Generation Of Clinical Grade, Highly Cytotoxic NK Cells Using Feeder Cell Free, Particle-Based Technology

NK cell immunotherapy shows exciting promise, but inconsistency and variability remain as a significant challenge. Since NK cells comprise a small fraction (∼5%) of the peripheral blood mononuclear cell fraction, expansion of NK cells in vivo or ex vivo is a critical requirement to attain therapeutically effective dosages and to observe consistent positive clinical outcomes. Most of currently developed ex vivo expansion protocols depend on co-culture with various engineered and/or cancer derived stimulator/feeder cells to induce the proliferation of NK cells. The use of accessory cells poses significant challenges to clinical transfer. Our laboratory has developed a nanoparticle-based expansion technology that utilizes particles, few hundred nanometers in size, derived from the plasma membrane (PM) of K562 feeder cells expressing IL-15 and 41BBL on their surface (PM-mb15-41BBL). These particles in combination with low concentration of IL-2 induce selective and efficient expansion of NK cells within human peripheral blood mononuclear cells (PBMC). When PBMC are stimulated with PM-mb15-41BBL over 21 days the NK cell numbers increase exponentially between days 6 and 18 of culture. The numbers of NK cell increased on average 200 fold (range 104-557, n=11, 4 donors) after 12-13 days of culture in the presence of PM-mb15-41BBL particles (at 200 µg of membrane protein/mL). The expansions with the PM particles are comparable to those in the presence of live feeder cells that gave ∼200 fold (79-895, n=11, 4 donors). The PM-particle based NK expansion is far better in comparison to NK stimulation with soluble purified 41BBL, IL-15 and IL-2, at matching concentrations, that yielded only 3 fold (1-4, n=6, 3 donors) increase in NK cells. Furthermore, the NK cells expand selectively under these conditions where they initially consisted only about 10% of the population of PBMC isolated from fresh peripheral blood, but increased to more than 95% of the cell suspension after 14 days in culture. The extent of expansion and NK cell content on day 12 of culture was dependent on the concentration of PM particles used with 200 µg of PM protein/mL being the optimal dose. Thus, PM nanoparticles can expand NK cells as efficiently and selectively as feeder cells. Furthermore, the PM-particle based expansion is more reproducible between trials and with different donors as compared to NK cell expansion induced with feeder cells (coefficient of variation 63% vs. 88%, respectively). The NK cells expanded in presence of PM-particles were highly cytotoxic against several leukemia cell lines and also against patient derived AML blasts. Expanded NK cells were 4 to 9 times more potent against AML cell lines K562, KG1 and HL-60 as compared to freshly isolated NK cells that were pre-activated with a high dose of IL-2. The PM-particle expanded NK cells also were selectively cytotoxic where they efficiently killed patient derived CD34+ leukemia blasts while sparing healthy CD34- peripheral blood cells. The expanded NK cells were observed to have an increase in the expression of major activating receptors such as NKG2D, NKp44, NKp30 and of the death receptor ligand FasL. This expression difference corresponds well with the activated cytotoxic phenotype and is likely responsible for their increased cytotoxicity against AML cells. Pilot trials in NSG mice are currently ongoing. Disclosures: Solh: Celgene: Speakers Bureau.

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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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Ex Vivo Expansion of Cord Blood NK Cell Have In Vivo Efficacy Against Leukemia.

Blood ◽

10.1182/blood.v110.11.2741.2741 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2741-2741 ◽

Cited By ~ 3

Author(s):

Dongxia Xing ◽

Wendy Fang ◽

William K. Decker ◽

Sufang Li ◽

Simon N. Robinson ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Cell Lines ◽

