Highly efficient IL-21 and feeder cell-driven ex vivo expansion of human NK cells with therapeutic activity in a xenograft mouse model of melanoma

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

Download Full-text

Significant Ex-Vivo Expansion of Cord Blood (CB) Natural Killer (NK) Cells and Concomitant Decrease in CB T-Cells by Genetically Reengineered K562 Cells (K562-mbIL15-41BBL)

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2008.12.135 ◽

2009 ◽

Vol 15 (2) ◽

pp. 44-45

Author(s):

J. Hochberg ◽

B. Mar ◽

J. Ayello ◽

N. Day ◽

C. van de Ven ◽

...

Keyword(s):

T Cells ◽

Cord Blood ◽

Nk Cells ◽

Natural Killer ◽

Ex Vivo ◽

K562 Cells ◽

Ex Vivo Expansion ◽

Concomitant Decrease

Download Full-text

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

International Journal of Hematology ◽

10.1007/s12185-008-0028-0 ◽

2008 ◽

Vol 87 (2) ◽

pp. 217-224 ◽

Cited By ~ 5

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

Download Full-text

Ex Vivo Expansion of Highly Purified Human NK Cells..

Blood ◽

10.1182/blood.v114.22.2157.2157 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2157-2157

Author(s):

Chunji Gao ◽

Xiaohong Li ◽

Jian Ma ◽

Xiaoxiong Wu ◽

Feifei Wang ◽

...

Keyword(s):

Nk Cells ◽

High Purity ◽

Cell Expansion ◽

Ex Vivo ◽

Granzyme B ◽

Ex Vivo Expansion ◽

Biological Functions ◽

Hematopoietic Stem ◽

Significant Difference ◽

Ifn Γ

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

Download Full-text

22: Ex-vivo expansion (EvE) of previously cryopreserved cord blood (CB) into natural killer (NK) cells with enhanced AML and neuroblastoma cytotoxicity

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2006.12.025 ◽

2007 ◽

Vol 13 (2) ◽

pp. 10-11

Author(s):

J. Ayello ◽

P. Satwani ◽

C. van de Ven ◽

E. Shereck ◽

E. Roman ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Natural Killer ◽

Ex Vivo ◽

Ex Vivo Expansion

Download Full-text

Lycopene Prolongs the Lifespan and Enhances the Cytotoxicity of NK Cells after Ex Vivo Expansion

International Journal of Research in Science ◽

10.24178/ijrs.2015.1.1.08 ◽

2015 ◽

Vol 1 (1) ◽

pp. 8

Author(s):

Qi Li ◽

Ting Huyan ◽

Lin-Jie Ye ◽

Jun-Ling Shi ◽

Qing-Sheng Huang

Keyword(s):

Nk Cells ◽

Fruits And Vegetables ◽

Mononuclear Cells ◽

Ex Vivo ◽

Biological Effects ◽

Ex Vivo Expansion ◽

Peripheral Blood Mononuclear ◽

Study Results ◽

Ifn Γ ◽

Positive Effect

Lycopene is a nonprovitamin A carotenoid mainly found in fruits and vegetables, which has been reported to possess a variety of biological effects. The properties of lycopene on human natural killer (NK) cells after ex vivo expansion were assessed in the present study. Results showed that lycopene has a positive effect on NK cells viability and cytotoxicity. Aging and apoptosis started from the fourth week onwards in the cultured NK cells which were obtained from the peripheral blood mononuclear cells (PBMC). Supplemented with lycopene (5μM) can restore the decreased viability and cytotoxicity of NK cells and reduce NK cells apoptosis caused by aging during fourth-sixth week culture. Its anti-apoptosis effect in NK cells may be related to lycopene which can decrease the expression of caspase 3 and 9 genes. Furthermore, lycopene can enhance the IFN-γ expression in gene and protein level after 7d treatment. However, lycopene did not affect the functional receptor’s (NKG2A, NKG2D, NKp30 and NKp44) expression on NK cells. These results indicated that lycopene has a positive effect on NK cells. As a health product, it may help to prolong the lifespan and enhance the cytotoxicity of NK cells after ex vivo expansion.

Download Full-text

An improved protocol for ex vivo expansion of highly lytic NK cells from human umbilical cord blood CD34+ cells

Protocol Exchange ◽

10.1038/protex.2013.025 ◽

2013 ◽

Cited By ~ 2

Author(s):

Haiming Wei ◽

Fang Ni ◽

Rui Sun ◽

Binqing Fu ◽

Fuyan Wang ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Cd34 Cells ◽

Cord Blood Cd34

Download Full-text

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

Blood ◽

10.1182/blood-2018-99-118804 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2061-2061

Author(s):

Enli Liu ◽

Sonny Ang ◽

Yijiu Tong ◽

Li Li ◽

Rafet Basar ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Cell Activation ◽

Nk Cell ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

High Avidity ◽

Clinical Grade ◽

Antigen Presenting ◽

Promising Source

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

Download Full-text

Significant Ex-Vivo Expansion and Functional Activation of Cord Blood (CB) Natural Killer (NK) Cells with A Concomittant Significant Decrease in CB T Cells Following Stimulation with Genetically Engineered K562 Cells (K562-mb15-41BBL).

