Clinical‐scale production of cGMP compliant CD3/CD19 cell‐depleted NK cells in the evolution of NK cell immunotherapy at a single institution

Williams, Shelly M.; Sumstad, Darin; Kadidlo, Diane; Curtsinger, Julie; Luo, Xianghua; Miller, Jeffrey S.; McKenna, David H.

doi:10.1111/trf.14564

Cited by 21 publications

(13 citation statements)

References 51 publications

(123 reference statements)

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“…We further showed that NK cells isolated by CD3/CD19-depletion exerted significantly higher cytotoxic activity than those isolated by CD56-enrichment. These findings are coherent to findings published by Williams et al (59). In addition to the common 2D cytotoxicity tests, we performed lysis experiments with tumor spheroids.…”

Section: Discussionsupporting

confidence: 93%

“…The median cell dose given in this study was with 2 × 10 8 NK cells kg/BW ~10–20 times higher compared to previous studies leading to a significant reduction in leukemia progression (68). Experience with NK cell expansion from CD3/CD19-depleted primary material was also published by Williams et al and van Ostaijen-ten Dam et al, however their cultivation with IL-2 and/or IL-15 was limited to overnight and 5 days, resulting in lower cell expansion compared to our protocol (59, 69).…”

Section: Discussionmentioning

confidence: 79%

See 1 more Smart Citation

The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

et al. 2019

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Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite therapeutic progress, prognosis in high-risk NB is poor and innovative therapies are urgently needed. Therefore, we addressed the potential cytotoxic capacity of interleukin (IL)-activated natural killer (NK) cells compared to cytokine-induced killer (CIK) cells for the treatment of NB. NK cells were isolated from peripheral blood mononuclear cells (PBMCs) by indirect CD56-enrichment or CD3/CD19-depletion and expanded with different cytokine combinations, such as IL-2, IL-15, and/or IL-21 under feeder-cell free conditions. CIK cells were generated from PBMCs by ex vivo stimulation with interferon-γ, IL-2, OKT-3, and IL-15. Comparative analysis of expansion rate, purity, phenotype and cytotoxicity was performed. CD56-enriched NK cells showed a median expansion rate of 4.3-fold with up to 99% NK cell content. The cell product after CD3/CD19-depletion consisted of a median 43.5% NK cells that expanded significantly faster reaching also 99% of NK cell purity. After 10–12 days of expansion, both NK cell preparations showed a significantly higher median cytotoxic capacity against NB cells relative to CIK cells. Remarkably, these NK cells were also capable of efficiently killing NB spheroidal 3D culture in long-term cytotoxicity assays. Further optimization using a novel NK cell culture medium and a prolonged culturing procedure after CD3/CD19-depletion for up to 15 days enhanced the expansion rate up to 24.4-fold by maintaining the cytotoxic potential. Addition of an IL-21 boost prior to harvesting significantly increased the cytotoxicity. The final cell product consisted for the major part of CD16−, NCR-expressing, poly-functional NK cells with regard to cytokine production, CD107a degranulation and antitumor capacity. In summary, our study revealed that NK cells have a significantly higher cytotoxic potential to combat NB than CIK cell products, especially following the synergistic use of IL-15 and IL-21 for NK cell activation. Therefore, the use of IL-15+IL-21 expanded NK cells generated from CD3/CD19-depleted apheresis products seems to be highly promising as an immunotherapy in combination with haploidentical stem cell transplantation (SCT) for high-risk NB patients.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 79%

The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

et al. 2019

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“…The donors were HLA haploidentical family members, who underwent unstimulated mononuclear cell collections by apheresis, with the product enriched for NK cells using large-scale immunomagnetic selection (CliniMACS, Miltenyi Biotech, Bergisch Gladbach, Germany) by depletion of CD3+ T cells, and CD19+ B cells in some protocols. NK-cellular products were prepared following good manufacturing practice conditions in the University of Minnesota Molecular and Cellular Therapy Facility, as previously described 12 . The donor NK cells were activated by incubation with IL-2 (or IL-15 in one protocol) overnight.…”

Section: Methodsmentioning

confidence: 99%

Natural Killer Cell Homing and Persistence in the Bone Marrow After Adoptive Immunotherapy Correlates With Better Leukemia Control