Mononuclear Cells ◽

Ex Vivo ◽

Leukemia Cell ◽

Ex Vivo Expansion ◽

Human Leukemia ◽

Feeder Cells

Abstract Introduction Allogeneic stem cell transplantation has demonstrated the ability to prevent leukemic relapse via an immune-mediated graft-vs.-leukemia effect. Natural killer (NK) cells have been shown to comprise a significant component of this anti-leukemia effect and have been reported to enhance engraftment and reduce graft-vs.-host disease. Ex-vivo expansion of peripheral blood-derived NK cells has been demonstrated. Cord blood (CB) is a promising alternate source of NK cells with enhanced cytokine/antigen-responsiveness, proliferation and increasing availability. We studied expansion conditions for the generation of CB NK cells for clinical use, and evaluated them in vivo in NOD-SCID/IL2Rgnull mice engrafted with human leukemia. Methods Two expansion protocols were compared: CD56+ NK cells were isolated and cultured with irradiated CD56− feeder cells in RPMI-1640 supplemented with 100ng/mL IL-2 for 21 days. CB mononuclear cells were depleted of CD3+ cells prior to culture. In vivo NK cytolytic activity was measured via chromium release assays on leukemia cell lines (K562, Nalm6) and fresh patient leukemia blasts. Functionality was confirmed in vivo as well by injecting 106 K562 cells or 2.5 ×104 Nalm6 cells into NOD-SCID/IL2Rgnull mice. 2 ×106 CB-expanded NK cells were administered on day +1, and an additional 5×106 CB NK cells were administered on day +7. NK killing of leukemic targets was then confirmed by flow cytometry. Results Feeder cells mediated >30-fold expansion of CB NK cells, generating a nearly pure population that contained 97% CD3−CD56+ cells. Expanded CB NK could lyse >90% of K562 targets at an E:T ratio of 10:1, as well as >40% of patient leukemia blast. In CD3− depleted cultures, similar expansions were observed, however only 55% of these cells were CD3−CD56+ with the remainder being 10% CD3+CD56+ NK/T and 35% CD3+ T lymphocytes. In the NOD-SCID~/IL2Rgnull model, ex vivo expanded CB NK cells demonstrated the ability to reduce the leukemia burden of both AML (K562) and ALL (Nalm6) cells >50%. Conclusions We have demonstrated the feasibility of expanding CB NK ex vivo to clinically-relevant doses with minimal manipulation. These expanded CB NK cells demonstrated cytotoxic activity in vitro and in vivo against a variety of human leukemic cell lines and patient leukemia blasts. These results provide rationale for immunotherapy of leukemia with CB-derived NK cells.

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Antigen Presenting Cell-Mediated Ex Vivo Expansion of Human Umbilical Cord Blood Cells Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity

Blood ◽

10.1182/blood.v116.21.2100.2100 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2100-2100

Author(s):

Nina Shah ◽

Dongxia Xing ◽

Dean A. Lee ◽

Laurence J.N. Cooper ◽

William Decker ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Cell Lines ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Research Funding ◽

Plasma Cells ◽

Nk Cell ◽

Ex Vivo ◽

Feeder Cells

Abstract Abstract 2100 Background: Multiple myeloma (MM) is the second most common hematologic malignancy in adults and, to date, is incurable. Allogeneic natural killer (NK) cells are active in various hematologic malignancies and may have a role against MM. Umbilical cord blood is a potential source for allogeneic NK cells and ex vivo expanded umbilical cord blood-derived NK (UCB-NK) cells demonstrate activity comparable to that of peripheral blood-derived NK cells. Here we demonstrate the anti-myeloma activity of UCB-NK cells expanded to clinical grade by a novel technique using artificial antigen presenting feeder cells (“K562 Clone 9” cells) modified to express IL-21 (“K562-cl9-mIL21”). Methods: Cord blood mononuclear cells (CBMCs) were cultured in 10% human serum albumin media with IL-2 (500 IU/ml) and K562-cl9-mIL21 feeder cells (2:1 feeder: CBMC ratio) for 21 days. Thereafter, cells were subjected to CD3-immunomagnetic depletion. CD3-negative cells were then used as effector cells in functional assays. Flow cytometry was used to confirm NK cell purity (C56+/CD3- cells) and a standard chromium-51 assay was performed to determine NK cell cytotoxicity. Targets included K562 cells, MM cell lines RPMI 8226, ARP-1 and U266, autologous non-neoplastic UCB cells (negative control) and bone marrow-derived CD138+ plasma cells from myeloma patients. Results: Expansion of CBMCs with K562-cl9-mIL21 yielded a >2000 fold expansion of NK cells, compared with 47 fold expansion of CD56-selected cells cultured with IL-2 alone (p <0.05). After CD3 depletion, UCB-NK cultures were comprised of 92% CD56+/CD3- cells. K562-cl9-mIL21-expanded UCB-NK cells demonstrated cytotoxicity against the classic NK cell target K562 as well as MM cell lines RPMI 8226, ARP-1 and U266 (Fig 1). In addition, these UCB-NK cells were also active against primary, bone marrow-derived CD138+ plasma cells from myeloma patients, an effect which was augmented by pre-incubation of UCB-NK cells with lenalidomide (Figure 2, NK=UCB-NK cells, NKR=UCB-NK cells+lenalidomide). Conclusions: UCB-NK cells can be expanded ex vivo to clinically relevant doses for allogeneic NK cell therapy via co-culture with K562-cl9-mIL21 feeder cells. Expanded UCB-NK cells are cytotoxic to myeloma cell lines and primary myeloma cells. Further development of UCB-NK cells as an adjunct therapy in stem cell transplantation for myeloma is warranted. Disclosures: Wang: Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding.

