Abstract
Efficacious and accessible sources of natural killer (NK) cells would widen their use as immunotherapeutics, particularly for solid cancers. Here, we show that human somatic cells can be directly reprogrammed into NK cells with a CD56brightCD16bright phenotype using pluripotency transcription factors and an optimized reprogramming medium. The directly reprogrammed NK cells have strong innate–adaptive immunomodulatory activity and are highly potent against a wide range of cancer cells, including difficult-to-treat solid cancers and cancer stem cells. Both directly reprogrammed NK cells bearing a cancer-specific chimeric antigen receptor and reprogrammed NK cells in combination with antibodies competent for antibody-dependent cell-mediated cytotoxicity led to selective anticancer effects with augmented potency. The direct reprogramming of human somatic cells into NK cells is amenable to the production of autologous and allogeneic NK cells, and will facilitate the design and testing of cancer immunotherapies and combination therapies.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. All data generated in this study, including source data and the data used to make the figures, are available from Figshare at https://figshare.com/s/93edb79d917acacdcd20. The microarray data are available from the Gene Expression Omnibus under accession number GSE132907. Source data are provided with this paper.
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Acknowledgements
We are grateful to I. Choi for insightful advice during the course of this investigation. This work was supported by the National Research Foundation of Korea (2020R1A2B5B02002252 and 2019M3A9H1103797), the National Research Council of Science and Technology (CRC-15-02-KRIBB) and the KRIBB Research Initiative Program (1711134084/KGM5502113).
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H.-S.K. and Y.S.C. conceived of the study idea. H.-S.K., B.S. and J.Y.K. developed the methodology. H.-S.K., B.S., J.Y.K., C.L.S., J.E.J. and Y.S.C. performed the investigation. H.-S.K., B.S. and J.Y.K. performed the statistical analyses. Y.S.C. provided resources. H.-S.K. and Y.S.C. wrote the original draft of the manuscript. H.-S.K. and Y.S.C. reviewed and edited the manuscript. Y.S.C. acquired funding.
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Extended data
Extended Data Fig. 1 Gene-expression profile in the reprogramming procedure.
a-c, RT-qPCR analysis of hematopoietic progenitor-related genes [C-KIT(CD117), CD27, CD34, CD38, CD49, and CD90] (a), NK-related genes (CD16, CD56, NCR1/NKP46, and NCR3/NKP30) (b), and T cell-related genes (CD3E, GATA3, HES1, and TCF1) (c) at the indicated days of reprogramming from CD3+ T cells. Mean ± SD (n = 3). Two-tailed Student’s t-test. *P < 0.001 (vs the starting cells, day 0).
Extended Data Fig. 2 Comparative cytolytic and cytokine-secretion activities of iNK cells, PBMC-NK cells and UCB-NK cells.
a, The cytolytic activity of the drNK cells, PBMC-NK cells, and UCB-NK cells against K562 blood cancer cells at the indicated E:T ratios. b, The cytolytic activity of the drNK cells, PBMC-NK cells, and UCB-NK cells against HepG2 liver cancer cells at the indicated E:T ratios. c, The cytokine (IFNγ, granzyme B, and TNF-α) secretion by drNK cells, PBMC-NK cells, and UCB-NK cells stimulated with or without cancer cells (K562 and HepG2) for 16 h at an E:T ratio of 1:1. Mean ± SD (n = 3 biological replicates for each sample). Two-tailed Student’s t-test. *P < 0.001 (vs no target cells).
Extended Data Fig. 3 T cells recruited by iNK cells invade solid tumours in vivo.
a, b, In T cell recruitment test in vivo (Fig. 6i), RT-qPCR analysis of T cell-specific genes (CD3D) (a), chemokines (CCL3, CCL4, and CCL5), and the cytokine IFN-γ in tumor specimens at day 8 (b). Mean ± SD (n = 3). Two-tailed Student’s t-test. *P < 0.001 (vs control).
Extended Data Fig. 4 Anticancer effects of iNK cells in the xenograft model.
a, Quantitative graph of the results shown in Fig. 8b. Quantification of bioluminescence signals at the indicated days (c). b, Representative images of tumors from the mice injected with PBS control, drNK-low (5.0 × 106), drNK cells (1.5 ×107), and doxorubicin (2 mg/kg) on day 28 post SW620-Luc inoculation. c, Anticancer effects of the indicated treatments were observed by assessing tumor volume in (b). Each symbol represents the tumor volume from the mouse in each group. Horizontal bars show the mean values, and error bars show the SD (n = 12 biological replicates for each sample). Two-tailed Student’s t-test. *P < 0.001 (vs no drNK cells). d, Infiltration of drNK cells in tumor xenografts, analyzed through both bioluminescence (tumor xenograft) and biofluorescence (DiR-drNK cells) 5 days after injection of drNK cells or PBS control.
Extended Data Fig. 5 Anticancer effects of CAR-iNK cells in the xenograft model.
a, b, Quantitative graph of the results shown in Fig. 8g. Quantification of bioluminescence signals at the indicated days (a) and on day 21 (b). Horizontal bars show the mean values, and error bars show the SD. Each symbol represents one tumor xenograft in each group. Mean ± SD (n = 8-10 biological replicates for each sample). Two-tailed Student’s t-test. *P < 0.001 (vs the control).c, Schematic of the in vivo anticancer activity assay of HER2-drNK cells. Mice bearing luciferase-expressing COLO 205 (COLO 205-Luc) xenografts were i.v. injected with either PBS, drNK cells, HER2-drNK cells, NK92 or HER2 CAR expressing NK-92 (HER2-NK92) on day 1. d, Representative bioluminescence images of the mice receiving the indicated treatments at the indicated days after COLO 205-Luc inoculation. Mean ± SD (n = 12 biological replicates for each sample). Mice bearing luciferase-expressing COLO 205 (COLO 205-Luc) xenografts were i.v. injected with either PBS, drNK cells, HER2-NK cells, NK-92 or HER2 CAR expressing NK-92 (HER2-NK-92) on day 1. e, f, Quantitative graph of the results shown in (d). Quantification of bioluminescence signals at the indicated days (e) and on day 21 (f). Horizontal bars show the mean values, and error bars show the SD. Each symbol represents one tumor xenograft in each group. Mean ± SD (n = 10-20 biological replicates for each sample). Two-tailed Student’s t-test. *P < 0.001 (vs the control).
Extended Data Fig. 6 T cells recruited by iNK cells promote iNK-cell antitumour effects.
a, Representative bioluminescence images of the mice receiving the indicated treatments at the indicated days after SW620-Luc inoculation. Mean ± SD (n = 6-12). Experimental design of in vivo anticancer activity assays of T cells recruited by drNK cells. The mice s.c. injected with luciferase-expressing SW620 cells (SW620-Luc) were i.v. injected with PBS or 1×107 drNK cells, activated T cells, or drNK cells and 5×106 activated T cells (n = 6 per group) on day 4. b, c, Quantitative graph of the results shown in (a). Quantification of bioluminescence signals at the indicated days (b) and on day 21 (c). Horizontal bars show the mean values, and error bars show the SD. Each symbol represents one mouse in each group. Mean ± SD (n = 6-8 biological replicates for each sample). Two-tailed Student’s t-test. *P < 0.001 (vs control).
Supplementary information
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Kim, HS., Kim, J.Y., Seol, B. et al. Directly reprogrammed natural killer cells for cancer immunotherapy. Nat Biomed Eng 5, 1360–1376 (2021). https://doi.org/10.1038/s41551-021-00768-z
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DOI: https://doi.org/10.1038/s41551-021-00768-z
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