml and 10μg/ml, respectively). Since CsA changed the expression of NK cell inhibitory and activating receptors, we tested whether this drug would influence the expression of other receptors important in NK cell function. To do this, multiplex PCR was used to analyze the expression of the chemokine receptors SDF-1, CCR 1–4 and CXCR 1–5. Relative to a GADPH control, there was no significant change in chemokine receptor expression after culture with CsA. Lastly, we investigated the effect of CsA on NK cell cytokine synthesis and secretion. Fewer IFN-γ secreting NK cells were present after PMA/ionmycin treatment in CsA containing cultures compared to EtOH controls. Using multiplex PCR, we consistently found that CsA treatment lead to either an induction or an increase in IL-5, IL-6, IL-8, IL-13 and TGF-β transcripts. Taken together these results demonstrate that CsA alters NK cells by inhibiting expansion, changing the density of NK cell inhibitory and activating receptors and shifts cytokine synthesis to a Th2 like pattern.

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Stromal Cells Support a Myeloid Pathway of Human NK Cell Differentiation.

Blood ◽

10.1182/blood.v110.11.1336.1336 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1336-1336

Author(s):

Bartosz J. Grzywacz ◽

Nandini Kataria ◽

Jeffrey S. Miller ◽

Michael R. Verneris

Keyword(s):

Nk Cells ◽

Stromal Cells ◽

Cell Biology ◽

Stromal Cell ◽

Nk Cell ◽

Specific Antigen ◽

Developmental Trajectory ◽

Receptor Expression ◽

Cd34 Cells ◽

Monocytic Lineage

Abstract Natural killer (NK) cells belong to the lymphocyte lineage; however a myeloid origin has been debated in the past based on nascent experimental evidence. We studied the in vitro development of human NK cells from UCB-derived CD34+ cells following culture with cytokines (IL15, IL7, SCF, FLT3L, IL3) on a murine fetal stromal cell line EL08.1D2 (Blood, 2006; 108: 3824–3833). We investigated the differential requirement of CD34+ subsets for stromal cell support. Limiting dilution experiments showed that CD34+ cells negative for phenotypic markers of NK commitment (CD7, CD161, integrin B7, CD122, CD45RA) absolutely require stromal cells and/or addition of hydrocortisone (HC) to differentiate into functional NK cells. Without stromal cells or HC those progenitors give rise to myeloid lineage cells, but not NK cells. Thus, we hypothesized that stromal cells could instruct myeloid precursors to convert to the NK lineage. Indeed, CD56+ cells generated in stroma supported cultures frequently co-express CD33 and CD13. To determine whether myeloid cells developing from CD34+ cells after 2–3 wk cultures could give rise to NK cells, we FACS sorted the CD56−CD33+CD13high and CD56−CD14+ populations. Such CD33+CD13high and CD14+ cells express macrosialin (CD68) and acquire lyzozyme (by FACS), confirming their myeloid characteristics. Sorted cells cultured further in cytokines alone (IL15, IL7, SCF, FLT3L) did not give rise to NK cells. However, in the presence of cytokines, stromal cells and HC, NK cells were generated. To exclude the possibility of NK cell contamination, CD33+CD13high and CD14+ cells were isolated from cultures of CD34+ cells in conditions not supportive of NK cell development (GM-CSF, IL3, FLT3L, SCF, without stroma, IL15 or IL7). Such cells gave the same results as above (i.e., NK cells developed only with stroma and HC). In additional studies, a fraction (∼16%) of CFU-GM colonies isolated from methylocellulose cultures could generate NK cells only in the presence of stromal cells, HC and cytokines, but not cytokines alone. As more of a definitive marker of the monocytic lineage, we used the surface expression of M-CSF receptor (CD115) on hematopoietic precursors. CD56−CD117+CD115+ and CD56−CD117+CD115− fractions were FACS sorted from 2–3 wk cultures of CD34+ cells. While both populations could differentiate into NK cells, only the CD115+ monocytic precursors required stromal cells. Quantitatively the CD117+CD115− cells were the main source of NK cells in this culture system. Notably the NK cells derived from CD115+ precursors were remarkably different, showing significantly higher expression of Killer Immunoglobulin-like Receptors (KIR: CD158a, CD158b and CD158e) than their CD115− derived counterparts (52% vs 15% KIR+, n=3, p=0.002). With respect to the repertoire of HLA-specific inhibitory receptors, NK cells derived from monocytic precursors resemble the dominant fraction of peripheral blood NK cells, including potentially alloreactive NK cells (KIR+CD94/NKG2A−). Collectively we present evidence that NK cells can be derived from developmental intermediates of the monocytic lineage and this differentiation pathway is dependent upon interaction with stroma. Our data indicate that the developmental trajectory shapes the pattern of inhibitory receptor expression on mature NK cells. Such findings have bearing on our understanding of NK cell biology, post transplant NK cell reconstitution and could explain the paucity of recognized immature NK cell leukemias coinciding with the occurrence of AML variants with NK specific antigen expression.