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High Anti-Tumor Activity of Natural Killer Cells after Non-Myeloablative Conditioning and Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v108.11.5146.5146 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5146-5146

Author(s):

Lars Fischer ◽

Olaf Penack ◽

Andrea Stroux ◽

Chiara Gentilini ◽

Eckhard Thiel ◽

...

Keyword(s):

Stem Cell ◽

Nk Cells ◽

Cell Lines ◽

Nk Cell ◽

Normal Values ◽

Leukemia Cell ◽

Myeloablative Conditioning ◽

Conventional Dose ◽

Leukemia Cell Lines ◽

Anti Tumor Activity

Abstract Background: It is hypothesized that enhanced graft-versus-leukemia activity of natural killer (NK) cells contributes to the clinical efficacy of non-myeloablative allogeneic hemotopoietic stem cell transplantation (HSCT). We compared NK cell cytotoxic activity after transplantation with full and reduced dose regimens with normal values obtained from healthy volunteers. Methods&Material: Using a flow cytometric assay that detects CD107a expression after coincubation with leukemia cell lines as a marker for NK cell degranulation we prospectively quantified and characterized NK cells mediating anti-tumor activity in patients after HSCT. Mononuclear cells (MNC) were isolated from peripheral blood of 17 healthy individuals and 31 patients transplanted with G-CSF mobilized peripheral blood stem cells at day +30. MNC were incubated with leukemia cell lines HL60 and K562 (effector/target ratio 1:1) and expression of CD107a was measured after 3 hours. The absolute number of degranulating (CD107a+) NK cells was calculated. Results: Thirty one patients (five with ALL, four with NHL, one with Aplastic Anemia and 21 with AML) were enrolled, of whome 22 were transplanted from a matched related donor, seven from a matched unrelated donor, and two from a haploidentical donor. Nine patients had been transplanted after conditioning with 2 Gy TBI only, whereas 22 patients had received various conventional dose regimens. NK cell counts at day +30 after HSCT with conventional conditioning were comparable to normal values, but percentage and number of degranulating cells were significantly reduced (2.4% and 7/μl vs. 6.2% and 18/μl; p<0.0001 and p=0.0003). In contrast, in patients after 2 Gy TBI the percentage of degranulating NK cells was comparable to healthy donors (7.6%; p=0.9), and interestingly, absolute numbers of all and of degranulating NK cells (41/μl; p=0.004) were higher than normal values and consequently higher than in patients with conventional dose conditioning. Conclusions: Using this new method we were able to exactly quantify tumor-reactive NK cells after HSCT in a feasible way. We found a high cytotoxic activity of NK cells towards leukemia cell lines in patients undergoing non-myeloablative conditioning with 2Gy TBI. Further studies to determine the clinical impact of these findings are needed.

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NK-Cell Reconstitution after HLA-Identical Blood and Marrow Transplantations for CML: The Recovery of NKG2D Is Inversely Correlated with NKG2A and It Promotes the GVL Effect.

Blood ◽

10.1182/blood.v110.11.4879.4879 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4879-4879

Author(s):

Juan Tong ◽

Huilan Liu ◽

Liangquan Geng ◽

Zimin Sun ◽

Baolin Tang ◽

...

