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1040-F Novel phytochemicals-based media supports NK cell expansion using neither feeder cells nor IL-2, while achieving high cytotoxicity against cancer cells
  1. Matthew Forsberg1,
  2. Ankita Shahi De1,
  3. Marc A Gillig2,
  4. Rachit Ohri3 and
  5. Christian Capitini1
  1. 1University of Wisconsin-Madison, Madison, WI, USA
  2. 2Enable Life Sciences LLC, Farmington, CT, USA
  3. 3Onkologic INC., Worcester, MA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Feeder-free NK cell expansion reduces cost and simplifies the approval of downstream therapies. Eliminating the use of IL-2 has the added benefit of reducing cellular exhaustion, leading to enhanced cytotoxicity.1 2 As previously reported, the novel phytochemicals-based media Enable-NK consistently exhibits enhanced proliferation and cytotoxic activity of NK cells compared to multiple standard medias for NK cells. Here, optimization of NK cell expansion using Enable-NK was achieved in the absence of IL-2 and feeder cells.

Methods NK cells from three different donors were activated by overnight culture in the presence of IL-12 (10 ng/ml), IL-15 (50 ng/nl), and IL-18 (50 ng/ml), and half were non-activated. In the first experiment, activated and non-activated cells were expanded in Enable-NK media with only IL-15; this was compared to an expansion protocol using both IL-2 (100 IU/ml) and IL-15 (10 ng/ml). In the second experiment, expansion in Enable-NK was compared to control medium following activation, with IL-15 (10 ng/ml) only. Additionally, phenotype was assessed using flow cytometry, and cytotoxicity against the neuroblastoma cell line CHLA-20 was assessed using an Incucyte imager. The anti-GD2 monoclonal antibody Ch14.18 was added to assess the antibody-dependent cellular cytotoxicity (ADCC) capacity of the expanded NK cells.

Results In the first experiment, activated NK cells achieved greater expansion than non-activated cells. The greatest proliferation was in the IL-15-only activated culture, which expanded 54-fold in 21 days. In the second experiment, activated cells from 3 separate donors cultured in Enable-NK expanded an average of 34-fold in 13 days, with the activated control culture only expanding 15-fold (figure 1). Activated NK cells expanded using Enable-NK media limited the growth of CHLA-20 to 30.3% (± 2.19 SEM) of what was observed with CHLA-20 alone over the course of 68 hours (figure 2). This growth was further limited to 21.1% (± 3.52 SEM) with the addition of the anti-GD2 monoclonal antibody. In comparison, activated control expanded NK cells limited growth to 50.2% (± 2.38 SEM) and 37.8% (± 5.64 SEM) (without and with the anti-GD2 antibody, respectively). On average, 25% of activated Enable-NK expanded cells expressed the memory associated marker CD57, and 80% retained expression of CD16, which correlates with observed ADCC.

Conclusions Here we report a novel NK expansion method without the use of feeder cells or IL-2. With this method we have achieved robust expansion over the course of 2 to 3 weeks with enhanced cytotoxicity, including retention of ADCC functionality.


  1. Kundu S, Gurney M, O’Dwyer M. Generating natural killer cells for adoptive transfer: expanding horizons. Cytotherapy. 2021;23(7):559–566

  2. Hüber CM, Doisne JM, Colucci F. IL-12/15/18-preactivated NK cells suppress GvHD in a mouse model of mismatched hematopoietic cell transplantation. Eur J Immunol. 2015;45(6):1727–1735

Ethics Approval This study obtained ethics approval from the institutional review board MRR IRB for the University of Wisconsin-Madison (ID number 2017-1070-CR006). All participants gave informed consent before taking part in this study.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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