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41BBL-Based Activation and Expansion of Autologous Natural Killer Cells Results in Enhanced Activity Against Leukemia Including ALL

Blood ◽

10.1182/blood.v124.21.2293.2293 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2293-2293

Author(s):

Ekta Kapadia ◽

Elad Jacoby ◽

Mark Kohler ◽

Waleed Haso ◽

Christopher Daniel Chien ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Myelogenous Leukemia ◽

Hematopoietic Stem ◽

Enhanced Activity

Abstract Childhood leukemia is the most common pediatric malignancy. There are now excellent cure rates for these patients, however outcomes remain poor for those with refractory disease and for those who relapse after standard salvage therapies, with a disease recurrence of approximately 50%. Therefore, development of novel cellular therapies is essential to treat these refractory patients. Natural Killer (NK) cells generated from an allograft contribute to improved disease free survival after Hematopoietic Stem Cell Transplantation for leukemia when there is a KIR mismatch. This effect appears to be particularly potent in the setting of Acute Myelogenous Leukemia (AML) with less benefit demonstrated in Acute Lymphoblastic Leukemia (ALL). Preclinical studies have also suggested that activation and expansion of resting NK cells can enhance NK cell cytotoxicity and eliminate the need for KIR mismatch due to up-regulation of activating receptors. We are currently testing this approach in the clinic following a fully matched allogeneic transplant platform for leukemia. Our aim is to explore whether 41BB ligand (41BBL) and recombinant IL-15 (rIL-15) mediated ex vivo expansion of autologous NK cells results in enhanced activity against AML and ALL. The activation/expansion process may allow for the use of autologous NK cell infusions, thus eliminating the need for allogeneic NK cell donors. To test this hypothesis, we ex vivo expanded and activated NK cells derived from C57BL/6J (B6) mice using artificial Antigen Presenting Cells (aAPCs) containing 41BBL and rIL-15 for 7-14 days. NK cells were co-cultured with murine AML cells (C1498) and murine ALL cells (E2A-PBX) – both on B6 background. Controls included YAC cells (murine T-cell lymphoma cell line sensitive to NK cell killing) as well as Phorbol Myristate Acetate (PMA)/ionomycin. All cells were co-cultured for 5 hours prior to functional assessment of NK cells via CD107a degranulation. NK cells cultured with 41BBL aAPCs and rIL-15 had a 30-fold expansion in numbers (Figure 1) and an increase in purity to approximately 95-98% (NK1.1+, CD3–) by Day 7. In the absence of cytokine or aAPCs, cultured NK cells underwent rapid apoptosis. Functionally, although resting NK cells (harvested prior to assessment) expressed CD107a when cultured with YAC cells and PMA, only minimal degranulation was observed in the presence of autologous AML cells or ALL cells. In contrast, activated and expanded autologous NK cells displayed enhanced activity against ALL, AML, as well as YAC cells, while only minimal levels of CD107a were seen in the absence of targets (Figure 2). In vivo experiments with a single injection of activated and expanded NK cells did not result in prolonged survival of mice bearing either AML or ALL. Assessment of adoptively transferred NK cells demonstrated very transient persistence (<2 days) with no in vivo expansion, suggesting that repeated injections may be necessary for leukemia eradication. Future murine experiments will investigate the effect repeated injections of activated/expanded NK cells and/or the administration of rIL-15 will have on survival and leukemia eradication. In addition, the ability to activate and expand NK cells in culture provides an opportunity for lentiviral-based transduction with chimeric antigen receptor (CAR) vectors. We are currently testing this with a murine CD19 CAR. These experiments suggest that autologous activated and expanded NK cells may serve as a viable cellular therapy for pediatric patients with refractory/relapsed leukemia. As demonstrated in these in vitro experiments, autologous activated/expanded NK cells still show increased targeting of mouse AML and ALL cell lines despite the lack of KIR mismatch. Thus, they may serve as a potential platform for leukemia therapy, including ALL, which appear to be poor targets for resting NK cells. In addition, these cells demonstrate transient persistence in vivo, a potential advantage in the context of redirected cytotoxicity using CAR constructs that target antigens with broader expression in the hematopoietic compartment. Figure 1: <![if !vml]><![endif]> Figure 1:. <![if !vml]><![endif]> Figure 2: Figure 2:. Disclosures No relevant conflicts of interest to declare.