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Nk Cell ◽

Linear Regression Analysis ◽

Cell Activity ◽

Target Cells ◽

Peripheral Blood Stem Cells ◽

Peripheral Blood Mononuclear ◽

Blood Stem Cells

Abstract Natural killer (NK) cell alloreactivity is reported to mediate strong graft versus leukemia (GVL) effect in patients after allogeneic stem-cell transplantation. NKG2D receptors recognize human MHC class Ichain related A and B (MICA/B) and UL16-binding protein 1∼4(ULBP 1∼4) on target cells, thereby regulating NK cell activity. To examine the recovery of NKG2D, NKG2A and other receptors expression by NK cells, we used flow cytometry to evaluate samples from 11 chronic myeloid leukemia patients and their donors in the year following unmanipulated HLA completely matched peripheral blood stem cells plus bone marrow transplantation. Peripheral blood mononuclear cells from patients and their donors were tested in standard 51Cr release assays against cultured K562 targets to determine the cytotoxicity of the NK cells in the same intervals. There is no mismatched immunoglobulin-like receptor (KIR) ligand in both GVH and HVG direction. The reconstitution of KIR2DL1 (CD158a) after this transplantation protocol was very slow and these receptors didn’t reach normal value in the year and KIR2DL2 (CD158b) was much better. The NKG2D increased and the NKG2A decreased quickly at the same time after engraftment, and used linear regression analysis we demonstrated that NKG2A recovery was inversely correlated with NKG2D recovery in the year following transplantation. The ratio of NKG2D/NKG2A was directly associated with the capacity of NK-cell cytotoxicity. Thus, the reconstitution of NKG2D makes contribution to the recovery of the NK cytotoxicity. These results reveals that the NK cells generated after HLA matched blood plus bone morrow transplantation of CML patients are promoted at an immature state characterized by specific phenotypic features and enhanced functioning, having potential impact for immune responsiveness and transplantation outcome.

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Co-Transfusion of Donor NK Cells in Haploidentical Stem Cell Transplantation

Blood ◽

10.1182/blood.v112.11.2907.2907 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2907-2907

Author(s):

Peter J. Lang ◽

Matthias Pfeiffer ◽

Heiko-Manuel Teltschik ◽

Ingo Mueller ◽

Tobias Feuchtinger ◽

...

Keyword(s):

