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

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2741-2741 ◽  
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.

Ex Vivo Expansion of Frozen Cord Blood Products without Selection.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2844-2844
Author(s):  
Ian K. McNiece ◽  
Jenny Harrington ◽  
Joshua Kellner ◽  
Jennifer Turney ◽  
Elizabeth J. Shpall
Keyword(s):  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
In Vivo Studies ◽  
Adherent Cells ◽  
Blood Products ◽  
Cd34 Cells ◽  
Duke University

Abstract Ex vivo expansion of cord blood products (CB) has been proposed as an approach to increase the number of cells available from a single CB unit. We and others have reported the requirement of CD34 selection for optimal expansion of CB products, however, the selection of frozen CB products results in significant losses of CD34+ cells with a median recovery of 43% (range 6 to 203%, N=40) and low purities resulting in decreased expansion. Therefore we explored approaches to expand CB without prior selection and have described the use of co-culture of CB mononuclear cells (MNC) on mesenchymal stem cells (MSC). In the present study we have evaluated the expansion of clinical CB products (provided by Duke University CB Bank CB). MNC were obtained after ficol separation of RBCs and 10% of the CB product was cultured on preformed layers of MSC in T150 flasks containing 50ml of defined media (Sigma Aldrich) plus 100 ng/ml each of rhSCF, rhG-CSF and rhTpo. After 6 days of culture, the non adherent cells were transferred to a Teflon bag and a further 50 ml of media and GFs added to the flask. Again at day 10, non adherent cells were transferred to the Teflon bag and media and growth factors replaced. At day 12 to 13 of incubation the cells were harvested, washed and total nucleated cell (TNC) counts and progenitor assays performed. In three separate experiments we have achieved greater than 20 fold expansion of TNC with a median of 22, and a median expansion of GM-CFC of 37 fold. Morphologic analysis demonstrated the expanded cells contained high levels of mature neutrophils and neutrophil precursors. In vivo studies in NOD/SCID mice also demonstrated that the expanded cells maintained in vivo engraftment potential. Clinical studies are being designed to evaluate the in vivo potential of CB MNC products expanded on MSC.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4503-4503
Author(s):  
Jerremiah Oyer ◽  
Igarashi Y Robert ◽  
Colosimo Dominic ◽  
Melhem M. Solh ◽  
Yasser Khaled ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
Free Particle ◽  
Leukemia Cell ◽  
High Dose ◽  
Feeder Cells ◽  
Peripheral Blood Mononuclear

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.

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 ◽  
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.

Ex vivo expansion of CD34+ and T and NK cells from umbilical cord blood for leukemic BALB/C nude mouse transplantation

2008 ◽  
Vol 87 (2) ◽  
pp. 217-224 ◽  
Author(s):  
Yaming Wei ◽  
Yinfeng Huang ◽  
Yinze Zhang ◽  
Huayou Zhou ◽  
Qiong Cao ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Nude Mouse ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion

Biomimetic Macroporous PCL Scaffolds for Ex Vivo Expansion of Cord Blood-Derived CD34+Cells with Feeder Cells Support

2017 ◽  
Vol 17 (8) ◽  
pp. 1700054 ◽  
Author(s):  
Xiuwei Pan ◽  
Qiong Sun ◽  
Yuanhao Zhang ◽  
Haibo Cai ◽  
Yun Gao ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Feeder Cells ◽  
Cd34 Cells

Influence Of DNA Methyltransferese Inhibitors On The Anti-Leukemic Effect Of Umbilical Cord Blood Derived NK Cells Against Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4496-4496
Author(s):  
Harry Dolstra ◽  
Jeannette Cany ◽  
Anniek B. van der Waart ◽  
Marleen Tordoir ◽  
Basav Nagaraj Hangalapura ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
Dnmt Inhibitors ◽  
Nsg Mice ◽  
Drug Concentrations

Natural killer (NK) cell-based immunotherapy is a promising adjuvant, relatively non-toxic therapy approach for AML. However, further improvement of NK cell-based therapy is needed to increase the clinical effect. In this regard, NK cells generated ex vivo from hematopoietic progenitor cells (HPC) may have significant clinical benefits over enriched NK cells from adult donors, including the ability to choose an appropriate killer-cell immunoglobuline-like receptor (KIR)-ligand or KIR B haplotype alloreactive donor, as well as the capacity to reach high therapeutic dosages. Previously, we reported a GMP-compliant, cytokine/heparin-based culture protocol for the ex vivo generation of highly active NK cells from CD34+ HPC isolated from cryopreserved umbilical cord blood (UCB) units. Expansion in closed, large-scale bioreactors yields a clinically relevant dose of NK cells with high purity and cytolytic activity against AML cells in vitro. Currently, a clinical phase I trial with these HPC-NK cells is ongoing in our hospital. Trafficking studies in NOD/SCID/IL2Rgnull (NSG) mice demonstrated that these HPC-NK cells migrate to the bone marrow (BM) as well as to lymphoid organs where in vivo expansion and maturation can take place. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Importantly, a single HPC-NK cell infusion combined with supportive IL-15 administration was shown to efficiently inhibit growth of K562 leukemia cells implanted in the femur of NSG mice, resulting in significant prolongation of mice survival. Furthermore, we investigated whether modulation by the DNA methyltransferase (DNMT) inhibitors Azacytidine (Aza) and Decitabine (Deci) could further potentiate the antileukemic effect of HPC-NK cells against AML cells. In concordance with previous reports, we observed a dose-dependent effect of Aza and Deci on the growth of the AML cell lines THP1 and KG1a. In subsequent NK cell killing assays, we used clinical relevant low drug concentrations to pre-treat AML cells that did not affect HPC-NK cell viability and cytolytic function. Interestingly, increased killing of pre-treated THP1 and KG1a cells by HPC-NK cells could be observed, which was correlated with an increase in the NKG2D ligand ULBP2, the DNAM-1 ligands CD112 and CD155 as well as TRAIL-R2. Notably, maintenance of low-dose DNMT inhibitors during the KG1a/NK co-culture resulted in pronounced AML growth inhibition. To examine the effect of DNMT inhibitors in vivo, THP1.LucGFP-bearing NSG mice were treated with increasing dose of both agents, which were administered according to current standard protocols applied in humans. Data indicated that treatment with Aza or Deci at dosage equivalent in human to 12.5 and 5 mg/m2 respectively was well tolerated with minimal and/or transient weight loss, and efficiently reduced the progression of THP-1.LucGFP cells in vivo. Currently, we explore whether HPC-NK cells and DNMT inhibitors can work together to combat AML in our xenograft models. These preclinical studies may provide a rationale to investigate the possible additive and/or synergistic anti-AML effects of adoptive HPC-NK cell transfer in combination with these DNMT inhibitors in AML patients. Disclosures: Tordoir: Glycostem Therapeutics: Employment. Spanholtz:Glycostem Therapeutics: Employment.

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