Blood ◽

10.1182/blood.v114.22.499.499 ◽

2009 ◽

Vol 114 (22) ◽

pp. 499-499

Author(s):

Jessica Hochberg ◽

Janet Ayello ◽

Carmella VandeVen ◽

Jeremy Gold ◽

Evan Cairo ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

Fold Increase ◽

Ex Vivo Expansion ◽

Genetically Engineered ◽

Cellular Immunotherapy ◽

Adoptive Cellular Immunotherapy

Abstract Abstract 499 Introduction: CD56+ NK subsets exhibit differential NK receptors (NKR) such as cytotoxicity profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors (NCR) involved with tumor target recognition, which, in part, may play a role in adoptive cellular immunotherapy (ACI) for malignancies (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by triggering receptors such as NCR (i.e. NKp46), and NKG2 surface receptors like NKG2D (Moretta et al, Curr Opin in Immunol, 2004, Marcenaro et al, Eur J Immunol, 2003). The major limitations of the use of NK cells in ACI include lack of tumor recognition and/or limited numbers of viable and functionally active NK cells (Shereck/Cairo et al. PBC, 2007). To circumvent these limitations, methods to expand and activate PB NK cells by genetic reengineering have been developed (Imai/Campana et al. Blood, 2005). It has been demonstrated that PB NK cells expanded with modified K562 cells expressing membrane bound IL-15 and 4-1BBL (K562-mb15-41BBL; Imai et al Blood, 2005) are significantly increased in number and maintain heterogeneous KIR expression (Fusaki/Campana et al, BJH, 2009) .We have previously reported the ex-vivo expansion, activation and cytolytic activity of CB NK cells with a cocktail of antibody and cytokines (Ayello/Cairo et al, BBMT, 2006; Ayello/Cairo, Exp Hem, 2009, In Press). Objective: In this study, we compared CB NK expansion and activation following stimulation with genetically engineered K562 cells (K562-mb15-41BBL, generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, TN) with wild-type (WT) K562 cells and NK cell characterization expressing inhibiting and activating KIRs, c-lectin, NCRs and NK cytolytic activation. Methods: Following irradiation with 100Gy, K562-mb15-41BBL or WTK562 were incubated at a 1:1 ratio with fresh CB MNCs at 37C, 5% CO2 for 7 days in RPMI-1640+10IU IL-2. NKR expression (KIR2DS4, NKG2D, NKG2A, CD94, KIR3DL1, KIR2DL2, Nkp46) and LAMP-1 (CD107a) receptor expression and NK cell phenotype (CD56 dim and bright subsets) were determined by flow cytometry. Results: On Day 0, NK cells population was 3.9±1.3%. After 7 days in culture, CB NK cells were significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). Concomitantly, there was a significant decrease in CB T cells vs WTK562 or media alone (15±2 vs 36±2 vs 51±7%, p<0.001),respectively. There was a significant increase in CD56bright vs CD56dim populations (67 vs 33%, p<0.01) following stimulation with K562-mb15-41BBL. Also, there was a 10-fold increase in CB NK cells expressing KIR3DL1 following stimulation with K562-mb15-41BBL vs WTK562 (p<0.01) and a 5-fold increase in NK KIR2DS4 expression (p<0.05), respectively. There was a significant increase in the expression of NK activation marker, CD107a, compared to WTK562 (51±0.7 vs 32±1.1,p<0.05). There was no change in CB NK cell expression of the c-lectin receptor, CD94/NKG2A and CD94/NKG2D after stimulation with K562-mb15-41BBL. A standard cryopreserved CB unit (25 ml) contains approximately 750×106 MNC. By using the smaller 5-ml aliquot (20%) of a two-aliquot bag (150×106 MNCs × 3.9%=5.8×106 NK cells), this expansion method would hypothetically yield 200×106 CB NK cells after 7 days stimulation with K562-mb15-41BBL. Conclusion: These results suggest that CB MNC can be ex-vivo expanded with K562-mb15-41BBL resulting in specific expansion of CB NK cells with increased NK KIR expression (KIR2DS4 and KIR3DL1) and NK activation (CD107a), along with a significant decrease in CB T cells. This expansion provides a means to enhance specific CB NK cell expansion for possible use for adoptive cellular immunotherapy in the post UCBT setting Disclosures: No relevant conflicts of interest to declare.

Download Full-text