Nk Cells ◽

Relapse Rate ◽

Peripheral Blood ◽

Nk Cell ◽

Ex Vivo ◽

Thymidine Uptake ◽

Nk Activity ◽

Specific Lysis ◽

The Mean

Abstract 39 pedatric patients with acute leukemias (ALL (n=19), AML (n=14) and MDS (n=6)) received T and B cell depleted grafts from full haplotype mismatched related donors. Depletion of the G-CSF stimulated leukapheresis products was carried out with CD3/CD19 coated magnetic microbeads and the CliniMACS device and resulted in a median number of 15.9×106 CD34 (2.5–41) stem cells, 147×106 CD56 NK-cells (9–552) and 413×106 CD14 monocytes (101–1100) per kg body weight. Median numbers of residual T and B cells were 56 000 (10 000–192 000) and 26 000 (2000–149 000) respectively. A reduced intensity regimen (melphalan (140mg/m2), thiotepa (10mg/kg), fludarabine (160mg/m2), OKT3 (0.1mg/kg)) was given in most patients. Co-transfused, HLA mismatched NK cells were traced in peripheral blood of 26 patients starting on day +1 with flow cytometry and appropriate HLA antibodies. Mean numbers of donor derived CD56+ cells/μl were: 3 (day 1), 22 (d 3), 17 (d 7), 75 (d 10), 197 (d 14). Theoretically, the mean absolute number of 4.8×106 co-transfused NK cells should have resulted in a mean number of 2000 cells/μl in peripheral blood of the patients. Comparison of this expected amount with the mean number of NK cells measured within the first week postransplant (25/μl, n=17 data points) showed, that only 1.2% of the cells remained in circulating blood. Thus, the majority of donor NKs did not circulate and probably homed to other compartments (bone marrow, lymph nodes). The number of NK cells cotransfused at day 0 partially influenced the speed of NK cell recovery: patients, who received > 100×106 donor NK cells/kg had significantly higher amounts of circulating cells at day 14 than patients, who received <100×106 donor NKs (240 vs. 140/μl, p<0.05). No significant difference was observed after d 14. Recovery of T cells was not influenced. Graft rejection occurred in 13%. This rate was similar to that of a historical control group (15% in patients who received CD34 positive selected grafts and standard conditioning regimens), although our study patients mainly received an intensity reduced regimen. We conclude, that co-transfused cells facilitated hematopoietic engraftment. Our approach resulted in low TRM (10% at d 365) and in a low relapse rate (20% at 2 years) in patients with microscopical remission (<5% blasts), but was insufficient in patients with active disease (80% relapse rate). We therefore investigated options to increase NK cell activity. Cytotoxicity against K562 cells and thymidine-uptake after PHA stimulation were measured prior and post depletion in 30 procedures. Median specific lysis at E:T ratio = 20:1 was 15% prior and 23% post depletion. Thus, NK activity was not hampered by the procedure. Specific lysis was significantly enhanced by pre-incubation with 1000 U/ml Interleukin (IL) 2 (44%, median) or 2ng/ml IL 12 (40%, median) or 1ng/ml IL 15 ( 53%, median) in vitro. In contrast, thymidine-uptake was reduced from 170 000 to 3000 counts due to profound T-cell-depletion. NK activity was weak against patient derived cryopreserved leukemic blasts without stimulation, but could be significantly increased by cytokine incubation in vitro. Therefore, a pilot study with infusions of IL 15 stimulated NK cells in vivo was started. Up to now, 6 patients received a total of 8 infusions with 12×106 - 150×106 ex vivo stimulated NK cells per kg bw without any side effects. Conclusions: co-transfusion of donor NK cells in haploidentical transplantation is feasible. Only a small portion of cells remained in circulating blood and homing to other organs is likely. NK activity could be increased by cytokines; the use of ex vivo IL 15 stimulated NK cells is currently evaluated. Clinical results suggest antileukemic and graft facilitating effects of donor NK cells.

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Early Evaluation Of Natural Killer Cell Reconstitution Following Unmanipulated Haploidentical Transplantation Compared With HLA-Identical Sibling Transplantation

Blood ◽

10.1182/blood.v122.21.5480.5480 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5480-5480

Author(s):

Isabel Gonzalez-Gascon y Marin ◽

Ana María Pérez-Corral ◽

Jorge Gayoso ◽

Javier Anguita ◽

Ana Carolina Franco ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cyclosporine A ◽

