Establishment of an efficient ex vivo expansion strategy for human natural killer cells stimulated by defined cytokine cocktail and antibodies against natural killer cell activating receptors

Nakazawa, Tsutomu; Morimoto, Takayuki; Maeoka, Ryosuke; Matsuda, Ryosuke; Nakamura, Mitsutoshi; Nishimura, Fusanori; Yamada, Shuichi; Nakagawa, Ichiro; Park, Young Soo; Nakase, Hiroyuki; Tsujimura, Takahiro

doi:10.1016/j.reth.2022.07.001

Cited by 15 publications

(16 citation statements)

References 45 publications

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“…The present study showed that CBMCs expanded by our system included 0.05–0.09% and 0.00–0.03% of T cells at day 7 and 14, respectively. Using the same method to culture PBMCs, 0.22–3.5% of T cells were observed at day 14 [ 29 ]. We considered that a reason for the low T cell contamination in the expanded-CBNKCs was that T cells in CB had not exposed to specific antigens, which were almost virgin T cells.…”

Section: Discussionmentioning

confidence: 99%

“…The specific approach for human NKC expansion was carried out as previously outlined [ 29 ]. In brief, frozen CBMCs were sourced from RIKEN BRC derived from three volunteers (0 years old, 1 male, and 2 females).…”

Section: Methodsmentioning

confidence: 99%

“…The inhibition of growth in the NK activity-sensitive GBM cell line, T98G, by expanded NKC-containing populations was examined using xCELLigence RTCA DP (real-time cell analysis dual purpose) instruments (ACEA Biosciences, San Diego, CA, USA), as previously detailed [ 29 , 30 , 32 ]. In brief, 100 μL of complete medium was added to each well on an E-plate 16 (ACEA Biosciences).…”

Section: Methodsmentioning

confidence: 99%

“…We have previously established an effective ex vivo expansion system for highly purified NKCs derived from human PB, employing a combination of cytokines and antibodies targeting NK receptors in a feeder-free environment [ 28 , 29 ]. Moreover, its anti-tumor effect in allogeneic glioblastoma (GBM) was also reported for clinical application [ 28 , 30 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

An efficient feeder-free and chemically-defined expansion strategy for highly purified natural killer cells derived from human cord blood

Nakazawa

Maeoka

Morimoto

et al. 2023

Regenerative Therapy

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

An efficient feeder-free and chemically-defined expansion strategy for highly purified natural killer cells derived from human cord blood

Nakazawa

Maeoka

Morimoto

et al. 2023

Regenerative Therapy

Self Cite

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“…CD16 ligation also affects NK cell proliferation by enhancing the entry of NK cells into division 16 . Although several clones of anti-CD16 antibodies have been used in some studies as an effective method to expand NK cells 17 , 18 , a direct comparison of anti-CD16 Ab clones for the expansion and activation of primary NK cells has not been reported.…”

Section: Introductionmentioning

confidence: 99%

Comparison of the different anti-CD16 antibody clones in the activation and expansion of peripheral blood NK cells

Kim

Phan

Hwang³

et al. 2023

Sci Rep

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Natural killer (NK) cells are promising tool for cancer treatment. Methods have been developed for large-scale NK cell expansion, including feeder cell-based methods or methods involving stimulation with NK cell activating signals, such as anti-CD16 antibodies. Different clones of anti-CD16 antibodies are available; however, a comprehensive comparison of their differential effects on inducing NK cell activation and expansion has not been conducted among these various clones under the same experimental conditions. Herein, we found that the NK cell expansion rate differed depending on the various anti-CD16 antibodies (CB16, 3G8, B73.1, and MEM-154) coated on microbeads when stimulated with genetically engineered feeder cells, K562‑membrane-bound IL‑18, and mbIL‑21 (K562‑mbIL‑18/-21). Only the CB16 clone combination caused enhanced NK cell expansion over K562‑mbIL‑18/-21 stimulation alone with similar NK cell functionality. Treatment with the CB16 clone once on the initial day of NK cell expansion was sufficient to maximize the combination effect. Overall, we developed a more enhanced NK expansion system by merging a feeder to effectively stimulate CD16 with the CB16 clone.

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A “Prime and Expand” strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy

Shrestha,

Dee,

Chaturvedi

et al. 2024

Cancer Immunol Immunother

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Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming (“Prime” step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 (“Expand” step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting “Prime and Expand” ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the “Prime and Expand” strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

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Establishment of an efficient ex vivo expansion strategy for human natural killer cells stimulated by defined cytokine cocktail and antibodies against natural killer cell activating receptors

Cited by 15 publications

References 45 publications

An efficient feeder-free and chemically-defined expansion strategy for highly purified natural killer cells derived from human cord blood

An efficient feeder-free and chemically-defined expansion strategy for highly purified natural killer cells derived from human cord blood

Comparison of the different anti-CD16 antibody clones in the activation and expansion of peripheral blood NK cells

A “Prime and Expand” strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy

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