Grzywacz¹,

Moench²,

McKenna³

et al. 2019

Journal of Immunotherapy

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Cellular immunotherapy using allogeneic natural killer (NK) cells may overcome chemotherapyrefractory acute myeloid leukemia (rAML). Our goal was to document NK cell homing/ persistence in the bone marrow following adoptive immunotherapy. Our cohort included 109 patients who received NK cell therapy for rAML following lymphodepleting conditioning +/− denileukin diftitox, +/− low-dose total body irradiation. We evaluated the NK cell density in bone marrow core biopsies performed an average of 14 days after NK cell transfer using a CD56 immunohistochemical stain. The NK cell density in core biopsies showed only moderate correlation with NK cell percentage in bone marrow aspirates evaluated by flow cytometry (r s = 0.48) suggesting that distribution of CD56 positive cells in the bone marrow niche offers unique insight into NK cell homing. Better leukemia control was associated with increased NK cell density, such that patients with <5% blasts had a higher NK cell density (p=0.01). As well, NK cell density above the median of reference group was significantly associated with morphologic remission of leukemia (p=0.01). Moreover, the NK cell density varied significantly between conditioning protocols. Our findings suggest that the use of low dose irradiation or CD25-targeting immunocytokine (denileukin diftitox, IL2DT) as part of conditioning results in increased NK cell homing/persistence in the bone marrow. These novel results will help guide future immunotherapy with NK cells.

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“…However, current NK-based immunotherapy products face hurdles imposed mainly by relatively high variability in the efficacy of NKs derived from different donors. Furthermore, allogenic NK products are usually prepared from donor blood by apheretic depletion of CD3 and CD19 cells, resulting in a monocyte/NK cell mixture containing approximately only 30-40% of NK cells in the final product ( 150 ). Therefore, immunotherapy approaches may leverage iPSCs to generate an off-the-shelf supply of NK cells with a known haplotype in a highly standardized manner.…”

Section: Immune Cell Programming and Reprogrammingmentioning

confidence: 99%

Cell Fate Reprogramming in the Era of Cancer Immunotherapy

et al. 2021

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Advances in understanding how cancer cells interact with the immune system allowed the development of immunotherapeutic strategies, harnessing patients’ immune system to fight cancer. Dendritic cell-based vaccines are being explored to reactivate anti-tumor adaptive immunity. Immune checkpoint inhibitors and chimeric antigen receptor T-cells (CAR T) were however the main approaches that catapulted the therapeutic success of immunotherapy. Despite their success across a broad range of human cancers, many challenges remain for basic understanding and clinical progress as only a minority of patients benefit from immunotherapy. In addition, cellular immunotherapies face important limitations imposed by the availability and quality of immune cells isolated from donors. Cell fate reprogramming is offering interesting alternatives to meet these challenges. Induced pluripotent stem cell (iPSC) technology not only enables studying immune cell specification but also serves as a platform for the differentiation of a myriad of clinically useful immune cells including T-cells, NK cells, or monocytes at scale. Moreover, the utilization of iPSCs allows introduction of genetic modifications and generation of T/NK cells with enhanced anti-tumor properties. Immune cells, such as macrophages and dendritic cells, can also be generated by direct cellular reprogramming employing lineage-specific master regulators bypassing the pluripotent stage. Thus, the cellular reprogramming toolbox is now providing the means to address the potential of patient-tailored immune cell types for cancer immunotherapy. In parallel, development of viral vectors for gene delivery has opened the door for in vivo reprogramming in regenerative medicine, an elegant strategy circumventing the current limitations of in vitro cell manipulation. An analogous paradigm has been recently developed in cancer immunotherapy by the generation of CAR T-cells in vivo. These new ideas on endogenous reprogramming, cross-fertilized from the fields of regenerative medicine and gene therapy, are opening exciting avenues for direct modulation of immune or tumor cells in situ, widening our strategies to remove cancer immunotherapy roadblocks. Here, we review current strategies for cancer immunotherapy, summarize technologies for generation of immune cells by cell fate reprogramming as well as highlight the future potential of inducing these unique cell identities in vivo, providing new and exciting tools for the fast-paced field of cancer immunotherapy.

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Clinical‐scale production of cGMP compliant CD3/CD19 cell‐depleted NK cells in the evolution of NK cell immunotherapy at a single institution

Cited by 21 publications

References 51 publications

The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

Natural Killer Cell Homing and Persistence in the Bone Marrow After Adoptive Immunotherapy Correlates With Better Leukemia Control

Cell Fate Reprogramming in the Era of Cancer Immunotherapy

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