Peripheral Blood ◽

Nk Cell ◽

Lymphoid Cells ◽

High Dose ◽

Color Flow ◽

Activating Receptors ◽

Activating Receptor

Abstract Background The main functions of Natural Killer (NK) cells are early protection against viruses or tumour cells and production of cytokines that regulate immune functions. NK cells are the first lymphoid cells to repopulate the marrow after Stem Cell Transplantation (SCT) and reach normal levels within 1 month after transplant. Acquisition of both, inhibiting and activating receptors on developing NK cells is an important step in their functional maturation. Previous studies showed the beneficial effect of NK alloreactivity in prevention of relapse, especially in the setting of haploidentical SCT. The aim of this study is to compare the reconstitution of the NK cell compartment during the first 3 months after unmanipulated haploidentical peripheral blood SCT (Haplo) and HLA-identical sibling peripheral blood SCT (HLA-id). Patients and Methods 11 adult patients received SCT (7 Haplo and 4 HLA-id) at Gregorio Marañón Hospital (Madrid-Spain) from November 2012 to April 2013. Conditioning regimen comprised fludarabine, cyclophosphamide and busulfan for Haplo SCT and fludarabine and busulfan or fludaribine and melphalan for HLA-id SCT. Prophylaxis for acute graft-versus-host disease consisted of high dose cyclophosphamide on days +3 and +4, cyclosporine A and mycophenolate mofetil for Haplo and Cyclosporine A and methotrexate for HLA-id. Patient´s characteristics and transplant outcomes are shown in table 1. We analysed reconstitution patterns and phenotype of NK at day +15, +30, +60, and +90 after transplantation by multi-color flow cytometry on FC500 Beckman Coulter® cytometer using the following anti-human monoclonal antibodies: CD3 FITC, CD56 ECD, CD45 PC7, NKG2A PC7, NKp30 PC5, NKp44 PE, Nkp46 PC5, and NKG2D PE (Beckman Coulter®). For comparison between the two groups Mann–Whitney U-test was used. Results 2/7 patients who received Haplo SCT died early in the post-transplantation period (day +50 and +66), and were excluded of the analysis because NK cells were not recovered by those days. NK cells reached normal levels by day +30: median 71 cells/µl (21-1089)) after Haplo; median 213.5 cells/µl (113-499) after HLA-id, and remained at high levels through follow up, with no significant differences between the two groups. Similarly to previous studies, a large percentage of NKbright cells was observed at day +30 after Haplo (median 89% of NK cells (55-97%)), a percentage that tended to decrease at day +60 (30% (7-38%)) and +90 (35% (10-45%)). Interestingly the percentage of NKbright cells after HLA-id SCT at day +30 (median 14.5% of NK cells (6-30%)) compared with Haplo, was significantly lower (p=0.016). This was accompanied by a significantly lower expression of inhibitory receptor NKG2A after HLA-id SCT than after Haplo: 59.5% (50-62%) versus 92.5% (50-62%) at day +30; 54% (38-61%) versus 86% (70-88%) versus at day +60 (p=0.016). Activating receptors NKp44 and NKp30 showed a low expression after both types of SCT throughout the first 3 months after transplantation. By contrast, activating receptor NKp46 levels were significantly higher at day +30 after Haplo than after HLA-id SCT (93% (87-98%) versus 50% (37-51%)) (p=0.016). Finally, high and similar proportions of activating receptor NKG2D were observed in both types of SCT. Figure 1 illustrates the recovery of the NK cell receptor phenotype for each type of SCT. Conclusions Our data showed an early and fast recovery of NK cells after Haplo and HLA-id SCT. However, phenotypic maturation of NK cells appears to be different for each type of transplant. NK cells generated after Haplo exhibit a more immature phenotype, characterized by a higher proportion of NKbright cells, and a higher expression of NKG2A at day +30. Interestingly expression of NKp46 was significantly higher after Haplo than after HLA-id SCT. Other authors have reported cytotoxic activity of these NK cells with high expression of NKp46, suggesting that cytotoxicity may be preserved in these immature NK cells. NKp30, NKG2D and NKp44 expression is less affected by the type of SCT. Acknowledgments This work has been partially supported by Project “Evaluación de la reconstitución inmune después del trasplante haploidéntico de progenitores hemopoyéticos sin depleción T” from Fundación Mutua Madrileña. Disclosures: No relevant conflicts of interest to declare.

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Cord Blood Derived Natural Killer Cells Engineered with a Chimeric Antigen Receptor Targeting CD19 and Expressing IL-15 Have Long Term Persistence and Exert Potent Anti-Leukemia Activity

Blood ◽

10.1182/blood.v126.23.3091.3091 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3091-3091 ◽

Cited By ~ 2

Author(s):

Enli Liu ◽

Yijiu Tong ◽

Gianpietro Dotti ◽

Barbara Savoldo ◽

Muharrem Muftuoglu ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Phase 1 ◽