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Induction of NK Cell Reactivity Against Myeloid Leukemia By a Novel Fc-Optimized CD133 Antibody

Blood ◽

10.1182/blood.v126.23.3793.3793 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3793-3793 ◽

Cited By ~ 3

Author(s):

Kathrin Rothfelder ◽

Samuel Koerner ◽

Maya Andre ◽

Julia Leibold ◽

Philaretos Kousis ◽

...

Keyword(s):

Nk Cells ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Conflicts Of Interest ◽

Residual Disease ◽

Ex Vivo ◽

Leukemic Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor

Abstract NK cells largely contribute to the success of monoclonal antibody (mAb) application in cancer due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), a feature considered critical for therapeutic success. Up to now, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Recently, we reported on the development of mAb targeting CD133, which is expressed on a wide variety of tumor cells (Koerner et al., Blood 2014 124:2309). Here we extend our analyses and provide further data on the preclinical characterization of an Fc-engineered CD133 mAb for the treatment of myeloid leukemia. Compared to two other anti-human CD133 mAb (clones AC133 and W6B3), which both bound to primary AML and CML cells in 15/25 and 7/10 cases, respectively, clone 293C3 recognized the leukemic cells in 22/25 AML cases and 7/10 CML cases. Based on these results, clone 293C3 was chosen to generate chimeric mAb with either a wildtype Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance affinity to the activating Fc receptor CD16 on NK cells (293C3-SDIE). Treatment with 293C3-SDIE resulted in significantly enhanced activation, degranulation and lysis of primary CD133-positive AML cells by NK cells in allogeneic and autologous experimental ex vivo settings as compared to its wildtype counterpart. Considering the expression of CD133 on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow (BM) cells. In line with the observed lower expression levels of CD133 on healthy compared to malignant hematopoietic cells no relevant toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells was observed. Moreover, 293C3-SDIE did not induce lysis of of healthy BM cells by allogeneic or autologous NK cells. In a NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG) xenotransplantation model, induction of ADCC by treatment with 293C3-SDIE resulted in the elimination of patient AML cells by NK cells from a matched human donor. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound, in particular for the elimination of minimal residual disease in CD133 bearing leukemia in the context of allogenic SCT. Disclosures No relevant conflicts of interest to declare.

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