Leukemia Cell ◽

Antigen Presenting Cells ◽

Caspase 9 ◽

Antigen Presenting

Abstract Natural killer (NK) cells are a major component of the innate immune system, possessing the ability to lyse their targets without the need for prior sensitization or specificity for antigen. Besides their classical role in providing potent antitumor and antiviral immunity, NK cells can reduce the risk of graft-versus-host disease (GVHD) by targeting host antigen-presenting cells, as well as activated alloreactive donor T cells, indicating that NK-mediated graft-versus-leukemia (GVL) responses may occur in the absence of GVHD. Although most groups have relied on autologous or adult peripheral blood donor-derived NK cells, we have studied umbilical CB as a potential source of NK cells because of their availability as an "off-the-shelf" frozen product and their potent preclinical activity against leukemia cells. To overcome the obstacle of limited numbers of NK cells in a single CB unit, we have established GMP-compliant conditions for the ex vivo expansion of clinically relevant doses of such cells. By using GMP grade K562-based artificial antigen-presenting cells (aAPCs), which express membrane-bound IL-21 (clone 9.mbIL21), to numerically expand highly functional and mature CB-derived NK cells. To further enhance the GVL effect independent of KIR-ligand mismatch, we have genetically modified human CB-derived NK cells with a retroviral vector, CAR19-CD28-zeta-2A-IL15 (CAR19/IL15), which incorporates the genes for CAR-CD19, IL-15 to enhance proliferation and survival, and the inducible caspase-9 molecule. CB-NK cells could be stably transduced with CAR19/IL15, proliferated efficiently in vitro and maintained superior effector function against CD19-expressing leukemia cell lines and primary CLL cells. Moreover, the effector functions of both NK-CAR and nontransduced NK cells against K562 were comparable, indicating that the genetic modification of CB-NK cells does not alter their intrinsic cytotoxicity against NK-sensitive targets. Because of concerns over autonomous, uncontrolled NK cell growth due to autocrine production of IL15, we also incorporated into our construct a suicide gene based on the inducible caspase-9 (IC9) gene. The addition of as little as 10 nM of the small molecule dimerizer CID AP20187 to cultures of iC9/CAR19/IL15+ NK cells induced apoptosis/necrosis of >60% of transgenic cells within 4 hours as assessed by annexin-V-7AAD staining. The infusion of CAR.CD19.IL15-transduced CB-NK cells into a NOD-SCID-gamma null model of lymphoblastic lymphoma (Raji model) resulted in impressive anti-tumor responses (Fig. 1). Moreover, adoptively infused CAR-transduced CB NK persisted for up to 70 days post-infusion (Fig. 2), supporting our hypothesis that IL-15 enhances the proliferation and survival of the engineered CB-NK cells. Based on these promising data, we now propose to manufacture a GMP-grade CAR19-CD28-zeta-2A-IL15 vector for a phase 1 dose escalation trial in patients with high risk B-cell leukemia. Disclosures Wierda: Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Research Funding. Rezvani:Pharmacyclics: Research Funding.

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Interleukin-15 is associated with disease severity in viral bronchiolitis

European Respiratory Journal ◽

10.1183/13993003.00642-2015 ◽

2015 ◽

Vol 47 (1) ◽

pp. 212-222 ◽

Cited By ~ 10

Author(s):

T. Ronan Leahy ◽

Ross McManus ◽

Derek G. Doherty ◽

Robert Grealy ◽

Tanya Coulter ◽

...

Keyword(s):

Nk Cells ◽

Disease Severity ◽

Peripheral Blood ◽

Nk Cell ◽

Severe Disease ◽

Single Centre ◽

Peripheral Blood Mononuclear ◽

Mirna Sequencing ◽

Interleukin 15 ◽

Study Participants

Disease severity in viral bronchiolitis in infancy is difficult to predict and has been linked to host innate immunity. The study aimed to investigate the innate cytokine interleukin-15 (IL-15) as a marker of disease severity.A prospective single-centre observational study was conducted in a university-affiliated paediatric teaching hospital, comparing children (0–18 months) hospitalised for viral bronchiolitis, those admitted to the paediatric intensive care unit with severe disease and healthy age-matched controls. IL-15-related parameters were compared between groups. PCR and microRNA (miRNA) sequencing was undertaken on natural killer (NK) cells collected from study participants.Samples from 88 children with viral bronchiolitis and 43 controls enrolled between 2009 and 2012 were analysed. Peripheral blood mononuclear cell (PBMC) IL-15 mRNA expression was significantly higher in those with moderate severity bronchiolitis compared with controls and those with severe disease. Serum IL-15 levels correlated with disease severity. The relative frequency of NK cells in peripheral blood was significantly reduced in participants with bronchiolitis. The NK cell miRNA transcriptome in bronchiolitis was distinct. Targets of de-regulated miRNA were differentially expressed in bronchiolitis, including JAK3, STAT5A and NFKB1 on the IL-15 signalling pathway.IL-15 is associated with disease severity in children hospitalised with viral bronchiolitis.

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CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Blood ◽

10.1182/blood-2018-99-118926 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3199-3199 ◽

Cited By ~ 2

Author(s):

Subhashis Sarkar ◽

Sachin Chauhan ◽

Arwen Stikvoort ◽

Alessandro Natoni ◽

John Daly ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Advisory Committees ◽

Cd38 Expression ◽

Rpmi 8226

Abstract Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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